Publications

Abstract

ABSTRACT:

Authors: Sebastian Müller, Clara Baldin, Marco Groth, Reinhard Guthke, Olaf Kniemeyer, Axel A Brakhage, Vito Valiante

Date Published: 2nd Oct 2012

Publication Type: Not specified

Abstract (Expand)

Fungi produce a multitude of low-molecular-mass compounds known as secondary metabolites, which have roles in a range of cellular processes such as transcription, development and intercellular communication. In addition, many of these compounds now have important applications, for instance, as antibiotics or immunosuppressants. Genome mining efforts indicate that the capability of fungi to produce secondary metabolites has been substantially underestimated because many of the fungal secondary metabolite biosynthesis gene clusters are silent under standard cultivation conditions. In this Review, I describe our current understanding of the regulatory elements that modulate the transcription of genes involved in secondary metabolism. I also discuss how an improved knowledge of these regulatory elements will ultimately lead to a better understanding of the physiological and ecological functions of these important compounds and will pave the way for a novel avenue to drug discovery through targeted activation of silent gene clusters.

Author: Axel A Brakhage

Date Published: 26th Nov 2012

Publication Type: Not specified

Abstract (Expand)

Non-invasive imaging techniques in microbial disease models have delivered valuable insights in the intimate pathogen-host interplay during infection. Here we describe evaluation and validation of a transgenic bioluminescence reporter strain of the human-pathogenic mold Aspergillus fumigatus, one of the main fungal pathogens affecting immunocompromised individuals. Expression and surface display of the Gaussia princeps luciferase allowed sensitive and rapid detection of luminescence emitted from this strain after substrate addition, with photon fluxes strongly correlating to the amounts of fungal conidia or germlings. The reporter strain allowed spatio-temporal monitoring of infection in a cutaneous model of aspergillosis, where neutropenic mice maintained the fungal burden while immunocompetent ones were able to clear it entirely. Most importantly, antifungal therapy could be followed in this type of disease model making use of the bioluminescent A. fumigatus strain. In conclusion, combining sensitivity of the Gaussia luciferase with a surface display expression system in the fungal host allows longitudinal infection studies on cutaneous forms of aspergillosis, providing perspective on drug screening approaches at high-throughput.

Authors: Stefanie Donat, Mike Hasenberg, Tina Schäfer, Knut Ohlsen, Matthias Gunzer, Hermann Einsele, Jürgen Löffler, Andreas Beilhack, Sven Krappmann

Date Published: 2012

Publication Type: Not specified

Abstract (Expand)

FungiDB (http://FungiDB.org) is a functional genomic resource for pan-fungal genomes that was developed in partnership with the Eukaryotic Pathogen Bioinformatic resource center (http://EuPathDB.org). FungiDB uses the same infrastructure and user interface as EuPathDB, which allows for sophisticated and integrated searches to be performed using an intuitive graphical system. The current release of FungiDB contains genome sequence and annotation from 18 species spanning several fungal classes, including the Ascomycota classes, Eurotiomycetes, Sordariomycetes, Saccharomycetes and the Basidiomycota orders, Pucciniomycetes and Tremellomycetes, and the basal 'Zygomycete' lineage Mucormycotina. Additionally, FungiDB contains cell cycle microarray data, hyphal growth RNA-sequence data and yeast two hybrid interaction data. The underlying genomic sequence and annotation combined with functional data, additional data from the FungiDB standard analysis pipeline and the ability to leverage orthology provides a powerful resource for in silico experimentation.

Authors: Jason E Stajich, Todd Harris, Brian P Brunk, John Brestelli, Steve Fischer, Omar S Harb, Jessica C Kissinger, Wei Li, Vishal Nayak, Deborah F Pinney, Chris J Stoeckert, David S Roos

Date Published: 7th Nov 2011

Publication Type: Not specified

Abstract (Expand)

The human pathogenic fungus Aspergillus fumigatus normally lives as a soil saprophyte. Its environment includes poorly oxygenated substrates that also occur during tissue invasive growth of the fungus in the human host. Up to now, few cellular factors have been identified that allow the fungus to efficiently adapt its energy metabolism to hypoxia. Here, we cultivated A. fumigatus in an O2 -controlled fermenter and analysed its responses to O2 limitation on a minute timescale. Transcriptome sequencing revealed several genes displaying a rapid and highly dynamic regulation. One of these genes was analysed in detail and found to encode fungoglobin, a previously uncharacterized member of the sensor globin protein family widely conserved in filamentous fungi. Besides low O2 , iron limitation also induced transcription, but regulation was not entirely dependent on the two major transcription factors involved in adaptation to iron starvation and hypoxia, HapX and SrbA respectively. The protein was identified as a functional haemoglobin, as binding of this cofactor was detected for the recombinant protein. Gene deletion in A. fumigatus confirmed that haem-binding fungoglobins are important for growth in microaerobic environments with O2 levels far lower than in hypoxic human tissue.

Authors: F. Hillmann, , N. Beckmann, M. Cyrulies, M. Strassburger, T. Heinekamp, H. Haas, , ,

Date Published: 7th Jul 2014

Publication Type: Not specified

Abstract (Expand)

Verticillium hemipterigenum (anamorph Torrubiella hemipterigena) is an entomopathogenic fungus and produces a broad range of secondary metabolites. Here, we present the draft genome sequence of the fungus, including gene structure and functional annotation. Genes were predicted incorporating RNA-Seq data and functionally annotated to provide the basis for further genome studies.

Authors: F. Horn, A. Habel, D. H. Scharf, J. Dworschak, , , C. Hertweck,

Date Published: 24th Jan 2015

Publication Type: Not specified

Abstract (Expand)

Streptomyces iranensis HM 35 has been shown to exhibit 72.7% DNA-DNA similarity to the important drug rapamycin (sirolimus)-producing Streptomyces rapamycinicus NRRL5491. Here, we report the genome sequence of HM 35, which represents a partially overlapping repertoire of secondary metabolite gene clusters with S. rapamycinicus, including the gene cluster for rapamycin biosynthesis.

Authors: F. Horn, V. Schroeckh, T. Netzker, , ,

Date Published: 19th Jul 2014

Publication Type: Not specified

Abstract (Expand)

Aspergillus fumigatus is a saprophytic mold that can cause life-threatening infections in immunocompromised patients. In the lung, inhaled conidia are confronted with immune effector cells that attack the fungus by various mechanisms such as phagocytosis, production of antimicrobial proteins or generation of reactive oxygen intermediates. Macrophages and neutrophils can also form nitric oxide (NO) and other reactive nitrogen intermediates (RNI) that potentially also contribute to killing of the fungus. However, fungi can produce several enzymes involved in RNI detoxification. Based on genome analysis of A. fumigatus, we identified two genes encoding flavohemoglobins, FhpA, and FhpB, which have been shown to convert NO to nitrate in other fungi, and a gene encoding S-nitrosoglutathione reductase GnoA reducing S-nitrosoglutathione to ammonium and glutathione disulphide. To elucidate the role of these enzymes in detoxification of RNI, single and double deletion mutants of FhpA, FhpB, and GnoA encoding genes were generated. The analysis of mutant strains using the NO donor DETA-NO indicated that FhpA and GnoA play the major role in defense against RNI. By generating fusions with the green fluorescence protein, we showed that both FhpA-eGFP and GnoA-eGFP were located in the cytoplasm of all A. fumigatus morphotypes, from conidia to hyphae, whereas FhpB-eGFP was localized in mitochondria. Because fhpA and gnoA mRNA was also detected in the lungs of infected mice, we investigated the role of these genes in fungal pathogenicity by using a murine infection model for invasive pulmonary aspergillosis. Remarkably, all mutant strains tested displayed wild-type pathogenicity, indicating that the ability to detoxify host-derived RNI is not essential for virulence of A. fumigatus in the applied mouse infection model. Consistently, no significant differences in killing of DeltafhpA, DeltafhpB, or DeltagnoA conidia by cells of the macrophage cell line MH-S were observed when compared to the wild type.

Authors: K. Lapp, M. Vodisch, K. Kroll, M. Strassburger, O. Kniemeyer, T. Heinekamp,

Date Published: 11th Sep 2014

Publication Type: Not specified

Abstract (Expand)

The Tor (target of rapamycin) kinase is one of the major regulatory nodes in eukaryotes. Here, we analyzed the Tor kinase in Aspergillus fumigatus, which is the most important airborne fungal pathogen of humans. Because deletion of the single tor gene was apparently lethal, we generated a conditional lethal tor mutant by replacing the endogenous tor gene by the inducible xylp-tor gene cassette. By both 2DE and gel-free LC-MS/MS, we found that Tor controls a variety of proteins involved in nutrient sensing, stress response, cell cycle progression, protein biosynthesis and degradation, but also processes in mitochondria, such as respiration and ornithine metabolism, which is required for siderophore formation. qRT-PCR analyses indicated that mRNA levels of ornithine biosynthesis genes were increased under iron limitation. When tor was repressed, iron regulation was lost. In a deletion mutant of the iron regulator HapX also carrying the xylp-tor cassette, the regulation upon iron deprivation was similar to that of the single tor inducible mutant strain. In line, hapX expression was significantly reduced when tor was repressed. Thus, Tor acts either upstream of HapX or independently of HapX as a repressor of the ornithine biosynthesis genes and thereby regulates the production of siderophores.

Authors: C. Baldin, V. Valiante, T. Kruger, L. Schafferer, H. Haas, O. Kniemeyer,

Date Published: 26th May 2015

Publication Type: Not specified

Abstract (Expand)

Aspergillus fumigatus is a saprotrophic filamentous fungus and also the most prevalent airborne fungal pathogen of humans. Depending on the host's immune status, the variety of diseases caused by A. fumigatus ranges from allergies in immunocompetent hosts to life-threatening invasive infections in patients with impaired immunity. In contrast to the majority of other Aspergillus species, which are in most cases nonpathogenic, A. fumigatus features an armory of virulence determinants to establish an infection. For example, A. fumigatus is able to evade the human complement system by binding or degrading complement regulators. Furthermore, the fungus interferes with lung epithelial cells, alveolar macrophages, and neutrophil granulocytes to prevent killing by these immune cells. This chapter summarizes the different strategies of A. fumigatus to manipulate the immune response. We also discuss the potential impact of recent advances in immunoproteomics to improve diagnosis and therapy of an A. fumigatus infection.

Authors: T. Heinekamp, H. Schmidt, K. Lapp, V. Pahtz, , N. Koster-Eiserfunke, T. Kruger, O. Kniemeyer,

Date Published: 18th Nov 2014

Publication Type: Not specified

Abstract (Expand)

Studying the pathobiology of the fungus Aspergillus fumigatus has gained a lot of attention in recent years. This is due to the fact that this fungus is a human pathogen that can cause severe diseases, like invasive pulmonary aspergillosis in immunocompromised patients. Because alveolar macrophages belong to the first line of defense against the fungus, here, we conduct an image-based study on the host-pathogen interaction between murine alveolar macrophages and A. fumigatus. This is achieved by an automated image analysis approach that uses a combination of thresholding, watershed segmentation and feature-based object classification. In contrast to previous approaches, our algorithm allows for the segmentation of individual macrophages in the images and this enables us to compute the distribution of phagocytosed and macrophage-adherent conidia over all macrophages. The novel automated image-based analysis provides access to all cell-cell interactions in the assay and thereby represents a framework that enables comprehensive computation of diverse characteristic parameters and comparative investigation for different strains. We here apply automated image analysis to confocal laser scanning microscopy images of the two wild-type strains ATCC 46645 and CEA10 of A. fumigatus and investigate the ability of macrophages to phagocytose the respective conidia. It is found that the CEA10 strain triggers a stronger response of the macrophages as revealed by a higher phagocytosis ratio and a larger portion of the macrophages being active in the phagocytosis process.

Authors: K. Kraibooj, , C. M. Svensson, ,

Date Published: 9th Jun 2015

Publication Type: Not specified

Abstract

Not specified

Authors: D. H. Scharf, T. Heinekamp,

Date Published: 30th Jan 2014

Publication Type: Not specified

Abstract (Expand)

Mitogen activated protein kinases (MAPKs) are highly conserved in eukaryotic organisms. In pathogenic fungi, their activities were assigned to different physiological functions including drug adaptation and resistance. Aspergillus fumigatus is a human pathogenic fungus, which causes life-threatening invasive infections. Therapeutic options against invasive mycoses are still limited. One of the clinically used drugs is caspofungin, which specifically targets the fungal cell wall biosynthesis. A systems biology approach, based on comprehensive transcriptome data sets and mathematical modeling, was employed to infer a regulatory network and identify key interactions during adaptation to caspofungin stress in A. fumigatus. Mathematical modeling and experimental validations confirmed an intimate cross talk occurring between the cell wall-integrity and the high osmolarity-glycerol signaling pathways. Specifically, increased concentrations of caspofungin promoted activation of these signalings. Moreover, caspofungin affected the intracellular transport, which caused an additional osmotic stress that is independent of glucan inhibition. High concentrations of caspofungin reduced this osmotic stress, and thus decreased its toxic activity. Our results demonstrated that MAPK signaling pathways play a key role during caspofungin adaptation and are contributing to the paradoxical effect exerted by this drug.

Authors: R. Altwasser, C. Baldin, J. Weber, , O. Kniemeyer, , , V. Valiante

Date Published: 10th Sep 2015

Publication Type: Not specified

Abstract (Expand)

The genus Penicillium belongs to the phylum Ascomycota and includes a variety of fungal species important for food and drug production. We report the draft genome sequence of Penicillium brasilianum MG11. This strain was isolated from soil, and it was reported to produce different secondary metabolites.

Authors: F. Horn, , D. J. Mattern, G. Walther, , , V. Valiante

Date Published: 5th Sep 2015

Publication Type: Not specified

Abstract (Expand)

Farnesol, produced by the polymorphic fungus Candida albicans, is the first quorum-sensing molecule discovered in eukaryotes. Its main function is control of C. albicans filamentation, a process closely linked to pathogenesis. In this study, we analyzed the effects of farnesol on innate immune cells known to be important for fungal clearance and protective immunity. Farnesol enhanced the expression of activation markers on monocytes (CD86 and HLA-DR) and neutrophils (CD66b and CD11b) and promoted oxidative burst and the release of proinflammatory cytokines (tumor necrosis factor alpha [TNF-alpha] and macrophage inflammatory protein 1 alpha [MIP-1alpha]). However, this activation did not result in enhanced fungal uptake or killing. Furthermore, the differentiation of monocytes to immature dendritic cells (iDC) was significantly affected by farnesol. Several markers important for maturation and antigen presentation like CD1a, CD83, CD86, and CD80 were significantly reduced in the presence of farnesol. Furthermore, farnesol modulated migrational behavior and cytokine release and impaired the ability of DC to induce T cell proliferation. Of major importance was the absence of interleukin 12 (IL-12) induction in iDC generated in the presence of farnesol. Transcriptome analyses revealed a farnesol-induced shift in effector molecule expression and a down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor during monocytes to iDC differentiation. Taken together, our data unveil the ability of farnesol to act as a virulence factor of C. albicans by influencing innate immune cells to promote inflammation and mitigating the Th1 response, which is essential for fungal clearance. IMPORTANCE: Farnesol is a quorum-sensing molecule which controls morphological plasticity of the pathogenic yeast Candida albicans. As such, it is a major mediator of intraspecies communication. Here, we investigated the impact of farnesol on human innate immune cells known to be important for fungal clearance and protective immunity. We show that farnesol is able to enhance inflammation by inducing activation of neutrophils and monocytes. At the same time, farnesol impairs differentiation of monocytes into immature dendritic cells (iDC) by modulating surface phenotype, cytokine release and migrational behavior. Consequently, iDC generated in the presence of farnesol are unable to induce proper T cell responses and fail to secrete Th1 promoting interleukin 12 (IL-12). As farnesol induced down-regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, desensitization to GM-CSF could potentially explain transcriptional reprofiling of iDC effector molecules. Taken together, our data show that farnesol can also mediate Candida-host communication and is able to act as a virulence factor.

Authors: I. Leonhardt, S. Spielberg, M. Weber, D. Albrecht-Eckardt, M. Blass, R. Claus, D. Barz, K. Scherlach, C. Hertweck, J. Loffler, ,

Date Published: 19th Mar 2015

Publication Type: Not specified

Abstract (Expand)

Polymorphonuclear neutrophilic granulocytes (PMN) as cellular components of innate immunity play a crucial role in the defense against systemic Candida albicans infection. To analyze stimuli that are required for PMN activity during C. albicans infection in a situation similar to in vivo, we used a human whole-blood infection model. In this model, PMN activation 10 min after C. albicans infection was largely dependent on the anaphylatoxin C5a. Most importantly, C5a enabled blood PMN to overcome filament-restricted recognition of C. albicans and allowed efficient elimination of nonfilamentous C. albicans cph1Delta/efg1Delta from blood. Major PMN effector mechanisms, including oxidative burst, release of secondary granule contents and initial fungal phagocytosis could be prevented by blocking C5a receptor signaling. Identical effects were achieved using a humanized Ab specifically targeting human C5a. Phagocytosis of C. albicans 10 min postinfection was mediated by C5a-dependent enhancement of CD11b surface expression on PMN, thus establishing the C5a-C5aR-CD11b axis as a major modulator of early anti-Candida immune responses in human blood. In contrast, phagocytosis of C. albicans by PMN 60 min postinfection occurred almost independently of C5a and mainly contributed to activation of phagocytically active PMN at later time points. Our results show that C5a is a critical mediator in human blood during C. albicans infection.

Authors: , K. Bieber, R. Martin, T. Lehnert, , J. Loffler, R. F. Guo, N. C. Riedemann,

Date Published: 24th Dec 2014

Publication Type: Not specified

Abstract (Expand)

BACKGROUND: Natural killer (NK) cells are innate lymphocytes with potent cytotoxic activity. Whereas activity of NK cells has been demonstrated against the fungal pathogens Aspergillus fumigatus and Cryptococcus neoformans, little was known about their interaction with Candida albicans. METHODS: Primary human NK cells were isolated from buffy coats, primed with a cytokine cocktail and used for confrontation assays with C. albicans. Interaction was monitored and quantified using live cell imaging, confocal microscopy, flow cytometry, and enzyme-linked immunosorbent assay. RESULTS: Human NK cells actively recognized C. albicans, resulting in degranulation and secretion of granulocyte-macrophage colony-stimulating factor, interferon gamma, and tumor necrosis factor alpha . Uniquely, activation of NK cells was triggered by actin-dependent phagocytosis. Antifungal activity of NK cells against C. albicans could be detected and mainly attributed to secreted perforin. However, NK cells were unable to inhibit filamentation of C. albicans. Human polymorphonuclear neutrophils (PMNs) counteracted the proinflammatory reaction of NK cells by preventing direct contact between NK cells and the fungal pathogen. Activation of PMNs was enhanced in the presence of NK cells, resulting in increased fungicidal activity. CONCLUSIONS: Our results show a unique pattern of NK cell interaction with C. albicans, which involves direct proinflammatory activation and modulation of PMN activity. For the first time, phagocytosis of a pathogen is shown to contribute to NK cell activation.

Authors: J. Voigt, , M. Bouzani, , D. Barz, , ,

Date Published: 25th Oct 2013

Publication Type: Not specified

Abstract (Expand)

OBJECTIVES: Candida albicans is an important fungal pathogen that can cause life-threatening disseminated infections. To determine the efficacy of therapy in murine models, a determination of renal fungal burden as cfu is commonly used. However, this approach provides only a snapshot of the current situation in an individual animal and cryptic sites of infection may easily be missed. Thus, we aimed to develop real-time non-invasive imaging to monitor infection in vivo. METHODS: Bioluminescent C. albicans reporter strains were developed based on a bioinformatical approach for codon optimization. The reporter strains were analysed in vitro and in vivo in the murine model of systemic candidiasis. RESULTS: Reporter strains allowed the in vivo monitoring of infection and a determination of fungal burden, with a high correlation between bioluminescence and cfu count. We confirmed the kidney as the main target organ but additionally observed the translocation of C. albicans to the urinary bladder. The treatment of infected mice with caspofungin and fluconazole significantly improved the clinical outcome and clearance of C. albicans from the kidneys; however, unexpectedly, viable fungal cells persisted in the gall bladder. Fungi were secreted with bile and detected in the faeces, implicating the gall bladder as a reservoir for colonization by C. albicans after antifungal therapy. Bile extracts significantly decreased the susceptibility of C. albicans to various antifungals in vitro, thereby probably contributing to its persistence. CONCLUSIONS: Using in vivo imaging, we identified cryptic sites of infection and persistence of C. albicans in the gall bladder during otherwise effective antifungal treatment. Bile appears to directly interfere with antifungal activity.

Authors: , A. Luttich, , ,

Date Published: 20th Jun 2014

Publication Type: Not specified

Abstract (Expand)

Nitrogen is one of the key nutrients for microbial growth. During infection, pathogenic fungi like C. albicans need to acquire nitrogen from a broad range of different and changing sources inside the host. Detecting the available nitrogen sources and adjusting the expression of genes for their uptake and degradation is therefore crucial for survival and growth as well as for establishing an infection. Here, we analyzed the transcriptional response of C. albicans to nitrogen starvation and feeding with the infection-relevant nitrogen sources arginine and bovine serum albumin (BSA), representing amino acids and proteins, respectively. The response to nitrogen starvation was marked by an immediate repression of protein synthesis and an up-regulation of general amino acid permeases, as well as an up-regulation of autophagal processes in its later stages. Feeding with arginine led to a fast reduction in expression of general permeases for amino acids and to resumption of protein synthesis. The response to BSA feeding was generally slower, and was additionally characterized by an up-regulation of oligopeptide transporter genes. From time-series data, we inferred network interaction models for genes relevant in nitrogen detection and uptake. Each individual network was found to be largely specific for the experimental condition (starvation or feeding with arginine or BSA). In addition, we detected several novel connections between regulator and effector genes, with putative roles in nitrogen uptake. We conclude that C. albicans adopts a particular nitrogen response network, defined by sets of specific gene-gene connections for each environmental condition. All together, they form a grid of possible gene regulatory networks, increasing the transcriptional flexibility of C. albicans.

Authors: S. Ramachandra, , , , , S. Brunke

Date Published: 20th Mar 2014

Publication Type: Not specified

Abstract (Expand)

Unlike induced Foxp3(+) regulatory T cells (Foxp3(+) iTreg) that have been shown to play an essential role in the development of protective immunity to the ubiquitous mold Aspergillus fumigatus, type-(1)-regulatory T cells (Tr1) cells have, thus far, not been implicated in this process. Here, we evaluated the role of Tr1 cells specific for an epitope derived from the cell wall glucanase Crf-1 of A. fumigatus (Crf-1/p41) in antifungal immunity. We identified Crf-1/p41-specific latent-associated peptide(+) Tr1 cells in healthy humans and mice after vaccination with Crf-1/p41+zymosan. These cells produced high amounts of interleukin (IL)-10 and suppressed the expansion of antigen-specific T cells in vitro and in vivo. In mice, in vivo differentiation of Tr1 cells was dependent on the presence of the aryl hydrocarbon receptor, c-Maf and IL-27. Moreover, in comparison to Tr1 cells, Foxp3(+) iTreg that recognize the same epitope were induced in an interferon gamma-type inflammatory environment and more potently suppressed innate immune cell activities. Overall, our data show that Tr1 cells are involved in the maintenance of antifungal immune homeostasis, and most likely play a distinct, yet complementary, role compared with Foxp3(+) iTreg.

Authors: , R. G. Iannitti, A. De Luca, G. Giovannini, F. Fallarino, C. Berges, J. P. Latge, H. Einsele, L. Romani,

Date Published: 13th May 2014

Publication Type: Not specified

Abstract (Expand)

Beyond its well-documented role in reproduction, embryogenesis and maintenance of body tissues, vitamin A has attracted considerable attention due to its immunomodulatory effects on both the innate and the adaptive immune responses. In infectious diseases, vitamin A has been shown to have a host-protective effect in infections of bacterial, viral or protozoan origin. Nevertheless, its impact in fungal infections remains unknown. Meanwhile, the frequency of invasive mycoses keeps on growing, with Candida albicans being the major opportunistic fungal pathogen and associated with high mortality. In the present work, we explored the impact of all-trans retinoic acid (atRA), the most active metabolite of vitamin A, on the innate immune response against C. albicans in human monocytes. Our results show a strong immunomodulatory role for atRA, leading to a significant down-regulation of the fungi-induced expression and secretion of the pro-inflammatory cytokines TNFalpha, IL6 and IL12. Moreover, atRA significantly suppressed the expression of Dectin-1, a major fungal pattern recognition receptor, as well as the Dectin-1-dependent cytokine production. Both RAR-dependent and RAR-independent mechanisms seem to play a role in the atRA-mediated immunomodulation. Our findings open a new direction to elucidate the role of vitamin A on the immune function during fungal infections.

Authors: , A. Hanisch, J. Brauer, E. Klaile, K. A. Heyl, M. K. Mansour, J. M. Tam, J. M. Vyas,

Date Published: 17th Aug 2014

Publication Type: Not specified

Abstract (Expand)

Plant hormones involving salicylic acid (SA), jasmonic acid (JA), ethylene (Et), and auxin, gibberellins, and abscisic acid (ABA) are known to regulate host immune responses. However, plant hormone cytokinin has the potential to modulate defense signaling including SA and JA. It promotes plant pathogen and herbivore resistance; underlying mechanisms are still unknown. Using systems biology approaches, we unravel hub points of immune interaction mediated by cytokinin signaling in Arabidopsis. High-confidence Arabidopsis protein-protein interactions (PPI) are coupled to changes in cytokinin-mediated gene expression. Nodes of the cellular interactome that are enriched in immune functions also reconstitute sub-networks. Topological analyses and their specific immunological relevance lead to the identification of functional hubs in cellular interactome. We discuss our identified immune hubs in light of an emerging model of cytokinin-mediated immune defense against pathogen infection in plants.

Authors: M. Naseem, M. Kunz,

Date Published: 13th Feb 2014

Publication Type: Not specified

Abstract (Expand)

The diploid, polymorphic yeast Candida albicans is one of the most important human pathogenic fungi. C. albicans can grow, proliferate and coexist as a commensal on or within the human host for a long time. However, alterations in the host environment can render C. albicans virulent. In this review, we describe the immunological cross-talk between C. albicans and the human innate immune system. We give an overview in form of pairs of human defense strategies including immunological mechanisms as well as general stressors such as nutrient limitation, pH, fever etc. and the corresponding fungal response and evasion mechanisms. Furthermore, Computational Systems Biology approaches to model and investigate these complex interactions are highlighted with a special focus on game-theoretical methods and agent-based models. An outlook on interesting questions to be tackled by Systems Biology regarding entangled defense and evasion mechanisms is given.

Authors: , S. Germerodt, , , ,

Date Published: 30th Jun 2015

Publication Type: Not specified

Abstract (Expand)

More than 80 years after its discovery, penicillin is still a widely used and commercially highly important antibiotic. Here, we analyse the metabolic network of penicillin synthesis in Penicillium chrysogenum based on the concept of elementary flux modes. In particular, we consider the synthesis of the invariant molecular core of the various subtypes of penicillin and the two major ways of incorporating sulfur: transsulfuration and direct sulfhydrylation. 66 elementary modes producing this invariant core are obtained. These show four different yields with respect to glucose, notably (1/2), 2/5, 1/3, and 2/7, with the highest yield of (1/2) occurring only when direct sulfhydrylation is used and alpha-aminoadipate is completely recycled. In the case of no recycling of this intermediate, we find the maximum yield to be 2/7. We compare these values with earlier literature values. Our analysis provides a systematic overview of the redundancy in penicillin synthesis and a detailed insight into the corresponding routes. Moreover, we derive suggestions for potential knockouts that could increase the average yield.

Authors: M. T. Prausse, S. Schauble, ,

Date Published: 19th Aug 2015

Publication Type: Not specified

Abstract (Expand)

Fungal microorganisms frequently lead to life-threatening infections. Within this group of pathogens, the commensal Candida albicans and the filamentous fungus Aspergillus fumigatus are by far the most important causes of invasive mycoses in Europe. A key capability for host invasion and immune response evasion are specific molecular interactions between the fungal pathogen and its human host. Experimentally validated knowledge about these crucial interactions is rare in literature and even specialized host-pathogen databases mainly focus on bacterial and viral interactions whereas information on fungi is still sparse. To establish large-scale host-fungi interaction networks on a systems biology scale, we develop an extended inference approach based on protein orthology and data on gene functions. Using human and yeast intraspecies networks as template, we derive a large network of pathogen-host interactions (PHI). Rigorous filtering and refinement steps based on cellular localization and pathogenicity information of predicted interactors yield a primary scaffold of fungi-human and fungi-mouse interaction networks. Specific enrichment of known pathogenicity-relevant genes indicates the biological relevance of the predicted PHI. A detailed inspection of functionally relevant subnetworks reveals novel host-fungal interaction candidates such as the Candida virulence factor PLB1 and the anti-fungal host protein APP. Our results demonstrate the applicability of interolog-based prediction methods for host-fungi interactions and underline the importance of filtering and refinement steps to attain biologically more relevant interactions. This integrated network framework can serve as a basis for future analyses of high-throughput host-fungi transcriptome and proteome data.

Authors: , C. H. Luther, J. Balkenhol, , ,

Date Published: 4th Aug 2015

Publication Type: Not specified

Abstract (Expand)

In this work, we investigate optimality principles behind synthesis strategies for protein complexes using a dynamic optimization approach. We show that the cellular capacity of protein synthesis has a strong influence on optimal synthesis strategies reaching from a simultaneous to a sequential synthesis of the subunits of a protein complex. Sequential synthesis is preferred if protein synthesis is strongly limited, whereas a simultaneous synthesis is optimal in situations with a high protein synthesis capacity. We confirm the predictions of our optimization approach through the analysis of the operonic organization of protein complexes in several hundred prokaryotes. Thereby, we are able to show that cellular protein synthesis capacity is a driving force in the dissolution of operons comprising the subunits of a protein complex. Thus, we also provide a tested hypothesis explaining why the subunits of many prokaryotic protein complexes are distributed across several operons despite the presumably less precise co-regulation.

Authors: J. Ewald, M. Kotzing, M. Bartl,

Date Published: 1st May 2015

Publication Type: Not specified

Abstract (Expand)

BACKGROUND: Adjusting the capacity of metabolic pathways in response to rapidly changing environmental conditions is an important component of microbial adaptation strategies to stochastic environments. In this work, we use advanced dynamic optimization techniques combined with theoretical models to study which reactions in pathways are optimally targeted by regulatory interactions in order to minimize the regulatory effort that is required to adjust the flux through a complex metabolic network. Moreover, we analyze how constraints in the speed at which an organism can respond on a proteomic level influences these optimal targets of pathway control. RESULTS: We find that limitations in protein biosynthetic rates have a strong influence. With increasing protein biosynthetic rates the regulatory effort targeting the initial enzyme in a pathway is reduced while the regulatory effort in the terminal enzyme is increased. Studying the impact of allosteric regulation for different pathway topologies, we find that the presence of feedback inhibition by products of metabolic pathways allows organisms to reduce the regulatory effort that is required to control a metabolic pathway in all cases. In a linear pathway this even leads to the case where the sole transcriptional regulatory control of the terminal enzyme is sufficient to control flux through the entire pathway. We confirm the utilization of these pathway regulation strategies through the large-scale analysis of transcriptional regulation in several hundred prokaryotes. CONCLUSIONS: This work expands our knowledge about optimal programs of pathway control. Optimal targets of pathway control strongly depend on the speed at which proteins can be synthesized. Moreover, post-translational regulation such as allosteric regulation allows to strongly reduce the number of transcriptional regulatory interactions required to control a metabolic pathway across different pathway topologies.

Authors: G. M. de Hijas-Liste, E. Balsa-Canto, J. Ewald, M. Bartl, P. Li, J. R. Banga,

Date Published: 16th May 2015

Publication Type: Not specified

Abstract (Expand)

Inference of inter-species gene regulatory networks based on gene expression data is an important computational method to predict pathogen-host interactions (PHIs). Both the experimental setup and the nature of PHIs exhibit certain characteristics. First, besides an environmental change, the battle between pathogen and host leads to a constantly changing environment and thus complex gene expression patterns. Second, there might be a delay until one of the organisms reacts. Third, toward later time points only one organism may survive leading to missing gene expression data of the other organism. Here, we account for PHI characteristics by extending NetGenerator, a network inference tool that predicts gene regulatory networks from gene expression time series data. We tested multiple modeling scenarios regarding the stimuli functions of the interaction network based on a benchmark example. We show that modeling perturbation of a PHI network by multiple stimuli better represents the underlying biological phenomena. Furthermore, we utilized the benchmark example to test the influence of missing data points on the inference performance. Our results suggest that PHI network inference with missing data is possible, but we recommend to provide complete time series data. Finally, we extended the NetGenerator tool to incorporate gene- and time point specific variances, because complex PHIs may lead to high variance in expression data. Sample variances are directly considered in the objective function of NetGenerator and indirectly by testing the robustness of interactions based on variance dependent disturbance of gene expression values. We evaluated the method of variance incorporation on dual RNA sequencing (RNA-Seq) data of Mus musculus dendritic cells incubated with Candida albicans and proofed our method by predicting previously verified PHIs as robust interactions.

Authors: S. Schulze, S. G. Henkel, D. Driesch, R. Guthke, J. Linde

Date Published: 6th Feb 2015

Publication Type: Not specified

Abstract (Expand)

Sepsis is a clinical syndrome that can be caused by bacteria or fungi. Early knowledge on the nature of the causative agent is a prerequisite for targeted anti-microbial therapy. Besides currently used detection methods like blood culture and PCR-based assays, the analysis of the transcriptional response of the host to infecting organisms holds great promise. In this study, we aim to examine the transcriptional footprint of infections caused by the bacterial pathogens Staphylococcus aureus and Escherichia coli and the fungal pathogens Candida albicans and Aspergillus fumigatus in a human whole-blood model. Moreover, we use the expression information to build a random forest classifier to classify if a sample contains a bacterial, fungal, or mock-infection. After normalizing the transcription intensities using stably expressed reference genes, we filtered the gene set for biomarkers of bacterial or fungal blood infections. This selection is based on differential expression and an additional gene relevance measure. In this way, we identified 38 biomarker genes, including IL6, SOCS3, and IRG1 which were already associated to sepsis by other studies. Using these genes, we trained the classifier and assessed its performance. It yielded a 96% accuracy (sensitivities >93%, specificities >97%) for a 10-fold stratified cross-validation and a 92% accuracy (sensitivities and specificities >83%) for an additional test dataset comprising Cryptococcus neoformans infections. Furthermore, the classifier is robust to Gaussian noise, indicating correct class predictions on datasets of new species. In conclusion, this genome-wide approach demonstrates an effective feature selection process in combination with the construction of a well-performing classification model. Further analyses of genes with pathogen-dependent expression patterns can provide insights into the systemic host responses, which may lead to new anti-microbial therapeutic advances.

Authors: , , M. Weber, , ,

Date Published: 11th Mar 2015

Publication Type: Not specified

Abstract (Expand)

Candida glabrata is the second most common pathogenic Candida species and has emerged as a leading cause of nosocomial fungal infections. Its reduced susceptibility to antifungal drugs and its close relationship to Saccharomyces cerevisiae make it an interesting research focus. Although its genome sequence was published in 2004, little is known about its transcriptional dynamics. Here, we provide a detailed RNA-Seq-based analysis of the transcriptomic landscape of C. glabrata in nutrient-rich media, as well as under nitrosative stress and during pH shift. Using RNA-Seq data together with state-of-the-art gene prediction tools, we refined the annotation of the C. glabrata genome and predicted 49 novel protein-coding genes. Of these novel genes, 14 have homologs in S. cerevisiae and six are shared with other Candida species. We experimentally validated four novel protein-coding genes of which two are differentially regulated during pH shift and interaction with human neutrophils, indicating a potential role in host-pathogen interaction. Furthermore, we identified 58 novel non-protein-coding genes, 38 new introns and condition-specific alternative splicing. Finally, our data suggest different patterns of adaptation to pH shift and nitrosative stress in C. glabrata, Candida albicans and S. cerevisiae and thus further underline a distinct evolution of virulence in yeast.

Authors: , S. Duggan, M. Weber, F. Horn, , D. Hellwig, , , R. Martin, ,

Date Published: 13th Jan 2015

Publication Type: Not specified

Abstract (Expand)

Pathogens manipulate the cellular mechanisms of host organisms via pathogen-host interactions (PHIs) in order to take advantage of the capabilities of host cells, leading to infections. The crucial role of these interspecies molecular interactions in initiating and sustaining infections necessitates a thorough understanding of the corresponding mechanisms. Unlike the traditional approach of considering the host or pathogen separately, a systems-level approach, considering the PHI system as a whole is indispensable to elucidate the mechanisms of infection. Following the technological advances in the post-genomic era, PHI data have been produced in large-scale within the last decade. Systems biology-based methods for the inference and analysis of PHI regulatory, metabolic, and protein-protein networks to shed light on infection mechanisms are gaining increasing demand thanks to the availability of omics data. The knowledge derived from the PHIs may largely contribute to the identification of new and more efficient therapeutics to prevent or cure infections. There are recent efforts for the detailed documentation of these experimentally verified PHI data through Web-based databases. Despite these advances in data archiving, there are still large amounts of PHI data in the biomedical literature yet to be discovered, and novel text mining methods are in development to unearth such hidden data. Here, we review a collection of recent studies on computational systems biology of PHIs with a special focus on the methods for the inference and analysis of PHI networks, covering also the Web-based databases and text-mining efforts to unravel the data hidden in the literature.

Authors: S. Durmus, T. Cakir, A. Ozgur,

Date Published: 9th Apr 2015

Publication Type: Not specified

Abstract (Expand)

Intestinal epithelial cells (IEC) form a tight barrier to the gut lumen. Paracellular permeability of the intestinal barrier is regulated by tight junction proteins and can be modulated by microorganisms and other stimuli. The polymorphic fungus Candida albicans, a frequent commensal of the human mucosa has the capacity of traversing this barrier and establishing systemic disease within the host. Infection of polarized C2BBe1 IEC with wild-type C. albicans led to a transient increase of transepithelial electric resistance (TEER) before subsequent barrier disruption, accompanied by a strong decline of junctional protein levels and substantial, but considerably delayed cytotoxicity. Time-resolved microarray-based transcriptome analysis of C. albicans challenged IEC revealed a prominent role of NF-kappaB and MAPK signaling pathways in the response to infection. Hence, we inferred a gene regulatory network based on differentially expressed NF-kappaB and MAPK pathway components and their predicted transcriptional targets. The network model predicted activation of GDF15 by NF-kappaB was experimentally validated. Furthermore, inhibition of NF-kappaB activation in C. albicans infected C2BBe1 cells led to enhanced cytotoxicity in the epithelial cells. Taken together our study identifies NF-kappaB activation as an important protective signaling pathway in the response of epithelial cells to C. albicans. This article is protected by copyright. All rights reserved.

Authors: M. Bohringer, S. Pohlers, , , J. Piegsa, M. Weber, R. Martin, , , ,

Date Published: 12th Jan 2016

Publication Type: Not specified

Abstract (Expand)

Candida albicans bloodstream infection is increasingly frequent and can result in disseminated candidiasis associated with high mortality rates. To analyze the innate immune response against C. albicans, fungal cells were added to human whole-blood samples. After inoculation, C. albicans started to filament and predominantly associate with neutrophils, whereas only a minority of fungal cells became attached to monocytes. While many parameters of host-pathogen interaction were accessible to direct experimental quantification in the whole-blood infection assay, others were not. To overcome these limitations, we generated a virtual infection model that allowed detailed and quantitative predictions on the dynamics of host-pathogen interaction. Experimental time-resolved data were simulated using a state-based modeling approach combined with the Monte Carlo method of simulated annealing to obtain quantitative predictions on a priori unknown transition rates and to identify the main axis of antifungal immunity. Results clearly demonstrated a predominant role of neutrophils, mediated by phagocytosis and intracellular killing as well as the release of antifungal effector molecules upon activation, resulting in extracellular fungicidal activity. Both mechanisms together account for almost [Formula: see text] of C. albicans killing, clearly proving that beside being present in larger numbers than other leukocytes, neutrophils functionally dominate the immune response against C. albicans in human blood. A fraction of C. albicans cells escaped phagocytosis and remained extracellular and viable for up to four hours. This immune escape was independent of filamentation and fungal activity and not linked to exhaustion or inactivation of innate immune cells. The occurrence of C. albicans cells being resistant against phagocytosis may account for the high proportion of dissemination in C. albicans bloodstream infection. Taken together, iterative experiment-model-experiment cycles allowed quantitative analyses of the interplay between host and pathogen in a complex environment like human blood.

Authors: , T. Lehnert, K. Bieber, R. Martin, ,

Date Published: 20th Feb 2014

Publication Type: Not specified

Abstract (Expand)

Time-lapse microscopy is an important technique to study the dynamics of various biological processes. The labor-intensive manual analysis of microscopy videos is increasingly replaced by automated segmentation and tracking methods. These methods are often limited to certain cell morphologies and/or cell stainings. In this paper, we present an automated segmentation and tracking framework that does not have these restrictions. In particular, our framework handles highly variable cell shapes and does not rely on any cell stainings. Our segmentation approach is based on a combination of spatial and temporal image variations to detect moving cells in microscopy videos. This method yields a sensitivity of 99% and a precision of 95% in object detection. The tracking of cells consists of different steps, starting from single-cell tracking based on a nearest-neighbor-approach, detection of cell-cell interactions and splitting of cell clusters, and finally combining tracklets using methods from graph theory. The segmentation and tracking framework was applied to synthetic as well as experimental datasets with varying cell densities implying different numbers of cell-cell interactions. We established a validation framework to measure the performance of our tracking technique. The cell tracking accuracy was found to be >99% for all datasets indicating a high accuracy for connecting the detected cells between different time points.

Authors: S. Brandes, Z. Mokhtari, F. Essig, , ,

Date Published: 8th Nov 2014

Publication Type: Not specified

Abstract (Expand)

Candida albicans and Candida glabrata account for the majority of candidiasis cases worldwide. Although both species are in the same genus, they differ in key virulence attributes. Within this work, live cell imaging was used to examine the dynamics of neutrophil activation after confrontation with either C. albicans or C. glabrata. Analyses revealed higher phagocytosis rates of C. albicans than C. glabrata that resulted in stronger PMN (polymorphonuclear cells) activation by C. albicans. Furthermore, we observed differences in the secretion of chemokines, indicating chemotactic differences in PMN signalling towards recruitment of further immune cells upon confrontation with Candida spp. Supernatants from co-incubations of neutrophils with C. glabrata primarily attracted monocytes and increased the phagocytosis of C. glabrata by monocytes. In contrast, PMN activation by C. albicans resulted in recruitment of more neutrophils. Two complex infection models confirmed distinct targeting of immune cell populations by the two Candida spp.: In a human whole blood infection model, C. glabrata was more effectively taken up by monocytes than C. albicans and histopathological analyses of murine model infections confirmed primarily monocytic infiltrates in C. glabrata kidney infection in contrast to PMN-dominated infiltrates in C. albicans infection. Taken together, our data demonstrate that the human opportunistic fungi C. albicans and C. glabrata are differentially recognized by neutrophils and one outcome of this differential recognition is the preferential uptake of C. glabrata by monocytes.

Authors: S. Duggan, F. Essig, , Z. Mokhtari, , T. Lehnert, S. Brandes, A. Hader, , R. Martin, ,

Date Published: 5th May 2015

Publication Type: Not specified

Abstract (Expand)

Opportunistic fungal pathogens can cause bloodstream infection and severe sepsis upon entering the blood stream of the host. The early immune response in human blood comprises the elimination of pathogens by antimicrobial peptides and innate immune cells, such as neutrophils or monocytes. Mathematical modeling is a predictive method to examine these complex processes and to quantify the dynamics of pathogen-host interactions. Since model parameters are often not directly accessible from experiment, their estimation is required by calibrating model predictions with experimental data. Depending on the complexity of the mathematical model, parameter estimation can be associated with excessively high computational costs in terms of run time and memory. We apply a strategy for reliable parameter estimation where different modeling approaches with increasing complexity are used that build on one another. This bottom-up modeling approach is applied to an experimental human whole-blood infection assay for Candida albicans. Aiming for the quantification of the relative impact of different routes of the immune response against this human-pathogenic fungus, we start from a non-spatial state-based model (SBM), because this level of model complexity allows estimating a priori unknown transition rates between various system states by the global optimization method simulated annealing. Building on the non-spatial SBM, an agent-based model (ABM) is implemented that incorporates the migration of interacting cells in three-dimensional space. The ABM takes advantage of estimated parameters from the non-spatial SBM, leading to a decreased dimensionality of the parameter space. This space can be scanned using a local optimization approach, i.e., least-squares error estimation based on an adaptive regular grid search, to predict cell migration parameters that are not accessible in experiment. In the future, spatio-temporal simulations of whole-blood samples may enable timely stratification of sepsis patients by distinguishing hyper-inflammatory from paralytic phases in immune dysregulation.

Authors: T. Lehnert, , J. Pollmacher, , ,

Date Published: 19th Jun 2015

Publication Type: Not specified

Abstract (Expand)

The successful treatment of infectious diseases requires interdisciplinary studies of all aspects of infection processes. The overarching combination of experimental research and theoretical analysis in a systems biology approach can unravel mechanisms of complex interactions between pathogens and the human immune system. Taking into account spatial information is especially important in the context of infection, since the migratory behavior and spatial interactions of cells are often decisive for the outcome of the immune response. Spatial information is provided by image and video data that are acquired in microscopy experiments and that are at the heart of an image-based systems biology approach. This review demonstrates how image-based systems biology improves our understanding of infection processes. We discuss the three main steps of this approach--imaging, quantitative characterization, and modeling--and consider the application of these steps in the context of studying infection processes. After summarizing the most relevant microscopy and image analysis approaches, we discuss ways to quantify infection processes, and address a number of modeling techniques that exploit image-derived data to simulate host-pathogen interactions in silico.

Authors: A. Medyukhina, , Z. Mokhtari,

Date Published: 29th Jan 2015

Publication Type: Not specified

Abstract (Expand)

Lichtheimia corymbifera is a ubiquitous soilborne zygomycete fungus, which is an opportunistic human pathogen in immunocompromised patients. The fungus can cause life-threatening diseases by attacking the lung during early stages of invasion and by disseminating during later phases causing systemic infection. Since infections have drastically increased during the last decades, it is a major goal to investigate the mechanisms underlying pathogenicity of L. corymbifera. One of the first barriers, which the fungus needs to cope with in the lung tissue, is phagocytosis by alveolar macrophages. Here, we report on phagocytosis assays for murine alveolar macrophages co-incubated with resting, swollen and opsonised spores of a virulent and an attenuated L. corymbifera strain. A major finding of this study is the significantly increased phagocytosis ratio of the virulent strain if compared to the attenuated strain. We quantify the phagocytosis by performing automated analysis of fluorescence microscopy images and by computing ratios for (i) fungal phagocytosis, (ii) fungal adhesion to phagocytes and (iii) fungal aggregation and spore cluster distribution in space. Automation of the image analysis yields objective results that overcome the disadvantages of manual analyses being time consuming, error-prone and subjective. Therefore, it can be expected that automated image analysis of confrontation assays will play a crucial role in future investigations of host-pathogen interactions.

Authors: , H. R. Park, H. M. Dahse, C. Skerka, K. Voigt,

Date Published: 1st Sep 2014

Publication Type: Not specified

Abstract (Expand)

Candida albicans is the most common opportunistic fungal pathogen of the human mucosal flora, frequently causing infections. The fungus is responsible for invasive infections in immunocompromised patients that can lead to sepsis. The yeast to hypha transition and invasion of host-tissue represent major determinants in the switch from benign colonizer to invasive pathogen. A comprehensive understanding of the infection process requires analyses at the quantitative level. Utilizing fluorescence microscopy with differential staining, we obtained images of C. albicans undergoing epithelial invasion during a time course of 6 h. An image-based systems biology approach, combining image analysis and mathematical modeling, was applied to quantify the kinetics of hyphae development, hyphal elongation, and epithelial invasion. The automated image analysis facilitates high-throughput screening and provided quantities that allow for the time-resolved characterization of the morphological and invasive state of fungal cells. The interpretation of these data was supported by two mathematical models, a kinetic growth model and a kinetic transition model, that were developed using differential equations. The kinetic growth model describes the increase in hyphal length and revealed that hyphae undergo mass invasion of epithelial cells following primary hypha formation. We also provide evidence that epithelial cells stimulate the production of secondary hyphae by C. albicans. Based on the kinetic transition model, the route of invasion was quantified in the state space of non-invasive and invasive fungal cells depending on their number of hyphae. This analysis revealed that the initiation of hyphae formation represents an ultimate commitment to invasive growth and suggests that in vivo, the yeast to hypha transition must be under exquisitely tight negative regulation to avoid the transition from commensal to pathogen invading the epithelium.

Authors: F. Mech, D. Wilson, T. Lehnert, , M. Thilo Figge

Date Published: 20th Nov 2013

Publication Type: Not specified

Abstract (Expand)

Only few Candida species, e.g., Candida albicans, Candida glabrata, Candida dubliniensis, and Candida parapsilosis, are successful colonizers of a human host. Under certain circumstances these species can cause infections ranging from superficial to life-threatening disseminated candidiasis. The success of C. albicans, the most prevalent and best studied Candida species, as both commensal and human pathogen depends on its genetic, biochemical, and morphological flexibility which facilitates adaptation to a wide range of host niches. In addition, formation of biofilms provides additional protection from adverse environmental conditions. Furthermore, in many host niches Candida cells coexist with members of the human microbiome. The resulting fungal-bacterial interactions have a major influence on the success of C. albicans as commensal and also influence disease development and outcome. In this chapter, we review the current knowledge of important survival strategies of Candida spp., focusing on fundamental fitness and virulence traits of C. albicans.

Authors: M. Polke, ,

Date Published: 24th Feb 2015

Publication Type: Not specified

Abstract (Expand)

Oral candidiasis remains one of the most common forms of Candida infections and occurs if the balance between host, Candida and microbiota is disturbed, e.g., by broad spectrum antibiotics or immunosuppression. In recent years, identification of fungal factors contributing to host cell damage and new insights into host defense mechanisms have significantly extended our understanding of the pathogenesis of oral candidiasis. In this review, we will provide an overview of the pathogenicity mechanisms during oral Candida infections and discuss some approaches by which this knowledge could be transferred into therapeutic approaches.

Authors: B. Hebecker, J. R. Naglik, ,

Date Published: 7th May 2014

Publication Type: Not specified

Abstract (Expand)

Following antifungal treatment, Candida albicans, and other human pathogenic fungi can undergo microevolution, which leads to the emergence of drug resistance. However, the capacity for microevolutionary adaptation of fungi goes beyond the development of resistance against antifungals. Here we used an experimental microevolution approach to show that one of the central pathogenicity mechanisms of C. albicans, the yeast-to-hyphae transition, can be subject to experimental evolution. The C. albicans cph1Delta/efg1Delta mutant is nonfilamentous, as central signaling pathways linking environmental cues to hyphal formation are disrupted. We subjected this mutant to constant selection pressure in the hostile environment of the macrophage phagosome. In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation. In addition, the evolved mutant exhibited hyper-virulence in a murine infection model and an altered cell wall composition compared to the cph1Delta/efg1Delta strain. Moreover, the transcriptional regulation of hyphae-associated, and other pathogenicity-related genes became re-responsive to environmental cues in the evolved strain. We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery. This mutation was responsible for the reconnection of the hyphal growth program with environmental signals in the evolved strain and was sufficient to bypass Efg1/Cph1-dependent filamentation. These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure.

Authors: A. Wartenberg, , R. Martin, M. Schreiner, F. Horn, , S. Jenull, , K. Kuchler, , , A. Forche, C. d'Enfert, S. Brunke,

Date Published: 4th Dec 2014

Publication Type: Not specified

Abstract (Expand)

Fungal pathogens must assimilate local nutrients to establish an infection in their mammalian host. We focus on carbon, nitrogen, and micronutrient assimilation mechanisms, discussing how these influence host-fungus interactions during infection. We highlight several emerging trends based on the available data. First, the perturbation of carbon, nitrogen, or micronutrient assimilation attenuates fungal pathogenicity. Second, the contrasting evolutionary pressures exerted on facultative versus obligatory pathogens have led to contemporary pathogenic fungal species that display differing degrees of metabolic flexibility. The evolutionarily ancient metabolic pathways are conserved in most fungal pathogen, but interesting gaps exist in some species (e.g., Candida glabrata). Third, metabolic flexibility is generally essential for fungal pathogenicity, and in particular, for the adaptation to contrasting host microenvironments such as the gastrointestinal tract, mucosal surfaces, bloodstream, and internal organs. Fourth, this metabolic flexibility relies on complex regulatory networks, some of which are conserved across lineages, whereas others have undergone significant evolutionary rewiring. Fifth, metabolic adaptation affects fungal susceptibility to antifungal drugs and also presents exciting opportunities for the development of novel therapies.

Authors: I. V. Ene, S. Brunke, A. J. Brown,

Date Published: 4th Sep 2014

Publication Type: Not specified

Abstract (Expand)

Little is known regarding the role of NK cells during primary and secondary disseminated Candida albicans infection. We assessed the role of NK cells for host defense against candidiasis in immunocompetent, as well as immunodeficient, hosts. Surprisingly, depletion of NK cells in immunocompetent WT mice did not increase susceptibility to systemic candidiasis, suggesting that NK cells are redundant for antifungal defense in otherwise immunocompetent hosts. NK-cell-depleted mice were found to be protected as a consequence of attenuation of systemic inflammation. In contrast, the absence of NK cells in T/B/NK-cell-deficient NSG (NOD SCID gamma) mice led to an increased susceptibility to both primary and secondary systemic C. albicans infections compared with T/B-cell-deficient SCID mice. In conclusion, this study demonstrates that NK cells are an essential and nonredundant component of anti-C. albicans host defense in immunosuppressed hosts with defective T/B-lymphocyte immunity, while contributing to hyperinflammation in immunocompetent hosts. The discovery of the importance of NK cells in hosts with severe defects of adaptive immunity might have important consequences for the design of adjunctive immunotherapeutic approaches in systemic C. albicans infections targeting NK-cell function.

Authors: J. Quintin, J. Voigt, R. van der Voort, , I. Verschueren, , E. J. Giamarellos-Bourboulis, J. W. van der Meer, L. A. Joosten, , M. G. Netea

Date Published: 27th May 2014

Publication Type: Not specified

Abstract

Not specified

Authors: G. P. Otto, K. Ludewig, , , ,

Date Published: 12th Jun 2013

Publication Type: Not specified

Abstract (Expand)

Human fungal pathogens are distributed throughout their kingdom, suggesting that pathogenic potential evolved independently. Candida albicans is the most virulent member of the CUG clade of yeasts and a common cause of both superficial and invasive infections. We therefore hypothesized that C. albicans possesses distinct pathogenicity mechanisms. In silico genome subtraction and comparative transcriptional analysis identified a total of 65 C. albicans-specific genes (ASGs) expressed during infection. Phenotypic characterization of six ASG-null mutants demonstrated that these genes are dispensable for in vitro growth but play defined roles in host-pathogen interactions. Based on these analyses, we investigated two ASGs in greater detail. An orf19.6688Delta mutant was found to be fully virulent in a mouse model of disseminated candidiasis and to induce higher levels of the proinflammatory cytokine interleukin-1beta (IL-1beta) following incubation with murine macrophages. A pga16Delta mutant, on the other hand, exhibited attenuated virulence. Moreover, we provide evidence that secondary filamentation events (multiple hyphae emerging from a mother cell and hyphal branching) contribute to pathogenicity: PGA16 deletion did not influence primary hypha formation or extension following contact with epithelial cells; however, multiple hyphae and hyphal branching were strongly reduced. Significantly, these hyphae failed to damage host cells as effectively as the multiple hypha structures formed by wild-type C. albicans cells. Together, our data show that species-specific genes of a eukaryotic pathogen can play important roles in pathogenicity.

Authors: D. Wilson, F. L. Mayer, P. Miramon, F. Citiulo, S. Slesiona, ,

Date Published: 7th Mar 2014

Publication Type: Not specified

Abstract (Expand)

The pathology of vulvovaginal candidiasis (VVC) caused by Candida albicans is associated with a nonprotective inflammatory response and is frequently treated with clotrimazole. We investigated the mechanisms by which clotrimazole resolves VVC. Low levels of clotrimazole, which do not block fungal growth, inhibit expression of a "danger response" transcription factor, c-Fos, block production of proinflammatory cytokines, and inhibit neutrophil infiltration to the site of infection.

Authors: D. Wilson, B. Hebecker, D. L. Moyes, P. Miramon, N. Jablonowski, S. Wisgott, S. Allert, J. R. Naglik,

Date Published: 29th Jul 2013

Publication Type: Not specified

Abstract (Expand)

Systemic infections of humans with the fungal pathogen Candida albicans are associated with a high mortality rate. Currently, efficient treatment of these infections is hampered by the relatively low number of available antifungal drugs. We recently identified the small heat shock protein Hsp21 in C. albicans and demonstrated its fundamental role for environmental stress adaptation and fungal virulence. Hsp21 was found in several pathogenic Candida species but not in humans. This prompted us to investigate the effects of a broad range of different antifungal drugs on an Hsp21-null C. albicans mutant strain. Our results indicate that combinatorial therapy targeting Hsp21, together with specific antifungal drug targets, has strong synergistic potential. In addition, we demonstrate that Hsp21 is required for tolerance to ethanol-induced stress and induction of filamentation in response to pharmacological inhibition of Hsp90. These findings might pave the way for the development of new treatment strategies against Candida infections.

Authors: F. L. Mayer, D. Wilson,

Date Published: 22nd Mar 2013

Publication Type: Not specified

Abstract (Expand)

Although morphological plasticity is a central virulence trait of Candida albicans, the number of filament-associated genes and the interplay of mechanisms regulating their expression remain unknown. By correlation-based network modeling of the transcriptional response to different defined external stimuli for morphogenesis we identified a set of eight genes with highly correlated expression patterns, forming a core filamentation response. This group of genes included ALS3, ECE1, HGT2, HWP1, IHD1 and RBT1 which are known or supposed to encode for cell- wall associated proteins as well as the Rac1 guanine nucleotide exchange factor encoding gene DCK1 and the unknown function open reading frame orf19.2457. The validity of network modeling was confirmed using a dataset of advanced complexity that describes the transcriptional response of C. albicans during epithelial invasion as well as comparing our results with other previously published transcriptome studies. Although the set of core filamentation response genes was quite small, several transcriptional regulators are involved in the control of their expression, depending on the environmental condition.

Authors: R. Martin, , S. Brunke, , ,

Date Published: 14th Mar 2013

Publication Type: Not specified

Abstract (Expand)

The amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms of cysteine tolerance in microbes remain largely obscure. Here we show that the human pathogenic yeast Candida albicans excretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed that sulfite formation from cysteine in C. albicans relies on cysteine dioxygenase Cdg1, an enzyme with similar functions in humans. Environmental cysteine induced not only the expression of the CDG1 gene in C. albicans, but also the expression of SSU1, encoding a putative sulfite efflux pump. Accordingly, the deletion of SSU1 resulted in enhanced sensitivity of the fungal cells to both cysteine and sulfite. To study the regulation of sulfite/cysteine tolerance in more detail, we screened a C. albicans library of transcription factor mutants in the presence of sulfite. This approach and subsequent independent mutant analysis identified the zinc cluster transcription factor Zcf2 to govern sulfite/cysteine tolerance, as well as cysteine-inducible SSU1 and CDG1 gene expression. cdg1Delta and ssu1Delta mutants displayed reduced hypha formation in the presence of cysteine, indicating a possible role of the newly proposed mechanisms of cysteine tolerance and sulfite secretion in the pathogenicity of C. albicans. Moreover, cdg1Delta mutants induced delayed mortality in a mouse model of disseminated infection. Since sulfite is toxic and a potent reducing agent, its production by C. albicans suggests diverse roles during host adaptation and pathogenicity.

Authors: F. Hennicke, M. Grumbt, U. Lermann, N. Ueberschaar, K. Palige, B. Bottcher, , C. Staib, J. Morschhauser, M. Monod, , C. Hertweck, P. Staib

Date Published: 15th Feb 2013

Publication Type: Not specified

Abstract (Expand)

Candida albicans is a major cause of bloodstream infection which may present as sepsis and septic shock - major causes of morbidity and mortality world-wide. After invasion of the pathogen, innate mechanisms govern the early response. Here, we outline the models used to study these mechanisms and summarize our current understanding of innate immune responses during Candida bloodstream infection. This includes protective immunity as well as harmful responses resulting in Candida induced sepsis. Neutrophilic granulocytes are considered principal effector cells conferring protection and recognize C. albicans mainly via complement receptor 3. They possess a range of effector mechanisms, contributing to elimination of the pathogen. Neutrophil activation is closely linked to complement and modulated by activated mononuclear cells. A thorough understanding of these mechanisms will help in creating an individualized approach to patients suffering from systemic candidiasis and aid in optimizing clinical management.

Authors: S. Duggan, I. Leonhardt, ,

Date Published: 18th Mar 2015

Publication Type: Not specified

Abstract (Expand)

In this work, we investigate optimality principles behind synthesis strategies for protein complexes using a dynamic optimization approach. We show that the cellular capacity of protein synthesis has a strong influence on optimal synthesis strategies reaching from a simultaneous to a sequential synthesis of the subunits of a protein complex. Sequential synthesis is preferred if protein synthesis is strongly limited, whereas a simultaneous synthesis is optimal in situations with a high protein synthesis capacity. We confirm the predictions of our optimization approach through the analysis of the operonic organization of protein complexes in several hundred prokaryotes. Thereby, we are able to show that cellular protein synthesis capacity is a driving force in the dissolution of operons comprising the subunits of a protein complex. Thus, we also provide a tested hypothesis explaining why the subunits of many prokaryotic protein complexes are distributed across several operons despite the presumably less precise co-regulation.

Authors: , M. Kotzing, M. Bartl,

Date Published: 1st May 2015

Publication Type: Not specified

Abstract (Expand)

BACKGROUND: Adjusting the capacity of metabolic pathways in response to rapidly changing environmental conditions is an important component of microbial adaptation strategies to stochastic environments. In this work, we use advanced dynamic optimization techniques combined with theoretical models to study which reactions in pathways are optimally targeted by regulatory interactions in order to minimize the regulatory effort that is required to adjust the flux through a complex metabolic network. Moreover, we analyze how constraints in the speed at which an organism can respond on a proteomic level influences these optimal targets of pathway control. RESULTS: We find that limitations in protein biosynthetic rates have a strong influence. With increasing protein biosynthetic rates the regulatory effort targeting the initial enzyme in a pathway is reduced while the regulatory effort in the terminal enzyme is increased. Studying the impact of allosteric regulation for different pathway topologies, we find that the presence of feedback inhibition by products of metabolic pathways allows organisms to reduce the regulatory effort that is required to control a metabolic pathway in all cases. In a linear pathway this even leads to the case where the sole transcriptional regulatory control of the terminal enzyme is sufficient to control flux through the entire pathway. We confirm the utilization of these pathway regulation strategies through the large-scale analysis of transcriptional regulation in several hundred prokaryotes. CONCLUSIONS: This work expands our knowledge about optimal programs of pathway control. Optimal targets of pathway control strongly depend on the speed at which proteins can be synthesized. Moreover, post-translational regulation such as allosteric regulation allows to strongly reduce the number of transcriptional regulatory interactions required to control a metabolic pathway across different pathway topologies.

Authors: G. M. de Hijas-Liste, E. Balsa-Canto, , M. Bartl, P. Li, J. R. Banga,

Date Published: 16th May 2015

Publication Type: Not specified

Abstract (Expand)

BACKGROUND: In System Biology, iterations of wet-lab experiments followed by modelling approaches and model-inspired experiments describe a cyclic workflow. This approach is especially useful for the inference of gene regulatory networks based on high-throughput gene expression data. Experiments can verify or falsify the predicted interactions allowing further refinement of the network model. Aspergillus fumigatus is a major human fungal pathogen. One important virulence trait is its ability to gain sufficient amounts of iron during infection process. Even though some regulatory interactions are known, we are still far from a complete understanding of the way iron homeostasis is regulated. RESULTS: In this study, we make use of a reverse engineering strategy to infer a regulatory network controlling iron homeostasis in A. fumigatus. The inference approach utilizes the temporal change in expression data after a change from iron depleted to iron replete conditions. The modelling strategy is based on a set of linear differential equations and offers the possibility to integrate known regulatory interactions as prior knowledge. Moreover, it makes use of important selection criteria, such as sparseness and robustness. By compiling a list of known regulatory interactions for iron homeostasis in A. fumigatus and softly integrating them during network inference, we are able to predict new interactions between transcription factors and target genes. The proposed activation of the gene expression of hapX by the transcriptional regulator SrbA constitutes a so far unknown way of regulating iron homeostasis based on the amount of metabolically available iron. This interaction has been verified by Northern blots in a recent experimental study. In order to improve the reliability of the predicted network, the results of this experimental study have been added to the set of prior knowledge. The final network includes three SrbA target genes. Based on motif searching within the regulatory regions of these genes, we identify potential DNA-binding sites for SrbA. Our wet-lab experiments demonstrate high-affinity binding capacity of SrbA to the promoters of hapX, hemA and srbA. CONCLUSIONS: This study presents an application of the typical Systems Biology circle and is based on cooperation between wet-lab experimentalists and in silico modellers. The results underline that using prior knowledge during network inference helps to predict biologically important interactions. Together with the experimental results, we indicate a novel iron homeostasis regulating system sensing the amount of metabolically available iron and identify the binding site of iron-related SrbA target genes. It will be of high interest to study whether these regulatory interactions are also important for close relatives of A. fumigatus and other pathogenic fungi, such as Candida albicans.

Authors: , P. Hortschansky, E. Fazius, , , H. Haas

Date Published: 19th Jan 2012

Publication Type: Not specified

Abstract (Expand)

Aspergillus fumigatus is the species that most commonly causes the opportunistic infection invasive aspergillosis (IA) in patients being treated for hematological malignancies. Little is known about the A. fumigatus proteins that trigger the production of Aspergillus-specific IgG antibodies during the course of IA. To characterize the serological response to A. fumigatus protein antigens, mycelial proteins were separated by 2-D gel electrophoresis. The gels were immunoblotted with sera from patients with probable and proven IA and control patients without IA. We identified 49 different fungal proteins, which gave a positive IgG antibody signal. Most of these antigens play a role in primary metabolism and stress responses. Overall, our analysis identified 18 novel protein antigens from A. fumigatus. To determine whether these antigens can be used as diagnostic or prognostic markers or exhibit a protective activity, we employed supervised machine learning with decision trees. We identified two candidates for further analysis, the protein antigens CpcB and Shm2. Heterologously produced Shm2 induced a strongly proinflammatory response in human peripheral blood mononuclear cells after in vitro stimulation. In contrast, CpcB did not activate the immune response of PBMCs. These findings could serve as the basis for the development of an immunotherapy of IA.

Authors: J. Teutschbein, S. Simon, J. Lother, J. Springer, P. Hortschansky, C. O. Morton, , , E. Conneally, T. R. Rogers, , ,

Date Published: 15th Mar 2016

Publication Type: Not specified

Abstract (Expand)

Aspergillus fumigatus is the predominant airborne pathogenic fungus causing invasive aspergillosis in immunocompromised patients. During infection A. fumigatus has to adapt to oxygen-limiting conditions in inflammatory or necrotic tissue. Previously, we identified a mitochondrial protein to be highly up-regulated during hypoxic adaptation. Here, this protein was found to represent the novel oxidoreductase HorA. In Saccharomyces cerevisiae a homologue was shown to play a role in biosynthesis of coenzyme Q. Consistently, reduced coenzyme Q content in the generated DeltahorA mutant indicated a respective function in A. fumigatus. Since coenzyme Q is involved in cellular respiration and maintaining cellular redox homeostasis, the strain DeltahorA displayed an impaired response to both oxidative and reductive stress, a delay in germination and an accumulation of NADH. Moreover, an increased resistance against antifungal drugs was observed. All phenotypes were completely reversed by the addition of the synthetic electron carrier menadione. The deletion strain DeltahorA showed significantly attenuated virulence in two murine infection models of invasive pulmonary aspergillosis. Therefore, the biosynthesis of coenzyme Q and, particularly, the fungal-specific protein HorA play a crucial role in virulence of A. fumigatus. Due to its absence in mammals, HorA might represent a novel therapeutic target against fungal infections. This article is protected by copyright. All rights reserved.

Authors: K. Kroll, E. Shekhova, D. J. Mattern, A. Thywissen, , M. Strassburger, T. Heinekamp, , ,

Date Published: 19th Mar 2016

Publication Type: Not specified

Abstract (Expand)

Organisms constantly interact with other species through physical contact which leads to changes on the molecular level, for example the transcriptome. These changes can be monitored for all genes, with the help of high-throughput experiments such as RNA-seq or microarrays. The adaptation of the gene expression to environmental changes within cells is mediated through complex gene regulatory networks. Often, our knowledge of these networks is incomplete. Network inference predicts gene regulatory interactions based on transcriptome data. An emerging application of high-throughput transcriptome studies are dual transcriptomics experiments. Here, the transcriptome of two or more interacting species is measured simultaneously. Based on a dual RNA-seq data set of murine dendritic cells infected with the fungal pathogen Candida albicans, the software tool NetGenerator was applied to predict an inter-species gene regulatory network. To promote further investigations of molecular inter-species interactions, we recently discussed dual RNA-seq experiments for host-pathogen interactions and extended the applied tool NetGenerator (Schulze et al., 2015). The updated version of NetGenerator makes use of measurement variances in the algorithmic procedure and accepts gene expression time series data with missing values. Additionally, we tested multiple modeling scenarios regarding the stimuli functions of the gene regulatory network. Here, we summarize the work by Schulze et al. (2015) and put it into a broader context. We review various studies making use of the dual transcriptomics approach to investigate the molecular basis of interacting species. Besides the application to host-pathogen interactions, dual transcriptomics data are also utilized to study mutualistic and commensalistic interactions. Furthermore, we give a short introduction into additional approaches for the prediction of gene regulatory networks and discuss their application to dual transcriptomics data. We conclude that the application of network inference on dual-transcriptomics data is a promising approach to predict molecular inter-species interactions.

Authors: , J. Schleicher, ,

Date Published: 31st Mar 2016

Publication Type: Not specified

Abstract (Expand)

Recent and rapidly evolving progress on high-throughput measurement techniques and computational performance has led to the emergence of new disciplines, such as systems medicine and translational systems biology. At the core of these disciplines lies the desire to produce multiscale models: mathematical models that integrate multiple scales of biological organization, ranging from molecular, cellular and tissue models to organ, whole-organism and population scale models. Using such models, hypotheses can systematically be tested. In this review, we present state-of-the-art multiscale modelling of bacterial and fungal infections, considering both the pathogen and host as well as their interaction. Multiscale modelling of the interactions of bacteria, especially Mycobacterium tuberculosis, with the human host is quite advanced. In contrast, models for fungal infections are still in their infancy, in particular regarding infections with the most important human pathogenic fungi, Candida albicans and Aspergillus fumigatus. We reflect on the current availability of computational approaches for multiscale modelling of host-pathogen interactions and point out current challenges. Finally, we provide an outlook for future requirements of multiscale modelling.

Authors: J. Schleicher, , M. Gustafsson, G. Cedersund, ,

Date Published: 10th Feb 2016

Publication Type: Not specified

Abstract (Expand)

Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune responses. Such toxins have not been identified previously in human pathogenic fungi. Here we identify the first, to our knowledge, fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans. This secreted toxin directly damages epithelial membranes, triggers a danger response signalling pathway and activates epithelial immunity. Membrane permeabilization is enhanced by a positive charge at the carboxy terminus of the peptide, which triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name 'Candidalysin' for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans.

Authors: D. L. Moyes, D. Wilson, J. P. Richardson, S. Mogavero, S. X. Tang, J. Wernecke, S. Hofs, R. L. Gratacap, J. Robbins, M. Runglall, C. Murciano, M. Blagojevic, S. Thavaraj, , B. Hebecker, , G. Vizcay, S. I. Iancu, N. Kichik, A. Hader, , T. Luo, T. Kruger, O. Kniemeyer, E. Cota, O. Bader, R. T. Wheeler, T. Gutsmann, , J. R. Naglik

Date Published: 30th Mar 2016

Publication Type: Not specified

Abstract (Expand)

Invasive aspergillosis (IA) is a devastating opportunistic infection and its treatment constitutes a considerable burden for the health care system. Immunocompromised patients are at an increased risk for IA, which is mainly caused by the species Aspergillus fumigatus. An early and reliable diagnosis is required to initiate the appropriate antifungal therapy. However, diagnostic sensitivity and accuracy still needs to be improved, which can be achieved at least partly by the definition of new biomarkers. Besides the direct detection of the pathogen by the current diagnostic methods, the analysis of the host response is a promising strategy toward this aim. Following this approach, we sought to identify new biomarkers for IA. For this purpose, we analyzed gene expression profiles of hematological patients and compared profiles of patients suffering from IA with non-IA patients. Based on microarray data, we applied a comprehensive feature selection using a random forest classifier. We identified the transcript coding for the S100 calcium-binding protein B (S100B) as a potential new biomarker for the diagnosis of IA. Considering the expression of this gene, we were able to classify samples from patients with IA with 82.3% sensitivity and 74.6% specificity. Moreover, we validated the expression of S100B in a real-time reverse transcription polymerase chain reaction (RT-PCR) assay and we also found a down-regulation of S100B in A. fumigatus stimulated DCs. An influence on the IL1B and CXCL1 downstream levels was demonstrated by this S100B knockdown. In conclusion, this study covers an effective feature selection revealing a key regulator of the human immune response during IA. S100B may represent an additional diagnostic marker that in combination with the established techniques may improve the accuracy of IA diagnosis.

Authors: , K. Czakai, J. Springer, M. Fliesser, M. Bonin, , A. L. Schmitt, , ,

Date Published: 21st Mar 2016

Publication Type: Not specified

Abstract (Expand)

The human restricted pathogen Moraxella catarrhalis is an important causal agent for exacerbations in chronic obstructive lung disease (COPD) in adults. In such patients, increased numbers of granulocytes are present in the airways, which correlate with bacteria-induced exacerbations and severity of the disease. Our study investigated whether the interaction of M. catarrhalis with the human granulocyte-specific carcinoembryonic antigen-related cell adhesion molecule (CEACAM)-3 is linked to NF-kappaB activation, resulting in chemokine production. Granulocytes from healthy donors and NB4 cells were infected with M. catarrhalis in the presence of different inhibitors, blocking antibodies and siRNA. The supernatants were analysed by ELISA for chemokines. NF-kappaB activation was determined using a luciferase reporter gene assay and chromatin-immunoprecipitation. We found evidence that the specific engagement of CEACAM3 by Moraxella catarrhalis ubiquitous surface protein A1 (UspA1) results in the activation of pro-inflammatory events, such as degranulation of neutrophils, ROS production and chemokine secretion. The interaction of UspA1 with CEACAM3 induced the activation of the NF-kappaB pathway via Syk and the Card9 pathway and was dependent on the phosphorylation of the CEACAM3 ITAM -like motif. These findings suggest that the CEACAM3 signalling in neutrophils is able to specifically modulate airway inflammation caused by infection with M. catarrhalis.

Authors: A. Heinrich, K. A. Heyl, E. Klaile, , , A. Wiessner, K. Fischer, R. R. Schumann, U. Seifert, K. Riesbeck, A. Moter, B. B. Singer, S. Bachmann,

Date Published: 3rd Apr 2016

Publication Type: Not specified

Abstract

Not specified

Authors: S. Durmus, T. Cakir,

Date Published: 4th Feb 2016

Publication Type: Not specified

Abstract (Expand)

In systems biology, researchers aim to understand complex biological systems as a whole, which is often achieved by mathematical modelling and the analyses of high-throughput data. In this review, we give an overview of medical applications of systems biology approaches with special focus on host-pathogen interactions. After introducing general ideas of systems biology, we focus on (1) the detection of putative biomarkers for improved diagnosis and support of therapeutic decisions; (2) network modelling for the identification of regulatory interactions between cellular molecules to reveal putative drug targets; (3) module discovery for the detection of phenotype-specific modules in molecular interaction networks. Biomarker detection applies supervised machine learning methods utilising high-throughput data (e.g. SNP detection, RNA-seq, proteomics) and clinical data. We demonstrate structural analysis of molecular networks, especially by identification of disease modules as novel strategy, and discuss possible applications to host-pathogen interactions. Pioneering work was done to predict molecular host-pathogen interactions networks based on dual RNA-seq data. However, currently this network modelling is restricted to a small number of genes. With increasing number and quality of databases and data repositories, also the prediction of large-scale networks will be feasible that can used for multi-dimensional diagnosis and decision support for prevention and therapy of diseases. Finally, we outline further perspective issues such as support of personalised medicine with high-throughput data and generation of multi-scale host-pathogen interaction models.

Authors: , S. Vlaic, ,

Date Published: 27th Apr 2016

Publication Type: Not specified

Abstract (Expand)

In the emerging field of systems biology of fungal infection, one of the central roles belongs to the modeling of gene regulatory networks (GRNs). Utilizing omics-data, GRNs can be predicted by mathematical modeling. Here, we review current advances of data-based reconstruction of both small-scale and large-scale GRNs for human pathogenic fungi. The advantage of large-scale genome-wide modeling is the possibility to predict central (hub) genes and thereby indicate potential biomarkers and drug targets. In contrast, small-scale GRN models provide hypotheses on the mode of gene regulatory interactions, which have to be validated experimentally. Due to the lack of sufficient quantity and quality of both experimental data and prior knowledge about regulator-target gene relations, the genome-wide modeling still remains problematic for fungal pathogens. While a first genome-wide GRN model has already been published for Candida albicans, the feasibility of such modeling for Aspergillus fumigatus is evaluated in the present article. Based on this evaluation, opinions are drawn on future directions of GRN modeling of fungal pathogens. The crucial point of genome-wide GRN modeling is the experimental evidence, both used for inferring the networks (omics 'first-hand' data as well as literature data used as prior knowledge) and for validation and evaluation of the inferred network models.

Authors: , S. Gerber, , S. Vlaic, S. Durmus, T. Cakir, F. E. Sevilgen, ,

Date Published: 22nd Apr 2016

Publication Type: Not specified

Abstract (Expand)

Gene regulatory network inference is a systems biology approach which predicts interactions between genes with the help of high-throughput data. In this review, we present current and updated network inference methods focusing on novel techniques for data acquisition, network inference assessment, network inference for interacting species and the integration of prior knowledge. After the advance of Next-Generation-Sequencing of cDNAs derived from RNA samples (RNA-Seq) we discuss in detail its application to network inference. Furthermore, we present progress for large-scale or even full-genomic network inference as well as for small-scale condensed network inference and review advances in the evaluation of network inference methods by crowdsourcing. Finally, we reflect the current availability of data and prior knowledge sources and give an outlook for the inference of gene regulatory networks that reflect interacting species, in particular pathogen-host interactions.

Authors: , , S. G. Henkel,

Date Published: 2nd Mar 2015

Publication Type: Not specified

Abstract (Expand)

Fungal infections have increased dramatically in the last 2 decades, and fighting infectious diseases requires innovative approaches such as the combination of two drugs acting on different targets or even targeting a salvage pathway of one of the drugs. The fungal cell wall biosynthesis is inhibited by the clinically used antifungal drug caspofungin. This antifungal activity has been found to be potentiated by humidimycin, a new natural product identified from the screening of a collection of 20,000 microbial extracts, which has no major effect when used alone. An analysis of transcriptomes and selected Aspergillus fumigatus mutants indicated that humidimycin affects the high osmolarity glycerol response pathway. By combining humidimycin and caspofungin, a strong increase in caspofungin efficacy was achieved, demonstrating that targeting different signaling pathways provides an excellent basis to develop novel anti-infective strategies.

Authors: , M. C. Monteiro, J. Martin, R. Altwasser, N. El Aouad, I. Gonzalez, , E. Mellado, S. Palomo, N. de Pedro, I. Perez-Victoria, J. R. Tormo, F. Vicente, F. Reyes, O. Genilloud,

Date Published: 8th Jun 2015

Publication Type: Not specified

Abstract (Expand)

The ability to adapt to diverse micro-environmental challenges encountered within a host is of pivotal importance to the opportunistic fungal pathogen Candida albicans. We have quantified C. albicans and M. musculus gene expression dynamics during phagocytosis by dendritic cells in a genome-wide, time-resolved analysis using simultaneous RNA-seq. A robust network inference map was generated from this dataset using NetGenerator, predicting novel interactions between the host and the pathogen. We experimentally verified predicted interdependent sub-networks comprising Hap3 in C. albicans, and Ptx3 and Mta2 in M. musculus. Remarkably, binding of recombinant Ptx3 to the C. albicans cell wall was found to regulate the expression of fungal Hap3 target genes as predicted by the network inference model. Pre-incubation of C. albicans with recombinant Ptx3 significantly altered the expression of Mta2 target cytokines such as IL-2 and IL-4 in a Hap3-dependent manner, further suggesting a role for Mta2 in host-pathogen interplay as predicted in the network inference model. We propose an integrated model for the functionality of these sub-networks during fungal invasion of immune cells, according to which binding of Ptx3 to the C. albicans cell wall induces remodeling via fungal Hap3 target genes, thereby altering the immune response to the pathogen. We show the applicability of network inference to predict interactions between host-pathogen pairs, demonstrating the usefulness of this systems biology approach to decipher mechanisms of microbial pathogenesis.

Authors: L. Tierney, , S. Muller, S. Brunke, J. C. Molina, , U. Schock, , K. Kuchler

Date Published: 12th Mar 2012

Publication Type: Not specified

Abstract (Expand)

FungiFun assigns functional annotations to fungal genes or proteins and performs gene set enrichment analysis. Based on three different classification methods (FunCat, GO and KEGG), FungiFun categorizes genes and proteins for several fungal species on different levels of annotation detail. It is web-based and accessible to users without any programming skills. FungiFun is the first tool offering gene set enrichment analysis including the FunCat categorization. Two biological datasets for Aspergillus fumigatus and Candida albicans were analyzed using FungiFun, providing an overview of the usage and functions of the tool. FungiFun is freely accessible at https://www.omnifung.hki-jena.de/FungiFun/.

Authors: S. Priebe, , D. Albrecht, , A. A. Brakhage

Date Published: 10th Nov 2010

Publication Type: Not specified

Abstract (Expand)

Invasive fungal infections are associated with high mortality rates and are mostly caused by the opportunistic fungi Aspergillus fumigatus and Candida albicans. Immune responses against these fungi are still not fully understood. Dendritic cells (DCs) are crucial players in initiating innate and adaptive immune responses against fungal infections. The immunomodulatory effects of fungi were compared to the bacterial stimulus LPS to determine key players in the immune response to fungal infections. A genome wide study of the gene regulation of human monocyte-derived dendritic cells (DCs) confronted with A. fumigatus, C. albicans or LPS was performed and Kruppel-like factor 4 (KLF4) was identified as the only transcription factor that was down-regulated in DCs by both fungi but induced by stimulation with LPS. Downstream analysis demonstrated the influence of KLF4 on the interleukine-6 expression in human DCs. Furthermore, KLF4 regulation was shown to be dependent on pattern recognition receptor ligation. Therefore KLF4 was identified as a controlling element in the IL-6 immune response with a unique expression pattern comparing fungal and LPS stimulation.

Authors: K. Czakai, I. Leonhardt, , M. Bonin, , , ,

Date Published: 28th Jun 2016

Publication Type: Not specified

Abstract (Expand)

Here, we report the draft genome sequence of Aspergillus calidoustus (strain SF006504). The functional annotation of A. calidoustus predicts a relatively large number of secondary metabolite gene clusters. The presented genome sequence builds the basis for further genome mining.

Authors: F. Horn, , D. J. Mattern, G. Walther, , K. Scherlach, K. Martin, , L. Petzke,

Date Published: 12th Mar 2016

Publication Type: Not specified

Abstract (Expand)

As part of the innate immune system, natural killer (NK) cells are directly involved in the response to fungal infections. Perforin has been identified as the major effector molecule acting against many fungal pathogens. While several studies have shown that perforin mediated fungicidal effects can contribute to fungal clearance, neither the activation of NK cells by fungal pathogens nor the effects of perforin on fungal cells are well-understood. In a dual approach, we have studied the global gene expression pattern of primary and cytokine activated NK cells after co-incubation with Candida albicans and the transcriptomic adaptation of C. albicans to perforin exposure. NK cells responded to the fungal pathogen with an up-regulation of genes involved in immune signaling and release of cytokines. Furthermore, we observed a pronounced increase of genes involved in glycolysis and glycolysis inhibitor 2-deoxy-D-glucose impaired C. albicans induced NK cell activation. This strongly indicates that metabolic adaptation is a major part of the NK cell response to C. albicans infections. In the fungal pathogen, perforin induced a strong up-regulation of several fungal genes involved in the zinc depletion response, such as PRA1 and ZRT1. These data suggest that fungal zinc homeostasis is linked to the reaction to perforin secreted by NK cells. However, deletion mutants in PRA1 and ZRT1 did not show altered susceptibility to perforin.

Authors: , J. Voigt, M. Bouzani, , , , , R. Martin, ,

Date Published: 19th May 2016

Publication Type: Not specified

Abstract (Expand)

Humans are continuously exposed to airborne spores of the saprophytic fungus Aspergillus fumigatus. However, in healthy individuals pulmonary host defense mechanisms efficiently eliminate the fungus. In contrast, A. fumigatus causes devastating infections in immunocompromised patients. Host immune responses against A. fumigatus lung infections in immunocompromised conditions have remained largely elusive. Given the dynamic changes in immune cell subsets within tissues upon immunosuppressive therapy, we dissected the spatiotemporal pulmonary immune response after A. fumigatus infection to reveal basic immunological events that fail to effectively control invasive fungal disease. In different immunocompromised murine models, myeloid, notably neutrophils, and macrophages, but not lymphoid cells were strongly recruited to the lungs upon infection. Other myeloid cells, particularly dendritic cells and monocytes, were only recruited to lungs of corticosteroid treated mice, which developed a strong pulmonary inflammation after infection. Lymphoid cells, particularly CD4(+) or CD8(+) T-cells and NK cells were highly reduced upon immunosuppression and not recruited after A. fumigatus infection. Moreover, adoptive CD11b(+) myeloid cell transfer rescued cyclophosphamide immunosuppressed mice from lethal A. fumigatus infection but not cortisone and cyclophosphamide immunosuppressed mice. Our findings illustrate that CD11b(+) myeloid cells are critical for anti-A. fumigatus defense under cyclophosphamide immunosuppressed conditions.

Authors: , J. Amich, B. Arslan, S. Poreddy, K. Mattenheimer, Z. Mokhtari, , , ,

Date Published: 13th Jul 2016

Publication Type: Not specified

Abstract (Expand)

Mushrooms, such as Schizophyllum commune, have a specific odor. Whether this is linked to mating, prerequisite for mushroom formation, or also found in monokaryotic, unmated strains, was investigated with a comprehensive study on the transcriptome and proteome of this model organism. Mating interactions were investigated using a complete, cytosolic proteome map for unmated, monokaryotic, as well as for mated, dikaryotic mycelia. The regulations of the proteome were compared to transcriptional changes upon mating and to changes in smell by volatilome studies. We could show a good overlap between proteome and transcriptome data, but extensive posttranslational regulation was identified for more than 80% of transcripts. This suggests down-stream regulation upon interaction of mating partners and formation of the dikaryon that is competent to form fruiting bodies. The volatilome was shown to respond to mating by a broader spectrum of volatiles and increased emission of the mushroom smell molecules 3-octanone and 1-octen-3-ol, as well as ethanol and beta-bisabolol in the dikaryon. Putatively involved biosynthetic proteins like alcohol dehydrogenases, Ppo-like oxygenases, or sesquiterpene synthases showed correlating transcriptional regulation depending on either mono- or dikaryotic stages.

Authors: D. Freihorst, M. Brunsch, S. Wirth, K. Krause, , , M. Kunert, W. Boland, E. Kothe

Date Published: 7th Sep 2016

Publication Type: Not specified

Abstract (Expand)

A precise and rapid adjustment of fluxes through metabolic pathways is crucial for organisms to prevail in changing environmental conditions. Based on this reasoning, many guiding principles that govern the evolution of metabolic networks and their regulation have been uncovered. To this end, methods from dynamic optimization are ideally suited since they allow to uncover optimality principles behind the regulation of metabolic networks. We used dynamic optimization to investigate the influence of toxic intermediates in connection with the efficiency of enzymes on the regulation of a linear metabolic pathway. Our results predict that transcriptional regulation favors the control of highly efficient enzymes with less toxic upstream intermediates to reduce accumulation of toxic downstream intermediates. We show that the derived optimality principles hold by the analysis of the interplay between intermediate toxicity and pathway regulation in the metabolic pathways of over 5000 sequenced prokaryotes. Moreover, using the lipopolysaccharide biosynthesis in Escherichia coli as an example, we show how knowledge about the relation of regulation, kinetic efficiency and intermediate toxicity can be used to identify drug targets, which control endogenous toxic metabolites and prevent microbial growth. Beyond prokaryotes, we discuss the potential of our findings for the development of antifungal drugs.

Authors: J. Ewald, M. Bartl, T. Dandekar, C. Kaleta

Date Published: 18th Feb 2017

Publication Type: Not specified

Abstract (Expand)

Understanding optimality principles shaping the evolution of regulatory networks controlling metabolism is crucial for deriving a holistic picture of how metabolism is integrated into key cellular processes such as growth, adaptation and pathogenicity. While in the past the focus of research in pathway regulation was mainly based on stationary states, more recently dynamic optimization has proved to be an ideal tool to decipher regulatory strategies for metabolic pathways in response to environmental cues. In this short review, we summarize recent advances in the elucidation of optimal regulatory strategies and identification of optimal control points in metabolic pathways. We discuss biological implications of the discovered optimality principles on genome organization and provide examples how the derived knowledge can be used to identify new treatment strategies against pathogens. Furthermore, we briefly discuss the variety of approaches for solving dynamic optimization problems and emphasize whole-cell resource allocation models as an important emerging area of research that will allow us to study the regulation of metabolism on the whole-cell level.

Authors: J. Ewald, M. Bartl, C. Kaleta

Date Published: 30th Jul 2017

Publication Type: Not specified

Abstract (Expand)

Candida albicans colonizes human mucosa, including the gastrointestinal tract, as a commensal. In immunocompromised patients, C. albicans can breach the intestinal epithelial barrier and cause fatal invasive infections. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1; CD66a), CEACAM5 (CEA), and CEACAM6 (CD66c) are immunomodulatory receptors expressed on human mucosa and are recruited by bacterial and viral pathogens. Here we show for the first time that a fungal pathogen (i.e., C. albicans) also binds directly to the extracellular domain of human CEACAM1, CEACAM3, CEACAM5, and CEACAM6. Binding was specific for human CEACAMs and mediated by the N-terminal IgV-like domain. In enterocytic C2BBe1 cells, C. albicans caused a transient tyrosine phosphorylation of CEACAM1 and induced higher expression of membrane-bound CEACAM1 and soluble CEACAM6. Lack of the CEACAM1 receptor after short hairpin RNA (shRNA) knockdown abolished CXCL8 (interleukin-8) secretion by C2BBe1 cells in response to C. albicans In CEACAM1-competent cells, the addition of recombinant soluble CEACAM6 reduced the C. albicans-induced CXCL8 secretion.IMPORTANCE The present study demonstrates for the first time that fungal pathogens can be recognized by at least four members of the immunomodulatory CEACAM receptor family: CEACAM1, -3, -5, and -6. Three of the four receptors (i.e., CEACAM1, -5, and -6) are expressed in mucosal cells of the intestinal tract, where they are implicated in immunomodulation and control of tissue homeostasis. Importantly, the interaction of the major fungal pathogen in humans Candida albicans with CEACAM1 and CEACAM6 resulted in an altered epithelial immune response. With respect to the broad impact of CEACAM receptors on various aspects of the innate and the adaptive immune responses, in particular epithelial, neutrophil, and T cell behavior, understanding the role of CEACAMs in the host response to fungal pathogens might help to improve management of superficial and systemic fungal infections.

Authors: E. Klaile, M. M. Muller, M. R. Schafer, A. K. Clauder, S. Feer, K. A. Heyl, M. Stock, T. E. Klassert, P. F. Zipfel, B. B. Singer, H. Slevogt

Date Published: 16th Mar 2017

Publication Type: Not specified

Abstract (Expand)

Helicobacter pylori specifically colonizes the human gastric epithelium and is the major causative agent for ulcer disease and gastric cancer development. Here, we identify members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family as receptors of H. pylori and show that HopQ is the surface-exposed adhesin that specifically binds human CEACAM1, CEACAM3, CEACAM5 and CEACAM6. HopQ-CEACAM binding is glycan-independent and targeted to the N-domain. H. pylori binding induces CEACAM1-mediated signalling, and the HopQ-CEACAM1 interaction enables translocation of the virulence factor CagA into host cells and enhances the release of pro-inflammatory mediators such as interleukin-8. Based on the crystal structure of HopQ, we found that a beta-hairpin insertion (HopQ-ID) in HopQ's extracellular 3+4 helix bundle domain is important for CEACAM binding. A peptide derived from this domain competitively inhibits HopQ-mediated activation of the Cag virulence pathway, as genetic or antibody-mediated abrogation of the HopQ function shows. Together, our data suggest the HopQ-CEACAM1 interaction to be a potentially promising novel therapeutic target to combat H. pylori-associated diseases.

Authors: A. Javaheri, T. Kruse, K. Moonens, R. Mejias-Luque, A. Debraekeleer, C. I. Asche, N. Tegtmeyer, B. Kalali, N. C. Bach, S. A. Sieber, D. J. Hill, V. Koniger, C. R. Hauck, R. Moskalenko, R. Haas, D. H. Busch, E. Klaile, H. Slevogt, A. Schmidt, S. Backert, H. Remaut, B. B. Singer, M. Gerhard

Date Published: 18th Oct 2016

Publication Type: Not specified

Abstract (Expand)

Candida albicans is an important human opportunistic fungal pathogen which is frequently found as part of the normal human microbiota. It is well accepted that the fungus interacts with other components of the resident microbiota and that this impacts the commensal or pathogenic outcome of C. albicans colonization. Different types of interactions, including synergism or antagonism, contribute to a complex balance between the multitude of different species. Mixed biofilms of C. albicans and streptococci are a well-studied example of a mutualistic interaction often potentiating the virulence of the individual members. In contrast, other bacteria like lactobacilli are known to antagonize C. albicans, and research has just started elucidating the mechanisms behind these interactions. This scenario is even more complicated by a third player, the host. This review focuses on interactions between C. albicans and gram-positive bacteria whose investigation will without doubt ultimately help understanding C. albicans infections.

Editor:

Date Published: No date defined

Publication Type: Not specified

Abstract (Expand)

BACKGROUND: Although Candida albicans and Candida dubliniensis are most closely related, both species behave significantly different with respect to morphogenesis and virulence. In order to gain further insight into the divergent routes for morphogenetic adaptation in both species, we investigated qualitative along with quantitative differences in the transcriptomes of both organisms by cDNA deep sequencing. RESULTS: Following genome-associated assembly of sequence reads we were able to generate experimentally verified databases containing 6016 and 5972 genes for C. albicans and C. dubliniensis, respectively. About 95% of the transcriptionally active regions (TARs) contain open reading frames while the remaining TARs most likely represent non-coding RNAs. Comparison of our annotations with publically available gene models for C. albicans and C. dubliniensis confirmed approximately 95% of already predicted genes, but also revealed so far unknown novel TARs in both species. Qualitative cross-species analysis of these databases revealed in addition to 5802 orthologs also 399 and 49 species-specific protein coding genes for C. albicans and C. dubliniensis, respectively. Furthermore, quantitative transcriptional profiling using RNA-Seq revealed significant differences in the expression of orthologs across both species. We defined a core subset of 84 hyphal-specific genes required for both species, as well as a set of 42 genes that seem to be specifically induced during hyphal morphogenesis in C. albicans. CONCLUSIONS: Species-specific adaptation in C. albicans and C. dubliniensis is governed by individual genetic repertoires but also by altered regulation of conserved orthologs on the transcriptional level.

Authors: C. Grumaz, S. Lorenz, P. Stevens, E. Lindemann, U. Schock, J. Retey, S. Rupp, K. Sohn

Date Published: 4th Apr 2013

Publication Type: Not specified

Abstract (Expand)

Candida albicans is the most important fungal pathogen of humans, causing severe infections, especially in nosocomial and immunocompromised settings. However, it is also the most prevalent fungus of the normal human microbiome, where it shares its habitat with hundreds of trillions of other microbial cells. Despite weak organic acids (WOAs) being among the most abundant metabolites produced by bacterial microbiota, little is known about their effect on C. albicans. Here we used a sequencing-based profiling strategy to systematically investigate the transcriptional stress response of C. albicans to lactic, acetic, propionic, and butyric acid at several time points after treatment. Our data reveal a complex transcriptional response, with individual WOAs triggering unique gene expression profiles and with important differences between acute and chronic exposure. Despite these dissimilarities, we found significant overlaps between the gene expression changes induced by each WOA, which led us to uncover a core transcriptional response that was largely unrelated to other previously published C. albicans transcriptional stress responses. Genes commonly up-regulated by WOAs were enriched in several iron transporters, which was associated with an overall decrease in intracellular iron concentrations. Moreover, chronic exposure to any WOA lead to down-regulation of RNA synthesis and ribosome biogenesis genes, which resulted in significant reduction of total RNA levels and of ribosomal RNA in particular. In conclusion, this study suggests that gastrointestinal microbiota might directly influence C. albicans physiology via production of WOAs, with possible implications of how this fungus interacts with its host in both health and disease.

Authors: F. Cottier, A. S. Tan, J. Chen, J. Lum, F. Zolezzi, M. Poidinger, N. Pavelka

Date Published: 1st Feb 2015

Publication Type: Not specified

Abstract (Expand)

UNLABELLED: Single-celled organisms have different strategies to sense and utilize nutrients in their ever-changing environments. The opportunistic fungal pathogen Candida albicans is a common member of the human microbiota, especially that of the gastrointestinal (GI) tract. An important question concerns how C. albicans gained a competitive advantage over other microbes to become a successful commensal and opportunistic pathogen. Here, we report that C. albicans uses N-acetylglucosamine (GlcNAc), an abundant carbon source present in the GI tract, as a signal for nutrient availability. When placed in water, C. albicans cells normally enter the G0 phase and remain viable for weeks. However, they quickly lose viability when cultured in water containing only GlcNAc. We term this phenomenon GlcNAc-induced cell death (GICD). GlcNAc triggers the upregulation of ribosomal biogenesis genes, alterations of mitochondrial metabolism, and the accumulation of reactive oxygen species (ROS), followed by rapid cell death via both apoptotic and necrotic mechanisms. Multiple pathways, including the conserved cyclic AMP (cAMP) signaling and GlcNAc catabolic pathways, are involved in GICD. GlcNAc acts as a signaling molecule to regulate multiple cellular programs in a coordinated manner and therefore maximizes the efficiency of nutrient use. This adaptive behavior allows C. albicans' more efficient colonization of the gut. IMPORTANCE: The ability to rapidly and appropriately respond to nutrients in the environment is crucial to free-living microorganisms. To maximize the use of available nutrients, microorganisms often use a limiting nutritional component as a signal to coordinate multiple biological processes. The human fungal pathogen Candida albicans uses N-acetylglucosamine (GlcNAc) as a signal for the availability of external nutrient resources. GlcNAc induces rapid cell death in C. albicans due to the constitutive activation of oxidative metabolism and accumulation of reactive oxygen species (ROS), and multiple pathways are involved in its regulation. This study sheds light on the mechanisms of niche specialization of pathogenic fungi and raises the possibility that this cell death pathway could be an unexplored therapeutic target.

Authors: H. Du, G. Guan, X. Li, M. Gulati, L. Tao, C. Cao, A. D. Johnson, C. J. Nobile, G. Huang

Date Published: 10th Sep 2015

Publication Type: Not specified

Abstract (Expand)

Despite their classical role as transcriptional repressors, several histone deacetylases, including the baker's yeast Set3/Hos2 complex (Set3C), facilitate gene expression. In the dimorphic human pathogen Candida albicans, the homologue of the Set3C inhibits the yeast-to-filament transition, but the precise molecular details of this function have remained elusive. Here, we use a combination of ChIP-Seq and RNA-Seq to show that the Set3C acts as a transcriptional co-factor of metabolic and morphogenesis-related genes in C. albicans. Binding of the Set3C correlates with gene expression during fungal morphogenesis; yet, surprisingly, deletion of SET3 leaves the steady-state expression level of most genes unchanged, both during exponential yeast-phase growth and during the yeast-filament transition. Fine temporal resolution of transcription in cells undergoing this transition revealed that the Set3C modulates transient expression changes of key morphogenesis-related genes. These include a transcription factor cluster comprising of NRG1, EFG1, BRG1, and TEC1, which form a regulatory circuit controlling hyphal differentiation. Set3C appears to restrict the factors by modulating their transcription kinetics, and the hyperfilamentous phenotype of SET3-deficient cells can be reverted by mutating the circuit factors. These results indicate that the chromatin status at coding regions represents a dynamic platform influencing transcription kinetics. Moreover, we suggest that transcription at the coding sequence can be transiently decoupled from potentially conflicting promoter information in dynamic environments.

Authors: D. Hnisz, A. F. Bardet, C. J. Nobile, A. Petryshyn, W. Glaser, U. Schock, A. Stark, K. Kuchler

Date Published: 14th Dec 2012

Publication Type: Not specified

Abstract (Expand)

Candida albicans demonstrates three main growth morphologies: yeast, pseudohyphal and true hyphal forms. Cell separation is distinct in these morphological forms and the process of separation is closely linked to the completion of mitosis and cytokinesis. In Saccharomyces cerevisiae the small GTPase Tem1 is known to initiate the mitotic exit network, a signalling pathway involved in signalling the end of mitosis and initiating cytokinesis and cell separation. Here we have characterised the role of Tem1 in C. albicans, and demonstrate that it is essential for mitotic exit and cytokinesis, and that this essential function is signalled through the kinase Cdc15. Cells depleted of Tem1 displayed highly polarised growth but ultimately failed to both complete cytokinesis and re-enter the cell cycle following nuclear division. Consistent with its role in activating the mitotic exit network Tem1 localises to spindle pole bodies in a cell cycle-dependent manner. Ultimately, the mitotic exit network in C. albicans appears to co-ordinate the sequential processes of mitotic exit, cytokinesis and cell separation.

Authors: S. W. Milne, J. Cheetham, D. Lloyd, S. Shaw, K. Moore, K. H. Paszkiewicz, S. J. Aves, S. Bates

Date Published: 29th Jun 2014

Publication Type: Not specified

Abstract (Expand)

Modes of sexual reproduction in eukaryotic organisms are extremely diverse. The human fungal pathogen Candida albicans undergoes a phenotypic switch from the white to the opaque phase in order to become mating-competent. In this study, we report that functionally- and morphologically-differentiated white and opaque cells show a coordinated behavior during mating. Although white cells are mating-incompetent, they can produce sexual pheromones when treated with pheromones of the opposite mating type or by physically interacting with opaque cells of the opposite mating type. In a co-culture system, pheromones released by white cells induce opaque cells to form mating projections, and facilitate both opposite- and same-sex mating of opaque cells. Deletion of genes encoding the pheromone precursor proteins and inactivation of the pheromone response signaling pathway (Ste2-MAPK-Cph1) impair the promoting role of white cells (MTLa) in the sexual mating of opaque cells. White and opaque cells communicate via a paracrine pheromone signaling system, creating an environment conducive to sexual mating. This coordination between the two different cell types may be a trade-off strategy between sexual and asexual lifestyles in C. albicans.

Authors: L. Tao, C. Cao, W. Liang, G. Guan, Q. Zhang, C. J. Nobile, G. Huang

Date Published: 21st Oct 2014

Publication Type: Not specified

Abstract (Expand)

As a successful commensal and pathogen of humans, Candida albicans encounters a wide range of environmental conditions. Among them, ambient pH, which changes frequently and affects many biological processes in this species, is an important factor, and the ability to adapt to pH changes is tightly linked with pathogenesis and morphogenesis. In this study, we report that pH has a profound effect on white-opaque switching and sexual mating in C. albicans. Acidic pH promotes white-to-opaque switching under certain culture conditions but represses sexual mating. The Rim101-mediated pH-sensing pathway is involved in the control of pH-regulated white-opaque switching and the mating response. Phr2 and Rim101 could play a major role in acidic pH-induced opaque cell formation. Despite the fact that the cyclic AMP (cAMP) signaling pathway does not play a major role in pH-regulated white-opaque switching and mating, white and opaque cells of the cyr1/cyr1 mutant, which is defective in the production of cAMP, showed distinct growth defects under acidic and alkaline conditions. We further discovered that acidic pH conditions repressed sexual mating due to the failure of activation of the Ste2-mediated alpha-pheromone response pathway in opaque A: cells. The effects of pH changes on phenotypic switching and sexual mating could involve a balance of host adaptation and sexual reproduction in C. albicans.

Authors: Y. Sun, C. Cao, W. Jia, L. Tao, G. Guan, G. Huang

Date Published: 6th Sep 2015

Publication Type: Not specified

Abstract (Expand)

Candida albicans is associated with humans as both a harmless commensal organism and a pathogen. Cph2 is a transcription factor whose DNA binding domain is similar to that of mammalian sterol response element binding proteins (SREBPs). SREBPs are master regulators of cellular cholesterol levels and are highly conserved from fungi to mammals. However, ergosterol biosynthesis is regulated by the zinc finger transcription factor Upc2 in C. albicans and several other yeasts. Cph2 is not necessary for ergosterol biosynthesis but is important for colonization in the murine gastrointestinal (GI) tract. Here we demonstrate that Cph2 is a membrane-associated transcription factor that is processed to release the N-terminal DNA binding domain like SREBPs, but its cleavage is not regulated by cellular levels of ergosterol or oxygen. Chromatin immunoprecipitation sequencing (ChIP-seq) shows that Cph2 binds to the promoters of HMS1 and other components of the regulatory circuit for GI tract colonization. In addition, 50% of Cph2 targets are also bound by Hms1 and other factors of the regulatory circuit. Several common targets function at the head of the glycolysis pathway. Thus, Cph2 is an integral part of the regulatory circuit for GI colonization that regulates glycolytic flux. Transcriptome sequencing (RNA-seq) shows a significant overlap in genes differentially regulated by Cph2 and hypoxia, and Cph2 is important for optimal expression of some hypoxia-responsive genes in glycolysis and the citric acid cycle. We suggest that Cph2 and Upc2 regulate hypoxia-responsive expression in different pathways, consistent with a synthetic lethal defect of the cph2 upc2 double mutant in hypoxia.

Authors: S. Lane, P. Di Lena, K. Tormanen, P. Baldi, H. Liu

Date Published: 6th Sep 2015

Publication Type: Not specified

Abstract (Expand)

BACKGROUND: Invasive aspergillosis is started after germination of Aspergillus fumigatus conidia that are inhaled by susceptible individuals. Fungal hyphae can grow in the lung through the epithelial tissue and disseminate hematogenously to invade into other organs. Low fungaemia indicates that fungal elements do not reside in the bloodstream for long. RESULTS: We analyzed whether blood represents a hostile environment to which the physiology of A. fumigatus has to adapt. An in vitro model of A. fumigatus infection was established by incubating mycelium in blood. Our model allowed to discern the changes of the gene expression profile of A. fumigatus at various stages of the infection. The majority of described virulence factors that are connected to pulmonary infections appeared not to be activated during the blood phase. Three active processes were identified that presumably help the fungus to survive the blood environment in an advanced phase of the infection: iron homeostasis, secondary metabolism, and the formation of detoxifying enzymes. CONCLUSIONS: We propose that A. fumigatus is hardly able to propagate in blood. After an early stage of sensing the environment, virtually all uptake mechanisms and energy-consuming metabolic pathways are shut-down. The fungus appears to adapt by trans-differentiation into a resting mycelial stage. This might reflect the harsh conditions in blood where A. fumigatus cannot take up sufficient nutrients to establish self-defense mechanisms combined with significant growth.

Authors: H. Irmer, S. Tarazona, C. Sasse, P. Olbermann, J. Loeffler, S. Krappmann, A. Conesa, G. H. Braus

Date Published: 28th Aug 2015

Publication Type: Not specified

Abstract (Expand)

The identification of novel transcription factors associated with antifungal response may allow the discovery of fungus-specific targets for new therapeutic strategies. A collection of 241 Candida albicans transcriptional regulator mutants was screened for altered susceptibility to fluconazole, caspofungin, amphotericin B, and 5-fluorocytosine. Thirteen of these mutants not yet identified in terms of their role in antifungal response were further investigated, and the function of one of them, a mutant of orf19.6102 (RCA1), was characterized by transcriptome analysis. Strand-specific RNA sequencing and phenotypic tests assigned Rca1 as the regulator of hyphal formation through the cyclic AMP/protein kinase A (cAMP/PKA) signaling pathway and the transcription factor Efg1, but also probably through its interaction with a transcriptional repressor, most likely Tup1. The mechanisms responsible for the high level of resistance to caspofungin and fluconazole observed resulting from RCA1 deletion were investigated. From our observations, we propose that caspofungin resistance was the consequence of the deregulation of cell wall gene expression and that fluconazole resistance was linked to the modulation of the cAMP/PKA signaling pathway activity. In conclusion, our large-scale screening of a C. albicans transcription factor mutant collection allowed the identification of new effectors of the response to antifungals. The functional characterization of Rca1 assigned this transcription factor and its downstream targets as promising candidates for the development of new therapeutic strategies, as Rca1 influences host sensing, hyphal development, and antifungal response.

Authors: P. Vandeputte, S. Pradervand, F. Ischer, A. T. Coste, S. Ferrari, K. Harshman, D. Sanglard

Date Published: No date defined

Publication Type: Not specified

Abstract (Expand)

Candida albicans is a common cause of life-threatening fungal bloodstream infections. In the murine model of systemic candidiasis, the kidney is the primary target organ while the fungal load declines over time in liver and spleen. To better understand these organ-specific differences in host-pathogen interaction, we performed gene expression profiling of murine kidney, liver and spleen and determined the fungal transcriptome in liver and kidney. We observed a delayed transcriptional immune response accompanied by late induction of fungal stress response genes in the kidneys. In contrast, early upregulation of the proinflammatory response in the liver was associated with a fungal transcriptome resembling response to phagocytosis, suggesting that phagocytes contribute significantly to fungal control in the liver. Notably, C. albicans hypha-associated genes were upregulated in the absence of visible filamentation in the liver, indicating an uncoupling of gene expression and morphology and a morphology-independent effect by hypha-associated genes in this organ. Consistently, integration of host and pathogen transcriptional data in an inter-species gene regulatory network indicated connections of C. albicans cell wall remodelling and metabolism to the organ-specific immune responses.

Authors: B. Hebecker, S. Vlaic, T. Conrad, M. Bauer, S. Brunke, M. Kapitan, J. Linde, B. Hube, I. D. Jacobsen

Date Published: No date defined

Publication Type: Not specified

Abstract (Expand)

Human fungal pathogens like Candida albicans respond to host immune surveillance by rapidly adapting their transcriptional programs. Chromatin assembly factors are involved in the regulation of stress genes by modulating the histone density at these loci. Here, we report a novel role for the chromatin assembly-associated histone acetyltransferase complex NuB4 in regulating oxidative stress resistance, antifungal drug tolerance and virulence in C. albicans. Strikingly, depletion of the NuB4 catalytic subunit, the histone acetyltransferase Hat1, markedly increases resistance to oxidative stress and tolerance to azole antifungals. Hydrogen peroxide resistance in cells lacking Hat1 results from higher induction rates of oxidative stress gene expression, accompanied by reduced histone density as well as subsequent increased RNA polymerase recruitment. Furthermore, hat1Delta/Delta cells, despite showing growth defects in vitro, display reduced susceptibility to reactive oxygen-mediated killing by innate immune cells. Thus, clearance from infected mice is delayed although cells lacking Hat1 are severely compromised in killing the host. Interestingly, increased oxidative stress resistance and azole tolerance are phenocopied by the loss of histone chaperone complexes CAF-1 and HIR, respectively, suggesting a central role for NuB4 in the delivery of histones destined for chromatin assembly via distinct pathways. Remarkably, the oxidative stress phenotype of hat1Delta/Delta cells is a species-specific trait only found in C. albicans and members of the CTG clade. The reduced azole susceptibility appears to be conserved in a wider range of fungi. Thus, our work demonstrates how highly conserved chromatin assembly pathways can acquire new functions in pathogenic fungi during coevolution with the host.

Authors: M. Tscherner, F. Zwolanek, S. Jenull, F. J. Sedlazeck, A. Petryshyn, I. E. Frohner, J. Mavrianos, N. Chauhan, A. von Haeseler, K. Kuchler

Date Published: No date defined

Publication Type: Not specified

Abstract (Expand)

Aspergillus fumigatus is a common airborne fungal pathogen of humans and a significant source of mortality in immunocompromised individuals. Here, we provide the most extensive cell wall proteome profiling to date of A. fumigatus resting conidia, the fungal morphotype pertinent to first contact with the host. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified proteins within the conidial cell wall by hydrogen-fluoride (HF)-pyridine extraction and proteins exposed on the surface using a trypsin-shaving approach. One protein, designated conidial cell wall protein A (CcpA), was identified by both methods and was found to be nearly as abundant as hydrophobic rodlet layer-forming protein RodA. CcpA, an amphiphilic protein, like RodA, peaks in expression during sporulation on resting conidia. Despite high cell wall abundance, the cell surface structure of DeltaccpA resting conidia appeared normal. However, trypsin shaving of DeltaccpA conidia revealed novel surface-exposed proteins not detected on conidia of the wild-type strain. Interestingly, the presence of swollen DeltaccpA conidia led to higher activation of neutrophils and dendritic cells than was seen with wild-type conidia and caused significantly less damage to epithelial cells in vitro In addition, virulence was highly attenuated when cortisone-treated, immunosuppressed mice were infected with DeltaccpA conidia. CcpA-specific memory T cell responses were detectable in healthy human donors naturally exposed to A. fumigatus conidia, suggesting a role for CcpA as a structural protein impacting conidial immunogenicity rather than possessing a protein-intrinsic immunosuppressive effect. Together, these data suggest that CcpA serves as a conidial stealth protein by altering the conidial surface structure to minimize innate immune recognition.IMPORTANCE The mammalian immune system relies on recognition of pathogen surface antigens for targeting and clearance. In the absence of immune evasion strategies, pathogen clearance is rapid. In the case of Aspergillus fumigatus, the successful fungus must avoid phagocytosis in the lung to establish invasive infection. In healthy individuals, fungal spores are cleared by immune cells; however, in immunocompromised patients, clearance mechanisms are impaired. Here, using proteome analyses, we identified CcpA as an important fungal spore protein involved in pathogenesis. A. fumigatus lacking CcpA was more susceptible to immune recognition and prompt eradication and, consequently, exhibited drastically attenuated virulence. In infection studies, CcpA was required for virulence in infected immunocompromised mice, suggesting that it could be used as a possible immunotherapeutic or diagnostic target in the future. In summary, our report adds a protein to the list of those known to be critical to the complex fungal spore surface environment and, more importantly, identifies a protein important for conidial immunogenicity during infection.

Authors: V. Voltersen, M. G. Blango, S. Herrmann, F. Schmidt, T. Heinekamp, M. Strassburger, T. Kruger, P. Bacher, J. Lother, E. Weiss, K. Hunniger, H. Liu, P. Hortschansky, A. Scheffold, J. Loffler, S. Krappmann, S. Nietzsche, O. Kurzai, H. Einsele, O. Kniemeyer, S. G. Filler, U. Reichard, A. A. Brakhage

Date Published: No date defined

Publication Type: Not specified

Abstract (Expand)

Super-resolution microscopy has evolved as a powerful method for subdiffraction-resolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.

Authors: R. Gotz, S. Panzer, N. Trinks, J. Eilts, J. Wagener, D. Turra, A. Di Pietro, M. Sauer, U. Terpitz

Date Published: 23rd Apr 2020

Publication Type: Not specified

Abstract (Expand)

Candida albicans is a leading cause of life-threatening hospital-acquired infections and can lead to Candidemia with sepsis-like symptoms and high mortality rates. We reconstructed a genome-scale C. albicans metabolic model to investigate bacterial-fungal metabolic interactions in the gut as determinants of fungal abundance. We optimized the predictive capacity of our model using wild type and mutant C. albicans growth data and used it for in silico metabolic interaction predictions. Our analysis of more than 900 paired fungal-bacterial metabolic models predicted key gut bacterial species modulating C. albicans colonization levels. Among the studied microbes, Alistipes putredinis was predicted to negatively affect C. albicans levels. We confirmed these findings by metagenomic sequencing of stool samples from 24 human subjects and by fungal growth experiments in bacterial spent media. Furthermore, our pairwise simulations guided us to specific metabolites with promoting or inhibitory effect to the fungus when exposed in defined media under carbon and nitrogen limitation. Our study demonstrates that in silico metabolic prediction can lead to the identification of gut microbiome features that can significantly affect potentially harmful levels of C. albicans.

Authors: M. H. Mirhakkak, S. Schauble, T. E. Klassert, S. Brunke, P. Brandt, D. Loos, R. V. Uribe, F. Senne de Oliveira Lino, Y. Ni, S. Vylkova, H. Slevogt, B. Hube, G. J. Weiss, M. O. A. Sommer, G. Panagiotou

Date Published: 15th Dec 2020

Publication Type: Not specified

Abstract (Expand)

BACKGROUND: Antibiotic treatment has a well-established detrimental effect on the gut bacterial composition, but effects on the fungal community are less clear. Bacteria in the lumen of the gastrointestinal tract may limit fungal colonization and invasion. Antibiotic drugs targeting bacteria are therefore seen as an important risk factor for fungal infections and induced allergies. However, antibiotic effects on gut bacterial-fungal interactions, including disruption and resilience of fungal community compositions, were not investigated in humans. We analysed stool samples collected from 14 healthy human participants over 3 months following a 6-day antibiotic administration. We integrated data from shotgun metagenomics, metatranscriptomics, metabolomics, and fungal ITS2 sequencing. RESULTS: While the bacterial community recovered mostly over 3 months post treatment, the fungal community was shifted from mutualism at baseline to competition. Half of the bacterial-fungal interactions present before drug intervention had disappeared 3 months later. During treatment, fungal abundances were associated with the expression of bacterial genes with functions for cell growth and repair. By extending the metagenomic species approach, we revealed bacterial strains inhibiting the opportunistic fungal pathogen Candida albicans. We demonstrated in vitro how C. albicans pathogenicity and host cell damage might be controlled naturally in the human gut by bacterial metabolites such as propionate or 5-dodecenoate. CONCLUSIONS: We demonstrated that antibacterial drugs have long-term influence on the human gut mycobiome. While bacterial communities recovered mostly 30-days post antibacterial treatment, the fungal community was shifted from mutualism towards competition. Video abstract.

Authors: B. Seelbinder, J. Chen, S. Brunke, R. Vazquez-Uribe, R. Santhaman, A. C. Meyer, F. S. de Oliveira Lino, K. F. Chan, D. Loos, L. Imamovic, C. C. Tsang, R. P. Lam, S. Sridhar, K. Kang, B. Hube, P. C. Woo, M. O. A. Sommer, G. Panagiotou

Date Published: 12th Sep 2020

Publication Type: Not specified

Abstract (Expand)

High-throughput RNA sequencing (RNA-seq) is routinely applied to study diverse biological processes; however, when performed separately on interacting organisms, systemic noise intrinsic to RNA extraction, library preparation, and sequencing hampers the identification of cross-species interaction nodes. Here, we develop triple RNA-seq to simultaneously detect transcriptomes of monocyte-derived dendritic cells (moDCs) infected with the frequently co-occurring pulmonary pathogens Aspergillus fumigatus and human cytomegalovirus (CMV). Comparing expression patterns after co-infection with those after single infections, our data reveal synergistic effects and mutual interferences between host responses to the two pathogens. For example, CMV attenuates the fungus-mediated activation of pro-inflammatory cytokines through NF-kappaB (nuclear factor kappaB) and NFAT (nuclear factor of activated T cells) cascades, while A. fumigatus impairs viral clearance by counteracting viral nucleic acid-induced activation of type I interferon signaling. Together, the analytical power of triple RNA-seq proposes molecular hubs in the differential moDC response to fungal/viral single infection or co-infection that contribute to our understanding of the etiology and, potentially, clearance of post-transplant infections.

Authors: B. Seelbinder, J. Wallstabe, L. Marischen, E. Weiss, S. Wurster, L. Page, C. Loffler, L. Bussemer, A. L. Schmitt, T. Wolf, J. Linde, L. Cicin-Sain, J. Becker, U. Kalinke, J. Vogel, G. Panagiotou, H. Einsele, A. J. Westermann, S. Schauble, J. Loeffler

Date Published: 17th Nov 2020

Publication Type: Not specified

Abstract (Expand)

Investigating metabolic functional capability of a human gut microbiome enables the quantification of microbiome changes, which can cause a phenotypic change of host physiology and disease. One possible way to estimate the functional capability of a microbial community is through inferring metagenomic content from 16S rRNA gene sequences. Genome-scale models (GEMs) can be used as scaffold for functional estimation analysis at a systematic level, however up to date, there is no integrative toolbox based on GEMs for uncovering metabolic functions. Here, we developed the MetGEMs (metagenome-scale models) toolbox, an open-source application for inferring metabolic functions from 16S rRNA gene sequences to facilitate the study of the human gut microbiome by the wider scientific community. The developed toolbox was validated using shotgun metagenomic data and shown to be superior in predicting functional composition in human clinical samples compared to existing state-of-the-art tools. Therefore, the MetGEMs toolbox was subsequently applied for annotating putative enzyme functions and metabolic routes related in human disease using atopic dermatitis as a case study.

Authors: P. Patumcharoenpol, M. Nakphaichit, G. Panagiotou, A. Senavonge, N. Suratannon, W. Vongsangnak

Date Published: 6th Jan 2021

Publication Type: Journal

Abstract (Expand)

Pathogenic microorganisms exploit host metabolism for sustained survival by rewiring its metabolic interactions. Therefore, several metabolic changes are induced in both pathogen and host cells in the course of infection. A systems-based approach to elucidate those changes includes the integrative use of genome-scale metabolic networks and molecular omics data, with the overall goal of better characterizing infection mechanisms for novel treatment strategies. This review focuses on novel aspects of metabolism-oriented systems-based investigation of pathogen-human interactions. The reviewed approaches are the generation of dual-omics data for the characterization of metabolic signatures of pathogen-host interactions, the reconstruction of pathogen-host integrated genome-scale metabolic networks, which has a high potential to be applied to pathogen-gut microbiota interactions, and the structure-based analysis of enzymes playing role in those interactions. The integrative use of those approaches will pave the way for the identification of novel biomarkers and drug targets for the prediction and prevention of infectious diseases.

Authors: T. Cakir, G. Panagiotou, R. Uddin, S. Durmus

Date Published: 3rd Mar 2020

Publication Type: Not specified

Abstract (Expand)

Rhinovirus (RV) and influenza virus are the most frequently detected respiratory viruses among adult patients with community acquired pneumonia. Previous clinical studies have identified major differences in the clinical presentations and inflammatory or immune response during these infections. A systematic transcriptomic analysis directly comparing influenza and RV is lacking. Here, we sought to compare the transcriptomic response to these viral infections. Human airway epithelial Calu-3 cells were infected with contemporary clinical isolates of RV, influenza A virus (IAV), or influenza B virus (IBV). Host gene expression was determined using RNA-seq. Differentially expressed genes (DEGs) with respect to mock-infected cells were identified using the overlapping gene-set of four different statistical models. Transcriptomic analysis showed that RV-infected cells have a more blunted host response with fewer DEGs than IAV or IBV-infected cells. IFNL1 and CXCL10 were among the most upregulated DEGs during RV, IAV, and IBV infection. Other DEGs that were highly expressed for all 3 viruses were mainly genes related to type I or type III interferons (RSAD2, IDO1) and chemokines (CXCL11). Notably, ICAM5, a known receptor for enterovirus D68, was highly expressed during RV infection only. Gene Set Enrichment Analysis (GSEA) confirmed that pathways associated with interferon response, innate immunity, or regulation of inflammatory response, were most perturbed for all three viruses. Network analysis showed that steroid-related pathways were enriched. Taken together, our data using contemporary virus strains suggests that genes related to interferon and chemokine predominated the host response associated with RV, IAV, and IBV infection. Several highly expressed genes, especially ICAM5 which is preferentially-induced during RV infection, deserve further investigation.

Authors: T. K. Dissanayake, S. Schauble, M. H. Mirhakkak, W. L. Wu, A. C. Ng, C. C. Y. Yip, A. G. Lopez, T. Wolf, M. L. Yeung, K. H. Chan, K. Y. Yuen, G. Panagiotou, K. K. To

Date Published: 28th Aug 2020

Publication Type: Not specified

Abstract (Expand)

Invasive pulmonary aspergillosis (IPA) is a severe infection that is difficult to diagnose due to the ubiquitous presence of fungal spores, the underlying diseases of risk patients, and limitations of currently available markers. In this study, we performed a comprehensive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based identification of host and fungal proteins expressed during IPA in mice and humans. The proteomic analysis of bronchoalveolar lavage samples of individual IPA and control cases allowed the description of common host factors that had significantly increased abundance in both infected animals and IPA patients compared to their controls. Although increased levels of these individual host proteins might not be sufficient to distinguish bacterial from fungal infection, a combination of these markers might be beneficial to improve diagnosis. We also identified 16 fungal proteins that were specifically detected during infection and may be valuable candidates for biomarker evaluation.

Authors: S. Machata, M. M. Muller, R. Lehmann, P. Sieber, G. Panagiotou, A. Carvalho, C. Cunha, K. Lagrou, J. Maertens, H. Slevogt, I. D. Jacobsen

Date Published: 12th Oct 2020

Publication Type: Not specified

Abstract (Expand)

Only four species, Candida albicans, C. glabrata, C. parapsilosis, and C. tropicalis, together account for about 90% of all Candida bloodstream infections and are among the most common causes of invasive fungal infections of humans. However, virulence potential varies among these species, and the phylogenetic tree reveals that their pathogenicity may have emerged several times independently during evolution. We therefore tested these four species in a human whole-blood infection model to determine, via comprehensive dual-species RNA-sequencing analyses, which fungal infection strategies are conserved and which are recent evolutionary developments. The ex vivo infection progressed from initial immune cell interactions to nearly complete killing of all fungal cells. During the course of infection, we characterized important parameters of pathogen-host interactions, such as fungal survival, types of interacting immune cells, and cytokine release. On the transcriptional level, we obtained a predominantly uniform and species-independent human response governed by a strong upregulation of proinflammatory processes, which was downregulated at later time points after most of the fungal cells were killed. In stark contrast, we observed that the different fungal species pursued predominantly individual strategies and showed significantly different global transcriptome patterns. Among other findings, our functional analyses revealed that the fungal species relied on different metabolic pathways and virulence factors to survive the host-imposed stress. These data show that adaptation of Candida species as a response to the host is not a phylogenetic trait, but rather has likely evolved independently as a prerequisite to cause human infections.IMPORTANCE To ensure their survival, pathogens have to adapt immediately to new environments in their hosts, for example, during the transition from the gut to the bloodstream. Here, we investigated the basis of this adaptation in a group of fungal species which are among the most common causes of hospital-acquired infections, the Candida species. On the basis of a human whole-blood infection model, we studied which genes and processes are active over the course of an infection in both the host and four different Candida pathogens. Remarkably, we found that, while the human host response during the early phase of infection is predominantly uniform, the pathogens pursue largely individual strategies and each one regulates genes involved in largely disparate processes in the blood. Our results reveal that C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis all have developed individual strategies for survival in the host. This indicates that their pathogenicity in humans has evolved several times independently and that genes which are central for survival in the host for one species may be irrelevant in another.

Authors: P. Kammer, S. McNamara, T. Wolf, T. Conrad, S. Allert, F. Gerwien, K. Hunniger, O. Kurzai, R. Guthke, B. Hube, J. Linde, S. Brunke

Date Published: 6th Oct 2020

Publication Type: Not specified

Abstract (Expand)

Sepsis remains a major cause of death despite advances in medical care. Metabolic deregulation is an important component of the survival process. Metabolomic analysis allows profiling of critical metabolic functions with the potential to classify patient outcome. Our prospective longitudinal characterization of 33 septic and non-septic critically ill patients showed that deviations, independent of direction, in plasma levels of lipid metabolites were associated with sepsis mortality. We identified a coupling of metabolic signatures between liver and plasma of a rat sepsis model that allowed us to apply a human kinetic model of mitochondrial beta-oxidation to reveal differing enzyme concentrations for medium/short-chain hydroxyacyl-CoA dehydrogenase (elevated in survivors) and crotonase (elevated in non-survivors). These data suggest a need to monitor cellular energy metabolism beyond the available biomarkers. A loss of metabolic adaptation appears to be reflected by an inability to maintain cellular (fatty acid) metabolism within a "corridor of safety".

Authors: W. Khaliq, P. Grossmann, S. Neugebauer, A. Kleyman, R. Domizi, S. Calcinaro, D. Brealey, M. Graler, M. Kiehntopf, S. Schauble, M. Singer, G. Panagiotou, M. Bauer

Date Published: 11th Dec 2020

Publication Type: Not specified

Abstract (Expand)

Antibiotic resistance is an increasing threat to human health. In the case of Aspergillus fumigatus, which is both an environmental saprobe and an opportunistic human fungal pathogen, resistance is suggested to arise from fungicide use in agriculture, as the azoles used for plant protection share the same molecular target as the frontline antifungals used clinically. However, limiting azole fungicide use on crop fields to preserve their activity for clinical use could threaten the global food supply via a reduction in yield. In this study, we clarify the link between azole fungicide use on crop fields and resistance in a prototypical human pathogen through systematic soil sampling on farms in Germany and surveying fields before and after fungicide application. We observed a reduction in the abundance of A. fumigatus on fields following fungicide treatment in 2017, a finding that was not observed on an organic control field with only natural plant protection agents applied. However, this finding was less pronounced during our 2018 sampling, indicating that the impact of fungicides on A. fumigatus population size is variable and influenced by additional factors. The overall resistance frequency among agricultural isolates is low, with only 1 to 3% of isolates from 2016 to 2018 displaying resistance to medical azoles. Isolates collected after the growing season and azole exposure show a subtle but consistent decrease in susceptibility to medical and agricultural azoles. Whole-genome sequencing indicates that, despite the alterations in antifungal susceptibility, fungicide application does not significantly affect the population structure and genetic diversity of A. fumigatus in fields. Given the low observed resistance rate among agricultural isolates as well the lack of genomic impact following azole application, we do not find evidence that azole use on crops is significantly driving resistance in A. fumigatus in this context.IMPORTANCE Antibiotic resistance is an increasing threat to human health. In the case of Aspergillus fumigatus, which is an environmental fungus that also causes life-threatening infections in humans, antimicrobial resistance is suggested to arise from fungicide use in agriculture, as the chemicals used for plant protection are almost identical to the antifungals used clinically. However, removing azole fungicides from crop fields threatens the global food supply via a reduction in yield. In this study, we survey crop fields before and after fungicide application. We find a low overall azole resistance rate among agricultural isolates, as well as a lack of genomic and population impact following fungicide application, leading us to conclude azole use on crops does not significantly contribute to resistance in A. fumigatus.

Authors: A. E. Barber, J. Riedel, T. Sae-Ong, K. Kang, W. Brabetz, G. Panagiotou, H. B. Deising, O. Kurzai

Date Published: 24th Nov 2020

Publication Type: Not specified

Abstract (Expand)

BACKGROUND: Viruses are important components of microbial communities modulating community structure and function; however, only a couple of tools are currently available for phage identification and analysis from metagenomic sequencing data. Here we employed the random forest algorithm to develop VirMiner, a web-based phage contig prediction tool especially sensitive for high-abundances phage contigs, trained and validated by paired metagenomic and phagenomic sequencing data from the human gut flora. RESULTS: VirMiner achieved 41.06% +/- 17.51% sensitivity and 81.91% +/- 4.04% specificity in the prediction of phage contigs. In particular, for the high-abundance phage contigs, VirMiner outperformed other tools (VirFinder and VirSorter) with much higher sensitivity (65.23% +/- 16.94%) than VirFinder (34.63% +/- 17.96%) and VirSorter (18.75% +/- 15.23%) at almost the same specificity. Moreover, VirMiner provides the most comprehensive phage analysis pipeline which is comprised of metagenomic raw reads processing, functional annotation, phage contig identification, and phage-host relationship prediction (CRISPR-spacer recognition) and supports two-group comparison when the input (metagenomic sequence data) includes different conditions (e.g., case and control). Application of VirMiner to an independent cohort of human gut metagenomes obtained from individuals treated with antibiotics revealed that 122 KEGG orthology and 118 Pfam groups had significantly differential abundance in the pre-treatment samples compared to samples at the end of antibiotic administration, including clustered regularly interspaced short palindromic repeats (CRISPR), multidrug resistance, and protein transport. The VirMiner webserver is available at http://sbb.hku.hk/VirMiner/ . CONCLUSIONS: We developed a comprehensive tool for phage prediction and analysis for metagenomic samples. Compared to VirSorter and VirFinder-the most widely used tools-VirMiner is able to capture more high-abundance phage contigs which could play key roles in infecting bacteria and modulating microbial community dynamics. TRIAL REGISTRATION: The European Union Clinical Trials Register, EudraCT Number: 2013-003378-28 . Registered on 9 April 2014.

Authors: T. Zheng, J. Li, Y. Ni, K. Kang, M. A. Misiakou, L. Imamovic, B. K. C. Chow, A. A. Rode, P. Bytzer, M. Sommer, G. Panagiotou

Date Published: 19th Mar 2019

Publication Type: Not specified

Abstract (Expand)

Invasive aspergillosis (IA) is a life-threatening complication among allogeneic hematopoietic stem cell transplant (alloSCT) recipients. Despite well known risk factors and different available assays, diagnosis of invasive aspergillosis remains challenging. 103 clinical variables from patients with hematological malignancies and subsequent alloSCT were collected. Associations between collected variables and patients with (n = 36) and without IA (n = 36) were investigated by applying univariate and multivariable logistic regression. The predictive power of the final model was tested in an independent patient cohort (23 IA cases and 25 control patients). Findings were investigated further by in vitro studies, which analysed the effect of etanercept on A. fumigatus-stimulated macrophages at the gene expression and cytokine secretion. Additionally, the release of C-X-C motif chemokine ligand 10 (CXCL10) in patient sera was studied. Low monocyte concentration (p = 4.8 x 10(-06)), severe GvHD of the gut (grade 2-4) (p = 1.08 x 10(-02)) and etanercept treatment of GvHD (p = 3.5 x 10(-03)) were significantly associated with IA. Our studies showed that etanercept lowers CXCL10 concentrations in vitro and ex vivo and down-regulates genes involved in immune responses and TNF-alpha signaling. Our study offers clinicians new information regarding risk factors for IA including low monocyte counts and administration of etanercept. After necessary validation, such information may be used for decision making regarding antifungal prophylaxis or closely monitoring patients at risk.

Authors: T. Zoran, M. Weber, J. Springer, P. L. White, J. Bauer, A. Schober, C. Loffler, B. Seelbinder, K. Hunniger, O. Kurzai, A. Scherag, S. Schauble, C. O. Morton, H. Einsele, J. Linde, J. Loffler

Date Published: 21st Nov 2019

Publication Type: Not specified

Abstract (Expand)

The identification of disease-associated modules based on protein-protein interaction networks (PPINs) and gene expression data has provided new insights into the mechanistic nature of diverse diseases. However, their identification is hampered by the detection of protein communities within large-scale, whole-genome PPINs. A presented successful strategy detects a PPIN's community structure based on the maximal clique enumeration problem (MCE), which is a non-deterministic polynomial time-hard problem. This renders the approach computationally challenging for large PPINs implying the need for new strategies. We present ModuleDiscoverer, a novel approach for the identification of regulatory modules from PPINs and gene expression data. Following the MCE-based approach, ModuleDiscoverer uses a randomization heuristic-based approximation of the community structure. Given a PPIN of Rattus norvegicus and public gene expression data, we identify the regulatory module underlying a rodent model of non-alcoholic steatohepatitis (NASH), a severe form of non-alcoholic fatty liver disease (NAFLD). The module is validated using single-nucleotide polymorphism (SNP) data from independent genome-wide association studies and gene enrichment tests. Based on gene enrichment tests, we find that ModuleDiscoverer performs comparably to three existing module-detecting algorithms. However, only our NASH-module is significantly enriched with genes linked to NAFLD-associated SNPs. ModuleDiscoverer is available at http://www.hki-jena.de/index.php/0/2/490 (Others/ModuleDiscoverer).

Authors: S. Vlaic, T. Conrad, C. Tokarski-Schnelle, M. Gustafsson, U. Dahmen, R. Guthke, S. Schuster

Date Published: 11th Jan 2018

Publication Type: Not specified

Abstract (Expand)

Alternative splicing (AS) is an important regulatory mechanism in eukaryotes but only little is known about its impact in fungi. Human fungal pathogens are of high clinical interest causing recurrent or life-threatening infections. AS can be well-investigated genome-wide and quantitatively with the powerful technology of RNA-Seq. Here, we systematically studied AS in human fungal pathogens based on RNA-Seq data. To do so, we investigated its effect in seven fungi during conditions simulating ex vivo infection processes and during in vitro stress. Genes undergoing AS are species-specific and act independently from differentially expressed genes pointing to an independent mechanism to change abundance and functionality. Candida species stand out with a low number of introns with higher and more varying lengths and more alternative splice sites. Moreover, we identified a functional difference between response to host and other stress conditions: During stress, AS affects more genes and is involved in diverse regulatory functions. In contrast, during response-to-host conditions, genes undergoing AS have membrane functionalities and might be involved in the interaction with the host. We assume that AS plays a crucial regulatory role in pathogenic fungi and is important in both response to host and stress conditions.

Authors: P. Sieber, K. Voigt, P. Kammer, S. Brunke, S. Schuster, J. Linde

Date Published: 19th Oct 2018

Publication Type: Not specified

Abstract (Expand)

BACKGROUND: Omics data provide deep insights into overall biological processes of organisms. However, integration of data from different molecular levels such as transcriptomics and proteomics, still remains challenging. Analyzing lists of differentially abundant molecules from diverse molecular levels often results in a small overlap mainly due to different regulatory mechanisms, temporal scales, and/or inherent properties of measurement methods. Module-detecting algorithms identifying sets of closely related proteins from protein-protein interaction networks (PPINs) are promising approaches for a better data integration. RESULTS: Here, we made use of transcriptome, proteome and secretome data from the human pathogenic fungus Aspergillus fumigatus challenged with the antifungal drug caspofungin. Caspofungin targets the fungal cell wall which leads to a compensatory stress response. We analyzed the omics data using two different approaches: First, we applied a simple, classical approach by comparing lists of differentially expressed genes (DEGs), differentially synthesized proteins (DSyPs) and differentially secreted proteins (DSePs); second, we used a recently published module-detecting approach, ModuleDiscoverer, to identify regulatory modules from PPINs in conjunction with the experimental data. Our results demonstrate that regulatory modules show a notably higher overlap between the different molecular levels and time points than the classical approach. The additional structural information provided by regulatory modules allows for topological analyses. As a result, we detected a significant association of omics data with distinct biological processes such as regulation of kinase activity, transport mechanisms or amino acid metabolism. We also found a previously unreported increased production of the secondary metabolite fumagillin by A. fumigatus upon exposure to caspofungin. Furthermore, a topology-based analysis of potential key factors contributing to drug-caused side effects identified the highly conserved protein polyubiquitin as a central regulator. Interestingly, polyubiquitin UbiD neither belonged to the groups of DEGs, DSyPs nor DSePs but most likely strongly influenced their levels. CONCLUSION: Module-detecting approaches support the effective integration of multilevel omics data and provide a deep insight into complex biological relationships connecting these levels. They facilitate the identification of potential key players in the organism's stress response which cannot be detected by commonly used approaches comparing lists of differentially abundant molecules.

Authors: T. Conrad, O. Kniemeyer, S. G. Henkel, T. Kruger, D. J. Mattern, V. Valiante, R. Guthke, I. D. Jacobsen, A. A. Brakhage, S. Vlaic, J. Linde

Date Published: 20th Oct 2018

Publication Type: Not specified

Abstract (Expand)

Within the last two decades, the incidence of invasive fungal infections has been significantly increased. They are characterized by high mortality rates and are often caused by Candida albicans and Aspergillus fumigatus. The increasing number of infections underlines the necessity for additional anti-fungal therapies, which require extended knowledge of gene regulations during fungal infection. MicroRNAs are regulators of important cellular processes, including the immune response. By analyzing their regulation and impact on target genes, novel therapeutic and diagnostic approaches may be developed. Here, we examine the role of microRNAs in human dendritic cells during fungal infection. Dendritic cells represent the bridge between the innate and the adaptive immune systems. Therefore, analysis of gene regulation of dendritic cells is of particular significance. By applying next-generation sequencing of small RNAs, we quantify microRNA expression in monocyte-derived dendritic cells after 6 and 12 h of infection with C. albicans and A. fumigatus as well as treatment with lipopolysaccharides (LPS). We identified 26 microRNAs that are differentially regulated after infection by the fungi or LPS. Three and five of them are specific for fungal infections after 6 and 12 h, respectively. We further validated interactions of miR-132-5p and miR-212-5p with immunological relevant target genes, such as FKBP1B, KLF4, and SPN, on both RNA and protein level. Our results indicate that these microRNAs fine-tune the expression of immune-related target genes during fungal infection. Beyond that, we identified previously undiscovered microRNAs. We validated three novel microRNAs via qRT-PCR. A comparison with known microRNAs revealed possible relations with the miR-378 family and miR-1260a/b for two of them, while the third one features a unique sequence with no resemblance to known microRNAs. In summary, this study analyzes the effect of known microRNAs in dendritic cells during fungal infections and proposes novel microRNAs that could be experimentally verified.

Authors: A. Dix, K. Czakai, I. Leonhardt, K. Schaferhoff, M. Bonin, R. Guthke, H. Einsele, O. Kurzai, J. Loffler, J. Linde

Date Published: 11th Mar 2017

Publication Type: Not specified

Abstract (Expand)

Organisms do not exist isolated from each other, but constantly interact. Cells can sense the presence of interaction partners by a range of receptors and, via complex regulatory networks, specifically react by changing the expression of many of their genes. Technological advances in next-generation sequencing over the recent years now allow us to apply RNA sequencing to two species at the same time (dual RNA-seq), and thus to directly study the gene expression of two interacting species without the need to physically separate cells or RNA. In this review, we give an overview over the latest studies in interspecies interactions made possible by dual RNA-seq, ranging from pathogenic to symbiotic relationships. We summarize state-of-the-art experimental techniques, bioinformatic data analysis and data interpretation, while also highlighting potential problems and pitfalls starting from the selection of meaningful time points and number of reads to matters of rRNA depletion. A short outlook on new trends in the field of dual RNA-seq concludes this review, looking at sequencing of non-coding RNAs during host-pathogen interactions and the prediction of molecular interspecies interactions networks.

Authors: T. Wolf, P. Kammer, S. Brunke, J. Linde

Date Published: 29th Sep 2017

Publication Type: Not specified

Abstract (Expand)

Recent technological advancements have made time-resolved, quantitative, multi-omics data available for many model systems, which could be integrated for systems pharmacokinetic use. Here, we present large-scale simulation modeling (LASSIM), which is a novel mathematical tool for performing large-scale inference using mechanistically defined ordinary differential equations (ODE) for gene regulatory networks (GRNs). LASSIM integrates structural knowledge about regulatory interactions and non-linear equations with multiple steady state and dynamic response expression datasets. The rationale behind LASSIM is that biological GRNs can be simplified using a limited subset of core genes that are assumed to regulate all other gene transcription events in the network. The LASSIM method is implemented as a general-purpose toolbox using the PyGMO Python package to make the most of multicore computers and high performance clusters, and is available at https://gitlab.com/Gustafsson-lab/lassim. As a method, LASSIM works in two steps, where it first infers a non-linear ODE system of the pre-specified core gene expression. Second, LASSIM in parallel optimizes the parameters that model the regulation of peripheral genes by core system genes. We showed the usefulness of this method by applying LASSIM to infer a large-scale non-linear model of naive Th2 cell differentiation, made possible by integrating Th2 specific bindings, time-series together with six public and six novel siRNA-mediated knock-down experiments. ChIP-seq showed significant overlap for all tested transcription factors. Next, we performed novel time-series measurements of total T-cells during differentiation towards Th2 and verified that our LASSIM model could monitor those data significantly better than comparable models that used the same Th2 bindings. In summary, the LASSIM toolbox opens the door to a new type of model-based data analysis that combines the strengths of reliable mechanistic models with truly systems-level data. We demonstrate the power of this approach by inferring a mechanistically motivated, genome-wide model of the Th2 transcription regulatory system, which plays an important role in several immune related diseases.

Authors: R. Magnusson, G. P. Mariotti, M. Kopsen, W. Lovfors, D. R. Gawel, R. Jornsten, J. Linde, T. E. M. Nordling, E. Nyman, S. Schulze, C. E. Nestor, H. Zhang, G. Cedersund, M. Benson, A. Tjarnberg, M. Gustafsson

Date Published: 24th Jun 2017

Publication Type: Not specified

Abstract (Expand)

During somatic hypermutation (SHM) of Ig genes in germinal center B cells, lesions introduced by activation-induced cytidine deaminase are processed by multiple error-prone repair pathways. Although error-free repair by homologous recombination (HR) is crucial to prevent excessive DNA strand breakage at activation-induced cytidine deaminase off-target genes, its role at the hypermutating Ig locus in the germinal center is unexplored. Using B cell-specific inactivation of the critical HR factor Brca2, we detected decreased proliferation, survival, and thereby class switching of ex vivo-activated B cells. Intriguingly, an HR defect allowed for a germinal center reaction and affinity maturation in vivo, albeit at reduced amounts. Analysis of SHM revealed that a certain fraction of DNA lesions at C:G bp was indeed repaired in an error-free manner via Brca2 instead of being processed by error-prone translesion polymerases. By applying a novel pseudo-time in silico analysis of mutational processes, we found that the activity of A:T mutagenesis during SHM increased during a germinal center reaction, but this was in part defective in Brca2-deficient mice. These mutation pattern changes in Brca2-deficient B cells were mostly specific for the Ig V region, suggesting a local or time-dependent need for recombination repair to survive high rates of SHM and especially A:T mutagenesis.

Authors: G. Hirth, C. M. Svensson, K. Bottcher, S. Ullrich, M. T. Figge, B. Jungnickel

Date Published: 15th Sep 2019

Publication Type: Not specified

Abstract (Expand)

Migration and interactions of immune cells are routinely studied by time-lapse microscopy of in vitro migration and confrontation assays. To objectively quantify the dynamic behavior of cells, software tools for automated cell tracking can be applied. However, many existing tracking algorithms recognize only rather short fragments of a whole cell track and rely on cell staining to enhance cell segmentation. While our previously developed segmentation approach enables tracking of label-free cells, it still suffers from frequently recognizing only short track fragments. In this study, we identify sources of track fragmentation and provide solutions to obtain longer cell tracks. This is achieved by improving the detection of low-contrast cells and by optimizing the value of the gap size parameter, which defines the number of missing cell positions between track fragments that is accepted for still connecting them into one track. We find that the enhanced track recognition increases the average length of cell tracks up to 2.2-fold. Recognizing cell tracks as a whole will enable studying and quantifying more complex patterns of cell behavior, e.g. switches in migration mode or dependence of the phagocytosis efficiency on the number and type of preceding interactions. Such quantitative analyses will improve our understanding of how immune cells interact and function in health and disease.

Authors: N. Al-Zaben, A. Medyukhina, S. Dietrich, A. Marolda, K. Hunniger, O. Kurzai, M. T. Figge

Date Published: 1st Mar 2019

Publication Type: Not specified

Abstract (Expand)

The condition of neutropenia, i.e., a reduced absolute neutrophil count in blood, constitutes a major risk factor for severe infections in the affected patients. Candida albicans and Candida glabrata are opportunistic pathogens and the most prevalent fungal species in the human microbiota. In immunocompromised patients, they can become pathogenic and cause infections with high mortality rates. In this study, we use a previously established approach that combines experiments and computational models to investigate the innate immune response during blood stream infections with the two fungal pathogens C. albicans and C. glabrata. First, we determine immune-reaction rates and migration parameters under healthy conditions. Based on these findings, we simulate virtual patients and investigate the impact of neutropenic conditions on the infection outcome with the respective pathogen. Furthermore, we perform in silico treatments of these virtual patients by simulating a medical treatment that enhances neutrophil activity in terms of phagocytosis and migration. We quantify the infection outcome by comparing the response to the two fungal pathogens relative to non-neutropenic individuals. The analysis reveals that these fungal infections in neutropenic patients can be successfully cleared by cytokine treatment of the remaining neutrophils; and that this treatment is more effective for C. glabrata than for C. albicans.

Authors: S. Timme, T. Lehnert, M. T. E. Prausse, K. Hunniger, I. Leonhardt, O. Kurzai, M. T. Figge

Date Published: 20th Apr 2018

Publication Type: Not specified

Abstract (Expand)

Life-threatening systemic infections often occur due to the translocation of pathogens across the gut barrier and into the bloodstream. While the microbial and host mechanisms permitting bacterial gut translocation are well characterized, these mechanisms are still unclear for fungal pathogens such as Candida albicans, a leading cause of nosocomial fungal bloodstream infections. In this study, we dissected the cellular mechanisms of translocation of C. albicans across intestinal epithelia in vitro and identified fungal genes associated with this process. We show that fungal translocation is a dynamic process initiated by invasion and followed by cellular damage and loss of epithelial integrity. A screen of >2,000 C. albicans deletion mutants identified genes required for cellular damage of and translocation across enterocytes. Correlation analysis suggests that hypha formation, barrier damage above a minimum threshold level, and a decreased epithelial integrity are required for efficient fungal translocation. Translocation occurs predominantly via a transcellular route, which is associated with fungus-induced necrotic epithelial damage, but not apoptotic cell death. The cytolytic peptide toxin of C. albicans, candidalysin, was found to be essential for damage of enterocytes and was a key factor in subsequent fungal translocation, suggesting that transcellular translocation of C. albicans through intestinal layers is mediated by candidalysin. However, fungal invasion and low-level translocation can also occur via non-transcellular routes in a candidalysin-independent manner. This is the first study showing translocation of a human-pathogenic fungus across the intestinal barrier being mediated by a peptide toxin.IMPORTANCECandida albicans, usually a harmless fungus colonizing human mucosae, can cause lethal bloodstream infections when it manages to translocate across the intestinal epithelium. This can result from antibiotic treatment, immune dysfunction, or intestinal damage (e.g., during surgery). However, fungal processes may also contribute. In this study, we investigated the translocation process of C. albicans using in vitro cell culture models. Translocation occurs as a stepwise process starting with invasion, followed by epithelial damage and loss of epithelial integrity. The ability to secrete candidalysin, a peptide toxin deriving from the hyphal protein Ece1, is key: C. albicans hyphae, secreting candidalysin, take advantage of a necrotic weakened epithelium to translocate through the intestinal layer.

Authors: S. Allert, T. M. Forster, C. M. Svensson, J. P. Richardson, T. Pawlik, B. Hebecker, S. Rudolphi, M. Juraschitz, M. Schaller, M. Blagojevic, J. Morschhauser, M. T. Figge, I. D. Jacobsen, J. R. Naglik, L. Kasper, S. Mogavero, B. Hube

Date Published: 5th Jun 2018

Publication Type: Not specified

Abstract (Expand)

Bloodstream infections by the human-pathogenic fungi Candida albicans and Candida glabrata increasingly occur in hospitalized patients and are associated with high mortality rates. The early immune response against these fungi in human blood comprises a concerted action of humoral and cellular components of the innate immune system. Upon entering the blood, the majority of fungal cells will be eliminated by innate immune cells, i.e., neutrophils and monocytes. However, recent studies identified a population of fungal cells that can evade the immune response and thereby may disseminate and cause organ dissemination, which is frequently observed during candidemia. In this study, we investigate the so far unresolved mechanism of fungal immune evasion in human whole blood by testing hypotheses with the help of mathematical modeling. We use a previously established state-based virtual infection model for whole-blood infection with C. albicans to quantify the immune response and identified the fungal immune-evasion mechanism. While this process was assumed to be spontaneous in the previous model, we now hypothesize that the immune-evasion process is mediated by host factors and incorporate such a mechanism in the model. In particular, we propose, based on previous studies that the fungal immune-evasion mechanism could possibly arise through modification of the fungal surface by as of yet unknown proteins that are assumed to be secreted by activated neutrophils. To validate or reject any of the immune-evasion mechanisms, we compared the simulation of both immune-evasion models for different infection scenarios, i.e., infection of whole blood with either C. albicans or C. glabrata under non-neutropenic and neutropenic conditions. We found that under non-neutropenic conditions, both immune-evasion models fit the experimental data from whole-blood infection with C. albicans and C. glabrata. However, differences between the immune-evasion models could be observed for the infection outcome under neutropenic conditions with respect to the distribution of fungal cells across the immune cells. Based on these predictions, we suggested specific experimental studies that might allow for the validation or rejection of the proposed immune-evasion mechanism.

Authors: M. T. E. Prausse, T. Lehnert, S. Timme, K. Hunniger, I. Leonhardt, O. Kurzai, M. T. Figge

Date Published: 6th Apr 2018

Publication Type: Not specified

Abstract (Expand)

The healthy state of an organism is constantly threatened by external cues. Due to the daily inhalation of hundreds of particles and pathogens, the immune system needs to constantly accomplish the task of pathogen clearance in order to maintain this healthy state. However, infection dynamics are highly influenced by the peculiar anatomy of the human lung. Lung alveoli that are packed in alveolar sacs are interconnected by so called Pores of Kohn. Mainly due to the lack of in vivo methods, the role of Pores of Kohn in the mammalian lung is still under debate and partly contradicting hypotheses remain to be investigated. Although it was shown by electron microscopy that Pores of Kohn may serve as passageways for immune cells, their impact on the infection dynamics in the lung is still unknown under in vivo conditions. In the present study, we apply a hybrid agent-based infection model to quantitatively compare three different scenarios and discuss the importance of Pores of Kohn during infections of Aspergillus fumigatus. A. fumigatus is an airborne opportunistic fungus with rising incidences causing severe infections in immunocompromised patients that are associated with high mortality rates. Our hybrid agent-based model incorporates immune cell dynamics of alveolar macrophages - the resident phagocytes in the lung - as well as molecular dynamics of diffusing chemokines that attract alveolar macrophages to the site of infection. Consequently, this model allows a quantitative comparison of three different scenarios and to study the importance of Pores of Kohn. This enables us to demonstrate how passaging of alveolar macrophages and chemokine diffusion affect A. fumigatus infection dynamics. We show that Pores of Kohn alter important infection clearance mechanisms, such as the spatial distribution of macrophages and the effect of chemokine signaling. However, despite these differences, a lack of passageways for alveolar macrophages does impede infection clearance only to a minor extend. Furthermore, we quantify the importance of recruited macrophages in comparison to resident macrophages.

Authors: M. Blickensdorf, S. Timme, M. T. Figge

Date Published: 9th Sep 2020

Publication Type: Not specified

Abstract (Expand)

Aspergillus fumigatus is a ubiquitous opportunistic fungal pathogen that can cause severe infections in immunocompromised patients. Conidia that reach the lower respiratory tract are confronted with alveolar macrophages, which are the resident phagocytic cells, constituting the first line of defense. If not efficiently removed in time, A. fumigatus conidia can germinate causing severe infections associated with high mortality rates. Mice are the most extensively used model organism in research on A. fumigatus infections. However, in addition to structural differences in the lung physiology of mice and the human host, applied infection doses in animal experiments are typically orders of magnitude larger compared to the daily inhalation doses of humans. The influence of these factors, which must be taken into account in a quantitative comparison and knowledge transfer from mice to humans, is difficult to measure since in vivo live cell imaging of the infection dynamics under physiological conditions is currently not possible. In the present study, we compare A. fumigatus infection in mice and humans by virtual infection modeling using a hybrid agent-based model that accounts for the respective lung physiology and the impact of a wide range of infection doses on the spatial infection dynamics. Our computer simulations enable comparative quantification of A. fumigatus infection clearance in the two hosts to elucidate (i) the complex interplay between alveolar morphometry and the fungal burden and (ii) the dynamics of infection clearance, which for realistic fungal burdens is found to be more efficiently realized in mice compared to humans.

Authors: M. Blickensdorf, S. Timme, M. T. Figge

Date Published: 27th Feb 2019

Publication Type: Not specified

Abstract (Expand)

BACKGROUND: Roux-en-Y gastric bypass (RYGB) surgery is a last-resort treatment to induce substantial and sustained weight loss in cases of severe obesity. This anatomical rearrangement affects the intestinal microbiota, but so far, little information is available on how it interferes with microbial functionality and microbial-host interactions independently of weight loss. METHODS: A rat model was employed where the RYGB-surgery cohort is compared to sham-operated controls which were kept at a matched body weight by food restriction. We investigated the microbial taxonomy and functional activity using 16S rRNA amplicon gene sequencing, metaproteomics, and metabolomics on samples collected from theileum, the cecum, and the colon, and separately analysed the lumen and mucus-associated microbiota. RESULTS: Altered gut architecture in RYGB increased the relative occurrence of Actinobacteria, especially Bifidobacteriaceae and Proteobacteria, while in general, Firmicutes were decreased although Streptococcaceae and Clostridium perfringens were observed at relative higher abundances independent of weight loss. A decrease of conjugated and secondary bile acids was observed in the RYGB-gut lumen. The arginine biosynthesis pathway in the microbiota was altered, as indicated by the changes in the abundance of upstream metabolites and enzymes, resulting in lower levels of arginine and higher levels of aspartate in the colon after RYGB. CONCLUSION: The anatomical rearrangement in RYGB affects microbiota composition and functionality as well as changes in amino acid and bile acid metabolism independently of weight loss. The shift in the taxonomic structure of the microbiota after RYGB may be mediated by the resulting change in the composition of the bile acid pool in the gut and by changes in the composition of nutrients in the gut. Video abstract.

Authors: S. B. Haange, N. Jehmlich, U. Krugel, C. Hintschich, D. Wehrmann, M. Hankir, F. Seyfried, J. Froment, T. Hubschmann, S. Muller, D. K. Wissenbach, K. Kang, C. Buettner, G. Panagiotou, M. Noll, U. Rolle-Kampczyk, W. Fenske, M. von Bergen

Date Published: 7th Feb 2020

Publication Type: Not specified

Abstract (Expand)

The gut microbiota has the potential to influence the efficacy of cancer therapy. Here, we investigated the contribution of the intestinal microbiome on treatment outcomes in a heterogeneous cohort that included multiple cancer types to identify microbes with a global impact on immune response. Human gut metagenomic analysis revealed that responder patients had significantly higher microbial diversity and different microbiota compositions compared to non-responders. A machine-learning model was developed and validated in an independent cohort to predict treatment outcomes based on gut microbiota composition and functional repertoires of responders and non-responders. Specific species, Bacteroides ovatus and Bacteroides xylanisolvens, were positively correlated with treatment outcomes. Oral gavage of these responder bacteria significantly increased the efficacy of erlotinib and induced the expression of CXCL9 and IFN-gamma in a murine lung cancer model. These data suggest a predictable impact of specific constituents of the microbiota on tumor growth and cancer treatment outcomes with implications for both prognosis and therapy.

Authors: Y. Heshiki, R. Vazquez-Uribe, J. Li, Y. Ni, S. Quainoo, L. Imamovic, J. Li, M. Sorensen, B. K. C. Chow, G. J. Weiss, A. Xu, M. O. A. Sommer, G. Panagiotou

Date Published: 5th Mar 2020

Publication Type: Not specified

Abstract (Expand)

Fungal spores and hyphal fragments play an important role as allergens in respiratory diseases. In this study, we performed trypsin shaving and secretome analyses to identify the surface-exposed proteins and secreted/shed proteins of Aspergillus fumigatus conidia, respectively. We investigated the surface proteome under different conditions, including temperature variation and germination. We found that the surface proteome of resting A. fumigatus conidia is not static but instead unexpectedly dynamic, as evidenced by drastically different surface proteomes under different growth conditions. Knockouts of two abundant A. fumigatus surface proteins, ScwA and CweA, were found to function only in fine-tuning the cell wall stress response, implying that the conidial surface is very robust against perturbations. We then compared the surface proteome of A. fumigatus to other allergy-inducing molds, including Alternaria alternata, Penicillium rubens, and Cladosporium herbarum, and performed comparative proteomics on resting and swollen conidia, as well as secreted proteins from germinating conidia. We detected 125 protein ortholog groups, including 80 with putative catalytic activity, in the extracellular region of all four molds, and 42 nonorthologous proteins produced solely by A. fumigatus. Ultimately, this study highlights the dynamic nature of the A. fumigatus conidial surface and provides targets for future diagnostics and immunotherapy.

Authors: M. G. Blango, A. Pschibul, F. Rivieccio, T. Kruger, M. Rafiq, L. J. Jia, T. Zheng, M. Goldmann, V. Voltersen, J. Li, G. Panagiotou, O. Kniemeyer, A. A. Brakhage

Date Published: 1st May 2020

Publication Type: Not specified

Abstract (Expand)

BACKGROUND: The selection of bioengineering platform strains and engineering strategies to improve the stress resistance of Saccharomyces cerevisiae remains a pressing need in bio-based chemical production. Thus, a systematic effort to exploit genotypic and phenotypic diversity to boost yeast's industrial value is still urgently needed. RESULTS: We analyzed 5,400 growth curves obtained from 36 S. cerevisiae strains and comprehensively profiled their resistances against 13 industrially relevant stresses. We observed that bioethanol and brewing strains exhibit higher resistance against acidic conditions; however, plant isolates tend to have a wider range of resistance, which may be associated with their metabolome and fluxome signatures in the tricarboxylic acid cycle and fatty acid metabolism. By deep genomic sequencing, we found that industrial strains have more genomic duplications especially affecting transcription factors, showing that they result from disparate evolutionary paths in comparison with the environmental strains, which have more indels, gene deletions, and strain-specific genes. Genome-wide association studies coupled with protein-protein interaction networks uncovered novel genetic determinants of stress resistances. CONCLUSIONS: These resistance-related engineering targets and strain rankings provide a valuable source for engineering significantly improved industrial platform strains.

Authors: K. Kang, B. Bergdahl, D. Machado, L. Dato, T. L. Han, J. Li, S. Villas-Boas, M. J. Herrgard, J. Forster, G. Panagiotou

Date Published: 1st Apr 2019

Publication Type: Not specified

Abstract (Expand)

Despite the documented antibiotic-induced disruption of the gut microbiota, the impact of antibiotic intake on strain-level dynamics, evolution of resistance genes, and factors influencing resistance dissemination potential remains poorly understood. To address this gap we analyzed public metagenomic datasets from 24 antibiotic treated subjects and controls, combined with an in-depth prospective functional study with two subjects investigating the bacterial community dynamics based on cultivation-dependent and independent methods. We observed that short-term antibiotic treatment shifted and diversified the resistome composition, increased the average copy number of antibiotic resistance genes, and altered the dominant strain genotypes in an individual-specific manner. More than 30% of the resistance genes underwent strong differentiation at the single nucleotide level during antibiotic treatment. We found that the increased potential for horizontal gene transfer, due to antibiotic administration, was approximately 3-fold stronger in the differentiated resistance genes than the non-differentiated ones. This study highlights how antibiotic treatment has individualized impacts on the resistome and strain level composition, and drives the adaptive evolution of the gut microbiota.

Authors: J. Li, E. A. Rettedal, E. van der Helm, M. Ellabaan, G. Panagiotou, M. O. A. Sommer

Date Published: 27th Apr 2019

Publication Type: Not specified

Abstract (Expand)

Exercise is an effective strategy for diabetes management but is limited by the phenomenon of exercise resistance (i.e., the lack of or the adverse response to exercise on metabolic health). Here, in 39 medication-naive men with prediabetes, we found that exercise-induced alterations in the gut microbiota correlated closely with improvements in glucose homeostasis and insulin sensitivity (clinicaltrials.gov entry NCT03240978). The microbiome of responders exhibited an enhanced capacity for biosynthesis of short-chain fatty acids and catabolism of branched-chain amino acids, whereas those of non-responders were characterized by increased production of metabolically detrimental compounds. Fecal microbial transplantation from responders, but not non-responders, mimicked the effects of exercise on alleviation of insulin resistance in obese mice. Furthermore, a machine-learning algorithm integrating baseline microbial signatures accurately predicted personalized glycemic response to exercise in an additional 30 subjects. These findings raise the possibility of maximizing the benefits of exercise by targeting the gut microbiota.

Authors: Y. Liu, Y. Wang, Y. Ni, C. K. Y. Cheung, K. S. L. Lam, Y. Wang, Z. Xia, D. Ye, J. Guo, M. A. Tse, G. Panagiotou, A. Xu

Date Published: 7th Jan 2020

Publication Type: Not specified

Abstract (Expand)

Filamentous fungi of the genus Aspergillus are of particular interest for biotechnological applications due to their natural capacity to secrete carbohydrate-active enzymes (CAZy) that target plant biomass. The presence of easily metabolizable sugars such as glucose, whose concentrations increase during plant biomass hydrolysis, results in the repression of CAZy-encoding genes in a process known as carbon catabolite repression (CCR), which is undesired for the purpose of large-scale enzyme production. To date, the C2H2 transcription factor CreA has been described as the major CC repressor in Aspergillus spp., although little is known about the role of posttranslational modifications in this process. In this work, phosphorylation sites were identified by mass spectrometry on Aspergillus nidulans CreA, and subsequently, the previously identified but uncharacterized site S262, the characterized site S319, and the newly identified sites S268 and T308 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was investigated. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 was not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. All sites were shown to be important for glycogen and trehalose metabolism. This study highlights the importance of CreA phosphorylation sites for the regulation of CCR. These sites are interesting targets for biotechnological strain engineering without the need to delete essential genes, which could result in undesired side effects.IMPORTANCE In filamentous fungi, the transcription factor CreA controls carbohydrate metabolism through the regulation of genes encoding enzymes required for the use of alternative carbon sources. In this work, phosphorylation sites were identified on Aspergillus nidulans CreA, and subsequently, the two newly identified sites S268 and T308, the previously identified but uncharacterized site S262, and the previously characterized site S319 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was characterized. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 is not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. This work characterized novel CreA phosphorylation sites under carbon catabolite-repressing conditions and showed that they are crucial for CreA protein turnover, control of carbohydrate utilization, and biotechnologically relevant enzyme production.

Authors: L. J. de Assis, L. P. Silva, O. Bayram, P. Dowling, O. Kniemeyer, T. Kruger, A. A. Brakhage, Y. Chen, L. Dong, K. Tan, K. H. Wong, L. N. A. Ries, G. H. Goldman

Date Published: 5th Jan 2021

Publication Type: Not specified

Abstract (Expand)

Polymorphonuclear granulocytes (PMNs) are indispensable for controlling life-threatening fungal infections. In addition to various effector mechanisms, PMNs also produce extracellular vesicles (EVs). Their contribution to antifungal defense has remained unexplored. We reveal that the clinically important human-pathogenic fungus Aspergillus fumigatus triggers PMNs to release a distinct set of antifungal EVs (afEVs). Proteome analyses indicated that afEVs are enriched in antimicrobial proteins. The cargo and the release kinetics of EVs are modulated by the fungal strain confronted. Tracking of afEVs indicated that they associated with fungal cells and even entered fungal hyphae, resulting in alterations in the morphology of the fungal cell wall and dose-dependent antifungal effects. To assess as a proof of concept whether the antimicrobial proteins found in afEVs might contribute to growth inhibition of hyphae when present in the fungal cytoplasm, two human proteins enriched in afEVs, cathepsin G and azurocidin, were heterologously expressed in fungal hyphae. This led to reduced fungal growth relative to that of a control strain producing the human retinol binding protein 7. In conclusion, extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. This finding offers an intriguing, previously overlooked mechanism of antifungal defense against A. fumigatus IMPORTANCE Invasive fungal infections caused by the mold Aspergillus fumigatus are a growing concern in the clinic due to the increasing use of immunosuppressive therapies and increasing antifungal drug resistance. These infections result in high rates of mortality, as treatment and diagnostic options remain limited. In healthy individuals, neutrophilic granulocytes are critical for elimination of A. fumigatus from the host; however, the exact extracellular mechanism of neutrophil-mediated antifungal activity remains unresolved. Here, we present a mode of antifungal defense employed by human neutrophils against A. fumigatus not previously described. We found that extracellular vesicles produced by neutrophils in response to A. fumigatus infection are able to associate with the fungus, limit growth, and elicit cell damage by delivering antifungal cargo. In the end, antifungal extracellular vesicle biology provides a significant step forward in our understanding of A. fumigatus host pathogenesis and opens up novel diagnostic and therapeutic possibilities.

Authors: I. A. Shopova, I. Belyaev, P. Dasari, S. Jahreis, M. C. Stroe, Z. Cseresnyes, A. K. Zimmermann, A. Medyukhina, C. M. Svensson, T. Kruger, V. Szeifert, S. Nietzsche, T. Conrad, M. G. Blango, O. Kniemeyer, M. von Lilienfeld-Toal, P. F. Zipfel, E. Ligeti, M. T. Figge, A. A. Brakhage

Date Published: 14th Apr 2020

Publication Type: Not specified

Abstract (Expand)

Aspergillus fumigatus is an opportunistic fungal pathogen that can cause life-threatening invasive lung infections in immunodeficient patients. The cellular and molecular processes of infection during onset, establishment, and progression of A. fumigatus infections are highly complex and depend on both fungal attributes and the immune status of the host. Therefore, preclinical animal models are of paramount importance to investigate and gain better insight into the infection process. Yet, despite their extensive use, commonly employed murine models of invasive pulmonary aspergillosis are not well understood due to analytical limitations. Here, we present quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the local immune response in whole lungs at cellular resolution within its anatomical context. We analyzed three very common murine models of pulmonary aspergillosis based on immunosuppression with corticosteroids, chemotherapy-induced leukopenia, or myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial distribution, interaction, and activation of two key immune cell populations in antifungal defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the patterns of fungal growth correlated with the detected effects of the immunosuppressive regimens on the local immune cell populations. Moreover, LSFM demonstrates that the commonly used intranasal route of spore administration did not result in complete intra-alveolar deposition, as about 80% of fungal growth occurred outside the alveolar space. Hence, characterization by LSFM is more rigorous than by previously used methods employing murine models of invasive pulmonary aspergillosis and pinpoints their strengths and limitations.IMPORTANCE The use of animal models of infection is essential to advance our understanding of the complex host-pathogen interactions that take place during Aspergillus fumigatus lung infections. As in the case of humans, mice need to suffer an immune imbalance in order to become susceptible to invasive pulmonary aspergillosis (IPA), the most serious infection caused by A. fumigatus There are several immunosuppressive regimens that are routinely used to investigate fungal growth and/or immune responses in murine models of invasive pulmonary aspergillosis. However, the precise consequences of the use of each immunosuppressive model for the local immune populations and for fungal growth are not completely understood. Here, to pin down the scenarios involving commonly used IPA models, we employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular resolution. Our results will be valuable to optimize and refine animal models to maximize their use in future research.

Authors: J. Amich, Z. Mokhtari, M. Strobel, E. Vialetto, D. Sheta, Y. Yu, J. Hartweg, N. Kalleda, K. J. Jarick, C. Brede, A. L. Jordan-Garrote, S. Thusek, K. Schmiedgen, B. Arslan, J. Pinnecker, C. R. Thornton, M. Gunzer, S. Krappmann, H. Einsele, K. G. Heinze, A. Beilhack

Date Published: 4th Feb 2020

Publication Type: Not specified

Abstract (Expand)

BACKGROUND: Candida albicans and Candida glabrata are the 2 most prevalent Candida species causing bloodstream infections. Patterns of innate immune activation triggered by the 2 fungi differ considerably. METHODS: To analyze human natural killer (NK) cell activation by both species, we performed ex vivo whole-blood infection assays and confrontation assays with primary human NK cells. RESULTS: C. albicans was a stronger activator for isolated human NK cells than C. glabrata. In contrast, activation of blood NK cells, characterized by an upregulated surface exposure of early activation antigen CD69 and death receptor ligand TRAIL, as well as interferon-gamma (IFN-gamma) secretion, was more pronounced during C. glabrata infection. NK cell activation in blood is mediated by humoral mediators released by other immune cells and does not depend on direct activation by fungal cells. Cross-talk between Candida-confronted monocyte-derived dendritic cells (moDC) and NK cells resulted in the same NK activation phenotype as NK cells in human blood. Blocking experiments and cytokine substitution identified interleukin-12 as a critical mediator in regulation of primary NK cells by moDC. CONCLUSIONS: Activation of human NK cells in response to Candida in human blood mainly occurs indirectly by mediators released from monocytic cells.

Authors: A. Marolda, K. Hunniger, S. Bottcher, W. Vivas, J. Loffler, M. T. Figge, O. Kurzai

Date Published: 11th Jun 2020

Publication Type: Not specified

Abstract (Expand)

Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1alpha, MIP-1beta, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56(bright)CD16(dim) cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1alpha, MIP-1beta, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.

Authors: E. Weiss, J. Schlegel, U. Terpitz, M. Weber, J. Linde, A. L. Schmitt, K. Hunniger, L. Marischen, F. Gamon, J. Bauer, C. Loffler, O. Kurzai, C. O. Morton, M. Sauer, H. Einsele, J. Loeffler

Date Published: 5th Oct 2020

Publication Type: Not specified

Abstract (Expand)

Mucormycosis is an emergent, fatal fungal infection of humans and warm-blooded animals caused by species of the order Mucorales. Immune cells of the innate immune system serve as the first line of defence against inhaled spores. Alveolar macrophages were challenged with the mucoralean fungus Lichtheimia corymbifera and subjected to biotinylation and streptavidin enrichment procedures followed by LC-MS/MS analyses. A total of 28 host proteins enriched for binding to macrophage-L. corymbifera interaction. Among those, the HSP70-family protein Hspa8 was found to be predominantly responsive to living and heat-killed spores of a virulent and an attenuated strain of L. corymbifera. Confocal scanning laser microscopy of infected macrophages revealed colocalization of Hspa8 with phagocytosed spores of L. corymbifera. The amount of detectable Hspa8 was dependent on the multiplicity of infection. Incubation of alveolar macrophages with an anti-Hspa8 antibody prior to infection reduced their capability to phagocytose spores of L. corymbifera. In contrast, anti-Hspa8 antibodies did not abrogate the phagocytosis of Aspergillus fumigatus conidia by macrophages. These results suggest an important contribution of the heat-shock family protein Hspa8 in the recognition of spores of the mucoralean fungus L. corymbifera by host alveolar macrophages and define a potential immunomodulatory therapeutic target.

Authors: M. I. A. Hassan, J. M. Kruse, T. Kruger, H. M. Dahse, Z. Cseresnyes, M. G. Blango, H. Slevogt, F. Horhold, V. Ast, R. Konig, M. T. Figge, O. Kniemeyer, A. A. Brakhage, K. Voigt

Date Published: 26th Jun 2020

Publication Type: Not specified

Abstract (Expand)

Apart from some model organisms, the interactome of most organisms is largely unidentified. High-throughput experimental techniques to determine protein-protein interactions (PPIs) are resource intensive and highly susceptible to noise. Computational methods of PPI determination can accelerate biological discovery by identifying the most promising interacting pairs of proteins and by assessing the reliability of identified PPIs. Here we present a first in-depth study describing a global view of the ant Camponotus floridanus interactome. Although several ant genomes have been sequenced in the last eight years, studies exploring and investigating PPIs in ants are lacking. Our study attempts to fill this gap and the presented interactome will also serve as a template for determining PPIs in other ants in future. Our C. floridanus interactome covers 51,866 non-redundant PPIs among 6,274 proteins, including 20,544 interactions supported by domain-domain interactions (DDIs), 13,640 interactions supported by DDIs and subcellular localization, and 10,834 high confidence interactions mediated by 3,289 proteins. These interactions involve and cover 30.6% of the entire C. floridanus proteome.

Authors: S. K. Gupta, M. Srivastava, O. Osmanoglu, T. Dandekar

Date Published: 11th Feb 2020

Publication Type: Not specified

Abstract (Expand)

OBJECTIVE: The biological interpretation of gene expression measurements is a challenging task. While ordination methods are routinely used to identify clusters of samples or co-expressed genes, these methods do not take sample or gene annotations into account. We aim to provide a tool that allows users of all backgrounds to assess and visualize the intrinsic correlation structure of complex annotated gene expression data and discover the covariates that jointly affect expression patterns. RESULTS: The Bioconductor package covRNA provides a convenient and fast interface for testing and visualizing complex relationships between sample and gene covariates mediated by gene expression data in an entirely unsupervised setting. The relationships between sample and gene covariates are tested by statistical permutation tests and visualized by ordination. The methods are inspired by the fourthcorner and RLQ analyses used in ecological research for the analysis of species abundance data, that we modified to make them suitable for the distributional characteristics of both, RNA-Seq read counts and microarray intensities, and to provide a high-performance parallelized implementation for the analysis of large-scale gene expression data on multi-core computational systems. CovRNA provides additional modules for unsupervised gene filtering and plotting functions to ensure a smooth and coherent analysis workflow.

Authors: L. Urban, C. W. Remmele, M. Dittrich, R. F. Schwarz, T. Muller

Date Published: 24th Feb 2020

Publication Type: Not specified

Abstract (Expand)

The capacity of Candida albicans to reversibly change its morphology between yeast and filamentous stages is crucial for its virulence. Formation of hyphae correlates with the upregulation of genes ALS3 and ECE1, which are involved in pathogenicity processes such as invasion, iron acquisition, and host cell damage. The global repressor Tup1 and its cofactor Nrg1 are considered to be the main antagonists of hyphal development in C. albicans However, our experiments revealed that Tup1, but not Nrg1, was required for full expression of ALS3 and ECE1 In contrast to NRG1, overexpression of TUP1 was found to inhibit neither filamentous growth nor transcription of ALS3 and ECE1 In addition, we identified the transcription factor Ahr1 as being required for full expression of both genes. A hyperactive version of Ahr1 bound directly to the promoters of ALS3 and ECE1 and induced their transcription even in the absence of environmental stimuli. This regulation worked even in the absence of the crucial hyphal growth regulators Cph1 and Efg1 but was dependent on the presence of Tup1. Overall, our results show that Ahr1 and Tup1 are key contributors in the complex regulation of virulence-associated genes in the different C. albicans morphologies.IMPORTANCE Candida albicans is a major human fungal pathogen and the leading cause of systemic Candida infections. In recent years, Als3 and Ece1 were identified as important factors for fungal virulence. Transcription of both corresponding genes is closely associated with hyphal growth. Here, we describe how Tup1, normally a global repressor of gene expression as well as of filamentation, and the transcription factor Ahr1 contribute to full expression of ALS3 and ECE1 in C. albicans hyphae. Both regulators are required for high mRNA amounts of the two genes to ensure functional relevant protein synthesis and localization. These observations identified a new aspect of regulation in the complex transcriptional control of virulence-associated genes in C. albicans.

Authors: S. Ruben, E. Garbe, S. Mogavero, D. Albrecht-Eckardt, D. Hellwig, A. Hader, T. Kruger, K. Gerth, I. D. Jacobsen, O. Elshafee, S. Brunke, K. Hunniger, O. Kniemeyer, A. A. Brakhage, J. Morschhauser, B. Hube, S. Vylkova, O. Kurzai, R. Martin

Date Published: 28th Apr 2020

Publication Type: Not specified

Abstract (Expand)

Th17 cells provide protection at barrier tissues but may also contribute to immune pathology. The relevance and induction mechanisms of pathologic Th17 responses in humans are poorly understood. Here, we identify the mucocutaneous pathobiont Candida albicans as the major direct inducer of human anti-fungal Th17 cells. Th17 cells directed against other fungi are induced by cross-reactivity to C. albicans. Intestinal inflammation expands total C. albicans and cross-reactive Th17 cells. Strikingly, Th17 cells cross-reactive to the airborne fungus Aspergillus fumigatus are selectively activated and expanded in patients with airway inflammation, especially during acute allergic bronchopulmonary aspergillosis. This indicates a direct link between protective intestinal Th17 responses against C. albicans and lung inflammation caused by airborne fungi. We identify heterologous immunity to a single, ubiquitous member of the microbiota as a central mechanism for systemic induction of human anti-fungal Th17 responses and as a potential risk factor for pulmonary inflammatory diseases.

Authors: P. Bacher, T. Hohnstein, E. Beerbaum, M. Rocker, M. G. Blango, S. Kaufmann, J. Rohmel, P. Eschenhagen, C. Grehn, K. Seidel, V. Rickerts, L. Lozza, U. Stervbo, M. Nienen, N. Babel, J. Milleck, M. Assenmacher, O. A. Cornely, M. Ziegler, H. Wisplinghoff, G. Heine, M. Worm, B. Siegmund, J. Maul, P. Creutz, C. Tabeling, C. Ruwwe-Glosenkamp, L. E. Sander, C. Knosalla, S. Brunke, B. Hube, O. Kniemeyer, A. A. Brakhage, C. Schwarz, A. Scheffold

Date Published: 7th Mar 2019

Publication Type: Not specified

Abstract (Expand)

Phagosomes must maintain membrane integrity to exert their microbicidal function. Some microorganisms, however, survive and grow within phagosomes. In such instances, phagosomes must expand to avoid rupture and microbial escape. We studied whether phagosomes regulate their size to preserve integrity during infection with the fungal pathogen Candida albicans. Phagosomes release calcium as C. albicans hyphae elongate, inducing lysosome recruitment and insertion, thereby increasing the phagosomal surface area. As hyphae grow, the expanding phagosome consumes the majority of free lysosomes. Simultaneously, lysosome biosynthesis is stimulated by activation of TFEB, a transcriptional regulator of lysosomal biogenesis. Preventing lysosomal insertion causes phagosomal rupture, NLRP3 inflammasome activation, IL-1beta secretion and host-cell death. Whole-genome transcriptomic analysis demonstrate that stress responses elicited in C. albicans upon engulfment are reversed if phagosome expansion is prevented. Our findings reveal a mechanism whereby phagosomes maintain integrity while expanding, ensuring that growing pathogens remain entrapped within this microbicidal compartment.

Authors: J. Westman, G. F. W. Walpole, L. Kasper, B. Y. Xue, O. Elshafee, B. Hube, S. Grinstein

Date Published: 9th Dec 2020

Publication Type: Not specified

Abstract (Expand)

The dimorphic fungus Candida albicans is both a harmless commensal organism on mucosal surfaces and an opportunistic pathogen. Under certain predisposing conditions, the fungus can overgrow the mucosal microbiome and cause both superficial and life-threatening systemic infections after gaining access to the bloodstream. As the first line of defense of the innate immune response, infecting C. albicans cells face macrophages, which mediate the clearance of invading fungi by intracellular killing. However, the fungus has evolved sophisticated strategies to counteract macrophage antimicrobial activities and thus evade immune surveillance. The cytolytic peptide toxin, candidalysin, contributes to this fungal defense machinery by damaging immune cell membranes, providing an escape route from the hostile phagosome environment. Nevertheless, candidalysin also induces NLRP3 inflammasome activation, leading to an increased host-protective pro-inflammatory response in mononuclear phagocytes. Therefore, candidalysin facilitates immune evasion by acting as a classical virulence factor but also contributes to an antifungal immune response, serving as an avirulence factor. In this review, we discuss the role of candidalysin during C. albicans infections, focusing on its implications during C. albicans-macrophage interactions.

Authors: A. Konig, B. Hube, L. Kasper

Date Published: 24th Jul 2020

Publication Type: Not specified

Abstract (Expand)

Clearance of invading microbes requires phagocytes of the innate immune system. However, successful pathogens have evolved sophisticated strategies to evade immune killing. The opportunistic human fungal pathogen Candida albicans is efficiently phagocytosed by macrophages, but causes inflammasome activation, host cytolysis, and escapes after hypha formation. Previous studies suggest that macrophage lysis by C. albicans results from early inflammasome-dependent cell death (pyroptosis), late damage due to glucose depletion and membrane piercing by growing hyphae. Here we show that Candidalysin, a cytolytic peptide toxin encoded by the hypha-associated gene ECE1, is both a central trigger for NLRP3 inflammasome-dependent caspase-1 activation via potassium efflux and a key driver of inflammasome-independent cytolysis of macrophages and dendritic cells upon infection with C. albicans. This suggests that Candidalysin-induced cell damage is a third mechanism of C. albicans-mediated mononuclear phagocyte cell death in addition to damage caused by pyroptosis and the growth of glucose-consuming hyphae.

Authors: L. Kasper, A. Konig, P. A. Koenig, M. S. Gresnigt, J. Westman, R. A. Drummond, M. S. Lionakis, O. Gross, J. Ruland, J. R. Naglik, B. Hube

Date Published: 15th Oct 2018

Publication Type: Not specified

Abstract (Expand)

During infection, the human pathogenic fungus Candida albicans undergoes a yeast-to-hypha transition, secretes numerous proteins for invasion of host tissues, and modulates the host's immune response. Little is known about the interplay of C. albicans secreted proteins and the host adaptive immune system. Here, we applied a combined 2D gel- and LC-MS/MS-based approach for the characterization of C. albicans extracellular proteins during the yeast-to-hypha transition, which led to a comprehensive C. albicans secretome map. The serological responses to C. albicans extracellular proteins were investigated by a 2D-immunoblotting approach combined with MS for protein identification. On the basis of the screening of sera from candidemia and three groups of noncandidemia patients, a core set of 19 immunodominant antibodies against secreted proteins of C. albicans was identified, seven of which represent potential diagnostic markers for candidemia (Xog1, Lip4, Asc1, Met6, Tsa1, Tpi1, and Prx1). Intriguingly, some secreted, strongly glycosylated protein antigens showed high cross-reactivity with sera from noncandidemia control groups. Enzymatic deglycosylation of proteins secreted from hyphae significantly impaired sera antibody recognition. Furthermore, deglycosylation of the recombinantly produced, secreted aspartyl protease Sap6 confirmed a significant contribution of glycan epitopes to the recognition of Sap6 by antibodies in patient's sera.

Authors: T. Luo, T. Kruger, U. Knupfer, L. Kasper, N. Wielsch, B. Hube, A. Kortgen, M. Bauer, E. J. Giamarellos-Bourboulis, G. Dimopoulos, A. A. Brakhage, O. Kniemeyer

Date Published: 5th Aug 2016

Publication Type: Not specified

Abstract (Expand)

The protein kinase Snf1, a member of the highly conserved AMP-activated protein kinase family, is a central regulator of metabolic adaptation. In the pathogenic yeast Candida albicans, Snf1 is considered to be essential, as previous attempts by different research groups to generate homozygous snf1Delta mutants were unsuccessful. We aimed to elucidate why Snf1 is required for viability in C. albicans by generating snf1Delta null mutants through forced, inducible gene deletion and observing the terminal phenotype before cell death. Unexpectedly, we found that snf1Delta mutants were viable and could grow, albeit very slowly, on rich media containing the preferred carbon source glucose. Growth was improved when the cells were incubated at 37 degrees C instead of 30 degrees C, and this phenotype enabled us to isolate homozygous snf1Delta mutants also by conventional, sequential deletion of both SNF1 alleles in a wild-type C. albicans strain. All snf1Delta mutants could grow slowly on glucose but were unable to utilize alternative carbon sources. Our results show that, under optimal conditions, C. albicans can live and grow without Snf1. Furthermore, they demonstrate that inducible gene deletion is a powerful method for assessing gene essentiality in C. albicans IMPORTANCE Essential genes are those that are indispensable for the viability and growth of an organism. Previous studies indicated that the protein kinase Snf1, a central regulator of metabolic adaptation, is essential in the pathogenic yeast Candida albicans, because no homozygous snf1 deletion mutants of C. albicans wild-type strains could be obtained by standard approaches. In order to investigate the lethal consequences of SNF1 deletion, we generated conditional mutants in which SNF1 could be deleted by forced, inducible excision from the genome. Unexpectedly, we found that snf1 null mutants were viable and could grow slowly under optimal conditions. The growth phenotypes of the snf1Delta mutants explain why such mutants were not recovered in previous attempts. Our study demonstrates that inducible gene deletion is a powerful method for assessing gene essentiality in C. albicans.

Authors: A. Mottola, S. Schwanfelder, J. Morschhauser

Date Published: 19th Aug 2020

Publication Type: Not specified

Abstract (Expand)

The heterotrimeric protein kinase SNF1 plays a key role in the metabolic adaptation of the pathogenic yeast Candida albicans It consists of the essential catalytic alpha-subunit Snf1, the gamma-subunit Snf4, and one of the two beta-subunits Kis1 and Kis2. Snf4 is required to release the N-terminal catalytic domain of Snf1 from autoinhibition by the C-terminal regulatory domain, and snf4Delta mutants cannot grow on carbon sources other than glucose. In a screen for suppressor mutations that restore growth of a snf4Delta mutant on alternative carbon sources, we isolated a mutant in which six amino acids between the N-terminal kinase domain and the C-terminal regulatory domain of Snf1 were deleted. The deletion was caused by an intragenic recombination event between two 8-bp direct repeats flanking six intervening codons. In contrast to truncated forms of Snf1 that contain only the kinase domain, the Snf4-independent Snf1(Delta311 - 316) was fully functional and could replace wild-type Snf1 for normal growth, because it retained the ability to interact with the Kis1 and Kis2 beta-subunits via its C-terminal domain. Indeed, the Snf4-independent Snf1(Delta311 - 316) still required the beta-subunits of the SNF1 complex to perform its functions and did not rescue the growth defects of kis1Delta mutants. Our results demonstrate that a preprogrammed in-frame deletion event within the SNF1 coding region can generate a mutated form of this essential kinase which abolishes autoinhibition and thereby overcomes growth deficiencies caused by a defect in the gamma-subunit Snf4.IMPORTANCE Genomic alterations, including different types of recombination events, facilitate the generation of genetically altered variants and enable the pathogenic yeast Candida albicans to adapt to stressful conditions encountered in its human host. Here, we show that a specific recombination event between two 8-bp direct repeats within the coding sequence of the SNF1 gene results in the deletion of six amino acids between the N-terminal kinase domain and the C-terminal regulatory domain and relieves this essential kinase from autoinhibition. This preprogrammed deletion allowed C. albicans to overcome growth defects caused by the absence of the regulatory subunit Snf4 and represents a built-in mechanism for the generation of a Snf4-independent Snf1 kinase.

Authors: A. Mottola, J. Morschhauser

Date Published: 19th Jun 2019

Publication Type: Not specified

Abstract (Expand)

The clonal population structure of Candida albicans suggests that (para)sexual recombination does not play an important role in the lifestyle of this opportunistic fungal pathogen, an assumption that is strengthened by the fact that most C. albicans strains are heterozygous at the mating type locus (MTL) and therefore mating-incompetent. On the other hand, mating might occur within clonal populations and allow the combination of advantageous traits that were acquired by individual cells to adapt to adverse conditions. We have investigated if parasexual recombination may be involved in the evolution of highly drug-resistant strains exhibiting multiple resistance mechanisms against fluconazole, an antifungal drug that is commonly used to treat infections by C. albicans Growth of strains that were heterozygous for MTL and different fluconazole resistance mutations in the presence of the drug resulted in the emergence of derivatives that had become homozygous for the mutated allele and the mating type locus and exhibited increased drug resistance. When MTL a/a and MTLalpha/alpha cells of these strains were mixed in all possible combinations, we could isolate mating products containing the genetic material from both parents. The initial mating products did not exhibit higher drug resistance than their parental strains, but further propagation under selective pressure resulted in the loss of the wild-type alleles and increased fluconazole resistance. Therefore, fluconazole treatment not only selects for resistance mutations but also promotes genomic alterations that confer mating competence, which allows cells in an originally clonal population to exchange individually acquired resistance mechanisms and generate highly drug-resistant progeny.IMPORTANCE Sexual reproduction is an important mechanism in the evolution of species, since it allows the combination of advantageous traits of individual members in a population. The pathogenic yeast Candida albicans is a diploid organism that normally propagates in a clonal fashion, because heterozygosity at the mating type locus (MTL) inhibits mating between cells. Here we show that C. albicans cells that have acquired drug resistance mutations during treatment with the commonly used antifungal agent fluconazole rapidly develop further increased resistance by genome rearrangements that result in simultaneous loss of heterozygosity for the mutated allele and the mating type locus. This enables the drug-resistant cells of a population to switch to the mating-competent opaque morphology and mate with each other to combine different individually acquired resistance mechanisms. The tetraploid mating products reassort their merged genomes and, under selective pressure by the drug, generate highly resistant progeny that have retained the advantageous mutated alleles. Parasexual propagation, promoted by stress-induced genome rearrangements that result in the acquisition of mating competence in cells with adaptive mutations, may therefore be an important mechanism in the evolution of C. albicans populations.

Authors: C. Popp, B. Ramirez-Zavala, S. Schwanfelder, I. Kruger, J. Morschhauser

Date Published: 5th Feb 2019

Publication Type: Not specified

Abstract (Expand)

Gain-of-function mutations in the zinc cluster transcription factors Mrr1, Tac1, and Upc2, which result in constitutive overexpression of their target genes, are a frequent cause of fluconazole resistance in the pathogenic yeast Candida albicans In this study, we show that an activated form of another zinc cluster transcription factor, Stb5, confers resistance to the natural compound beauvericin via the overexpression of YOR1, encoding an efflux pump of the ATP-binding cassette transporter superfamily. Beauvericin was recently shown to potentiate the activity of azole drugs against C. albicans Although Yor1 did not contribute to fluconazole resistance when C. albicans cells were treated with the drug alone, Stb5-mediated YOR1 overexpression diminished the synergistic effect of the fluconazole-beauvericin combination, thereby enhancing fluconazole resistance in beauvericin-treated C. albicans cells. Stb5-mediated YOR1 overexpression also suppressed the inhibition of hyphal growth, an important virulence trait of C. albicans, by beauvericin. Therefore, activating mutations in Stb5, which result in constitutive YOR1 overexpression, may enable C. albicans to acquire resistance to beauvericin and thereby overcome both the sensitization to azole drugs and the inhibition of morphogenesis caused by this compound.

Authors: B. Ramirez-Zavala, H. Manz, F. Englert, P. D. Rogers, J. Morschhauser

Date Published: 27th Sep 2018

Publication Type: Not specified

Abstract (Expand)

The opportunistic fungal pathogen Candida albicans frequently produces genetically altered variants to adapt to environmental changes and new host niches in the course of its life-long association with the human host. Gain-of-function mutations in zinc cluster transcription factors, which result in the constitutive upregulation of their target genes, are a common cause of acquired resistance to the widely used antifungal drug fluconazole, especially during long-term therapy of oropharyngeal candidiasis. In this study, we investigated if C. albicans also can develop resistance to the antimicrobial peptide histatin 5, which is secreted in the saliva of humans to protect the oral mucosa from pathogenic microbes. As histatin 5 has been shown to be transported out of C. albicans cells by the Flu1 efflux pump, we screened a library of C. albicans strains that contain artificially activated forms of all zinc cluster transcription factors of this fungus for increased FLU1 expression. We found that a hyperactive Mrr1, which confers fluconazole resistance by upregulating the multidrug efflux pump MDR1 and other genes, also causes FLU1 overexpression. Similarly to the artificially activated Mrr1, naturally occurring gain-of-function mutations in this transcription factor also caused FLU1 upregulation and increased histatin 5 resistance. Surprisingly, however, Mrr1-mediated histatin 5 resistance was mainly caused by the upregulation of MDR1 instead of FLU1, revealing a previously unrecognized function of the Mdr1 efflux pump. Fluconazole-resistant clinical C. albicans isolates with different Mrr1 gain-of-function mutations were less efficiently killed by histatin 5, and this phenotype was reverted when MRR1 was deleted. Therefore, antimycotic therapy can promote the evolution of strains that, as a consequence of drug resistance mutations, simultaneously have acquired increased resistance against an innate host defense mechanism and are thereby better adapted to certain host niches.

Authors: I. A. I. Hampe, J. Friedman, M. Edgerton, J. Morschhauser

Date Published: 28th Sep 2017

Publication Type: Not specified

Abstract (Expand)

The metabolic flexibility of the opportunistic fungal pathogen Candida albicans is important for colonisation and infection of different host niches. Complex regulatory networks, in which protein kinases play central roles, link metabolism and other virulence-associated traits, such as filamentous growth and stress resistance, and thereby control commensalism and pathogenicity. By screening a protein kinase deletion mutant library that was generated in the present work using an improved SAT1 flipper cassette, we found that the previously uncharacterised kinase Sak1 is a key upstream activator of the protein kinase Snf1, a highly conserved regulator of nutrient stress responses that is essential for viability in C. albicans. The sak1Delta mutants failed to grow on many alternative carbon sources and were hypersensitive to cell wall/membrane stress. These phenotypes were mirrored in mutants lacking other subunits of the SNF1 complex and partially compensated by a hyperactive form of Snf1. Transcriptional profiling of sak1Delta mutants showed that Sak1 ensures basal expression of glyoxylate cycle and gluconeogenesis genes even in glucose-rich media and thereby contributes to the metabolic plasticity of C. albicans. In a mouse model of gastrointestinal colonisation, sak1Delta mutants were rapidly outcompeted by wild-type cells, demonstrating that Sak1 is essential for the in vivo fitness of C. albicans.

Authors: B. Ramirez-Zavala, A. Mottola, J. Haubenreisser, S. Schneider, S. Allert, S. Brunke, K. Ohlsen, B. Hube, J. Morschhauser

Date Published: 25th Mar 2017

Publication Type: Not specified

Abstract (Expand)

The fungal pathogen Candida albicans forms polymorphic biofilms where hyphal morphogenesis and metabolic adaptation are tightly coordinated by a complex intertwined network of transcription factors. The sensing and metabolism of amino acids play important roles during various phases of biofilm development - from adhesion to maturation. Stp2 is a transcription factor that activates the expression of amino acid permease genes and is required for environmental alkalinization and hyphal growth in vitro and during macrophage phagocytosis. While it is well established that Stp2 is activated in response to external amino acids, its role in biofilm formation remains unknown. In addition to widely used techniques, we applied newly developed approaches for automated image analysis to quantify Stp2-regulated filamentation and biofilm growth. Our results show that in the stp2Delta deletion mutant adherence to abiotic surfaces and initial germ tube formation were strongly impaired, but formed mature biofilms with cell density and morphological structures comparable to the control strains. Stp2-dependent nutrient adaptation appeared to play an important role in biofilm development: stp2Delta biofilms formed under continuous nutrient flow displayed an overall reduction in biofilm formation, whereas under steady conditions the mutant strain formed biofilms with lower metabolic activity, resulting in increased cell survival and biofilm longevity. A deletion of STP2 led to increased rapamycin susceptibility and transcriptional activation of GCN4, the transcriptional regulator of the general amino acid control pathway, demonstrating a connection of Stp2 to other nutrient-responsive pathways. In summary, the transcription factor Stp2 is important for C. albicans biofilm formation, where it contributes to adherence and induction of morphogenesis, and mediates nutrient adaption and cell longevity in mature biofilms.

Authors: B. Bottcher, B. Hoffmann, E. Garbe, T. Weise, Z. Cseresnyes, P. Brandt, S. Dietrich, D. Driesch, M. T. Figge, S. Vylkova

Date Published: 20th May 2020

Publication Type: Not specified

Abstract (Expand)

Host-fungus interactions have gained a lot of interest in the past few decades, mainly due to an increasing number of fungal infections that are often associated with a high mortality rate in the absence of effective therapies. These interactions can be studied at the genetic level or at the functional level via imaging. Here, we introduce a new image processing method that quantifies the interaction between host cells and fungal invaders, for example, alveolar macrophages and the conidia of Aspergillus fumigatus. The new technique relies on the information content of transmitted light bright field microscopy images, utilizing the Hessian matrix eigenvalues to distinguish between unstained macrophages and the background, as well as between macrophages and fungal conidia. The performance of the new algorithm was measured by comparing the results of our method with that of an alternative approach that was based on fluorescence images from the same dataset. The comparison shows that the new algorithm performs very similarly to the fluorescence-based version. Consequently, the new algorithm is able to segment and characterize unlabeled cells, thus reducing the time and expense that would be spent on the fluorescent labeling in preparation for phagocytosis assays. By extending the proposed method to the label-free segmentation of fungal conidia, we will be able to reduce the need for fluorescence-based imaging even further. Our approach should thus help to minimize the possible side effects of fluorescence labeling on biological functions. (c) 2017 International Society for Advancement of Cytometry.

Authors: Z. Cseresnyes, K. Kraibooj, M. T. Figge

Date Published: 16th Sep 2017

Publication Type: Not specified

Abstract (Expand)

Phagocytosis is series of steps where the pathogens and the immune cells interact during an invasion. This starts with the adhesion process between the host and pathogen cells, and is followed by the engulfment of the pathogens. Many analytical methods that are applied to characterize phagocytosis based on imaging the host-pathogen confrontation assays rely on the fluorescence labeling of cells. However, the potential effect of the membrane labeling on the quantitative results of the confrontation assays has not been studied in detail. In this study, we determine whether the fluorescence labeling processes themselves influence the results of the phagocytosis measurements. Here, alveolar macrophages, which form one of the most important compartments of the innate immune system, were used as an example of host cells, whereas Aspergillus fumigatus and Lichtheimia corymbifera that cause aspergillosis and mucormycosis, respectively, were studied as examples for pathogens. At first, our study investigated the importance of the sequence of steps of the fixation process when preparing the confrontation assay sample for microscopy studies. Here we showed that applying the fixation agent before the counter-staining causes miscalculations during the determination of the phagocytic measures. Furthermore, we also found that staining the macrophages with various concentrations of DID, as a typical membrane label, in most cases altered the capability of macrophages to phagocytose FITC-stained A. fumigatus and L. corymbifera spores in comparison with unlabeled macrophages. This effect of the DID staining showed a differential character dependent upon the labeling status and the specific type of pathogen. Moreover, labeling the spores of A. fumigatus and L. corymbifera with FITC increased the phagocytic measures during confrontation with unlabeled macrophages when compared to label-free spores. Overall, our study confirms that the staining process itself may significantly manipulate the quantitative outcome of the confrontation assay. As a result of our study, we also developed a user-friendly image analysis tool that analyses confrontation assays both with and without fluorescence labeling of the host cells and of the pathogens. Our image analysis algorithm saves experimental work effort and time, provides more precise results when calculating the phagocytic measures, and delivers a convenient analysis tool for the biologists to monitor host-pathogen interactions as they happen without the artifacts that fluorescence labeling imposes on biological interactions.

Authors: Z. Cseresnyes, M. I. A. Hassan, H. M. Dahse, K. Voigt, M. T. Figge

Date Published: 26th Jun 2020

Publication Type: Not specified

Abstract (Expand)

The opportunistic fungal pathogen Aspergillus fumigatus can cause life-threatening infections, particularly in immunocompromised patients. Most pathogenic microbes control host innate immune responses at the earliest time, already before infiltrating host immune cells arrive at the site of infection. Here, we identify Aspf2 as the first A. fumigatus Factor H-binding protein. Aspf2 recruits several human plasma regulators, Factor H, factor-H-like protein 1 (FHL-1), FHR1, and plasminogen. Factor H contacts Aspf2 via two regions located in SCRs6-7 and SCR20. FHL-1 binds via SCRs6-7, and FHR1 via SCRs3-5. Factor H and FHL-1 attached to Aspf2-maintained cofactor activity and assisted in C3b inactivation. A Deltaaspf2 knockout strain was generated which bound Factor H with 28% and FHL-1 with 42% lower intensity. In agreement with less immune regulator acquisition, when challenged with complement-active normal human serum, Deltaaspf2 conidia had substantially more C3b (>57%) deposited on their surface. Consequently, Deltaaspf2 conidia were more efficiently phagocytosed (>20%) and killed (44%) by human neutrophils as wild-type conidia. Furthermore, Aspf2 recruited human plasminogen and, when activated by tissue-type plasminogen activator, newly generated plasmin cleaved the chromogenic substrate S2251 and degraded fibrinogen. Furthermore, plasmin attached to conidia damaged human lung epithelial cells, induced cell retraction, and caused matrix exposure. Thus, Aspf2 is a central immune evasion protein and plasminogen ligand of A. fumigatus. By blocking host innate immune attack and by disrupting human lung epithelial cell layers, Aspf2 assists in early steps of fungal infection and likely allows tissue penetration.

Authors: P. Dasari, I. A. Shopova, M. Stroe, D. Wartenberg, H. Martin-Dahse, N. Beyersdorf, P. Hortschansky, S. Dietrich, Z. Cseresnyes, M. T. Figge, M. Westermann, C. Skerka, A. A. Brakhage, P. F. Zipfel

Date Published: 1st Sep 2018

Publication Type: Not specified

Abstract (Expand)

The opportunistic fungal pathogen Aspergillus fumigatus can cause severe infections, particularly in immunocompromised individuals. Upon infection, A. fumigatus faces the powerful and directly acting immune defense of the human host. The mechanisms on how A. fumigatus evades innate immune attack and complement are still poorly understood. Here, we identify A. fumigatus enolase, AfEno1, which was also characterized as fungal allergen, as a surface ligand for human plasma complement regulators. AfEno1 binds factor H, factor-H-like protein 1 (FHL-1), C4b binding protein (C4BP), and plasminogen. Factor H attaches to AfEno1 via two regions, via short conserved repeats (SCRs) 6-7 and 19-20, and FHL-1 contacts AfEno1 via SCRs 6-7. Both regulators when bound to AfEno1 retain cofactor activity and assist in C3b inactivation. Similarly, the classical pathway regulator C4BP binds to AfEno1 and bound to AfEno1; C4BP assists in C4b inactivation. Plasminogen which binds to AfEno1 via lysine residues is accessible for the tissue-type plasminogen activator (tPA), and active plasmin cleaves the chromogenic substrate S2251, degrades fibrinogen, and inactivates C3 and C3b. Plasmin attached to swollen A. fumigatus conidia damages human A549 lung epithelial cells, reduces the cellular metabolic activity, and induces cell retraction, which results in exposure of the extracellular matrix. Thus, A. fumigatus AfEno1 is a moonlighting protein and virulence factor which recruits several human regulators. The attached human regulators allow the fungal pathogen to control complement at the level of C3 and to damage endothelial cell layers and tissue components.

Authors: P. Dasari, N. Koleci, I. A. Shopova, D. Wartenberg, N. Beyersdorf, S. Dietrich, A. Sahagun-Ruiz, M. T. Figge, C. Skerka, A. A. Brakhage, P. F. Zipfel

Date Published: 12th Dec 2019

Publication Type: Not specified

Abstract (Expand)

Mucormycoses are life-threatening infections that affect patients suffering from immune deficiencies. We performed phagocytosis assays confronting various strains of Lichtheimia species with alveolar macrophages, which form the first line of defence of the innate immune system. To investigate 17 strains from four different continents in a comparative fashion, transmitted light and confocal fluorescence microscopy was applied in combination with automated image analysis. This interdisciplinary approach enabled the objective and quantitative processing of the big volume of image data. Applying machine-learning supported methods, a spontaneous clustering of the strains was revealed in the space of phagocytic measures. This clustering was not driven by measures of fungal morphology but rather by the geographical origin of the fungal strains. Our study illustrates the crucial contribution of machine-learning supported automated image analysis to the qualitative discovery and quantitative comparison of major factors affecting host-pathogen interactions. We found that the phagocytic vulnerability of Lichtheimia species depends on their geographical origin, where strains within each geographic region behaved similarly, but strongly differed amongst the regions. Based on this clustering, we were able to also classify clinical isolates with regard to their potential geographical origin.

Authors: M. I. A. Hassan, Z. Cseresnyes, N. Al-Zaben, H. M. Dahse, R. J. Vilela de Oliveira, G. Walther, K. Voigt, M. T. Figge

Date Published: 23rd Jul 2019

Publication Type: Not specified

Abstract (Expand)

Alterations of the microbial composition in the gut and the concomitant dysregulation of the mucosal immune response are associated with the pathogenesis of opportunistic infections, chronic inflammation, and inflammatory bowel disease. To create a platform for the investigation of the underlying mechanisms, we established a three-dimensional microphysiological model of the human intestine. This model resembles organotypic microanatomical structures and includes tissue resident innate immune cells exhibiting features of mucosal macrophages and dendritic cells. The model displays the physiological immune tolerance of the intestinal lumen to microbial-associated molecular patterns and can, therefore, be colonised with living microorganisms. Functional studies on microbial interaction between probiotic Lactobacillus rhamnosus and the opportunistic pathogen Candida albicans show that pre-colonization of the intestinal lumen of the model by L. rhamnosus reduces C. albicans-induced tissue damage, lowers its translocation, and limits fungal burden. We demonstrate that microbial interactions can be efficiently investigated using the in vitro model creating a more physiological and immunocompetent microenvironment. The intestinal model allows a detailed characterisation of the immune response, microbial pathogenicity mechanisms, and quantification of cellular dysfunction attributed to alterations in the microbial composition.

Authors: M. Maurer, M. S. Gresnigt, A. Last, T. Wollny, F. Berlinghof, R. Pospich, Z. Cseresnyes, A. Medyukhina, K. Graf, M. Groger, M. Raasch, F. Siwczak, S. Nietzsche, I. D. Jacobsen, M. T. Figge, B. Hube, O. Huber, A. S. Mosig

Date Published: 10th Aug 2019

Publication Type: Not specified

Abstract (Expand)

Pneumococcal hemolytic uremic syndrome (HUS) in children is caused by infections with Streptococcus pneumoniae. Because endothelial cell damage is a hallmark of HUS, we studied how HUS-inducing pneumococci derived from infant HUS patients during the acute phase disrupt the endothelial layer. HUS pneumococci efficiently bound human plasminogen. These clinical isolates of HUS pneumococci efficiently bound human plasminogen via the bacterial surface proteins Tuf and PspC. When activated to plasmin at the bacterial surface, the active protease degraded fibrinogen and cleaved C3b. Here, we show that PspC is a pneumococcal plasminogen receptor and that plasmin generated on the surface of HUS pneumococci damages endothelial cells, causing endothelial retraction and exposure of the underlying matrix. Thus, HUS pneumococci damage endothelial cells in the blood vessels and disturb local complement homeostasis. Thereby, HUS pneumococci promote a thrombogenic state that drives HUS pathology.

Authors: C. Meinel, G. Sparta, H. M. Dahse, F. Horhold, R. Konig, M. Westermann, S. M. Coldewey, Z. Cseresnyes, M. T. Figge, S. Hammerschmidt, C. Skerka, P. F. Zipfel

Date Published: 17th Jan 2018

Publication Type: Not specified

Abstract (Expand)

The complement system is part of the innate immune system and plays an important role in the host defense against infectious pathogens. One of the main effects is the opsonization of foreign invaders and subsequent uptake by phagocytosis. Due to the continuous default basal level of active complement molecules, a tight regulation is required to protect the body's own cells (self cells) from opsonization and from complement damage. A major complement regulator is Factor H, which is recruited from the fluid phase and attaches to cell surfaces where it effectively controls complement activation. Besides self cells, pathogens also have the ability to bind Factor H; they can thus escape opsonization and phagocytosis causing severe infections. In order to advance our understanding of the opsonization process at a quantitative level, we developed a mathematical model for the dynamics of the complement system-termed DynaCoSys model-that is based on ordinary differential equations for cell surface-bound molecules and on partial differential equations for concentration profiles of the fluid phase molecules in the environment of cells. This hybrid differential equation approach allows to model the complement cascade focusing on the role of active C3b in the fluid phase and on the cell surface as well as on its inactivation on the cell surface. The DynaCoSys model enables us to quantitatively predict the conditions under which Factor H mediated complement evasion occurs. Furthermore, investigating the quantitative impact of model parameters by a sensitivity analysis, we identify the driving processes of complement activation and regulation in both the self and non-self regime. The two regimes are defined by a critical Factor H concentration on the cell surface and we use the model to investigate the differential impact of complement model parameters on this threshold value. The dynamic modeling on the surface of pathogens are further relevant to understand pathophysiological situations where Factor H mutants and defective Factor H binding to target surfaces results in pathophysiology such as renal and retinal disease. In the future, this DynaCoSys model will be extended to also enable evaluating treatment strategies of complement-related diseases.

Authors: A. Tille, T. Lehnert, P. F. Zipfel, M. T. Figge

Date Published: 5th Oct 2020

Publication Type: Not specified

Abstract (Expand)

Rationale: The liver is a central organ not only for metabolism but also immune function. Life-threatening infections of both bacterial and fungal origin can affect liver function but it is yet unknown whether molecular changes differ depending on the pathogen. We aimed to determine whether the hepatic host response to bacterial and fungal infections differs in terms of hepatic metabolism and liver function. Methods: We compared murine models of infection, including bacterial peritoneal contamination and infection (PCI), intraperitoneal and systemic C. albicans infection, at 6 and 24 h post-infection, to sham controls. The molecular hepatic host response was investigated by the detection of regulatory modules based on large-scale protein-protein interaction networks and expression data. Topological analysis of these regulatory modules was used to reveal infection-specific biological processes and molecular mechanisms. Intravital microscopy and immunofluorescence microscopy were used to further analyze specific aspects of pathophysiology such as cholestasis. Results: Down-regulation of lipid catabolism and bile acid synthesis was observed after 6 h in all infection groups. Alterations in lipid catabolism were characterized by accumulation of long chain acylcarnitines and defective beta-oxidation, which affected metabolism by 6 h. While PCI led to an accumulation of unconjugated bile acids (BA), C. albicans infection caused accumulation of conjugated BA independent of the route of infection. Hepatic dye clearance and transporter expression revealed reduced hepatic uptake in fungal infections vs. defects in secretion following polybacterial infection. Conclusion: Molecular phenotypes of lipid accumulation and cholestasis allow differentiation between pathogens as well as routes of infection at early stages in mice. Targeted metabolomics could be a useful tool for the profiling of infected/septic patients and the type of pathoge