Abstract (Expand)

Gliotoxin and related epidithiodiketopiperazines (ETP) from diverse fungi feature highly functionalized hydroindole scaffolds with an array of medicinally and ecologically relevant activities. Mutation analysis, heterologous reconstitution, and biotransformation experiments revealed that a cytochrome P450 monooxygenase (GliF) from the human-pathogenic fungus Aspergillus fumigatus plays a key role in the formation of the complex heterocycle. In vitro assays using a biosynthetic precursor from a blocked mutant showed that GliF is specific to ETPs and catalyzes an unprecedented heterocyclization reaction that cannot be emulated with current synthetic methods. In silico analyses indicate that this rare biotransformation takes place in related ETP biosynthetic pathways.

Authors: D. H. Scharf, P. Chankhamjon, K. Scherlach, J. Dworschak, T. Heinekamp, M. Roth, A. A. Brakhage, C. Hertweck

Date Published: 15th Jan 2021

Journal: Chembiochem

Abstract (Expand)

Lipid rafts form signaling platforms on biological membranes with incompletely characterized role in immune response to infection. Here we report that lipid-raft microdomains are essential components of phagolysosomal membranes of macrophages and depend on flotillins. Genetic deletion of flotillins demonstrates that the assembly of both major defense complexes vATPase and NADPH oxidase requires membrane microdomains. Furthermore, we describe a virulence mechanism leading to dysregulation of membrane microdomains by melanized wild-type conidia of the important human-pathogenic fungus Aspergillus fumigatus resulting in reduced phagolysosomal acidification. We show that phagolysosomes with ingested melanized conidia contain a reduced amount of free Ca(2+) ions and that inhibition of Ca(2+)-dependent calmodulin activity led to reduced lipid-raft formation. We identify a single-nucleotide polymorphism in the human FLOT1 gene resulting in heightened susceptibility for invasive aspergillosis in hematopoietic stem cell transplant recipients. Collectively, flotillin-dependent microdomains on the phagolysosomal membrane play an essential role in protective antifungal immunity.

Authors: F. Schmidt, A. Thywissen, M. Goldmann, C. Cunha, Z. Cseresnyes, H. Schmidt, M. Rafiq, S. Galiani, M. H. Graler, G. Chamilos, J. F. Lacerda, A. Jr Campos, C. Eggeling, Marc Thilo Figge, Thorsten Heinekamp, S. G. Filler, A. Carvalho, Axel Brakhage

Date Published: 18th Aug 2020

Journal: Cell Rep

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, Kerstin Voigt, Marc Thilo Figge

Date Published: 26th Jun 2020

Journal: Front Microbiol

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: Prasad Dasari, Naile Koleci, Iordana Shopova, D. Wartenberg, Niklas Beyersdorf, Stefanie Dietrich, A. Sahagun-Ruiz, Marc Thilo Figge, Christine Skerka, Axel Brakhage, Peter Zipfel

Date Published: 12th Dec 2019

Journal: Front Immunol

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, Kerstin Voigt, Marc Thilo Figge

Date Published: 23rd Jul 2019

Journal: Environ Microbiol

Abstract (Expand)

The epidithiodioxopiperazine gliotoxin is a virulence factor of Aspergillus fumigatus, the most important airborne fungal pathogen of humans. Gliotoxin suppresses innate immunity in invasive aspergillosis, particularly by compromising neutrophils, but the underlying molecular mechanisms remain elusive. Neutrophils are the first responders among innate immune cells recruited to sites of infection by the chemoattractant leukotriene (LT)B4 that is biosynthesized by 5-lipoxygenase and LTA4 hydrolase (LTA4H). Here, we identified gliotoxin as inhibitor of LTA4H that selectively abrogates LTB4 formation in human leukocytes and in distinct animal models. Gliotoxin failed to inhibit the formation of other eicosanoids and the aminopeptidase activity of the bifunctional LTA4H. Suppression of LTB4 formation by gliotoxin required the cellular environment and/or reducing conditions, and only the reduced form of gliotoxin inhibited LTA4H activity. Conclusively, gliotoxin suppresses the biosynthesis of the potent neutrophil chemoattractant LTB4 by direct interference with LTA4H thereby impairing neutrophil functions in invasive aspergillosis.

Authors: S. Konig, S. Pace, H. Pein, Thorsten Heinekamp, J. Kramer, E. Romp, M. Strassburger, F. Troisi, A. Proschak, J. Dworschak, K. Scherlach, A. Rossi, L. Sautebin, J. Z. Haeggstrom, C. Hertweck, Axel Brakhage, J. Gerstmeier, E. Proschak, O. Werz

Date Published: 18th Apr 2019

Journal: Cell Chem Biol

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, Thorsten Heinekamp, M. Strassburger, Thomas Krüger, P. Bacher, Jasmin Lother, Esther Weiß, Kerstin Hünniger, H. Liu, P. Hortschansky, A. Scheffold, Jürgen Löffler, S. Krappmann, S. Nietzsche, Oliver Kurzai, Hermann Einsele, Olaf Kniemeyer, S. G. Filler, U. Reichard, Axel Brakhage

Date Published: 2nd Oct 2018

Journal: mBio

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: Prasad Dasari, Iordana Shopova, M. Stroe, D. Wartenberg, H. Martin-Dahse, Niklas Beyersdorf, P. Hortschansky, Stefanie Dietrich, Z. Cseresnyes, Marc Thilo Figge, M. Westermann, Christine Skerka, Axel Brakhage, Peter Zipfel

Date Published: 1st Sep 2018

Journal: Front Immunol

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, Olaf Kniemeyer, Jörg Linde, M. Kunert, W. Boland, E. Kothe

Date Published: 7th Sep 2016

Journal: Fungal Genet Biol

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: Natarajaswamy Kalleda, J. Amich, B. Arslan, S. Poreddy, K. Mattenheimer, Z. Mokhtari, Hermann Einsele, Matthias Brock, Katrin Heinze, Andreas Beilhack

Date Published: 13th Jul 2016

Journal: Front Microbiol

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, Andreas Dix, M. Bonin, Jörg Linde, Hermann Einsele, Oliver Kurzai, Jürgen Löffler

Date Published: 28th Jun 2016

Journal: Sci Rep

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: Dragana Slavkovic Lukic, J. Voigt, M. Bouzani, Jürgen Löffler, Daniela Albrecht-Eckardt, Michael Weber, Stefanie Allert, R. Martin, Oliver Kurzai, Kerstin Hünniger

Date Published: 19th May 2016

Journal: Front Microbiol

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, Tobias Müller, Tilman Klassert, A. Wiessner, K. Fischer, R. R. Schumann, U. Seifert, K. Riesbeck, A. Moter, B. B. Singer, S. Bachmann, Hortense Slevogt

Date Published: 3rd Apr 2016

Journal: Cell Microbiol

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: Andreas Dix, K. Czakai, J. Springer, M. Fliesser, M. Bonin, Reinhard Guthke, A. L. Schmitt, Hermann Einsele, Jörg Linde, Jürgen Löffler

Date Published: 21st Mar 2016

Journal: Front Microbiol

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, Reinhard Guthke, O. Kniemeyer, Axel Brakhage, Jörg Linde, V. Valiante

Date Published: 10th Sep 2015

Journal: PLoS One

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, Jörg Linde, D. J. Mattern, G. Walther, Reinhard Guthke, Axel Brakhage, V. Valiante

Date Published: 5th Sep 2015

Journal: Genome Announc

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

Journal: BMC Genomics

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, Hanno Schoeler, C. M. Svensson, Axel Brakhage, Marc Thilo Figge

Date Published: 9th Jun 2015

Journal: Front Microbiol

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

Date Published: 8th Jun 2015

Journal: Antimicrob Agents Chemother

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, Axel Brakhage

Date Published: 26th May 2015

Journal: Proteomics

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: Andreas Dix, Kerstin Hünniger, M. Weber, Reinhard Guthke, Oliver Kurzai, Jörg Linde

Date Published: 11th Mar 2015

Journal: Front Microbiol

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, Axel Brakhage, Reinhard Guthke, C. Hertweck, Jörg Linde

Date Published: 24th Jan 2015

Journal: Genome Announc

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, Iordana Shopova, N. Koster-Eiserfunke, T. Kruger, O. Kniemeyer, Axel Brakhage

Date Published: 18th Nov 2014

Journal: Semin Immunopathol

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, Axel Brakhage

Date Published: 11th Sep 2014

Journal: Front Microbiol

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: Tilman Klassert, A. Hanisch, J. Brauer, E. Klaile, K. A. Heyl, M. K. Mansour, J. M. Tam, J. M. Vyas, Hortense Slevogt

Date Published: 17th Aug 2014

Journal: Med Microbiol Immunol

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, Reinhard Guthke, Axel Brakhage, Jörg Linde

Date Published: 19th Jul 2014

Journal: Genome Announc

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, Jörg Linde, N. Beckmann, M. Cyrulies, M. Strassburger, T. Heinekamp, H. Haas, Reinhard Guthke, Olaf Kniemeyer, Axel Brakhage

Date Published: 7th Jul 2014

Journal: Mol Microbiol

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

Date Published: 13th May 2014

Journal: Immunol Cell Biol


Not specified

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

Date Published: 30th Jan 2014

Journal: PLoS Pathog

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, Kerstin Hünniger, M. Bouzani, Ilse Jacobsen, D. Barz, Bernhard Hube, Jürgen Löffler, Oliver Kurzai

Date Published: 25th Oct 2013

Journal: J Infect Dis

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

Journal: Nat. Rev. Microbiol.



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

Date Published: 2nd Oct 2012

Journal: BMC Genomics

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: Jörg Linde, P. Hortschansky, E. Fazius, Axel Brakhage, Reinhard Guthke, H. Haas

Date Published: 19th Jan 2012

Journal: BMC Syst Biol

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

Journal: Virulence

Powered by
Copyright © 2008 - 2019 The University of Manchester and HITS gGmbH