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MetGEMs Toolbox: Metagenome-scale models as integrative toolbox for uncovering metabolic functions and routes of human gut microbiome.
INF, B5

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, Gianni Panagiotou, A. Senavonge, N. Suratannon, W. Vongsangnak

Date Published: 6th Jan 2021

Journal: PLoS Comput Biol

Carbon Catabolite Repression in Filamentous Fungi Is Regulated by Phosphorylation of the Transcription Factor CreA.
FungiNet total, A1, Z2

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, Olaf Kniemeyer, Thomas Krüger, Axel Brakhage, Y. Chen, L. Dong, K. Tan, K. H. Wong, L. N. A. Ries, G. H. Goldman

Date Published: 5th Jan 2021

Journal: mBio

Metabolic modeling predicts specific gut bacteria as key determinants for Candida albicans colonization levels.
INF, A5, C1, C2, B5

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: Mohammad Mirhakkak, Sascha Schäuble, Tilman Klassert, S. Brunke, Philipp Brandt, D. Loos, R. V. Uribe, F. Senne de Oliveira Lino, Y. Ni, Slavena Vylkova, Hortense Slevogt, Bernhard Hube, Esther Weiß, M. O. A. Sommer, Gianni Panagiotou

Date Published: 15th Dec 2020

Journal: ISME J

Lipid metabolic signatures deviate in sepsis survivors compared to non-survivors.
INF, C5

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, Peter Großmann, S. Neugebauer, A. Kleyman, R. Domizi, S. Calcinaro, D. Brealey, M. Graler, M. Kiehntopf, Sascha Schäuble, M. Singer, Gianni Panagiotou, Michael Bauer

Date Published: 11th Dec 2020

Journal: Comput Struct Biotechnol J

Effects of Agricultural Fungicide Use on Aspergillus fumigatus Abundance, Antifungal Susceptibility, and Population Structure.
INF, B5

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, Gianni Panagiotou, H. B. Deising, Oliver Kurzai

Date Published: 24th Nov 2020

Journal: mBio

Triple RNA-Seq Reveals Synergy in a Human Virus-Fungus Co-infection Model.
INF, A2, B5

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: Bastian Seelbinder, J. Wallstabe, Lothar Marischen, Esther Weiß, S. Wurster, L. Page, C. Loffler, L. Bussemer, A. L. Schmitt, Thomas Wolf, Jörg Linde, L. Cicin-Sain, J. Becker, U. Kalinke, J. Vogel, Gianni Panagiotou, Hermann Einsele, A. J. Westermann, Sascha Schäuble, Jürgen Löffler

Date Published: 17th Nov 2020

Journal: Cell Rep

Proteome analysis of bronchoalveolar lavage fluids reveals host and fungal proteins highly expressed during invasive pulmonary aspergillosis in mice and humans.
INF, A5, C5

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, Wolfgang Müller, R. Lehmann, Patricia Sieber, Gianni Panagiotou, A. Carvalho, C. Cunha, K. Lagrou, J. Maertens, Hortense Slevogt, Ilse Jacobsen

Date Published: 12th Oct 2020

Journal: Virulence

Survival Strategies of Pathogenic Candida Species in Human Blood Show Independent and Specific Adaptations.
INF, B3 (E), C1, C3

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, Thomas Wolf, Theresia Conrad, Stefanie Allert, F. Gerwien, Kerstin Hünniger, Oliver Kurzai, Reinhard Guthke, Bernhard Hube, Jörg Linde, S. Brunke

Date Published: 6th Oct 2020

Journal: mBio

Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus.
INF, A2, A3, C3

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: Esther Weiß, J. Schlegel, Ulrich Terpitz, Michael Weber, Jörg Linde, A. L. Schmitt, Kerstin Hünniger, Lothar Marischen, F. Gamon, J. Bauer, C. Loffler, Oliver Kurzai, C. O. Morton, M. Sauer, Hermann Einsele, Jürgen Löffler

Date Published: 5th Oct 2020

Journal: Front Immunol

Antibiotics create a shift from mutualism to competition in human gut communities with a longer-lasting impact on fungi than bacteria.
INF, C1, B5

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: Bastian 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, Bernhard Hube, P. C. Woo, M. O. A. Sommer, Gianni Panagiotou

Date Published: 12th Sep 2020

Journal: Microbiome

Comparative Transcriptomic Analysis of Rhinovirus and Influenza Virus Infection.
INF, B5

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, Sascha Schäuble, Mohammad Mirhakkak, W. L. Wu, A. C. Ng, C. C. Y. Yip, A. G. Lopez, Thomas Wolf, M. L. Yeung, K. H. Chan, K. Y. Yuen, Gianni Panagiotou, K. K. To

Date Published: 28th Aug 2020

Journal: Front Microbiol

Functional surface proteomic profiling reveals the host heat-shock protein A8 as a mediator of Lichtheimia corymbifera recognition by murine alveolar macrophages.
A1, A5, B4, Z2, A6

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, Thomas Krüger, H. M. Dahse, Z. Cseresnyes, M. G. Blango, Hortense Slevogt, F. Horhold, V. Ast, R. Konig, Marc Thilo Figge, Olaf Kniemeyer, Axel Brakhage, Kerstin Voigt

Date Published: 26th Jun 2020

Journal: Environ Microbiol

Candida Species-Dependent Release of IL-12 by Dendritic Cells Induces Different Levels of NK Cell Stimulation.
A2, B4, C3

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, Kerstin Hünniger, S. Bottcher, W. Vivas, Jürgen Löffler, Marc Thilo Figge, Oliver Kurzai

Date Published: 11th Jun 2020

Journal: J Infect Dis

Dynamic Surface Proteomes of Allergenic Fungal Conidia.
A1, B5

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, Flora Rivieccio, Thomas Krüger, M. Rafiq, L. J. Jia, T. Zheng, M. Goldmann, V. Voltersen, J. Li, Gianni Panagiotou, Olaf Kniemeyer, Axel Brakhage

Date Published: 1st May 2020

Journal: J Proteome Res

Ahr1 and Tup1 Contribute to the Transcriptional Control of Virulence-Associated Genes in Candida albicans.
A1, C1, C2, C3, C5, Z2

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, Selene Mogavero, Daniela Albrecht-Eckardt, D. Hellwig, A. Hader, Thomas Krüger, K. Gerth, Ilse Jacobsen, O. Elshafee, S. Brunke, Kerstin Hünniger, Olaf Kniemeyer, Axel Brakhage, Joachim Morschhäuser, Bernhard Hube, Slavena Vylkova, Oliver Kurzai, R. Martin

Date Published: 28th Apr 2020

Journal: mBio

Human Neutrophils Produce Antifungal Extracellular Vesicles against Aspergillus fumigatus.
A1, B4, C6 (E), Z2

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: Iordana Shopova, I. Belyaev, Prasad Dasari, S. Jahreis, M. C. Stroe, Z. Cseresnyes, Ann-Kathrin Zimmermann, A. Medyukhina, Carl-Magnus Svensson, Thomas Krüger, V. Szeifert, S. Nietzsche, Theresia Conrad, M. G. Blango, Olaf Kniemeyer, M. von Lilienfeld-Toal, Peter Zipfel, E. Ligeti, Marc Thilo Figge, Axel Brakhage

Date Published: 14th Apr 2020

Journal: mBio

Predictable modulation of cancer treatment outcomes by the gut microbiota.
B5

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, Gianni Panagiotou

Date Published: 5th Mar 2020

Journal: Microbiome

Novel Approaches for Systems Biology of Metabolism-Oriented Pathogen-Human Interactions: A Mini-Review.
INF, B5

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, Gianni Panagiotou, R. Uddin, S. Durmus

Date Published: 3rd Mar 2020

Journal: Front Cell Infect Microbiol

covRNA: discovering covariate associations in large-scale gene expression data.
B2

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, Christian Remmele, Marcus Dittrich, R. F. Schwarz, Tobias Müller

Date Published: 24th Feb 2020

Journal: BMC Res Notes

Genome-wide inference of the Camponotus floridanus protein-protein interaction network using homologous mapping and interacting domain profile pairs.
B1

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: Shishir K Gupta, Mugdha Srivastava, O. Osmanoglu, Thomas Dandekar

Date Published: 11th Feb 2020

Journal: Sci Rep

Gastric bypass surgery in a rat model alters the community structure and functional composition of the intestinal microbiota independently of weight loss.
INF, B5

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, Gianni Panagiotou, M. Noll, U. Rolle-Kampczyk, W. Fenske, M. von Bergen

Date Published: 7th Feb 2020

Journal: Microbiome

Three-Dimensional Light Sheet Fluorescence Microscopy of Lungs To Dissect Local Host Immune-Aspergillus fumigatus Interactions.
A3

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, Zeinab Mokhtari, Marlene Strobel, E. Vialetto, D. Sheta, Y. Yu, J. Hartweg, N. Kalleda, K. J. Jarick, C. Brede, A. L. Jordan-Garrote, S. Thusek, K. Schmiedgen, Berkan Arslan, J. Pinnecker, C. R. Thornton, M. Gunzer, S. Krappmann, Hermann Einsele, Katrin Heinze, Andreas Beilhack

Date Published: 4th Feb 2020

Journal: mBio

Gut Microbiome Fermentation Determines the Efficacy of Exercise for Diabetes Prevention.
INF, B5

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, Gianni Panagiotou, A. Xu

Date Published: 7th Jan 2020

Journal: Cell Metab

Treatment with etanercept and low monocyte concentration contribute to the risk of invasive aspergillosis in patients post allogeneic stem cell transplantation.
INF, A2, C3

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, Michael Weber, J. Springer, P. L. White, J. Bauer, A. Schober, C. Loffler, B. Seelbinder, Kerstin Hünniger, Oliver Kurzai, A. Scherag, Sascha Schäuble, C. O. Morton, Hermann Einsele, Jörg Linde, Jürgen Löffler

Date Published: 21st Nov 2019

Journal: Sci Rep

Antibiotic Treatment Drives the Diversification of the Human Gut Resistome.
B5

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, Gianni Panagiotou, M. O. A. Sommer

Date Published: 27th Apr 2019

Journal: Genomics Proteomics Bioinformatics

Linking genetic, metabolic, and phenotypic diversity among Saccharomyces cerevisiae strains using multi-omics associations.
B5

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, Gianni Panagiotou

Date Published: 1st Apr 2019

Journal: Gigascience

Mining, analyzing, and integrating viral signals from metagenomic data.
INF, B5

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, Gianni Panagiotou

Date Published: 19th Mar 2019

Journal: Microbiome

Human Anti-fungal Th17 Immunity and Pathology Rely on Cross-Reactivity against Candida albicans.
A1, C1, Z2

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, Bernhard Hube, Olaf Kniemeyer, Axel Brakhage, C. Schwarz, A. Scheffold

Date Published: 7th Mar 2019

Journal: Cell

Module-detection approaches for the integration of multilevel omics data highlight the comprehensive response of Aspergillus fumigatus to caspofungin.
INF, A1, C5

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: Theresia Conrad, Olaf Kniemeyer, S. G. Henkel, T. Kruger, D. J. Mattern, V. Valiante, Reinhard Guthke, Ilse Jacobsen, Axel Brakhage, S. Vlaic, Jörg Linde

Date Published: 20th Oct 2018

Journal: BMC Syst Biol

Comparative Study on Alternative Splicing in Human Fungal Pathogens Suggests Its Involvement During Host Invasion.
INF, B1

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: Patricia Sieber, Kerstin Voigt, P. Kammer, S. Brunke, Stefan Schuster, Jörg Linde

Date Published: 19th Oct 2018

Journal: Front Microbiol

ModuleDiscoverer: Identification of regulatory modules in protein-protein interaction networks.
INF, B1

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, Theresia Conrad, C. Tokarski-Schnelle, M. Gustafsson, U. Dahmen, Reinhard Guthke, Stefan Schuster

Date Published: 11th Jan 2018

Journal: Sci Rep

Two's company: studying interspecies relationships with dual RNA-seq.
INF

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: Thomas Wolf, P. Kammer, S. Brunke, Jörg Linde

Date Published: 29th Sep 2017

Journal: Curr Opin Microbiol

LASSIM-A network inference toolbox for genome-wide mechanistic modeling.
INF

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, Jörg Linde, T. E. M. Nordling, E. Nyman, Sylvie Schulze, C. E. Nestor, H. Zhang, G. Cedersund, M. Benson, A. Tjarnberg, M. Gustafsson

Date Published: 24th Jun 2017

Journal: PLoS Comput Biol

Specific and Novel microRNAs Are Regulated as Response to Fungal Infection in Human Dendritic Cells.
INF, A2, B3 (E), C3

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: Andreas Dix, K. Czakai, I. Leonhardt, K. Schaferhoff, M. Bonin, Reinhard Guthke, Hermann Einsele, Oliver Kurzai, Jürgen Löffler, Jörg Linde

Date Published: 11th Mar 2017

Journal: Front Microbiol

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