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Proteome Analysis Reveals the Conidial Surface Protein CcpA Essential for Virulence of the Pathogenic Fungus Aspergillus fumigatus.
FungiNet total, Z2

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: No date defined

Journal: MBio

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

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

Proteome Analysis Reveals the Conidial Surface Protein CcpA Essential for Virulence of the Pathogenic Fungus Aspergillus fumigatus.
FungiNet A - Aspergillus projects

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

Playing 'hide-and-seek' with factor H: game-theoretical analysis of a single nucleotide polymorphism.
B1

Abstract (Expand)

As a part of the complement system, factor H regulates phagocytosis and helps differentiate between a body's own and foreign cells. Owing to mimicry efforts, some pathogenic microorganisms such as … Candida albicans are able to bind factor H on their cell surfaces and, thus, become similar to host cells. This implies that the decision between self and foreign is not clear-cut, which leads to a classification problem for the immune system. Here, two different alleles determining the binding affinity of factor H are relevant. Those alleles differ in the SNP Y402H; they are known to be associated with susceptibility to certain diseases. Interestingly, the fraction of both alleles differs in ethnic groups. The game-theoretical model proposed in this article explains the coexistence of both alleles by a battle of the sexes game and investigates the trade-off between pathogen detection and protection of host cells. Further, we discuss the ethnicity-dependent frequencies of the alleles. Moreover, the model elucidates the mimicry efforts by pathogenic microorganisms.

Authors: S. Hummert, C. Glock, S. N. Lang, C. Hummert, Christine Skerka, Peter Zipfel, S. Germerodt, Stefan Schuster

Date Published: 4th May 2018

Journal: J R Soc Interface

Predictive Virtual Infection Modeling of Fungal Immune Evasion in Human Whole Blood.
FungiNet C - Candida projects, B4

Abstract (Expand)

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

Authors: M. T. E. Prausse, Teresa Lehnert, Sandra Timme, Kerstin Hünniger, I. Leonhardt, Oliver Kurzai, Marc Thilo Figge

Date Published: 6th Apr 2018

Journal: Front Immunol

Proteomics of Aspergillus fumigatus Conidia-containing Phagolysosomes Identifies Processes Governing Immune Evasion.
B2

Abstract (Expand)

Invasive infections by the human pathogenic fungus Aspergillus fumigatus start with the outgrowth of asexual, airborne spores (conidia) into the lung tissue of immunocompromised patients. The resident … alveolar macrophages phagocytose conidia, which end up in phagolysosomes. However, A. fumigatus conidia resist phagocytic degradation to a certain degree. This is mainly attributable to the pigment 1,8-dihydroxynaphthalene (DHN) melanin located in the cell wall of conidia, which manipulates the phagolysosomal maturation and prevents their intracellular killing. To get insight in the underlying molecular mechanisms, we comparatively analyzed proteins of mouse macrophage phagolysosomes containing melanized wild-type (wt) or nonmelanized pksP mutant conidia. For this purpose, a protocol to isolate conidia-containing phagolysosomes was established and a reference protein map of phagolysosomes was generated. We identified 637 host and 22 A. fumigatus proteins that were differentially abundant in the phagolysosome. 472 of the host proteins were overrepresented in the pksP mutant and 165 in the wt conidia-containing phagolysosome. Eight of the fungal proteins were produced only in pksP mutant and 14 proteins in wt conidia-containing phagolysosomes. Bioinformatical analysis compiled a regulatory module, which indicates host processes affected by the fungus. These processes include vATPase-driven phagolysosomal acidification, Rab5 and Vamp8-dependent endocytic trafficking, signaling pathways, as well as recruitment of the Lamp1 phagolysosomal maturation marker and the lysosomal cysteine protease cathepsin Z. Western blotting and immunofluorescence analyses confirmed the proteome data and moreover showed differential abundance of the major metabolic regulator mTOR. Taken together, with the help of a protocol optimized to isolate A. fumigatus conidia-containing phagolysosomes and a potent bioinformatics algorithm, we were able to confirm A. fumigatus conidia-dependent modification of phagolysosomal processes that have been described before and beyond that, identify pathways that have not been implicated in A. fumigatus evasion strategy, yet.Mass spectrometry proteomics data are available via ProteomeXchange with identifiers PXD005724 and PXD006134.

Authors: H. Schmidt, S. Vlaic, T. Kruger, F. Schmidt, J. Balkenhol, T. Dandekar, R. Guthke, O. Kniemeyer, T. Heinekamp, A. A. Brakhage

Date Published: 7th Mar 2018

Journal: Mol Cell Proteomics

Proteomic Profiling of Serological Responses to Aspergillus fumigatus Antigens in Patients with Invasive Aspergillosis
Z2

Abstract (Expand)

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

Authors: J. Teutschbein, S. Simon, J. Lother, J. Springer, P. Hortschansky, C. O. Morton, Jürgen Löffler, Hermann Einsele, E. Conneally, T. R. Rogers, Reinhard Guthke, Axel Brakhage, Olaf Kniemeyer

Date Published: 15th Mar 2016

Journal: J Proteome Res

pH Regulates White-Opaque Switching and Sexual Mating in Candida albicans.
FungiNet C - Candida projects

Abstract (Expand)

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

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

Date Published: 6th Sep 2015

Journal: Eukaryot Cell

Pathogenicity mechanisms and host response during oral Candida albicans infections
FungiNet C - Candida projects

Abstract (Expand)

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

Authors: B. Hebecker, J. R. Naglik, Bernhard Hube, Ilse Jacobsen

Date Published: 7th May 2014

Journal: Expert Rev Anti Infect Ther

Probing the unknowns in cytokinin-mediated immune defense in Arabidopsis with systems biology approaches
FungiNet B - Bioinformatics projects

Abstract (Expand)

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

Authors: M. Naseem, M. Kunz, Thomas Dandekar

Date Published: 13th Feb 2014

Journal: Bioinform Biol Insights

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