Journal Article

Proteome Analysis Reveals the Conidial Surface Protein CcpA Essential for Virulence of the Pathogenic Fungus Aspergillus fumigatus

Bacher, Petra, Hünniger, Kerstin, Kurzai, Oliver, Reichard, Utz, Brakhage, Axel A., Hortschansky, Peter, Nietzsche, Sandor, Liu, Hong, Filler, Scott G., Schmidt, Franziska, Blango, Matthew G., Herrmann, Sahra, Kniemeyer, Olaf, Strassburger, Maria, Weiss, Esther, Voltersen, Vera, Löffler, Jürgen, Krappmann, Sven, Heinekamp, Thorsten, Lother, Jasmin, Scheffold, Alexander, Krüger, Thomas, Einsele, Hermann

2018

Overview

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. 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 c onidial c ell wall p rotein 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 Δ ccpA resting conidia appeared normal. However, trypsin shaving of Δ ccpA conidia revealed novel surface-exposed proteins not detected on conidia of the wild-type strain. Interestingly, the presence of swollen Δ ccpA 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 Δ ccpA 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.

• Publisher: American Society for Microbiology
• Subject: A549 Cells / Amino acids / Animals / Aspergillosis - immunology / Aspergillus fumigatus / Aspergillus fumigatus - genetics / Aspergillus fumigatus - pathogenicity / CcpA / Cell activation / Cell surface / Cell Wall - chemistry / Cell walls / Chromatography / Chromatography, Liquid / Conidia / dendritic cell / Dendritic cells / Dendritic Cells - immunology / Endocytosis / epithelial cell / Epithelial cells / Epithelial Cells - immunology / Female / Fungal Proteins - genetics / Fungal Proteins - metabolism / Heat shock proteins / Host-Microbe Biology / Humans / Hydrophobicity / Immune clearance / immune response / Immune system / Immunocompromised Host / Immunocompromised hosts / Immunogenicity / Immunological memory / Immunosuppression / Leukocytes (neutrophilic) / Liquid chromatography / Localization / Lymphocytes T / Mass spectroscopy / Membrane Proteins - genetics / Membrane Proteins - metabolism / Memory cells / Mice / Neutrophil Activation / Pathogens / Peptides / Phagocytosis / Proteome - analysis / Proteomes / Scanning electron microscopy / Spores, Fungal - pathogenicity / Sporulation / Surface antigens / T cells / T-Lymphocytes - immunology / Trypsin / Virulence / Virulence Factors - genetics / Virulence Factors - metabolism