Cell surface associated secreted aspartic proteases (saps) of Candida albicans
Zusammenfassung der Projektergebnisse
Candida albicans is the most important human-pathogenic yeast that can cause superficial to severe systemic fungal infections. The secreted aspartic proteases (Saps) are a set of virulence factors which contribute to C. albicans pathogenicity. Our previous data suggested that Sap9 and Sap10 process fungal proteins involved in cell surface integrity, cell separation, adhesion and infection. The biochemical characteristics and the protein substrates, however, were unknown. In the current project, Sap9 and Sap10 recombinant proteases were shown to exhibit substantial proteolytic activity over a broad pH range with an optimum at near-neutral pH. Inhibition of enzymatic activity by the aspartic protease inhibitor pepstatin A was less efficient for Sap9 and Sap10 in comparison to other Sap members. A systematic evaluation of peptide substrate cleavage by Sap9 and Sap10 showed that both enzymes prefer the processing of peptides containing basic or dibasic residues, dependent on the nature of neighbouring amino acids. These enzymatic properties distinguish Sap9 and Sap10 from other Sap proteases and reveal similarities to Saccharomyces cerevisiae yapsins. Recombinant Sap9 or Sap10 proteases cleaved distinct cell wall proteins present on isolated cell walls. The covalently linked cell wall proteins Cht2, Ywp1, Als2, Rhd3, Rbt5, Ecm33, and Pga4 were identified as protease substrates. Most of these proteins have known functions in cell wall integrity, cell separation or adhesion. Proteolytic cleavage of the chitinase Cht2 and the glucan-crosslinking protein Pir1 by Sap9 was further shown using hemagglutinin (HA) epitope-tagged versions of both proteins. Deletion of the SAP9 and SAP10 genes resulted in a reduction of cell-associated chitinase activity similar to that observed upon deletion of CHT2, indicating a direct influence of Sap9 and Sap10 on Cht2 function. Alterations on the fungal cell surface elicited by SAP9 and SAP10 deletion had no major impact phagocytosis or killing of C. albicans by human macrophages. However, Sap9 was shown to be important for neutrophil chemotaxis. Cleavage and inactivation of the human salivary antimicrobial peptide histatin 5 by Sap9 revealed a novel function of Sap9 in proteolysis of host factors as an immune evasion strategy. The data obtained in this project support the view that both proteases modulate specific cell wall functions by proteolytic cleavage of covalently linked cell wall proteins. Sap9 and Sap10 likely influence C. albicans-host interaction by targeting both, fungal cell surface proteins and host components. The main proteolytic activity of Sap9 and Sap10 was expected to target fungal proteins. Therefore, the major focus of the project was the identification of fungal substrates. Surprisingly, histatin 5, an antimicrobial peptide important during oral infection, was found as the first host molecule processed by Sap9. The experimental evidence for Sap9/10 processing in vivo appeared to more difficult than expected. It is possible, that Sap9 and Sap10 are only locally and transiently active on the cell surface.
Projektbezogene Publikationen (Auswahl)
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(2009) A novel immune evasion strategy of Candida albicans: proteolytic cleavage of a salivary antimicrobial peptide. PLoS One 4: e5039
Meiller TF, Hube B, Schild L, Shirtliff ME, Scheper MA, Winkler R, Ton A, Jabra-Rizk MA
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(2009) Proteolytic activity of GPI-anchored proteases from Candida albicans modulates cell wall functions and interaction with phagocytes. „Weimar Sepsis Update“, Weimar, Germany
Schild L, Heyken A, de Groot PW, Hiller E, Rupp S, Hube B
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(2009) The glycosylphosphatidylinositol-anchored protease Sap9 modulates the interaction of Candida albicans with human neutrophils. Infect Immun 77: 5216-5224
Hornbach A, Heyken A, Schild L, Hube B, Loffler J, Kurzai O
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(2009) The yeast Candida albicans evades human complement attack by secretion of aspartic proteases. Mol Immunol 47: 465-475
Gropp K, Schild L, Schindler S, Hube B, Zipfel PF, Skerka C
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(2011) Influence of cell surface-associated proteases on the cell biology and pathogenicity of Candida albicans. Friedrich Schiller University, Jena, Germany
Schild L
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(2011) Proteolytic cleavage of covalently linked cell wall proteins by Candida albicans Sap9 and Sap10. Eukaryot Cell 10: 98-109
Schild L, Heyken A, de Groot PW, Hiller E, Mock M, de Koster C, Horn U, Rupp S, Hube B
