Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis

Abstract

Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a ⌬sap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the ⌬sap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and ⌬sap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis. Candida albicans is a commensal fungus that is often part of the oral microflora of healthy people. Loss of host immunity, HIV infection, corticosteroid use, or alteration of the oral microflora following antibiotic therapies permits a pathogenic transition of C. albicans to cause oropharyngeal candidiasis (OPC) (1, 2). Acute pseudomembranous candidiasis is one of the most common forms of OPC, in which C. albicans forms white patches on the surface of the buccal mucosa, tongue, or soft palate. These superficial fungal plaques can be lifted from underlying tissues for purposes of clinical diagnosis and analysis (3).C. albicans expresses specific sets of virulence factors that promote hypha formation and adhesion and invasion of host tissues (4). Secreted aspartyl proteinases (Saps) are recognized virulence factors because they degrade host proteins to provide nitrogen for fungal cell metabolism, contribute to adherence, facilitate fungal epithelial and endothelial penetration, and are immunogenic during infection (5-7). Microbial proteinases are classified as serine, cysteine, metallo-, or aspartyl proteinases according to the site of catalytic hydrolysis of substrate peptide bonds; however, C. albicans produces only aspartyl proteinases (5, 6).C. albicans expresses a family of 10 SAP genes that are clustered into groups SAP1 to SAP3, SAP4 to SAP6, SAP7, SAP8, and SAP9 and SAP10 based upon their sequence homologies and pH activities (8, 9). Sap1 through Sap8 are processed and transported via the secreto...

Extracellular proteinases of Candida species pathogenic yeasts

“…Sap4‐Sap6 contain amino acid motifs Arg‐Gly‐Asp/Lys‐Gly‐Asp that mediate binding to integrins on epithelial cells and internalization into endosomes and lysosomes . This integrin‐binding motif (Arg‐Gly‐Asp) has also been suggested to be critical for a novel function that is unique to Sap6—cell‐cell aggregation in germinated C. albicans forms that promotes infection and virulence in oral candidiasis . Moreover, Sap6 was recently reported to contain four amyloidogenic regions that promote Sap6‐dependent C. albicans autoaggregation .…”

Candida albicans Sap6 amyloid regions function in cellular aggregation and zinc binding, and contribute to zinc acquisition

“…dubliniensisHIS1 / Δpra1:::C . maltosaLEU2 This Study SAP6 OE Δura3::imm434 Δura3::imm434 RPS1 Δrps1::CIp10-SAP6-URA3 3 Δals1 / Δals3 als1::hisG als1::hisG als3::dpl::200 als3::dpl200 56 Δint1 int1Δ::hisG / int1Δ::hisG-URA3-hisG 57 CJN1193 (ZAP1 complement) ∆ura3::imm434 arg4::hisG his1::hisG::pHIS1-ZAP1 zap1::ARG4 ∆ura3::imm434 arg4::hisG his1::hisG zap1::URA3 37 CJN1623(PRA1O/E) ∆ura3::imm434 arg4::hisG his1::hisG::pHIS1 zap1::ARG4 PRA1::pAgTEF1-NAT1-AgTEF1UTR-TDH3-PRA1 ∆ura3::imm434 arg4::hisG his1::hisG zap1::URA3 PRA1 37 CJN1651(ZRT1O/E) ∆ura3::imm434 arg4::hisG his1::hisG::pHIS1 zap1::ARG4 ZRT1::pAgTEF1-NAT1-AgTEF1UTR-TDH3-ZRT1 ∆ura3::mm434 arg4::hisG his1::hisG zap1::URA3 ZRT1 37 JFY348(ZAP1O/E) ura3∆::•imm434 ARG4:URA3:arg4::hisG his1::hisG::pHIS1 ZAP1:pAgTEF1-NAT1-AgTEF1UTR-TDH3-ZAP1 ura3∆::imm434arg4::his1 his1::hisG ZAP1 16 SF1280 (ECM331 complement) SC5314 – ECM331::pAgTEF1-NAT1-AgTEF1UTR-TDH3-ECM331 58 Δals5 See reference 45 Δals9 See reference 45 Δcsa1 See reference 45 Δcsa2 See reference 45 Δece1 See reference 45 Δecm331 See reference 58 Δhwp1 See reference 59 Δhwp2 See reference 59 Δhyr1 See reference 45 Δhyr3 See reference 45 Δiff4 See reference 45 Δpga10 See reference 45 Δpga13 See reference 45 Δpga17 See reference 60 Δpga18 See reference 45…”

Experimental Models of Vaginal Candidiasis and Their Relevance to Human Candidiasis

“…Data reported by Naglik's research group, using a similar mouse vaginal infection model, also highlighted the role of some Sap enzymes in contributing to infection, which is in keeping with SAP gene expression in infected women (33,34). Overall, among the many putative virulence factors of C. albicans, including the transition from dimorphic yeast to hypha, some adhesins (in particular, those of the agglutinin-like sequence [ALS] family) (6,35), and biofilm formation, Sap enzymes have been those most intensely studied and advocated for as having a role in vaginal candidiasis and perhaps also in some forms of oral infection (36).…”