The secreted aspartyl proteinases Sap1 and Sap2 cause tissue damage in an in vitro model of vaginal candidiasis based on reconstituted human vaginal epithelium.

Secreted aspartyl proteinases (Saps) contribute to the ability of Candida albicans to cause mucosal and disseminated infections. A model of vaginal candidiasis based on reconstituted human vaginal epithelium (RHVE) was used to study the expression and role of these C. albicans proteinases during infection and tissue damage of vaginal epithelium. Colonization of the RHVE by C. albicans SC5314 did not cause any visible epithelial damage 6 h after inoculation, although expression of SAP2, SAP9, and SAP10 was detected by reverse transcriptase PCR. However, significant epithelial damage was observed after 12 h, concomitant with the additional expression of SAP1, SAP4, and SAP5. Additional transcripts of SAP6 and SAP7 were detected at a later stage of the artificial infection (24 h). Similar SAP expression profiles were observed in three samples isolated from human patients with vaginal candidiasis. In experimental infection, secretion of antigens Sap1 to Sap6 by C. albicans was confirmed at the ultrastructural level by using polyclonal antisera raised against Sap1 to Sap6. Addition of the aspartyl proteinase inhibitors pepstatin A and the human immunodeficiency virus proteinase inhibitors ritonavir and amprenavir strongly reduced the tissue damage of the vaginal epithelia by C. albicans cells. Furthermore, SAP null mutants lacking either SAP1 or SAP2 had a drastically reduced potential to cause tissue damage even though SAP3, SAP4, and SAP7 were up-regulated in these mutants. In contrast the vaginopathic potential of mutants lacking SAP3 or SAP4 to SAP6 was not reduced compared to wild-type cells. These data provide further evidence for a crucial role of Sap1 and Sap2 in C. albicans vaginal infections.

Candida albicans hyphal formation and the expression of the Efg1-regulated proteinases Sap4 to Sap6 are required for the invasion of parenchymal organs.

The ability to change between yeast and hyphal cells (dimorphism) is known to be a virulence property of the human pathogen Candida albicans. The pathogenesis of disseminated candidosis involves adhesion and penetration of hyphal cells from a colonized mucosal site to internal organs. Parenchymal organs, such as the liver and pancreas, are invaded by C. albicans wild-type hyphal cells between 4 and 24 h after intraperitoneal (i.p.) infection of mice. In contrast, a hypha-deficient mutant lacking the transcription factor Efg1 was not able to invade or damage these organs. To investigate whether this was due to the inability to undergo the dimorphic transition or due to the lack of hypha-associated factors, we investigated the role of secreted aspartic proteinases during tissue invasion and their association with the different morphologies of C. albicans. Wild-type cells expressed a distinct pattern of SAP genes during i.p. infections. Within the first 72 h after infection, SAP1, SAP2, SAP4, SAP5, SAP6, and SAP9 were the most commonly expressed proteinase genes. Sap1 to Sap3 antigens were found on yeast and hyphal cells, while Sap4 to Sap6 antigens were predominantly found on hyphal cells in close contact with host cells, in particular, eosinophilic leukocytes. Mutants lacking EFG1 had either noticeably reduced or higher expressed levels of SAP4 to SAP6 transcripts in vitro depending on the culture conditions. During infection, efg1 mutants had a strongly reduced ability to produce hyphae, which was associated with reduced levels of SAP4 to SAP6 transcripts. Mutants lacking SAP1 to SAP3 had invasive properties indistinguishable from those of wild-type cells. In contrast, a triple mutant lacking SAP4 to SAP6 showed strongly reduced invasiveness but still produced hyphal cells. When the tissue damage of liver and pancreas caused by single sap4, sap5, and sap6 and double sap4 and -6, sap5 and -6, and sap4 and -5 double mutants was compared to the damage caused by wild-type cells, all mutants which lacked functional SAP6 showed significantly reduced tissue damage. These data demonstrate that strains which produce hyphal cells but lack hypha-associated proteinases, particularly that encoded by SAP6, are less invasive. In addition, it can be concluded that the reduced virulence of hypha-deficient mutants is not only due to the inability to form hyphae but also due to modified expression of the SAP genes normally associated with the hyphal morphology.