Secreted aspartic proteinase (Sap) activity contributes to tissue damage in a model of human oral candidosis

Secreted aspartic proteinases (Saps) are important virulence factors during Candida albicans mucosal or disseminated infections. A differential expres sion of individual SAP genes has been shown previously in a model of oral c andidosis based on reconstituted human epithelium (RHE), and in the oral ca vity of patients. In this study, the ultrastructural localization of distin ct groups of Sap isoenzymes expressed during RHE infection was shown by imm unoelectron microscopy using specific polyclonal antibodies directed agains t the gene products of SAP1-3 and SAP4-6, Large amounts of Sap1-3 antigen w ere found within C. albicans yeast and hyphal cell walls, often predominant ly in close contact with epithelial cells, whereas lower quantities of Sap4 -6 were detected in hyphal cells. To elucidate the relevance of the express ed Saps during oral infections, we examined the effect of the aspartic prot einase inhibitor, pepstatin A, during infection of the RHE. The extent of l esions caused by the strain SC5314 was found to be strongly reduced by the inhibitor, indicating that proteinase activity contributes to tissue damage in this model. To clarify which of the SAP genes are important for tissue necrosis, the histology of RHE infection with Delta sap1, Delta sap2 Delta sap3 Delta sap4-6 and three Delta sap1/3 double mutants were examined. Alth ough tissue damage was not blocked completely with these mutants, an attenu ated phenotype was observed for each of the single sap null mutants, and wa s more strongly attenuated in the Delta sap1/3 double null mutants. In cont rast, the lesions caused by the Delta sap4-6 triple mutant were at least as severe as those caused by SC5314, During infection with the mutants, we ob served that the SAP gene expression pattern of the Delta sap1 and the Delta sap1/3 mutants was altered in comparison with the wildtype strain. Express ion of SAP5 was observed only during infection with the Delta sap1/3 mutant , whereas upregulation of SAP2 and SAP8 transcripts was observed in the Del ta sap1 and the Delta sap1/3 mutants. These results suggest that Sap1-3, bu t not Sap4-8, contribute to tissue damage in this model. Furthermore, C. al bicans may compensate for the deletion of certain SAP genes by upregulation of alternative SAP genes.