THE EXPRESSION OF THE SECRETED ASPARTYL PROTEINASES SAP4 TO SAP6 FROMCANDIDA-ALBICANS IN MURINE MACROPHAGES

Medically important yeasts of the genus Candida secrete aspartyl prote inases (Sap), which are of particular interest as virulence factors. S ix closely related gene sequences, SAP1 to SAP6, for secreted proteina ses are present in Candida albicans. The methylotrophic yeast Pichia p astoris was chosen as an expression system for preparing substantial a mounts of each Sap isoenzyme. Interestingly, Sap4, Sap5 and Sap6, whic h have not yet been detected in C. albicans cultures in vitro, were pr oduced as active recombinant enzymes. Different Sap polyclonal antibod ies were raised in rabbits and tested before further application by en zyme-linked immunosorbent assay (ELISA) against each recombinant Sap. Two antisera recognized only Sap4 to Sap6. Using these antisera, toget her with sap null mutants obtained by targeted mutagenesis, we could d emonstrate a high production of Sap4, Sap5 and Sap6 by C. albicans cel ls after phagocytosis by murine peritoneal macrophages. Furthermore, a Delta sap4,5,6 null mutant was killed 53% more effectively after cont act with macrophages than the wild-type strain. These results support a role for Sap4 to Sap6 in pathogenicity.