OVEREXPRESSION OF CANDIDA-ALBICANS SECRETORY ASPARTYL PROTEINASE-2 AND ITS EXPRESSION IN SACCHAROMYCES-CEREVISIAE DO NOT AUGMENT VIRULENCE IN MICE

To elucidate the implications of secreted aspartyl proteinase (Sap)2p in the pathogenesis of Candida infections, the SAP2 gene was expressed in Saccharomyces cerevisiae and overexpressed in Candida albicans. Th e coding region of SAP2, including its signal sequence and propeptide, was amplified by PCR and cloned downstream of the S. cerevisiae or C. albicans ADH1 promoter. Plasmid expression of SAP2 in S. cerevisiae s howed that the signal peptide was functional. Integrative transformati on of 5. cerevisiae and C albicans was accomplished by homologous reco mbination within the URA3 locus for S. cerevisiae and the SAP2 locus f or C. albicans. Negative control transformants carried plasmids either without the SAP2 insert or with mutated sap2. 5 cerevisiae and C. alb icans transformants showed similar growth rates to their parental stra ins or negative controls, when grown in medium containing amino acids. However, in medium with BSA as sole nitrogen source, constitutive exp ression of SAP2 enabled S. cerevisiae to grow and increased the growth rate of C. albicans. In both media, only S. cerevisiae transformants harbouring SAP2 secreted the enzyme, as confirmed by proteinase activi ty assays and immunoblotting. When C. albicans was grown in amino acid s medium, the enzyme was detected exclusively in transformants constit utively expressing SAP2. However, in BSA medium these strains secreted enzyme earlier and secreted higher amounts of enzyme and total protei nase activity. In pathogenicity studies in intact mice, expression of Sap2p as a sole putative virulence factor did not cause S. cerevisiae to become virulent and constitutive overexpression of SAP2 did not aug ment virulence of C albicans in experimental oral or systemic infectio n.