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Overexpression of Candida albicans secretory aspartyl proteinase 2 and its expression in Saccharomyces cerevisiae do not augment virulence in mice.
- Source :
-
Microbiology (Reading, England) [Microbiology (Reading)] 1998 Aug; Vol. 144 ( Pt 8), pp. 2299-2310. - Publication Year :
- 1998
-
Abstract
- 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. The 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 showed that the signal peptide was functional. Integrative transformation of S. cerevisiae and C. albicans was accomplished by homologous recombination within the URA3 locus for S. cerevisiae and the SAP2 locus for C. albicans. Negative control transformants carried plasmids either without the SAP2 insert or with mutated sap2. S. cerevisiae and C. albicans transformants showed similar growth rates to their parental strains or negative controls, when grown in medium containing amino acids. However, in medium with BSA as sole nitrogen source, constitutive expression 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 activity assays and immunoblotting. When C. albicans was grown in amino acids medium, the enzyme was detected exclusively in transformants constitutively expressing SAP2. However, in BSA medium these strains secreted enzyme earlier and secreted higher amounts of enzyme and total proteinase 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 augment virulence of C. albicans in experimental oral or systemic infection.
- Subjects :
- Animals
Candida albicans genetics
Candidiasis microbiology
Drug Resistance, Microbial
Female
Hydrolases metabolism
Kinetics
Mice
Mice, Inbred ICR
Plasmids genetics
Plasmids metabolism
Saccharomyces cerevisiae physiology
Sodium Chloride metabolism
Sodium Chloride pharmacology
Transformation, Genetic
Virulence
Aspartic Acid Endopeptidases biosynthesis
Aspartic Acid Endopeptidases metabolism
Candida albicans enzymology
Candida albicans pathogenicity
Fungal Proteins
Saccharomyces cerevisiae enzymology
Saccharomyces cerevisiae genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1350-0872
- Volume :
- 144 ( Pt 8)
- Database :
- MEDLINE
- Journal :
- Microbiology (Reading, England)
- Publication Type :
- Academic Journal
- Accession number :
- 9720053
- Full Text :
- https://doi.org/10.1099/00221287-144-8-2299