Fluconazole induces phenotypic, genotypic and proteomic changes in C. glabrata

Abstract

Candida glabrata is a major pathogen in compromised patient populations due to its increased resistance to fluconazole (FL). Although the increase in fluconazole resistance is associated with changes in its genotype no parallel phenotypic or proteomic changes have been studied. OBJECTIVES: To observe specific phenotypic, chromosomal and proteomic alterations in a C. glabrata strain sequentially exposed to fluconazole. METHODS: C. glabrata was subjected to serial exposure of minimum concentrations (×MICs) of fluconazole in RPMI medium for a period of 55 days. Phenotypic changes (i.e., resistance to azole drugs, adhesion to BECs, phospholipase, haemolysin production, phenotypic switching) were evaluated at different time points using standard assays. The development of molecular/ proteomic changes in C. glabrata were analyzed by contour-clamped-homogeneous electrophoretic field (CHEF) electrophoresis, RT-PCR, 2-DE gel based methodology and mass spectrometric peptide finger print analysis of SDS-PAGE yeast separated protein fractions. RESULTS: C. glabrata demonstrated increased FL resistance (8->256µg/ml) accompanied with extensive cross-resistance to ketoconazole (0.38-3.0µg), itraconazole (8->32µg) and voriconazole (0.125-1.5µg). Variable colony morphology (size), a decrease in viable yeast counts, and inconsistent phenotypic switching on CuSO4 agar was observed. Further, seven randomly selected morphologically dissimilar yeast colonies also demonstrated pronounced differences in their chromosomal profiles compared with the control isolate. Stable changes in the chromosomal profiles of two yeast colonies were associated with significantly (p<0.05) higher transcript levels of the haemolysin gene relative to the control. Proteome analysis of one stable colony resulted in a total of 98 spots with 25 upregulated and 24 downregulated proteins. CONCLUSION: These findings show that sequential fluconazole exposure initiates the chance evolution of a new colonizing population with specific virulence traits. (Supported by the Research Grants Council, University of Hong Kong, SAR. We are grateful for the Pfizer Laboratories for supplying Fluconazole for this study).