Fluconazole exposure induces differential regulation of the Candida glabrata proteome

Abstract

BACKGROUND: Resistance to the azole antifungal fluconazole in C. glabrata is a global problem in compromised patient populations. OBJECTIVES: To evaluate quantitatively the specific protein profiles regulated by C. glabrata in response to acquired fluconazole resistance, and to validate the possible role of these proteins in fungal virulence. METHODS: A genotypically stable “off shoot” of C. glabrata ATCC2001 (CG) highly resistant to fluconazole (Cg) was obtained by sequential drug exposure. On subculture to RPMI/FL agar Cg produced a large number of petite yeast colonies yielding different chromosomal profiles (by CHEF analysis). Distinct differences in proteomic expression between a stable petite isolate (CgL) and the original C. glabrata strain (CG) were identified by mass spectrometry following high resolution two dimensional gel electrophoresis (2DE). Proteins implicated in virulence attributes were spotted and the expression of their mRNA transcript levels were also measured. Moreover we demonstrate the physiological role of TPR and MTI gene expression by conventional methodology. RESULTS: Eight petite colonies yielded different patterns of chromosomal profiles (some with minor and some with major chromosome size polymorphisms). Three small colonies and two of each of the large colonies demonstrated similar chromosomal patterns. Therefore two distinct genotypes were obtained for each of the colony groups. Proteomic analysis revealed distinct and reproducible differences in the protein profiles of one stable revertant and the original C. glabrata strain. Furthermore, the relative expression level of the following genes were affected; i.e., ERG11, MTI, MTIIa, TPR, EF2, MFS and VPS . Apart from the latter three, the mRNA transcript expression levels for the remaining five genes were significantly (p<0.001) upregulated. Subsequent phenotypic studies confirmed the higher expression of TPR and MTI. CONCLUSION: Further proteomic studies should prove an invaluable tool in understanding the biological complexicities associated with fluconazole resistance of Candida glabrata infections.