Date of Award

5-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Research Advisor

P. David Rogers, Pharm.D., Ph.D.

Committee

B. Keith English, M.D. Ramin Homayouni, Ph.D. Bernd Meibohm, Ph.D. Todd Reynolds, Ph.D.

Abstract

Azole antifungal resistance has emerged as a significant problem in the management of infections caused by fungi including Candida species. In recent years, Candida glabrata has become the second most common cause of mucosal and invasive fungal infections in humans second to Candida albicans. Not only are systemic C. glabrata infections characterized by high mortality rates, treatment failures to the azole class of antifungals, the most widely used antifungal for treatment of Candida infections, have been reported. Contributing to this problem, C. glabrata exhibits intrinsic reduced susceptibility to the azole antifungals, and the development of high-level azole resistance during therapy has been reported in oral as well as bloodstream C. glabrata isolates in immunocompromised patients. The azole antifungals are fungistatic against Candida species, thus C. glabrata also exhibits tolerance to the azoles which may contribute to both therapeutic failures and ultimately the development of high-level azole resistance.

In C. glabrata clinical isolates, the predominant mechanism behind azole resistance is upregulated expression of multidrug transporter genes CgCDR1 and CgPDH1. It was previously reported that azole-resistant mutants (MIC ≥ 64 μg/ml) of strain 66032 (MIC = 16 μg/ml) similarly show coordinate CDR1-PDH1 upregulation, and in one of these (F15) a putative gain-of-function mutation was identified in the single molecule homologue of Saccharomyces cerevisiae transcription factors Pdr1p–Pdr3p. Here we show that disruption of C. glabrata PDR1 conferred equivalent fluconazole hypersensitivity (MIC = 2 μg/ml) to both F15 and 66032 and eliminated both constitutive and fluconazole-induced CDR1-PDH1 expression. Reintroduction of wild-type or F15 PDR1 alleles fully reversed these effects; together these results demonstrate a role for this gene in both acquired and intrinsic azole resistance. CDR1 disruption had a partial effect, reducing fluconazole trailing in both strains while restoring wild-type susceptibility (MIC = 16 μg/ml) to F15. In an azole-resistant clinical isolate, PDR1 disruption reduced azole MICs eight- to 64-fold with no effect on sensitivity to other antifungals. To extend this analysis, C. glabrata gene expression microarrays were generated and used to analyze genome-wide expression in F15 relative to its parent. Homologues of 10 S. cerevisiae genes previously shown to be Pdr1p–Pdr3p targets were upregulated (YOR1, RTA1, RSB1, RPN4, YLR346c and YMR102c along with CDR1, PDH1 and PDR1 itself) or downregulated (PDR12); roles for these genes include small molecule transport and transcriptional regulation. However, expression of 99 additional genes was specifically altered in C. glabrata F15; their roles include transport (e.g. QDR2, YBT1), lipid metabolism (ATF2, ARE1), cell stress (HSP12, CTA1), DNA repair (YIM1, MEC3) and cell wall function (MKC7, MNT3). These azole resistance-associated changes could affect C. glabrata tissue-specific virulence; in support of this, we detected differences in F15 oxidant, alcohol and weak acid sensitivities. C. glabrata provides a promising model for studying the genetic basis of multidrug resistance and its impact on virulence.

We next examined the genome-wide gene expression profiles in four matched azole-susceptible and –resistant clinical isolate sets of C. glabrata in which CgCDR1 gene expression was upregulated in the resistant isolate. Of all the genes identified in the gene expression profiles for these four matched pairs, there were nine genes that were commonly upregulated with CgCDR1 in all four isolate sets (YOR1, LCB5, RTA1, YIM1, YIL077c, POG1, HFD1, GLK1, and FMS1). We then sequenced CgPDR1 from each susceptible and resistant isolate and found two alleles with novel gain-of-function mutations. A third isolate, and its susceptible parent, harbored a CgPDR1 allele with a frameshift mutation which presumably results in a truncated CgPdr1p. The final resistant isolate had noPDR1 mutation. CgPDR1 alleles with putative gain-of-function mutations were expressed in a common background strain in which CgPDR1 had been disrupted, and genome-wide gene expression profiles were examined to determine if different mutations inCgPDR1 result in different target gene activation and fluconazole MICs. Microarray analysis comparing these re-engineered strains to their respective parent strains identified a core set of commonly differentially-expressed genes as well as genes uniquely regulated by specific mutations.

DOI

10.21007/etd.cghs.2010.0045

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