Date of Award

12-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbial Pathogenesis, Immunology, and Inflammation

Research Advisor

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

Committee

James B. Dale, M.D. Ramin Homayouni, Ph.D. Richard E. Lee, Ph.D. Nathan Wiederhold, Pharm.D

Keywords

Antifungal, azole, Candida albicans, Rrsistance, transcriptional regulation

Abstract

Candida albicans is the most prevalent human fungal pathogen, found as a commensal organism in the mucosa, gastrointestinal, and urogenital tracts of humans. This pathogenic fungus causes a wide spectrum of diseases, including the mucosal infection oropharyngeal candidiasis (OPC) which frequently effects patients with human immunodeficiency virus (HIV). The azole antifungals (such as fluconazole) are the most widely used and important ergosterol biosynthesis inhibitors (EBIs) for the treatment of Candida infections, including OPC. However, the azoles are fungistatic against C. albicans and therefore have limited efficacy against this organism, especially for immunocompromised patients. In C. albicans, the transcription factor Upc2 is central to the regulation of ergosterol biosynthesis. UPC2 activating mutations contribute to azole resistance, whereas disruption increases azole susceptibility. We further investigated the relationship of UPC2 to fluconazole susceptibility, particularly in azole-resistant strains. In addition to the reduced fluconazole minimum inhibitory concentration (MIC) previously observed with UPC2 disruption, we observed a reduced minimum fungicidal concentration (MFC) in a upc2Δ/Δ mutant relative to its azole-susceptible parent SC5314. Moreover, upc2Δ/Δ was unable to grow on solid media containing 10 µg/mL fluconazole and exhibited increased susceptibility and a clear zone of inhibition when subjected to Etest. Time-kill analysis showed increased azole activity against upc2Δ/Δ compared to SC5314. UPC2 disruption in strains carrying specific resistance mutations also resulted in reduced MICs and MFCs. UPC2 disruption in a highly azole-resistant clinical isolate containing multiple resistance mechanisms likewise resulted in a reduced MIC and MFC. This mutant was unable to grow on solid media containing 10 µg/mL fluconazole and exhibited increased susceptibility and a clear zone of inhibition when subjected to Etest. Time-kill analysis showed increased azole activity against the upc2Δ/Δ mutant in the resistant background. Microarray analysis showed attenuated fluconazole induction of genes involved in sterol biosynthesis as well as iron transport and homeostasis in the absence of UPC2. Taken together, our results demonstrate that the UPC2 transcriptional network is universally essential for azole resistance in C. albicans and represents an attractive target for enhancing azole antifungal activity. Fungal survival in the presence of the azoles is permitted by specific signal transduction and transcriptional activation programs. In an effort to identify additional transcriptional pathways involved in fluconazole susceptibility, we sought to identify transcription factors (TFs) essential for this process. From a collection of C. albicans strains disrupted for genes encoding TFs (Homann et al., PLoS Genet. 2009;5:e1000783), 4 exhibited marked reduction in MFC in both RPMI and YPD media independent of any noted growth defect in medium alone as compared to the parent strain. In addition to UPC2, one gene of interest (GOI) (CAS5) was unable to recover from fluconazole exposure at concentrations as low as 2 µg/mL after 72 hours in YPD medium, showed reduced susceptibility and a clear zone of inhibition by Etest, was unable to grow on solid media containing 10 µg/mL fluconazole, and exhibited increased susceptibility by time-kill analysis. CAS5 disruption in highly azole resistant clinical isolates containing multiple resistance mechanisms did not alter susceptibility. However, CAS5 disruption in strains containing specific resistance mutations resulted in a moderate reduction in MIC and MFC. Genome-wide transcriptional analysis was performed in the presence of fluconazole and was consistent with the suggested role of CAS5 in cell wall organization while also suggesting a role in iron transport and homeostasis. These findings suggest that Cas5 regulates a transcriptional network that influences susceptibility of C. albicans to fluconazole. Further delineation of this transcriptional network may identify targets for potential cotherapeutic strategies to enhance the activity to the azole class of antifungals. Genome-wide expression profiling identified that ergosterol biosynthesis genes depend on Upc2 for their fluconazole-induceability, and cell wall maintenance genes depend on Cas5. In the presence of fluconazole, the expression of iron ion transport and homeostasis genes were also identified to be dependent on Upc2 as well as Cas5. The identification of these target genes validated the known and putative roles for Upc2 and Cas5, and also provided insight into novel roles for these transcription factors in azole susceptibility.

DOI

10.21007/etd.cghs.2013.0335

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