Date of Award

12-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbiology, Immunology, and Biochemistry

Research Advisor

Glen E. Palmer, Ph.D.

Committee

Ben Kelly, Ph.D. David R. Nelson, Ph.D. Brian M. Peters, Ph.D. P. David Rogers, Pharm.D., Ph.D.

Abstract

Several important antifungal drugs inhibit the synthesis of ergosterol, a lipid that modulates the thickness, fluidity and permeability of fungal cell membranes. These include the azole antifungals, which block ergosterol biosynthesis by inhibiting lanosterol demethylase (Erg11p). The resulting depletion of cellular ergosterol and the accumulation of ‘toxic’ sterol intermediates are both thought to cause plasma membrane dysfunction and ultimately growth arrest. However, the effects of ergosterol depletion upon the function of intracellular membranes and organelles are not well described. The purpose of this study was to characterize the effects of azole treatment upon the integrity of the Candida albicans vacuole, and determine if, in turn, vacuolar trafficking influences azole susceptibility. Profound fragmentation of the C. albicans vacuole was observed as an early consequence of azole treatment, before significant growth inhibition was observed. Using a genetic approach, we determined that vacuole fragmentation was a consequence of Erg11p inhibition rather than an off-target effect of azole treatment. Moreover, the degree of vacuole fragmentation following azole treatment was influenced by Erg3p, an enzyme in the ergosterol biosynthetic pathway involved in the production of toxic sterol intermediaries upon Erg11p inhibition. We also determined that vacuolar trafficking significantly impacts C. albicans susceptibility to azole antifungals and other ergosterol biosynthesis inhibitors. For instance, a vps21∆/∆ mutant, blocked in membrane trafficking through the pre-vacuolar compartment (PVC), grew significantly more than wild-type controls in the presence of several azole antifungals under standard susceptibility testing conditions. Furthermore, the vps21∆/∆ mutant was able to grow in the presence of the azoles despite depletion of cellular ergosterol. This phenotype resembles an exaggerated form of azole tolerance known as ‘trailing growth’, which has been described for some clinical isolates. In contrast, the vps21∆/∆ mutant is hypersensitive to drugs that block alternate steps in ergosterol biosynthesis. The azole tolerance phenotype of the C. albicans vps21Δ/Δ mutant was independent of known azole resistance mechanisms such as the efflux pumps Cdr1p and Mdr1p. Moreover, the azole tolerance of the vps21Δ/Δ mutant was influenced by both pH and incubation temperature, consistent with trailing growth phenotypes. The C. albicans vps21Δ/Δ mutant exhibits less plasma membrane permeabilization upon azole treatment, as determined by the release of a cytoplasmic luciferase reporter into the culture supernatant. Our results also reveal that the vps21Δ/Δ mutant has elevated levels of intracellular calcium and enhanced calcineurin activity, as evidenced by increased expression of a calcineurin responsive RTA2-GFP reporter construct in response to fluconazole. Furthermore, the azole tolerant phenotype of the vps21Δ/Δ mutant is dependent upon both calcium and calcineurin signaling. These findings underscore the importance of endosomal trafficking in determining the cellular consequences of azole treatment through modulation of intracellular calcium levels and calcineurin dependent responses. While we determined that deletion of VPS21 alone was not sufficient to confer a survival advantage upon C. albicans following azole treatment in the mouse model of vaginal candidiasis, the azole susceptibility of the vps21Δ/Δ mutant in the mouse model disseminated infection is yet to be tested. Moreover, it is unclear how, or if the azole tolerant phenotype of the vps21Δ/Δ mutant relates to that of trailing clinical isolates, or if these clinical isolates have abnormal endosomal trafficking.

ORCID

http://orcid.org/0000-0002-3129-4115

DOI

10.21007/etd.cghs.2016.0417

Comments

One year embargo expires October 2017.

Available for download on Monday, October 29, 2018

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