Functional Genomic Analysis of Fluconazole Susceptibility in the Pathogenic Yeast Candida glabrata : Roles of Calcium Signaling and Mitochondria

Abstract

The pathogenic yeast Candida glabrata exhibits innate resistance to fluconazole, the most commonly used antifungal agent. By screening a library of 9,216 random insertion mutants, we identified a set of 27 genes which upon mutation, confer altered fluconazole susceptibility in C. glabrata. Homologues of three of these genes have been implicated in azole and/or drug resistance in Saccharomyces cerevisiae: two of these belong to the family of ABC transporters (PDR5 and PDR16), and one is involved in retrograde signaling from mitochondria to nucleus (RTG2). The remaining 24 genes are involved in diverse cellular functions, including ribosomal biogenesis and mitochondrial function, activation of RNA polymerase II transcription, nuclear ubiquitin ligase function, cell wall biosynthesis, and calcium homeostasis. We characterized two sets of mutants in more detail. Strains defective in a putative plasma membrane calcium channel (Cch1-Mid1) were modestly more susceptible to fluconazole but showed a significant loss of viability upon prolonged fluconazole exposure, suggesting that calcium signaling is required for survival of azole stress in C. glabrata. These mutants were defective in calcium uptake in response to fluconazole exposure. The combined results suggest that, in the absence of Ca 2؉ signaling, fluconazole has a fungicidal rather than a fungistatic effect on C. glabrata. The second set of mutants characterized in detail were defective in mitochondrial assembly and organization, and these exhibited very high levels of fluconazole resistance. Further analysis of these mutants indicated that in C. glabrata a mechanism exists for reversible loss of mitochondrial function that does not involve loss of mitochondrial genome and that C. glabrata can switch between states of mitochondrial competence and incompetence in response to fluconazole exposure.

New Regulators of a High Affinity Ca2+ Influx System Revealed through a Genome-wide Screen in Yeast

“…An ecm7 mutant of yeast was one of many mutants reported with general cell wall defects (39). Additionally, an ecm7 mutant of the pathogenic yeast C. glabrata was one of several reported with hypersensitivity to the azole-class antifungal agent fluconazole (9). The Ecm7 proteins and their roles in Ca 2ϩ uptake have not been previously studied.…”

Mechanisms of azole resistance among clinical isolates of Candida glabrata in Poland

“…Interestingly, in contrast to C. albicans, most of the investigations carried out in different regions of the world have revealed that point mutations within the ERG11 gene of azole-resistant C. glabrata isolates are rarely identified or are not present at all (Berila & Subik 2010;Berila et al, 2009;Sanguinetti et al, 2005). There also exist many other possible, not fully recognized mechanisms affecting C. glabrata resistance to azoles, such as mitochondrial dysfunction (Ferrari et al, 2011) and calcium signalling (Kaur et al, 2004). However, they do not seem to be as important as overexpression of drug efflux proteins or enzymes involved in ergosterol synthesis (Rodrigues et al, 2014).…”

Endosomal Trafficking Defects Can Induce Calcium-Dependent Azole Tolerance in Candida albicans

“…The means Ϯ standard deviations of data from two biological replicates are shown for panels C and D. The means for each group were compared by using a two-tailed t test. *, P Ͻ 0.05. cellular Ca 2ϩ (29). Therefore, to determine if the vacuolar trafficking defects of the vps21⌬/⌬ mutant impact cellular Ca 2ϩ homeostasis, we compared the levels of intracellular free calcium using the calcium-responsive dye Fura 2-AM (30).…”