Evidence that Ergosterol Biosynthesis Modulates Activity of the Pdr1 Transcription Factor in Candida glabrata

Abstract

A crucial limitation in antifungal chemotherapy is the limited number of antifungal drugs currently available. Azole drugs represent the most commonly used chemotherapeutic, and loss of efficacy of these drugs is a major risk factor in successful treatment of a variety of fungal diseases. Candida glabrata is a pathogenic yeast that is increasingly found associated with bloodstream infections, a finding likely contributed to by its proclivity to develop azole drug resistance. C. glabrata often acquires azole resistance via gain-of-function (GOF) mutations in the transcription factor Pdr1. These GOF forms of Pdr1 drive elevated expression of target genes, including the ATP-binding cassette transporter-encoding CDR1 locus. GOF alleles of PDR1 have been extensively studied, but little is known of how Pdr1 is normally regulated. Here we test the idea that reduction of ergosterol biosynthesis (as occurs in the presence of azole drugs) might trigger activation of Pdr1 function. Using two different means of genetically inhibiting ergosterol biosynthesis, we demonstrated that Pdr1 activity and target gene expression are elevated in the absence of azole drug. Blocks at different points in the ergosterol pathway lead to Pdr1 activation as well as to induction of other genes in this pathway. Delivery of the signal from the ergosterol pathway to Pdr1 involves the transcription factor Upc2A, an ERG gene regulator. We show that Upc2A binds directly to the PDR1 and CDR1 promoters. Our studies argue for a physiological link between ergosterol biosynthesis and Pdr1-dependent gene regulation that is not restricted to efflux of azole drugs. IMPORTANCE A likely contributor to the increased incidence of non-albicans candidemias involving Candida glabrata is the ease with which this yeast acquires azole resistance, in large part due to induction of the ATP-binding cassette transporter-encoding gene CDR1. Azole drugs lead to induction of Pdr1 transactivation, with a central model being that this factor binds these drugs directly. Here we provide evidence that Pdr1 is activated without azole drugs by the use of genetic means to inhibit expression of azole drug target-encoding gene ERG11. These acute reductions in Erg11 levels lead to elevated Pdr1 activity even though no drug is present. A key transcriptional regulator of the ERG pathway, Upc2A, is shown to directly bind to the PDR1 and CDR1 promoters. We interpret these data as support for the view that Pdr1 function is responsive to ergosterol biosynthesis and suggest that this connection reveals the normal physiological circuitry in which Pdr1 participates.

Low level of antifungal resistance of Candida glabrata blood isolates in Turkey: Fluconazole minimum inhibitory concentration and FKS mutations can predict therapeutic failure

“…One of the two isolates with the FLZ MIC = 32 µg/mL harboured a unique novel mutation (L281V) (Figure 1, Tables 2 and 3, and Table ). Recent studies indicate that UPC2, a zinc cluster transcription factor, may contribute to FLZ resistance in C glabrata , 51,52 which may explain the high FLZ MIC for isolate G51 (32 µg/mL) without any exclusive amino acid changes in Pdr1p expected for this phenotype (Tables 2 and 3, and Table ). Although AFST did not reveal echinocandin resistance in vitro, previously reported mutations, including S629T 53 (in isolate G29 susceptible to both MCF and AND) and S663P 54 (in isolates G53 and G103 intermediate for susceptibility to MCF and AND), were detected in HS1‐Fks1 and HS1‐Fks2, respectively (Table 3).…”

The Quiet and Underappreciated Rise of Drug-Resistant Invasive Fungal Pathogens

“…In addition, according to a recent study, chemical and/or genetic inhibition of histone acetyltransferase, Cgn5p, is lethal in C. glabrata isolates harboring GOF mutations in PDR1; it showed a significantly reduced frequency of GOF mutations in CGN5-inhibited C. glabrata isolates compared with non-inhibited C. glabrata isolates in an evolutionary model of FLZR [100]. It was recently shown that the mechanism of azole resistance in C. glabrata involves a complicated circuitry of zinc cluster transcription factors other than Pdr1, such as Upc2A (Figure 3) [105,106]. Interestingly, double deletion of Upc2A (upc2AΔ) in both fluconazole-susceptible and FLZR C. glabrata isolates results in a 16-fold decrease of FLZ MIC, and downregulation of CDR1, PDH1, and PDR1 upon induction by FLZ [106].…”

“…It was recently shown that the mechanism of azole resistance in C. glabrata involves a complicated circuitry of zinc cluster transcription factors other than Pdr1, such as Upc2A (Figure 3) [105,106]. Interestingly, double deletion of Upc2A (upc2A∆) in both fluconazole-susceptible and FLZR C. glabrata isolates results in a 16-fold decrease of FLZ MIC, and downregulation of CDR1, PDH1, and PDR1 upon induction by FLZ [106].…”

“…Interestingly, double deletion of Upc2A (upc2AΔ) in both fluconazole-susceptible and FLZR C. glabrata isolates results in a 16-fold decrease of FLZ MIC, and downregulation of CDR1, PDH1, and PDR1 upon induction by FLZ [106]. Indeed, Upc2A directly binds to the CDR1 and PDR1 promoters, leading to the overexpression of these genes and FLZR ( Figure 4) [105]. However, PDR1 and CDR1 upregulation in the upc2AΔ isolate [105] may suggest that FLZR in C. glabrata is more complicated than currently thought, and may involve other zinc-cluster transcription factors.…”

LAM Genes Contribute to Environmental Stress Tolerance but Sensibilize Yeast Cells to Azoles

“…One of the key mechanisms that provide azole tolerance is the upregulation of PDR transporters that extrude xenobiotic compounds from cells (Prasad et al, 2015). An increase in azole resistance can be attributed to the upregulation or activation of these transporter proteins, which is triggered by sterol deficiency (Vu et al, 2019). To test whether azole tolerance in the LAM quadruple mutant is mediated by PDR transporters, we measured the accumulation of PDR substrates and the level of main ABC-transporter in the wild-type strain and in the Δlam1Δlam2Δlam3Δlam4 strain.…”