John-Paul Vermitsky

Azole Resistance in Candida glabrata: Coordinate Upregulation of Multidrug Transporters and Evidence for a Pdr1-Like Transcription Factor

ABSTRACT Candida glabrata has emerged as a common cause of fungal infection. This yeast has intrinsically low susceptibility to azole antifungals such as fluconazole, and mutation to frank azole resistance during treatment has been documented. Potential resistance mechanisms include changes in expression or sequence of ERG11 encoding the azole target. Alternatively, resistance could result from upregulated expression of multidrug transporter genes; in C. glabrata these include CDR1 and PDH1. By RNA hybridization, 10 of 12 azole-resistant clinical isolates showed 6- to 15-fold upregulation of CDR1 compared to susceptible strains. In 4 of these 10 isolates PDH1 was similarly upregulated, and in the remainder it was upregulated three- to fivefold, while ERG11 expression was minimally changed. Laboratory mutants were selected on fluconazole-containing medium with glycerol as carbon source (to eliminate mitochondrial mutants). Similar to the clinical isolates, six of seven laboratory mutants showed unchanged ERG11 expression but coordinate CDR1-PDH1 upregulation ranging from 2- to 20-fold. Effects of antifungal treatment on gene expression in susceptible C. glabrata strains were also studied: azole exposure induced CDR1-PDH1 expression 4- to 12-fold. These findings suggest that these transporter genes are regulated by a common mechanism. In support of this, a mutation associated with laboratory resistance was identified in the C. glabrata homolog of PDR1 which encodes a regulator of multidrug transporter genes in Saccharomyces cerevisiae. The mutation falls within a putative activation domain and was associated with PDR1 autoupregulation. Additional regulatory factors remain to be identified, as indicated by the lack of PDR1 mutation in a clinical isolate with coordinately upregulated CDR1-PDH1.

Pdr1 regulates multidrug resistance in Candida glabrata: gene disruption and genome‐wide expression studies

Candida glabrata emerged in the last decade as a common cause of mucosal and invasive fungal infection, in large part due to its intrinsic or acquired resistance to azole antifungals such as fluconazole. In C. glabrata clinical isolates, the predominant mechanism behind azole resistance is upregulated expression of multidrug transporter genes CDR1 and PDH1. We previously reported that azole‐resistant mutants (MIC ≥ 64 μg ml−1) of strain 66032 (MIC = 16 μg ml−1) similarly show coordinate CDR1‐PDH1 upregulation, and in one of these (F15) a putative gain‐of‐function mutation was identified in the single homologue of Saccharomyces cerevisiae transcription factors Pdr1–Pdr3. Here we show that disruption of C. glabrata PDR1 conferred equivalent fluconazole hypersensitivity (MIC = 2 μg ml−1) to both F15 and 66032 and eliminated both constitutive and fluconazole‐induced CDR1‐PDH1 expression. Reintroduction of wild‐type or F15 PDR1 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−1) 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 microarrays were generated and used to analyse genome‐wide expression in F15 relative to its parent. Homologues of 10 S. cerevisiae genes previously shown to be Pdr1–Pdr3 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.

Survey of vaginal-flora Candida species isolates from women of different age groups by use of species-specific PCR detection.

ABSTRACT A retrospective survey of 93,775 samples testing positive in Candida species-specific PCR tests performed on cervicovaginal swabs over a 4-year period demonstrated consistent yearly distributions of Candida albicans (89%), C. glabrata (7.9%), C. parapsilosis (1.7%), and C. tropicalis (1.4%). However, the species distributions among different age groups revealed increases in the percentages of non-albicans species with increases in age.

Promoter-dependent disruption of genes: simple, rapid, and specific PCR-based method with application to three different yeast

PCR product-based gene disruption has greatly accelerated molecular analysis of Saccharomyces cerevisiae. This approach involves amplification of a marker gene (e.g., URA3) including its flanking regulatory (promoter and polyadenylation) regions using primers that include at their 5′ ends about 50 bases of homology to the targeted gene. Unfortunately, this approach has proved less useful in organisms with higher rates of non-homologous recombination; e.g., in the yeast Candida glabrata, desired recombinants represent ≤2% of transformants. We modified the PCR-based approach by eliminating marker-flanking regions and precisely targeting recombination such that marker expression depends on the regulatory sequences of the disrupted gene. Application of this promoter-dependent disruption of genes (PRODIGE) method to three C. glabrata genes (SLT2, LEM3, and PDR1) yielded desired recombinants at frequencies of 20, 31, and 11%, the latter representing a weakly expressed gene. For Candida albicansLEM3 and RHO1, specificity was 79–95% for one or both alleles, >sixfold higher than the published results with conventional PCR-based gene disruption. All 5 C. glabrata and C. albicans mutants had predicted phenotypes of calcofluor hypersensitivity (slt2Δ and RHO1/rho1Δ), cycloheximide hypersensitivity (pdr1Δ), or miltefosine resistance (lem3Δ and lem3Δ/lem3Δ). PRODIGE application to the S. cerevisiaePDR5 gene in strains with and without the Pdr1–Pdr3 transcriptional activators of this gene confirmed that transformant yield and growth rate depend on promoter strength. Using this PDR5 promoter-URA3 recombinant, we further demonstrate a simple extension of the method that yields regulatory mutants via 5-fluoroorotic acid selection. PRODIGE warrants testing in other yeast, molds, and beyond.

Antifungal resistance of Candida glabrata vaginal isolates and development of a quantitative reverse transcription-PCR-based azole susceptibility assay.

ABSTRACT A multiplex quantitative reverse transcription-PCR assay was developed to detect azole resistance in Candida glabrata, an important opportunistic pathogen that develops resistance rapidly. Resistance was defined as a ≥3-fold increase in CDR1 expression by this assay, which proved to be 100% sensitive and 95% specific in comparison to the gold standard broth microdilution assay.

UPC2A Is Required for High-Level Azole Antifungal Resistance in Candida glabrata

ABSTRACT Candida glabrata, the second most common cause of Candida infections, is associated with high rates of mortality and often exhibits resistance to the azole class of antifungal agents. Upc2 and Ecm22 in Saccharomyces cerevisiae and Upc2 in Candida albicans are the transcriptional regulators of ERG11, the gene encoding the target of azoles in the ergosterol biosynthesis pathway. Recently two homologs for these transcription factors, UPC2A and UPC2B, were identified in C. glabrata. One of these, UPC2A, was shown to influence azole susceptibility. We hypothesized that due to the global role for Upc2 in sterol biosynthesis in S. cerevisiae and C. albicans, disruption of UPC2A would enhance the activity of fluconazole in both azole-susceptible dose-dependent (SDD) and -resistant C. glabrata clinical isolates. To test this hypothesis, we constructed mutants with disruptions in UPC2A and UPC2B alone and in combination in a matched pair of clinical azole-SDD and -resistant isolates. Disruption of UPC2A in both the SDD and resistant isolates resulted in increased susceptibility to sterol biosynthesis inhibitors, including a reduction in fluconazole MIC and minimum fungicidal concentration, enhanced azole activity by time-kill analysis, a decrease in ergosterol content, and downregulation of baseline and inducible expression of several sterol biosynthesis genes. Our results indicate that Upc2A is a key regulator of ergosterol biosynthesis and is essential for resistance to sterol biosynthesis inhibitors in C. glabrata. Therefore, the UPC2A pathway may represent a potential cotherapeutic target for enhancing azole activity against this organism.

Flucytosine Antagonism of Azole Activity versus Candida glabrata: Role of Transcription Factor Pdr1 and Multidrug Transporter Cdr1

ABSTRACT Infections with the opportunistic yeast Candida glabrata have increased dramatically in recent years. Antifungal therapy of yeast infections commonly employs azoles, such as fluconazole (FLC), but C. glabrata frequently develops resistance to these inhibitors of ergosterol biosynthesis. The pyrimidine analog flucytosine (5-fluorocytosine [5FC]) is highly active versus C. glabrata but is now rarely used clinically due to similar concerns over resistance and, a related concern, the toxicity associated with high doses used to counter resistance. Azole-5FC combination therapy would potentially address these concerns; however, previous studies suggest that 5FC may antagonize azole activity versus C. glabrata. Here, we report that 5FC at subinhibitory concentrations antagonized the activity of FLC 4- to 16-fold versus 8 of 8 C. glabrata isolates tested. 5FC antagonized the activity of other azoles similarly but had only indifferent effects in combination with unrelated antifungals. Since azole resistance in C. glabrata results from transcription factor Pdr1-dependent upregulation of the multidrug transporter gene CDR1, we reasoned that 5FC antagonism might be similarly mediated. Indeed, 5FC-FLC antagonism was abrogated in pdr1Δ and cdr1Δ strains. In further support of this hypothesis, 5FC exposure induced CDR1 expression 6-fold, and this upregulation was Pdr1 dependent. In contrast to azoles, 5FC is not a Cdr1 substrate and so its activation of Pdr1 was unexpected. We observed, however, that 5FC exposure readily induced petite mutants, which exhibit Pdr1-dependent CDR1 upregulation. Thus, mitochondrial dysfunction resulting in Pdr1 activation is the likely basis for 5FC antagonism of azole activity versus C. glabrata.

Evaluation of Polymorphic Locus Sequence Typing for Candida glabrata Epidemiology

ABSTRACT The opportunistic yeast Candida glabrata is increasingly refractory to antifungal treatment or prophylaxis and relatedly is increasingly implicated in health care-associated infections. To elucidate the epidemiology of these infections, strain typing is required. Sequence-based typing provides multiple advantages over length-based methods, such as pulsed-field gel electrophoresis (PFGE); however, conventional multilocus sequence typing (targeting 6 conserved loci) and whole-genome sequencing are impractical for routine use. A commercial sequence-based typing service for C. glabrata that targets polymorphic tandem repeat-containing loci has recently been developed. These CgMT-J and CgMT-M services were evaluated with 56 epidemiologically unrelated isolates, 4 to 7 fluconazole-susceptible or fluconazole-resistant isolates from each of 5 center A patients, 5 matched pairs of fluconazole-susceptible/resistant isolates from center B patients, and 7 isolates from a center C patient who responded to then failed caspofungin therapy. CgMT-J and CgMT-M generated congruent results, resolving isolates into 24 and 20 alleles, respectively. Isolates from all but one of the center A patients shared the same otherwise rare alleles, suggesting nosocomial transmission. Unexpectedly, Pdr1 sequencing showed that resistance arose independently in each patient. Similarly, most isolates from center B also clustered together; however, this may reflect a dominant clone since their alleles were shared by multiple unrelated isolates. Although distinguishable by their echinocandin susceptibilities, all isolates from the center C patient shared alleles, in agreement with the previously reported relatedness of these isolates based on PFGE. Finally, we show how phylogenetic clusters can be used to provide surrogate parents to analyze the mutational basis for antifungal resistance.

X-Plate Technology: a new method for detecting fluconazole resistance in Candida species

Candida species are responsible for many opportunistic fungal infections. Fluconazole is a well-tolerated antifungal drug, commonly used in the treatment of candidiasis. However, with fluconazole resistance ever increasing, rapid detection and antifungal susceptibility testing of Candida is imperative for proper patient treatment. This paper reports a cost-effective, simple and rapid chromogenic agar dilution method for simultaneous Candida species identification and fluconazole susceptibility testing. The results obtained by X-Plate Technology were in absolute concordance with standard microbroth dilution assays. Analysis of 1383 clinical patient samples with suspected vulvovaginal candidiasis revealed that this technology was able to detect and speciate the Candida isolate and determine the fluconazole susceptibility. The prevalence and susceptibility profiles of the clinical isolates using this method were highly similar to published reports using the microbroth dilution method.