1. STB5 is a negative regulator of azole resistance in C. glabrata The opportunistic yeast pathogen Candida glabrata is recognized for its ability to acquire resistance during prolonged treatment with azole antifungals. Resistance to azoles is largely mediated by the transcription factor PDR1, resulting in the upregulation of ATP-binding cassette (ABC) transporter proteins and drug efflux. Studies in the related yeast Saccharomyces cerevisiae have shown Pdr1p forms a heterodimer with another transcription factor, Stb5p. In C. glabrata the ORF designated CAGL0I02552g has 38.8% amino acid identity with STB5 (YHR178w), and shares an N-terminal Zn2Cys6 binuclear cluster domain and a fungal specific transcriptional factor domain, prompting us to test for homologous function and a possible role in azole resistance. Complementation of ∆yhr178w (∆stb5) with CAGL0I02552g resolved the increased sensitivity to cold, hydrogen peroxide and caffeine of the mutant, for which reason we designated CAGl0I02552g as CgSTB5. Overexpression of CgSTB5 in C. glabrata repressed azole resistance, whereas deletion of CgSTB5 increased resistance, both by a mechanism independent of CgPDR1. Expression analysis found that CgSTB5 shares many of the same transcriptional targets as CgPDR1 but, unlike the latter, is a negative regulator of pleiotropic drug resistance, including the ABC transporter genes CDR1, PDH1, and YOR1 2. Novel approach to assessing transcription in vivo Expression microarray C. glabrata following phagocytosis by human neutrophils was performed and compared with results from C. glabrata incubated under conditions of carbohydrate or nitrogen deprivation. Twenty genes were selected to represent the major cell processes altered by neutrophils or nutrient deprivation. Quantitative real-time PCR (qRT-PCR) with TaqMan chemistry was used to assess expression of the same genes in spleens of mice infected intravenously with Candida glabrata. The results in spleen closely paralleled gene expression in neutrophils or following carbohydrate deprivation. Fungal cells responded by upregulating alternative energy sources through gluconeogenesis, glyoxylate cycle and long chain fatty acid metabolism. Autophagy was likely employed to conserve intracellular resources. Aspartyl protease upregulation occurred and may represent defense against attacks on cell wall integrity. Down regulated genes were in the pathways of protein and ergosterol synthesis. Upregulation of the sterol transport gene, AUS1, suggested that murine cholesterol may have been used to replace ergosterol, as has been reported in vitro. C. glabrata in spleens of knock-out mice with reduced oxidative phagocyte defenses were grossly similar although with a slightly reduced level of response, closer to that of culture medium. These results are consistent with reported results of other fungi responding to phagocytosis, indicating that a rapid shift in metabolism is required for growth in nutrient-poor intracellular environment. The utility of qRT-PCR to assess gene expression directly in infected animal tissue was also shown. 3. Selection of appropriate controls is RT-PCR The selection of stable and suitable reference genes for real-time quantitative PCR (RTqPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata. Results: The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25) remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4) were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5-to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-∆∆CT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2∆∆CT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13) as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such experiments 4.Increased sterol uptake, sterol biosynthesis and azole efflux pumping act coordinately to mediate antifungal resistance in Candida glabrata under azole and environmental stresses. Pathogenic fungi including Candida glabrata develop strategies to grow and survive in vitro and in vivo under azole and environmental stresses. Three protective mechanisms by which yeast cells acquire antifungal tolerance are through the uptake of exogenous sterols and through the alteration of endogenous sterol biosynthesis, as well as through pumping antifungals out of the cells, thus counteracting the underlying inhibitory effect of azoles. Here we demonstrate that the expression of the ergosterol biosynthesis genes ERG2, ERG3, ERG4, ERG10 and ERG11 was strikingly up-regulated in C. glabrata following fluconazole treatment. Similarly, we showed that mRNA expression of the ABC transporter genes CDR1, PDR1 (Transcription factor not transporter), SNQ2 and YOR1 was markedly increased in C. glabrata in response to fluconazole. Blocking ergosterol biosynthesis by treatment with the drug also effected increases in the expression of AUS1, sterol influx transporter in the fungal cells. Likewise, exposure of C. glabrata to fluconazole significantly enhanced expression of the sterol metabolism regulators ECM22/UPC2A and SUT1A. Our microarray analysis using a C. glabrata mutant strain lacking ERG1 revealed that mRNA levels of AUS1 and ECM22/UPC2B expression were considerably elevated compared with its parental wild-type strain, indicating that the sterol uptake activity increases to compensate for the defective sterol biosynthesis in the cells. Furthermore, to assess whether sterol uptake affects yeast susceptibility to azoles, we generated a C. glabrata aus1 mutant strain. We found that inactivating this protective mechanism in C. glabrata (or loss of Aus1 in C. glabrata) sensitized the pathogen to azoles and obviously enhanced the efficacy of drug treatment during low oxygen tension. In contrast, the presence of exogenous cholesterol or ergosterol in the medium renders its C. glabrata aus1 wild-type strain highly resistant to a multitude of fluconazole and voriconazole, suggesting that the sterol uptake mechanism is augmented under hypoxic condition, thus allowing C. glabrata to survive in unfavorable environments. On the basis of these results, we conclude that sterol uptake, sterol synthesis, and efflux pumping may act coordinately and collaboratively to sustain growth and to mediate antifungal resistance in C. glabrata through dynamic gene expression in response to azole stress and environmental challenges.
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