1)STB5, a transcriptional repressor in Candida 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, is syntenic with STB5 (YHR178w) in S. cerevisiae and has 43.4% amino acid identity with STB5, including an N-terminal Zn2Cys6 binuclear cluster domain, causing us to test for homologous function and a possible role in azole resistance. CaGl0I02552g was able to restore vegetative growth rate, complemented increased sensitivity to cold, hydrogen peroxide and caffeine in a Δstb5 mutant, and was designated CgSTB5.Overexpression of Cg STB5 in C. glabrata repressed azole resistance, whereas deletion of CgSTB5 increased resistance, both by a mechanism independent of CgPDR1.Real time PCR analysis showed that overexpression of CgSTB5 repressed transcription of the transporter genes CDR1, PDH1 and YOR1. Transcriptional repression by CgSTB5 was not found to be due to competition with PDR1 for binding to the Pleomorphic Drug Response Element (PDRE), TCCGCCCA, but may be acting through binding to the transcriptional repressor complex, SIN3, as the S. cerevisiae homologue is conjectured to do. 2) Global transcriptional profiling of Candida glabrata during phagocytosis by neutrophils and in the infected mouse spleen Neutrophils provide an important host defense against deep C. glabrata infections but the effect neutrophils or the host environment have on the fungus transcriptome is unknown. We used microarray to study the transcriptome of C. glabrata cells after 30 and 60 minutes incubation with human neutrophils under conditions in which more than 80% of the cells were phagocytosed. A total of 519 genes were up-regulated and 360 down-regulated at 30 min incubation. Several highly up-regulated genes were involved in methionine biosynthesis (MET28, STR3), related to nitrogen depletion. We also found induction of processes involved in carboxylic acid transport (SFC1, MUP1, GAP1, ADY2), cellular carbohydrate metabolic processes (FBP1, PCK1, ICL1) and transcriptional factors which regulate gene expression for utilization of alternative carbohydrate sources instead of glucose (SIP4, CAT8, ADR1). This induction was strongly related to carbohydrate depletion . For our interest, multidrug resistance genes were also upregulated (PDR1, PDH1) , also seen as a response to carbohydrate depletion. Many of the down-regulated genes were involved in protein synthesis (RPL12A, GCD10) and ergosterol biosynthetic process (ERG 10, ERG9). These results suggested that C. glabrata cells were decreasing synthesis of proteins and cell membrane sterols because of nutrient depletion. We confirmed this by performing microarray on C. glabrata exposed to nitrogen or glucose deprivation. We hypothesized that C. glabrata in mouse spleen might be exposed to the same environmental conditions as in human neurophils. Therefore, we selected 20 target genes from our neutrophil experiments and tested expression in spleen 24 hours after infection of C57BL/6J mice, using quantitative real-time PCR. Our results indicated that expression of 15 genes was similar in neutrophils and mouse spleen, whereas 3 genes (AUS1, PCK1, CDC19) were significantly upregulated in C57BL/6J mouse spleen compared to neutrophils. These results suggested Cg cells are exposed to even more severe nutrient condition in mouse spleen compared with that in neutrophils. The 6-fold upregulation of AUS1 may indicate a need for importing exogenous sterol, such as cholesterol, in response to decreased sterol synthesis. As we have reported, C. glabrata can utilize cholesterol when ergosterol synthesis is impaired. To the extent that the cell can utilise cholesterol, azoles ability to block growth by blocking ergosterol synthesis is nullified. To clarify the effect of oxidative stress against C. glabrata cells in vivo, we also compared gene expression of C. glabrata in spleens of C57BL/6J and gp91phox- knock-out mice. The latter mice have reduced oxidative capacity for neutrophil killing of microorganisms. Expression of the 20 genes was similar in both mouse strains, including expression of CTA1, which codes for catalase and is upregulated under oxidative stress. This suggests that oxidative stress may not be the major mechanism by which the neutrophil inhibits growth. Response of C. glabrata to neutrophil phagocytosis reflects the lack of essential carbohydrate and nitrogen sources. The gene expression of C glabrata cells in mouse spleens showed the fungus was exposed to at least as severe nutrient depletion. Up-regulation of multidrug resistance genes, such as PDH1, in neutrophils and mouse spleens even in the absence of drugs is an integral part of C. glabrata response and this, as well as increased sterol transport, may contributes to azole resistance in vivo. 3)Evaluation of housekeeping genes for RT-PCR analysis of gene expression in Candida glabrata responding to azole stress Selection of stable and suitable reference genes for quantitative real-time PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions and environmental stimuli. The present study aimed at identifying and selecting housekeeping genes as internal controls for gene expression studies by RT-qPCR in fluconazole-stimulated Candida glabrata. The expression stability of 20 housekeeping genes was evaluated using the 2-∆∆CT Method. Our data revealed that the mRNA expression levels of the three ribosomal RNAs 5.8S rRNA, 18S rRNA, and 25S rRNA remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNA were relatively stable with only approximately 2.3-, 2.3-, and 1.9-fold induction, respectively. By contrast, mRNA levels of the other 14 housekeeping genes (ACT1, cyclophilin, EF-1α, GAPDH, RPL2A, RPL10, RPL13B, RPP2B, SDHA, TBP, TFRC, α-tubulin, β-tubulin, and UBC4) tested were significantly increased in C. glabrata following drug treatment, with changes ranging from 3.5 to 25 fold. We then validated the suitability of 6 candidate reference genes (5.8S rRNA, 18S rRNA, ACT1, PGK1, UBC7, and UBC13) as endogenous controls for 10 target genes in this system using the ∆∆CT Method. We chose to assess 5 ATP-binding cassette transporter genes (CDR1, PDH1, PDR1, SNQ2, and YOR1) and 5 sterol biosynthetic genes (ERG2, ERG3, ERG4, ERG10, and ERG11) as the targets in this study because these genes have been shown to be implicated in azole resistance in C. glabrata. Our validation experiments passed for all the 6 control genes analyzed except for 18S rRNA, where the absolute value of the slope of log input RNA vs. ∆CT of the targets was >0.1. Finally, we demonstrated that the relative quantification of the target gene expression varied according to the internal control used, thus highlighting the importance of the choice of endogenous controls in such experiments. 4)Infectious Disease Training Program This training program continues to attract 4-5 highly qualified physicians each year and remains fully accredited by the Adult Committee for Graduate Medical Education. The pass rate of graduates on the American Board of Internal Medicines Infectious Disease certifying exam remains 100% for the decade and 95% since inception of the examination in 1972.
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