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The most common nosocomial fungal infections are caused by several species of Candida, of which Candida glabrata is the second most frequently isolated species from bloodstream infections. C. glabrata displays relatively high minimal inhibitory concentration values (MIC) to the antifungal fluconazole and is associated with high mortality rates. To decrease mortality rates, the appropriate treatment must be administered promptly. C. glabrata contains in its genome several non-identical copies of species-specific sequences. We designed three pairs of C. glabrata-specific primers for endpoint PCR amplification that align to these species-specific sequences and amplify the different copies in the genome. Using these primers, we developed a fast, sensitive, inexpensive, and highly specific PCR-based method to positively detect C. glabrata DNA in a concentration-dependent manner from mixes of purified genomic DNA of several Candida species, as well as from hemocultures and urine clinical samples. This tool can be used for positive identification of C. glabrata in the clinic. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00584-2.

Candida albicans, Candida glabrata, Candida parapsilosis and Candida tropicalis are the four most common human fungal pathogens isolated that can cause superficial and invasive infections. It has been shown that specific metabolites present in the secretomes of these fungal pathogens are important for their virulence. C. glabrata is the second most common isolate world-wide and has an innate resistance to azoles, xenobiotics and oxidative stress that allows this fungal pathogen to evade the immune response and persist within the host. Here, we analyzed and compared the C. glabrata secretome with those of C. albicans, C. parapsilosis, C. tropicalis and the non-pathogenic yeast Saccharomyces cerevisiae. In C. glabrata, we identified a different number of metabolites depending on the growth media: 12 in synthetic complete media (SC), 27 in SC-glutamic acid and 23 in rich media (YPD). C. glabrata specific metabolites are 1-dodecene (0.09 ± 0.11%), 2,5-dimethylundecane (1.01 ± 0.19%), 3,7-dimethyldecane (0.14 ± 0.15%), and octadecane (0.4 ± 0.53%). The metabolites that are shared with C. albicans, C. glabrata, C. parapsilosis,&nbsp, C. tropicalis and S. cerevisiae are phenylethanol, which is synthesized from phenylalanine, and eicosane and nonanoic acid (identified as trimethylsilyl ester), which are synthesized from fatty acid metabolism. Phenylethanol is the most abundant metabolite in all fungi tested: 26.36 ± 17.42% (C. glabrata), 46.77 ± 15.58% (C. albicans), 49.76 ± 18.43% (C. tropicalis), 5.72 ± 0.66% (C. parapsilosis.) and 44.58 ± 27.91% (S. cerevisiae). The analysis of C. glabrata’s secretome will allow us to further our understanding of the possible role these metabolites could play in its virulence.

C. glabrata is an opportunistic fungal pathogen and the second most common cause of opportunistic fungal infections in humans, that has evolved virulence factors to become a successful pathogen: strong resistance to oxidative stress, capable to adhere and form biofilms in human epithelial cells as well as to abiotic surfaces and high resistance to xenobiotics. Hst1 (a NAD

An important virulence factor for the fungal pathogen Candida glabrata is the ability to adhere to the host cells, which is mediated by the expression of adhesins. Epa1 is responsible for ∼95% of the in vitro adherence to epithelial cells and is the founding member of the Epa family of adhesins. The majority of EPA genes are localized close to different telomeres, which causes transcriptional repression due to subtelomeric silencing. In C. glabrata there are three Sir proteins (Sir2, Sir3 and Sir4) that are essential for subtelomeric silencing. Among a collection of 79 clinical isolates, some display a hyperadherent phenotype to epithelial cells compared to our standard laboratory strain, BG14. These isolates also express several subtelomeric EPA genes simultaneously. We cloned the SIR2, SIR3 and SIR4 genes from the hyperadherent isolates and from the BG14 and the sequenced strain CBS138 in a replicative vector to complement null mutants in each of these genes in the BG14 background. All the SIR2 and SIR4 alleles tested from selected hyper-adherent isolates were functional and efficient to silence a URA3 reporter gene inserted in a subtelomeric region. The SIR3 alleles from these isolates were also functional, except the allele from isolate MC2 (sir3-MC2), which was not functional to silence the reporter and did not complement the hyperadherent phenotype of the BG14 sir3Δ. Consistently, sir3-MC2 allele is recessive to the SIR3 allele from BG14. Sir3 and Sir4 alleles from the hyperadherent isolates contain several polymorphisms and two of them are present in all the hyperadherent isolates analyzed. Instead, the Sir3 and Sir4 alleles from the BG14 and another non-adherent isolate do not display these polymorphisms and are identical to each other. The particular combination of polymorphisms in sir3-MC2 and in SIR4-MC2 could explain in part the hyperadherent phenotype displayed by this isolate.

Candida glabrata is an opportunistic fungal pathogen that can cause life-threatening infections in immunocompromised patients. To ensure a successful infection, C. glabrata has evolved a variety of strategies to avoid killing within the host. One of these strategies is the resistance to oxidative stress. Here we show that the sulfiredoxin Srx1 and the peroxiredoxins, Tsa1 and Tsa2, are implicated in the oxidative stress response (OSR) and required for virulence. We analyzed null mutations in SRX1, TSA1 and TSA2 and showed that TSA2 and SRX1 are required to respond to oxidative stress. While TSA1 expression is constitutive, SRX1 and TSA2 are induced in the presence of H2O2 in a process dependent on H2O2 concentration and on both transcription factors Yap1 and Skn7. Msn2 and Msn4 are not necessary for the regulation of SRX1, TSA1 and TSA2. Interestingly, TSA1 and TSA2, which are localized in the cytoplasm, are induced in the presence of neutrophils and required for survival in these phagocytic cells.

The fungal pathogen Candida glabrata has a well-defined oxidative stress response, is extremely resistant to oxidative stress and can survive inside phagocytic cells. In order to further our understanding of the oxidative stress response in C. glabrata, we characterized the superoxide dismutases (SODs) Cu,ZnSOD (Sod1) and MnSOD (Sod2). We found that Sod1 is the major contributor to total SOD activity and is present in cytoplasm, whereas Sod2 is a mitochondrial protein. Both SODs played a central role in the oxidative stress response but Sod1 was more important during fermentative growth and Sod2 during respiration and growth in non-fermentable carbon sources. Interestingly, C. glabrata cells lacking both SODs showed auxotrophy for lysine, a high rate of spontaneous mutation and reduced chronological lifespan. Thus, our study reveals that SODs play an important role in metabolism, lysine biosynthesis, DNA protection and aging in C. glabrata.

Candida glabrata has emerged as an important opportunistic pathogen in both mucosal and bloodstream infections. C. glabrata contains 67 adhesin-like glycosylphosphatidylinositol-cell-wall proteins (GPI-CWPs), which are classified into seven groups and the largest is the Epa family. Epa proteins are very diverse and their expression is differentially regulated. Like many of the EPA genes, EPA2 is localized in a subtelomeric region where it is subject to chromatin-based transcriptional silencing and its role remains largely unexplored. In this study, we show that EPA2 gene is induced specifically in vitro in the presence of oxidative stress generated by H2O2. This induction is dependent on both Yap1 and Skn7, whereas Msn4 represses EPA2 expression. Interestingly, EPA2 is not induced during phagocytosis, but its expression can be identified in the liver in a murine model of systemic infection. Epa2 has no effect on the virulence of C. glabrata. The work presented herein provides a foundation for future studies to dissect the molecular mechanism(s) by which EPA2 of C. glabrata can be induced in the presence of oxidative stress in a region subject to subtelomeric silencing.

Organisms have evolved different strategies to respond to oxidative stress generated as a by-product of aerobic respiration and thus maintain the redox homeostasis within the cell. In particular, fungal pathogens are exposed to reactive oxygen species (ROS) when they interact with the phagocytic cells of the host which are the first line of defense against fungal infections. These pathogens have co-opted the enzymatic (catalases, superoxide dismutases (SODs), and peroxidases) and non-enzymatic (glutathione) mechanisms used to maintain the redox homeostasis within the cell, to resist oxidative stress and ensure survival within the host. Several virulence factors have been related to the response to oxidative stress in pathogenic fungi. The opportunistic fungal pathogen Candida glabrata (C. glabrata) is the second most common cause of candidiasis after Candida albicans (C. albicans). C. glabrata has a well defined oxidative stress response (OSR), which include both enzymatic and non-enzymatic mechanisms. C. glabrata OSR is controlled by the well-conserved transcription factors Yap1, Skn7, Msn2 and Msn4. In this review, we describe the OSR of C. glabrata, what is known about its core elements, its regulation and how C. glabrata interacts with the host. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

In Candida glabrata, the sirtuins Sir2 and Hst1 control the expression of a wide number of genes including adhesins required for host colonization and niacin transporters needed for growth. Given that these sirtuins can be inactivated during infection, we asked if their inhibition could modify the response of C. glabrata to other stressful conditions. Here, we found that deletion of HST1 decreases susceptibility of C. glabrata to fluconazole and hydrogen peroxide. The transcription factor Pdr1 and the ABC transporter Cdr1 mediated the fluconazole resistance phenotype of the hst1Δ cells, whereas the transcriptional activator Msn4 and the catalase Cta1 are necessary to provide oxidative stress resistance. We show that the transcription factor Sum1 interacts with Hst1 and participate in the regulation of these genes. Interestingly, even though C. glabrata and Saccharomyces cerevisiae are closely related phylogenetically, deletion of HST1 decreased susceptibility to fluconazole and hydrogen peroxide only in C. glabrata but not in S. cerevisiae, indicating a different transcriptional control by two similar sirtuins. Our findings suggest that Hst1 acts as a regulator of stress resistance associated-genes.

We studied the effects of extracellular ATP and Ca2+ on uptake of bacteria (Staphylococcus aureus or Escherichia coli) and live yeast (Candida glabrata) by J774 macrophages to determine the role of endogenous P2X7 receptors in phagocytosis. Our findings show that phagocytosis of bio-particles coated with S. aureus or E. coli was blocked by ATP and the P2X7 receptor agonist BzATP, while yeast phagocytosis was not. A438079, an antagonist of P2X7 receptors, partially reverted the effects of ATP on bacterial phagocytosis. To determine if P2X7-mediated Ca2+ entry into macrophages was blocking the engulfment of bacteria, we measured phagocytic activity in the absence or presence of 2 mM extracellular Ca2+ with or without ATP. Ca2+, in the absence of ATP, was required for engulfment of E. coli and C. glabrata but not S. aureus. Adding ATP inhibited phagocytosis of S. aureus and E. coli regardless of Ca2+, suggesting that Ca2+ entry was not important for inhibiting phagocytosis. On the other hand, phagocytosis of normal or hyper-adherent C. glabrata mutants had an absolute requirement for extracellular Ca2+ due to yeast adhesion to macrophages mediated by Ca2+-dependent adhesion proteins. We conclude that unstimulated P2X7 from J774 cells act as scavenger receptor for the uptake of S. aureus and E. coli but not of yeast; Ca2+ entry via P2X7 receptors play no role in phagocytosis of S. aureus and E. coli; while the effect of Ca2+ on C. glabrata phagocytosis was mediated by the adhesins Epa1, Epa6 and Epa7.

Candida glabrata is a haploid opportunistic fungal pathogen that is phylogenetically related to Saccharomyces cerevisiae . Even though C. glabrata has no known sexual cycle, it contains, like S. cerevisiae , three mating type-like loci ( MTL ) called MTL1 , MTL2 , and MTL3 , as well as most of the genes required for mating, meiosis, and sporulation. MTL1 is localized at an internal position on chromosome B and is thought to be the locus corresponding to the MAT locus in S. cerevisiae . MTL2 and MTL3 are localized close to two telomeres on different chromosomes (29.4 kb from Chr E-L and 10.5 kb from Chr B-L, respectively). By using URA3 reporter gene insertions at the three MTL loci, we found that in contrast to the case for S. cerevisiae , only MTL3 is subject to transcriptional silencing while MTL2 is transcriptionally active, and this is in agreement with previously reported data. We found that the silencing of MTL3 is nucleated primarily at the left telomere of chromosome B and spreads over 12 kb to MTL3 , rather than nucleating at flanking, closely positioned cis -acting silencers, like those flanking HMR and HML of S. cerevisiae . Interestingly, the silencing of MTL3 absolutely requires the yKu70, yKu80, and Rif1 proteins, in sharp contrast to the silencing of the HM loci of S. cerevisiae . In addition, we found that several cell type-specific genes are expressed in C. glabrata regardless of the presence, or even absence, of mating type information at any of the MTL loci.

The human fungal pathogen Candida glabrata is causing more and more problems in hospitals, as this species shows an intrinsic antifungal drug resistance or rapidly becomes resistant when challenged with antifungals. C. glabrata only grows in the yeast form, so it is lacking a yeast-to-hyphae switch, which is one of the main virulence factors of C. albicans. An important virulence factor of C. glabrata is its capacity to strongly adhere to many different substrates. To achieve this, C. glabrata expresses a large number of adhesin-encoding genes and genome comparisons with closely related species, including the non-pathogenic S. cerevisiae, which revealed a correlation between the number of adhesin-encoding genes and pathogenicity. The adhesins are involved in the first steps during an infection; they are the first point of contact with the host. For several of these adhesins, their importance in adherence to different substrates and subsequent biofilm formation was demonstrated in vitro or in vivo. In this review, we provide an overview of the role of C. glabrata adhesins during adhesion and biofilm formation both, under in vitro and in vivo conditions ispartof: Journal of Fungi vol:4 issue:2 ispartof: location:Switzerland status: published

The opportunistic fungal pathogen Candida glabrata is the second most common isolate from bloodstream infections worldwide and is naturally less susceptible to the antifungal drug fluconazole than other Candida species. C. glabrata is a haploid yeast that contains three mating-type like loci (MTL), although no sexual cycle has been described. Strains containing both types of mating information at the MTL1 locus are found in clinical isolates, but it is thought that strains containing type a information are more common. Here we investigated if a particular combination of mating type information at each MTLlocus is more prevalent in clinical isolates from hospitalized patients in Mexico and if there is a correlation between mating information and resistance to fluconazole and 5-fluorocytosine. We found that while both types of information at MTL1 are equally represented in a collection of 64 clinical isolates, the vast majority of isolates contain a-type information at MTL2 and α-type at MTL3. We also found no correlation of the particular combination of mating type information at the three MTL loci and resistance to fluconazole.

The pathogenic yeast Candida glabrata is able to bind in vitro to human epithelial cells. This interaction depends on expression of the adhesin Epa1p. The genome contains a number of EPA1 paralogues which localize to the subtelomeric regions of the C. glabrata. We have identified three hyperadherent mutants of C. glabrata. The first has an insertion adjacent to EPA7, an EPA1-related adhesin. The others disrupt the SIR3 and RIF1 genes of C. glabrata. We show that SIR3 and RIF1 are required for subtelomeric silencing in C. glabrata and that RIF1 regulates telomere length in C. glabrata. We show that the hyperadherent phenotype of the sir3Delta and rif1Delta deletion strains depends primarily on derepression of two novel members of the EPA gene family -EPA6 and EPA7. The sir3Delta and rif1Delta mutants show increased colonization of the kidney in a murine model of disseminated infection and this hypercolonization depends, at least in part, on derepression of EPA6 and EPA7. The analysis here is the first evidence that multiple EPA genes encode adhesins and demonstrates that transcription of at least two of these adhesins is regulated by subtelomeric silencing.

Candida glabrata is a haploid yeast considered the second most common of the Candida species found in nosocomial infections, accounting for approximately 18% of candidemias worldwide. Even though molecular biology methods are easily adapted to study this organism, there are not enough vectors that will allow probing the transcriptional and translational activity of any gene of interest in C. glabrata. In this work we have generated a set of expression vectors to systematically tag any gene of interest at the carboxy-terminus with three different fluorophores (CFP, YFP and mCherry) or three epitopes (HA, FLAG or cMyc) independently. This system offers the possibility to generate translational fusions in three versions: under the gene’s own promoter integrated in its native locus in genome, on a replicative plasmid under its own promoter, or on a replicative plasmid under a strong promoter to overexpress the fusions. The expression of these translational fusions will allow determining the transcriptional and translational activity of the gene of interest as well as the intracellular localization of the protein. We have tested these expression vectors with two biosynthetic genes, HIS3 and TRP1. We detected fluorescence under the microscope and we were able to immunodetect the fusions using the three different versions of the system. These vectors permit coexpression of several different fusions simultaneously in the same cell, which will allow determining protein–protein and protein–DNA interactions. This set of vectors adds a new toolbox to study expression and protein interactions in the fungal pathogen C. glabrata.

Candida glabrata, a haploid and opportunistic fungal pathogen that has not known sexual cycle, has conserved the majority of the genes required for mating and cell type identity. The C. glabrata genome contains three mating-type-like loci called MTL1, MTL2 and MTL3. The three loci encode putative transcription factors, a1, α1 and α2 that regulate cell type identity and sexual reproduction in other fungi like the closely related Saccharomyces cerevisiae. MTL1 can contain either a or α information. MTL2, which contains a information and MTL3 with α information, are relatively close to two telomeres. MTL1 and MTL2 are transcriptionally active, while MTL3 is subject to an incomplete silencing nucleated at the telomere that depends on the silencing proteins Sir2, Sir3, Sir4, yKu70/80, Rif1, Rap1 and Sum1. C. glabrata does not seem to maintain cell type identity, as cell type-specific genes are expressed regardless of the type (or even absence) of mating information. These data highlight important differences in the control of mating and cell type identity between the non-pathogenic yeast S. cerevisiae and C. glabrata, which might explain the absence of a sexual cycle in C. glabrata. The fact that C. glabrata has conserved the vast majority of the genes involved in mating might suggest that some of these genes perhaps have been rewired to control other processes important for the survival inside the host as a commensal or as a human pathogen. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

The fungal pathogen Candida glabrata is a haploid asexual yeast. Candida glabrata contains orthologs of the genes that control mating and cell-type identity in other fungi, which encode putative transcription factors localized in the MAT locus in Saccharomyces cerevisiae or MTL in other fungi. Candida glabrata contains three copies of the CgMTL locus but only CgMTL1 correctly expresses the information encoded in it. CgMTL1 can encode the Cg a1 gene ( a information), or the Cg alpha1 and Cg alpha2 genes (alpha information). CgMTL2 contains an identical copy of the Cg a1 gene. CgMTL3 contains an identical copy of the Cg alpha1 gene but a longer variant of the Cg alpha2 gene that we termed Cg alpha3. In S. cerevisiae diploid cells, that express Sc a and Sc alpha information, Sc a1 and Sc alpha2 proteins form a heterodimer, which represses genes expressed only in haploid cells and some genes involved in stress response. We constructed C. glabrata strains that simultaneously express Cg a1 and Cg alpha2 or Cg a1 and Cg alpha3 genes. We did not find any phenotype in these strains when grown under a large variety of stress and nutritional conditions. However, we detected an interaction between Cg a1 and Cg alpha2 but not between Cg a1 and Cg alpha3 by Bimolecular Fluorescence Complementation and co-immunoprecipitation assays.

The opportunistic fungal pathogen Candida glabrata adheres tightly to epithelial cells in culture, mainly through the adhesin Epa1. EPA1 is the founding member of a family of up to 23 putative adhesin-encoding genes present in the C. glabrata genome. The majority of the EPA genes are localized close to the telomeres, where they are repressed by subtelomeric silencing that depends on the Sir, Ku, Rif1, and Rap1 proteins. EPA6 and EPA7 also encode functional adhesins that are repressed in vitro. EPA1 expression in vitro is tightly controlled both positively and negatively, and in addition, presents high cell-to-cell heterogeneity, which depends on Sir-mediated silencing. In this work, we characterized the ability to adhere to HeLa epithelial cells and the expression of several EPA genes in a collection of 79 C. glabrata clinical isolates from several hospitals in Mexico. We found 11 isolates that showed increased adherence to mammalian cells compared with our reference strain under conditions where EPA1 is not expressed. The majority of these isolates displayed over-expression of EPA1 and EPA6 or EPA7, but did not show increased biofilm formation. Sequencing of the SIR3 gene of several hyper-adherent isolates revealed that all of them contain several polymorphisms with respect to the reference strain. Interestingly, two isolates have polymorphisms in positions flanked by clusters of amino acids required for silencing in the Saccharomyces cerevisiae Sir3 protein. Our data show that there is a large variability in adhesin expression and adherence to epithelial cells among different C. glabrata clinical isolates.

Summary We report here the construction and analysis of insertional mutations in each of the three genes of the gltBDF operon and the nucleotide sequence of the region downstream from gltD. Two open reading frames were identified, the first of which corresponds to gltF. The gltB and gltD genes code for the large and small subunits, respectively, of the enzyme glutamate synthase (GOGAT). gltF codes for a protein, with a molecular mass of 26350Da, which is required for Ntr induction. Histidase synthesis was determined as a measure of Ntr function. First, insertions in gltB, gltD or gltF all prevent Ntr induction. Second, complementation analysis indicates that high-level expression of both the gltD and gltF genes is required for the induction of the Ntr enzymes under nitrogen-limiting conditions, indicating that the phenotype of the gltB insertion probably results from polarity on gltD and gltF. Third, glutamate-dependent repression of the glt operon appears to be mediated by the product of the gltF gene. Thus, the gltBDF operon of Escherichia coli is involved in induction of the so-called Ntr enzymes in response to nitrogen deprivation, as well as in glutamate biosynthesis.

Candida glabrata , a common opportunistic fungal pathogen, adheres efficiently to mammalian epithelial cells in culture. This interaction in vitro depends mainly on the adhesin Epa1, one of a large family of cell wall proteins. Most of the EPA genes are located in subtelomeric regions, where they are transcriptionally repressed by silencing. In order to better characterize the transcriptional regulation of the EPA family, we have assessed the importance of C. glabrata orthologues of known regulators of subtelomeric silencing in Saccharomyces cerevisiae. To this end, we used a series of strains containing insertions of the reporter URA3 gene within different intergenic regions throughout four telomeres of C. glabrata . Using these reporter strains, we have assessed the roles of SIR2 , SIR3 , SIR4 , HDF1 (yKu70), HDF2 (yKu80), and RIF1 in mediating silencing at four C. glabrata telomeres. We found that, whereas the SIR proteins are absolutely required for silencing of the reporter genes and the native subtelomeric EPA genes, the Rif1 and the Ku proteins regulate silencing at only a subset of the analyzed telomeres. We also mapped a cis element adjacent to the EPA3 locus that can silence a reporter gene when placed at a distance of 31 kb from the telomere. Our data show that silencing of the C. glabrata telomeres varies from telomere to telomere. In addition, recruitment of silencing proteins to the subtelomeres is likely, for certain telomeres, to depend both on the telomeric repeats and on particular discrete silencing elements.

Candida glabrata is an opportunistic fungal pathogen that can cause severe invasive infections and can evade phagocytic cell clearance. We are interested in understanding the virulence of this fungal pathogen, in particular its oxidative stress response. Here we investigated C. glabrata, Saccharomyces cerevisiae and Candida albicans responses to two different oxidants: menadione and cumene hydroperoxide (CHP). In log-phase, in the presence of menadione, C. glabrata requires Cta1p (catalase), while in a stationary phase (SP), Cta1p is dispensable. In addition, C. glabrata is less resistant to menadione than C. albicans in SP. The S. cerevisiae laboratory reference strain is less resistant to menadione than C. glabrata and C. albicans; however S. cerevisiaeclinical isolates (CIs) are more resistant than the lab reference strain. Furthermore, S. cerevisiae CIs showed an increased catalase activity. Interestingly, in SP C. glabrata and S. cerevisiae are more resistant to CHP than C. albicans and Cta1p plays no apparent role in detoxifying this oxidant.

We characterized the oxidative stress response of Candida glabrata to better understand the virulence of this fungal pathogen. C. glabrata could withstand higher concentrations of H 2 O 2 than Saccharomyces cerevisiae and even Candida albicans . Stationary-phase cells were extremely resistant to oxidative stress, and this resistance was dependent on the concerted roles of stress-related transcription factors Yap1p, Skn7p, and Msn4p. We showed that growing cells of C. glabrata were able to adapt to high levels of H 2 O 2 and that this adaptive response was dependent on Yap1p and Skn7p and partially on the general stress transcription factors Msn2p and Msn4p. C. glabrata has a single catalase gene, CTA1 , which was absolutely required for resistance to H 2 O 2 in vitro. However, in a mouse model of systemic infection, a strain lacking CTA1 showed no effect on virulence.

Candida glabrata is a fungal pathogen frequently found as a commensal in humans. To colonize and disseminate successfully in the mammalian host, C. glabrata must detect signals within the host and reprogram gene expression to respond appropriately to hostile environmental conditions. One of the layers of regulation of expression of many virulence-related genes (adhesin-encoding genes, genes involved in response to oxidative stress and xenobiotics) is achieved through epigenetic mechanisms. Local and regional silencing is mediated by the activity of two NAD(+)-dependent histone deacetylases, Hst1 and Sir2, respectively, repressing many virulence genes. Hst1 and Sir2 interact with different repressor complexes to achieve regional or local silencing. Sir2 can associate with Sir4, which is then recruited to the telomere by Rap1 and yKu. Deacetylation of the histone tails creates high affinity binding sites for new molecules of the Sir complex, thereby spreading the silent domain over >20 kb. Many of the adhesin-encoding EPA genes are subject to this regulation. Hst1 in turn associates with the Sum1-Rfm1 complex. Sum1 is a DNA-binding protein, which recognizes specific sites at individual promoters, recruiting Hst1 to specific genes involved in the response to oxidative stress and xenobiotics, which results in their repression.

The adherence of Candida glabrata to host cells is mediated, at least in part, by the EPA genes, a family of adhesins encoded at subtelomeric loci, where they are subject to transcriptional silencing. We show that normally silent EPA genes are expressed during murine urinary tract infection (UTI) and that the inducing signal is the limitation of nicotinic acid (NA), a precursor of nicotinamide adenine dinucleotide (NAD + ). C. glabrata is an NA auxotroph, and NA-induced EPA expression is likely the result of a reduction in NAD + availability for the NAD + -dependent histone deacetylase Sir2p. The adaptation of C. glabrata to the host, therefore, involves a loss of metabolic capacity and exploitation of the resulting auxotrophy to signal a particular host environment.

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.

A 2.0-kilobase DNA fragment carrying antibiotic resistance markers was inserted into the gltB gene of Escherichia coli previously cloned in a multicopy plasmid. Replacement of the chromosomal gltB+ gene by the gltB225::omega mutation led to cells unable to synthesize glutamate synthase, utilize growth rate-limiting nitrogen sources, or derepress their glutamine synthetase. The existence of a gltBDF operon encoding the large (gltB) and small (gltD) subunits of glutamate synthase and a regulatory peptide (gltF) at 69 min of the E. coli linkage map was deduced from complementation analysis. A plasmid carrying the entire gltB+D+F+ operon complemented cells for all three of the mutant phenotypes associated with the polar gltB225::omega mutation in the chromosome. By contrast, plasmids carrying gltB+ only complemented cells for glutamate synthase activity. A major tricistronic mRNA molecule was detected from Northern (RNA blot) DNA-RNA hybridization experiments with DNA probes containing single genes of the operon. A 30,200-dalton polypeptide was identified as the gltF product, the lack of which was responsible for the inability of cells to use nitrogen-limiting sources associated with gltB225::omega.