Characterization of the Trr/Trx system in the fungal pathogen Candida glabrata.

C. glabrata, an opportunistic fungal pathogen, can adapt and resist to different stress conditions. It is highly resistant to oxidant stress compared to other Candida spp and to the phylogenetically related but non-pathogen Saccharomyces cerevisiae. In this work, we describe the Trx/Trr system of C. glabrata composed of Trr1 and Trr2 (thioredoxin reductases) and Trx2 (thioredoxin) that are localized in the cytoplasm and Trx3 present in the mitochondrion. The transcriptional induction of TRR2 and TRX2 by oxidants depends on Yap1 and Skn7 and TRR1 and TRX3 have a low expression level. Both TRR2 and TRX2 play an important role in the oxidative stress response. The absence of TRX2 causes auxotrophy of methionine and cysteine. Trr1 and Trr2 are necessary for survival at high temperatures and for the chronological life span of C. glabrata. Furthermore, the Trx/Trr system is needed for survival in the presence of neutrophils. The role of TRR1 and TRX3 is not clear, but in the presence of neutrophils, they have non-overlapping functions with their TRR2 and TRX2 paralogues.

Chromatin Loop Formation Induced by a Subtelomeric Protosilencer Represses EPA Genes in Candida glabrata.

Adherence, an important virulence factor, is mediated by the EPA (Epithelial Adhesin) genes in the opportunistic pathogen Candida glabrata Expression of adhesin-encoding genes requires tight regulation to respond to harsh environmental conditions within the host. The majority of EPA genes are localized in subtelomeric regions regulated by subtelomeric silencing, which depends mainly on Rap1 and the Sir proteins. In vitro adhesion to epithelial cells is primarily mediated by Epa1. EPA1 forms a cluster with EPA2 and EPA3 in the right telomere of chromosome E (E-R). This telomere contains a cis-acting regulatory element, the protosilencer Sil2126 between EPA3 and the telomere. Interestingly, Sil2126 is only active in the context of its native telomere. Replacement of the intergenic regions between EPA genes in E-R revealed that cis-acting elements between EPA2 and EPA3 are required for Sil2126 activity when placed 32 kb away from the telomere (Sil@-32kb). Sil2126 contains several putative binding sites for Rap1 and Abf1, and its activity depends on these proteins. Indeed, Sil2126 binds Rap1 and Abf1 at its native position and also when inserted at -32 kb, a silencing-free environment in the parental strain. In addition, we found that Sil@-32kb and Sil2126 at its native position can physically interact with the intergenic regions between EPA1-EPA2 and EPA2-EPA3 respectively, by chromosome conformation capture assays. We speculate that Rap1 and Abf1 bound to Sil2126 can recruit the Silent Information Regulator complex, and together mediate silencing in this region, probably through the formation of a chromatin loop.

P2X7 from j774 murine macrophages acts as a scavenger receptor for bacteria but not yeast.

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.

The oxidative stress response of the opportunistic fungal pathogen Candida glabrata.

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).

Role of glutathione in the oxidative stress response in the fungal pathogen Candida glabrata.

Candida glabrata, an opportunistic fungal pathogen, accounts for 18-26 % of all Candida systemic infections in the US. C. glabrata has a robust oxidative stress response (OSR) and in this work we characterized the role of glutathione (GSH), an essential tripeptide-like thiol-containing molecule required to keep the redox homeostasis and in the detoxification of metal ions. GSH is synthesized from glutamate, cysteine, and glycine by the sequential action of Gsh1 (γ-glutamyl-cysteine synthetase) and Gsh2 (glutathione synthetase) enzymes. We first screened for suppressor mutations that would allow growth in the absence of GSH1 (gsh1∆ background) and found a single point mutation in PRO2 (pro2-4), a gene that encodes a γ-glutamyl phosphate reductase and catalyzes the second step in the biosynthesis of proline. We demonstrate that GSH is important in the OSR since the gsh1∆ pro2-4 and gsh2∆ mutant strains are more sensitive to oxidative stress generated by H2O2 and menadione. GSH is also required for Cadmium tolerance. In the absence of Gsh1 and Gsh2, cells show decreased viability in stationary phase. Furthermore, C. glabrata does not contain Saccharomyces cerevisiae high affinity GSH transporter ortholog, ScOpt1/Hgt1, however, our genetic and biochemical experiments show that the gsh1∆ pro2-4 and gsh2∆ mutant strains are able to incorporate GSH from the medium. Finally, GSH and thioredoxin, which is a second redox system in the cell, are not essential for the catalase-independent adaptation response to H2O2.

Sir3 Polymorphisms in Candida glabrata clinical isolates.

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.