Search

Chromatin architecture has an enormous impact on gene regulation, DNA replication, repair, and packaging. Chromatin is organized in a complex hierarchical manner in which distant fragments of DNA can interact with each other through DNA loops. DNA loops can interact between themselves to form topologically associated domains (TADs) that are further organized into functional compartments. In the last two decades, Chromatin Conformation Capture (3C technology) and its high-throughput derivatives allowed detailed analysis of the chromatin architecture. The 3C method is based on ligation of distant fragments brought together by DNA looping. The method analyzes a particular genomic region of interest and quantifies the interactions between a defined fragment with all the surrounding fragments of the region. It consists of four steps: (1) The long-distance interacting chromatin fragments are fixed with formaldehyde in whole cells which are then lysed; (2) the fixed chromatin is digested with a carefully chosen restriction enzymes to separate intervening DNA fragments; (3) the fragments brought into proximity by DNA looping are ligated in conditions favoring intramolecular ligation; and (4) the interactions are quantified by quantitative PCR using the TaqMan technology and unidirectional primers. Herein, we describe the use of this methodology to analyze the chromatin conformation at a subtelomeric locus containing three genes encoding adhesins and several cis-regulatory elements, in the pathogenic yeast Candida glabrata.

The renal artery is frequently involved in the pathogenesis of vasculorenal diseases, and it is a target in kidney surgery and therapeutic techniques for refractory hypertension. However, few detailed structural studies on the human renal artery have been conducted. Using histocytochemistry, immunohistochemistry, and quantitative image analysis, the wall thickness, structure, smooth muscle cells, extracellular matrix, and proportion of elastic tissue in the tunica media of main human renal arteries were used estimated. Ninety-six tissue samples were collected from sections of the right and left main renal arteries. The results showed that the renal artery changed from an elastic vessel in its proximal segment to a muscular artery in its distal part. A critical characteristic of the renal artery was the presence of longitudinal smooth muscle cell formations in the tunica adventitia of middle and distal segments but not in the proximal part of the artery. In addition, the tunica adventitia of the renal artery showed a rich vascularization and the presence of numerous nerves profiles. The artery's regional structural and morphometric features explain that a particular arterial pathology is more frequent in a specific vessel sector than in others. In addition, those characteristics could determine a different therapeutic response attending to the arterial sector.

Accurate DNA replication and segregation is key to reproduction and cell viability in all organisms. Autonomously replicating sequence-binding factor 1 (Abf1) is a multifunctional protein that has essential roles in replication, transcription, and regional silencing in the model yeast Saccharomyces cerevisiae. In the opportunistic pathogenic fungus Candida glabrata, which is closely related to S. cerevisiae, these processes are important for survival within the host, for example, the regulation of transcription of virulence-related genes like those involved in adherence. Here, we describe that CgABF1 is an essential gene required for cell viability and silencing near the telomeres, where many adhesin-encoding genes reside. CgAbf1 mediated subtelomeric silencing depends on the 43 C-terminal amino acids. We also found that abnormal expression, depletion, or overexpression of Abf1, results in defects in nuclear morphology, nuclear segregation, and transit through the cell cycle. In the absence of ABF1, cells are arrested in G2 but start cycling again after 9 h, coinciding with the loss of cell viability and the appearance of cells with higher DNA content. Overexpression of CgABF1 causes defects in nuclear segregation and cell cycle progression. We suggest that these effects could be due to the deregulation of DNA replication.

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.

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.

A fundamental question in biology is to understand how appropriate transcriptional regulation and dense packaging of the genetic material within the eukaryotic nucleus are achieved. The exquisite gene expression control and other metabolic processes of DNA require a highly complex, multilayered, three-dimensional architecture of the chromatin and its specific compartmentalization within the nucleus. Some of these architectural and sub-nuclear positioning mechanisms have been extensively co-opted by eukaryotic pathogens to keep fine expression control and expansion of virulence-related gene families in Plasmodium falciparum, Trypanosoma brucei and Candida glabrata. For example non-linear interactions between distant cis-acting regions and the formation of chromatin loops are required for appropriate regulation of the expression of virulence-related multi-gene families encoding cell surface proteins. These gene families are located near the chromosome ends and tethered to the nuclear periphery. Consequently, only one or very few genes of the family are expressed at a time. These genes are involved in antigenic variation in parasites and the generation of subpopulations of cells with diverse antigenic proteins at the surface in some pathogenic fungi, making them highly efficient pathogens.

Candida glabrata is the second most common cause of candidemia, and its ability to adhere to different host cell types, to microorganisms, and to medical devices are important virulence factors. Here, we consider three characteristics that confer extraordinary advantages to C. glabrata within the host. (1) C. glabrata has a large number of genes encoding for adhesins most of which are localized at subtelomeric regions. The number and sequence of these genes varies substantially depending on the strain, indicating that C. glabrata can tolerate high genomic plasticity; (2) The largest family of CWPs (cell wall proteins) is the EPA (epithelial adhesin) family of adhesins. Epa1 is the major adhesin and mediates adherence to epithelial, endothelial and immune cells. Several layers of regulation like subtelomeric silencing, cis-acting regulatory regions, activators, nutritional signaling, and stress conditions tightly regulate the expression of many adhesin-encoding genes in C. glabrata, while many others are not expressed. Importantly, there is a connection between acquired resistance to xenobiotics and increased adherence; (3) Other subfamilies of adhesins mediate adherence to Candida albicans, allowing C. glabrata to efficiently invade the oral epithelium and form robust biofilms. It is noteworthy that every C. glabrata strain analyzed presents a unique pattern of CWPs at the cell surface. ispartof: Journal of Fungi vol:4 issue:2 ispartof: location:Switzerland status: published

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.

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.

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.

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.

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.

Genetic deletion of the essential GTPase Gpn1 or replacement of the endogenous gene by partial loss of function mutants in yeast is associated with multiple cellular phenotypes, including in all cases a marked cytoplasmic retention of RNA polymerase II (RNAPII). Global inhibition of RNAPII-mediated transcription due to malfunction of Gpn1 precludes the identification and study of other cellular function(s) for this GTPase. In contrast to the single Gpn protein present in Archaea, eukaryotic Gpn1 possesses an extension of approximately 100 amino acids at the C-terminal end of the GTPase domain. To determine the importance of this C-terminal extension in Saccharomyces cerevisiae Gpn1, we generated yeast strains expressing either C-terminal truncated (gpn1ΔC) or full-length ScGpn1. We found that ScGpn1ΔC was retained in the cell nucleus, an event physiologically relevant as gpn1ΔC cells contained a higher nuclear fraction of the RNAPII CTD phosphatase Rtr1. gpn1ΔC cells displayed an increased size, a delay in mitosis exit, and an increased sensitivity to the microtubule polymerization inhibitor benomyl at the cell proliferation level and two cellular events that depend on microtubule function: RNAPII nuclear targeting and vacuole integrity. These phenotypes were not caused by inhibition of RNAPII, as in gpn1ΔC cells RNAPII nuclear targeting and transcriptional activity were unaffected. These data, combined with our description here of a genetic interaction between GPN1 and BIK1, a microtubule plus-end tracking protein with a mitotic function, strongly suggest that the ScGpn1 C-terminal tail plays a critical role in microtubule dynamics and mitotic progression in an RNAPII-independent manner.

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.

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.

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 an important opportunistic pathogen causing both mucosal and bloodstream infections. C. glabrata is able to adhere avidly to mammalian cells, an interaction that depends on the Epa1p lectin. EPA1 is shown here to be a member of a larger family of highly related genes encoded in subtelomeric clusters. Subtelomeric clustering of large families of surface glycoprotein-encoding genes is a hallmark of several pathogens, including Plasmodium, Trypanosoma, and Pneumocystis. In these other pathogens, a single surface glycoprotein is expressed, whereas other genes in the family are transcriptionally silent. Similarly, whereas EPA1 is expressed in vitro, EPA2-5 are transcriptionally repressed. This repression is shown to be due to regional silencing of the subtelomeric loci. In Saccharomyces cerevisiae, subtelomeric silencing is initiated by Rap1p binding to the telomeric repeats and subsequent recruitment of the Sir complex by protein-protein interaction. We demonstrate here that silencing of the subtelomeric EPA loci also depends on functional Sir3p and Rap1p. This identification and analysis of the EPA gene family provides a compelling example in an ascomycete of chromatin-based silencing of natural subtelomeric genes and provides for the first time in a pathogen, molecular insight into the transcriptional silencing of large subtelomeric gene families.

Candida glabrata (CG) is an opportunistic fungal pathogen that initiates infection by binding to host cells via specific lectin-like adhesin proteins. We have previously shown the importance of lectin-oligosaccharide binding in cardiac responses to flow and agonists. Because of the lectinic-oligosaccharide nature of CG binding, we tested the ability of CG to alter the agonist- and flow-induced changes in cardiac function in isolated perfused guinea pig hearts. Both transmission and scanning electron microscopy showed strong attachment of CG to the coronary endothelium, even after extensive washing. CG shifted the coronary flow vs. auricular-ventricular (AV) delay relationship upward, indicating that greater flow was required to achieve the same AV delay. This effect was completely reversed with mannose, partially reversed with galactose and N-acetylgalactosamine, but hyaluronan had no effect. Western blot analysis was used to determine binding of CG to isolated coronary endothelial luminal membrane (CELM) receptors, and the results indicate that flow-sensitive CELM receptors, ANG II type I, α-adrenergic 1A receptor, endothelin-2, and VCAM-1 bind to CG. In addition, CG inhibited agonist-induced effects of bradykinin, angiotensin, and phenylephrine on AV delay, coronary perfusion pressure, and left ventricular pressure. Mannose reversed the inhibitory effects of CG on the agonist responses. These results suggest that CG directly binds to flow-sensitive CELM receptors via lectinic-oligosaccharide interactions with mannose and disrupts the lectin-oligosaccharide binding necessary for flow-induced cardiac responses.

In fungi, the cell wall plays a primary role contributing to the structural integrity of the cell; in addition to this structural role, the cell wall is, by definition, the interface between the yeast and the environment. This chapter examines the overlapping and divergent function and regulation of some surface glycoprotein gene families in Saccharomyces cerevisiae and its pathogenic cousins Candida glabrata and Candida albicans. S. cerevisiae, C. albicans, and C. glabrata encode a variety of glycosylphosphatidylinositol-linked cell wall proteins (GPI-CWPs) that play accessory roles, functioning primarily as adhesins, facilitating yeast-yeast interactions or yeast adherence to a variety of surfaces. Analysis of adhesin gene regulation in S. cerevisiae and in Candida species has revealed significant overlap in the signals that induce transcription of these gene families as well as the mechanistic basis for that regulation. Analysis of FLO gene transcription reveals unexpected complexity in chromatin regulation in yeast. While the epigenetic regulation of FLO10 and FLO11 expression appears similar and some key regulators (Sfl1) are shared, the requirement for chromatinmodifying proteins which underlie the silencing mechanism is quite different—Hda1 in the case of FLO11 and Hst1-Hst2 and Sir3 in the case of FLO10. This complexity probably foreshadows similar complexity in the regulation of other fungal adhesin families.

[Abstract] Commensal and pathogenic fungi are exposed to hydrogen peroxide (H2O2) produced by macrophages of the host. Pathogenic fungi counteract the harmful effects of H2O2 with the enzyme catalase (EC 1.11.1.6), which decomposes two molecules of H2O2 to two molecules of H2O and O2. Contribution of antioxidant systems on fungal virulence is actively studied. Measurement of catalase activity can contribute to the elucidation of the factors that influence the regulation of this pivotal enzyme. Here we describe a simple spectrophotometric method in which the activity of catalase is measured in total yeast extracts. Decomposition of H2O2 by the yeast extract is followed by the decrease in absorbance at 240 nm. The difference in absorbance through time (A240) is inferred as the measure of catalase activity.

The aim of this study was to document similarities and differences between veins from human diabetic patients and an experimentally induced diabetic animal model. The saphenous vein and posterior tibial vein from diabetic patients and the femoral vein from rats were studied. An increase in the extracellular matrix with migration of smooth muscle cells and endothelial alterations were observed in the intima of all specimens. These findings demonstrate that there is a high degree of similarity between the pathological changes in the venous wall during human diabetes mellitus and streptozotocin (STZ) induced-diabetes. This finding validates STZ induced-diabetes in rats as a model for further experimental study to clarify the fate of the diabetic venous wall when used as a graft.

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.

Purpose To develop a digital image processing method to quantify structural components (smooth muscle fibers and extracellular matrix) in the vessel wall stained with Masson’s trichrome, and a statistical method suitable for small sample sizes to analyze the results previously obtained. Methods The quantification method comprises two stages. The pre-processing stage improves tissue image appearance and the vessel wall area is delimited. In the feature extraction stage, the vessel wall components are segmented by grouping pixels with a similar color. The area of each component is calculated by normalizing the number of pixels of each group by the vessel wall area. Statistical analyses are implemented by permutation tests, based on resampling without replacement from the set of the observed data to obtain a sampling distribution of an estimator. The implementation can be parallelized on a multicore machine to reduce execution time. Results The methods have been tested on 48 vessel wall samples of the internal saphenous vein stained with Masson’s trichrome. The results show that the segmented areas are consistent with the perception of a team of doctors and demonstrate good correlation between the expert judgments and the measured parameters for evaluating vessel wall changes. Conclusion The proposed methodology offers a powerful tool to quantify some components of the vessel wall. It is more objective, sensitive and accurate than the biochemical and qualitative methods traditionally used. The permutation tests are suitable statistical techniques to analyze the numerical measurements obtained when the underlying assumptions of the other statistical techniques are not met.