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Candida glabrata (Nakaseomyces glabrata) is an emergent and opportunistic fungal pathogen that colonizes and persists in different niches within its human host. In this work, we studied five clinical isolates from one patient (P7), that have a clonal origin, and all of which come from blood cultures except one, P7-3, obtained from a urine culture. We found phenotypic variation such as sensitivity to high temperature, oxidative stress, susceptibility to two classes of antifungal agents, and cell wall porosity. Only isolate P7-3 is highly resistant to the echinocandin caspofungin while the other four isolates from P7 are sensitive. However, this same isolate P7-3, is the only one that displays susceptibility to fluconazole (FLC), while the rest of the isolates are resistant to this antifungal. We sequenced the PDR1 gene which encodes a transcription factor required to induce the expression of several genes involved in the resistance to FLC and found that all the isolates encode for the same Pdr1 amino acid sequence except for the last isolate P7-5, which contains a single amino acid change, G1099C in the putative Pdr1 transactivation domain. Consistent with the resistance to FLC, we found that the CDR1 gene, encoding the main drug efflux pump in C. glabrata, is highly overexpressed in the FLC-resistant isolates, but not in the FLC-sensitive P7-3. In addition, the resistance to FLC observed in these isolates is dependent on the PDR1 gene. Additionally, we found that all P7 isolates have a different proportion of cell wall carbohydrates compared to our standard strains CBS138 and BG14. In P7 isolates, mannan is the most abundant cell wall component, whereas ß-glucan is the most abundant component in our standard strains. Consistently, all P7 isolates have a relatively low cell wall porosity compared to our standard strains. These data show phenotypic and genotypic variability between clonal isolates from different niches within a single host, suggesting microevolution of C. glabrata during an infection.

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Inflammatory and antimicrobial diseases constitute a major burden for society, and fighting them is a WHO strategic priority. Most of the treatments available to fight inflammatory diseases are anti-inflammatory drugs, such as corticosteroids or immunomodulators that lack cellular specificity and lead to numerous side effects. In addition to suppressing undesired inflammation and reducing disease progression, these drugs lessen the immune system protective functions. Furthermore, treating infectious diseases is more and more challenging due to the rise of microbial resistance to antimicrobial drugs. Thus, controlling the inflammatory process locally without compromising the ability to combat infections is an essential feature in the treatment of inflammatory diseases. We isolated three forms (DRS-DA2N, DRS-DA2NE, and DRS-DA2NEQ) of the same peptide, DRS-DA2, which belongs to the dermaseptin family, from the Mexican tree frog Pachymedusa dacnicolor. Interestingly, DRS-DA2N and DRS-DA2NEQ exhibit a dual activity by inducing the death of leukocytes as well as that of Gram-negative and Gram-positive bacteria, including multiresistant strains, without affecting other cells such as epithelial cells or erythrocytes. We showed that the death of both immune cells and bacteria is induced rapidly by DRS-DA2 and that the membrane is permeabilized, leading to the loss of membrane integrity. We also validated the capacity of DRS-DA2 to regulate the pool of inflammatory cells in vivo in a mouse model of noninfectious peritonitis. After the induction of peritonitis, a local injection of DRS-DA2N could decrease the number of inflammatory cells locally in the peritoneal cavity without inducing a systemic effect, as no changes in the number of inflammatory cells could be detected in blood or in the bone marrow. Collectively, these data suggest that this peptide could be a promising tool in the treatment of inflammatory diseases, such as inflammatory skin diseases, as it could reduce the number of inflammatory cells locally without suppressing the ability to combat infections.

RESUMO

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.

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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+-dependent histone deacetylase), Sum1 (putative DNA binding protein) and Rfm1 (connector protein) form a complex (HRS-C) and control the resistance to oxidative stress, to xenobiotics (the antifungal fluconazole), and adherence to epithelial cells. Hst1 is functionally conserved within the Saccharomycetaceae family, Rfm1 shows a close phylogenetic relation within the Saccharomycetaceae family while Sum1 displays a distant phylogenetic relation with members of the family and is not conserved functionally. CDR1 encodes for an ABC transporter (resistance to fluconazole) negatively controlled by HRS-C, for which its binding site is located within 223 bp upstream from the ATG of CDR1. The absence of Hst1 and Sum1 renders the cells hyper-adherent, possibly due to the overexpression of AED1, EPA1, EPA22 and EPA6, all encoding for adhesins. Finally, in a neutrophil survival assay, HST1 and SUM1, are not required for survival. We propose that Sum1 in the HRS-C diverged functionally to control a set of genes implicated in virulence: adherence, resistance to xenobiotics and oxidative stress.

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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.

RESUMO

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, 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.

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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.

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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.

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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.

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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.