The Hog1 MAPK substrate governs Candida glabrata-epithelial cell adhesion via the histone H2A variant.

CgHog1, terminal kinase of the high-osmolarity glycerol signalling pathway, orchestrates cellular response to multiple external stimuli including surplus-environmental iron in the human fungal pathogen Candida glabrata (Cg). However, CgHog1 substrates remain unidentified. Here, we show that CgHog1 adversely affects Cg adherence to host stomach and kidney epithelial cells in vitro, but promotes Cg survival in the iron-rich gastrointestinal tract niche. Further, CgHog1 interactome and in vitro phosphorylation analysis revealed CgSub2 (putative RNA helicase) to be a CgHog1 substrate, with CgSub2 also governing iron homeostasis and host adhesion. CgSub2 positively regulated EPA1 (encodes a major adhesin) expression and host adherence via its interactor CgHtz1 (histone H2A variant). Notably, both CgHog1 and surplus environmental iron had a negative impact on CgSub2-CgHtz1 interaction, with CgHTZ1 or CgSUB2 deletion reversing the elevated adherence of Cghog1Δ to epithelial cells. Finally, the surplus-extracellular iron led to CgHog1 activation, increased CgSub2 phosphorylation, elevated CgSub2-CgHta (canonical histone H2A) interaction, and EPA1 transcriptional activation, thereby underscoring the iron-responsive, CgHog1-induced exchange of histone partners of CgSub2. Altogether, our work mechanistically defines how CgHog1 couples Epa1 adhesin expression with iron abundance, and point towards specific chromatin composition modification programs that probably aid fungal pathogens align their adherence to iron-rich (gut) and iron-poor (blood) host niches.

Diverse mechanisms control amino acid-dependent environmental alkalization by Candida albicans.

Candida albicans has the capacity to neutralize acidic growth environments by releasing ammonia derived from the catabolism of amino acids. The molecular components underlying alkalization and its physiological significance remain poorly understood. Here, we present an integrative model with the cytosolic NAD+-dependent glutamate dehydrogenase (Gdh2) as the principal ammonia-generating component. We show that alkalization is dependent on the SPS-sensor-regulated transcription factor STP2 and the proline-responsive activator Put3. These factors function in parallel to derepress GDH2 and the two proline catabolic enzymes PUT1 and PUT2. Consistently, a double mutant lacking STP2 and PUT3 exhibits a severe alkalization defect that nearly phenocopies that of a gdh2-/- strain. Alkalization is dependent on mitochondrial activity and in wild-type cells occurs as long as the conditions permit respiratory growth. Strikingly, Gdh2 levels decrease and cells transiently extrude glutamate as the environment becomes more alkaline. Together, these processes constitute a rudimentary regulatory system that counters and limits the negative effects associated with ammonia generation. These findings align with Gdh2 being dispensable for virulence, and based on a whole human blood virulence assay, the same is true for C. glabrata and C. auris. Using a transwell co-culture system, we observed that the growth and proliferation of Lactobacillus crispatus, a common component of the acidic vaginal microenvironment and a potent antagonist of C. albicans, is unaffected by fungal-induced alkalization. Consequently, although Candida spp. can alkalinize their growth environments, other fungal-associated processes are more critical in promoting dysbiosis and virulent fungal growth.

Decoding the role of oxidative stress resistance and alternative carbon substrate assimilation in the mature biofilm growth mode of Candida glabrata.

BACKGROUND: Biofilm formation is viewed as a vital mechanism in C. glabrata pathogenesis. Although, it plays a significant role in virulence but transcriptomic architecture and metabolic pathways governing the biofilm growth mode of C. glabrata remain elusive. The present study intended to investigate the genes implicated in biofilm growth phase of C. glabrata through global transcriptomic approach. RESULTS: Functional analysis of Differentially expressed genes (DEGs) using gene ontology and pathways analysis revealed that upregulated genes are involved in the glyoxylate cycle, carbon-carbon lyase activity, pre-autophagosomal structure membrane and vacuolar parts whereas, down- regulated genes appear to be associated with glycolysis, ribonucleoside biosynthetic process, ribosomal and translation process in the biofilm growth condition. The RNA-Seq expression of eight selected DEGs (CgICL1, CgMLS1, CgPEP1, and CgNTH1, CgERG9, CgERG11, CgTEF3, and CgCOF1) was performed with quantitative real-time PCR (RT-qPCR). The gene expression profile of selected DEGs with RT-qPCR displayed a similar pattern of expression as observed in RNA-Seq. Phenotype screening of mutant strains generated for genes CgPCK1 and CgPEP1, showed that Cgpck1∆ failed to grow on alternative carbon substrate (Glycerol, Ethanol, Oleic acid) and similarly, Cgpep1∆ unable to grow on YPD medium supplemented with hydrogen peroxide. Our results suggest that in the absence of glucose, C. glabrata assimilate glycerol, oleic acid and generate acetyl coenzyme-A (acetyl-CoA) which is a central and connecting metabolite between catabolic and anabolic pathways (glyoxylate and gluconeogenesis) to produce glucose and fulfil energy requirements. CONCLUSIONS: The study was executed using various approaches (transcriptomics, functional genomics and gene deletion) and it revealed that metabolic plasticity of C. glabrata (NCCPF-100,037) in biofilm stage modulates its virulence and survival ability to counter the stress and may promote its transition from commensal to opportunistic pathogen. The observations deduced from the present study along with future work on characterization of the proteins involved in this intricate process may prove to be beneficial for designing novel antifungal strategies.

Purification and characterization of Cdr1, the drug-efflux pump conferring azole resistance in Candida species.

Candida albicans and C. glabrata express exporters of the ATP-binding cassette (ABC) superfamily and address them to their plasma membrane to expel azole antifungals, which cancels out their action and allows the yeast to become multidrug resistant (MDR). In a way to understand this mechanism of defense, we describe the purification and characterization of Cdr1, the membrane ABC exporter mainly responsible for such phenotype in both species. Cdr1 proteins were functionally expressed in the baker yeast, tagged at their C-terminal end with either a His-tag for the glabrata version, cgCdr1-His, or a green fluorescent protein (GFP) preceded by a proteolytic cleavage site for the albicans version, caCdr1-P-GFP. A membrane Cdr1-enriched fraction was then prepared to assay several detergents and stabilizers, probing their level of extraction and the ATPase activity of the proteins as a functional marker. Immobilized metal-affinity and size-exclusion chromatographies (IMAC, SEC) were then carried out to isolate homogenous samples. Overall, our data show that although topologically and phylogenetically close, both proteins display quite distinct behaviors during the extraction and purification steps, and qualify cgCdr1 as a good candidate to characterize this type of proteins for developing future inhibitors of their azole antifungal efflux activity.

Complement receptor 3-dependent engagement by Candida glabrata ß-glucan modulates dendritic cells to induce regulatory T-cell expansion.

Candida glabrata is an important pathogen causing invasive infection associated with a high mortality rate. One mechanism that causes the failure of Candida eradication is an increase in regulatory T cells (Treg), which play a major role in immune suppression and promoting Candida pathogenicity. To date, how C. glabrata induces a Treg response remains unclear. Dendritic cells (DCs) recognition of fungi provides the fundamental signal determining the fate of the T-cell response. This study investigated the interplay between C. glabrata and DCs and its effect on Treg induction. We found that C. glabrata ß-glucan was a major component that interacted with DCs and consequently mediated the Treg response. Blocking the binding of C. glabrata ß-glucan to dectin-1 and complement receptor 3 (CR3) showed that CR3 activation in DCs was crucial for the induction of Treg. Furthermore, a ligand-receptor binding assay showed the preferential binding of C. glabrata ß-glucan to CR3. Our data suggest that C. glabrata ß-glucan potentially mediates the Treg response, probably through CR3-dependent activation in DCs. This study contributes new insights into immune modulation by C. glabrata that may lead to a better design of novel immunotherapeutic strategies for invasive C. glabrata infection.

The secreted acid trehalase encoded by the CgATH1 gene is involved in Candida glabrata virulence

BACKGROUND Candida glabrata yeast is the second cause of candidiasis worldwide. Differs from other yeasts since assimilates only glucose and trehalose (a characteristic used in rapid identification tests for this pathogen) by secreting into the medium a highly active acid trehalase encoded by the CgATH1 gene. OBJECTIVE This study aimed to characterise the function of the acid trehalase in the physiopathology of C. glabrata. METHODS Gene deletion was performed to obtain a mutant ath1Δ strain, and the ability of the ath1Δ strain to grow in trehalase, or the presence of trehalase activity in the ath1Δ yeast cells, was verified. We also tested the virulence of the ath1Δ strain in a murine model of infection. FINDINGS The ath1Δ mutant strain grows normally in the presence of glucose, but loses its ability to grow in trehalose. Due to the high acid trehalase activity present in wild-type cells, the cytoplasmic neutral trehalase activity is only detected in the ath1Δ strain. We also observed a significantly lower virulence of the ath1Δ strain in a murine model of infection with either normal or immunocompromised mice. MAIN CONCLUSIONS The acid trehalase is involved in the hydrolysis of external trehalose by C. glabrata, and the enzyme also plays a major virulence role during infectivity.

Characterisation of an ABC transporter of a resistant Candida glabrata clinical isolate

BACKGROUND Candida glabrata ranks second in epidemiological surveillance studies, and is considered one of the main human yeast pathogens. Treatment of Candida infections represents a contemporary public health problem due to the limited availability of an antifungal arsenal, toxicity effects and increasing cases of resistance. C. glabrata presents intrinsic fluconazole resistance and is a significant concern in clinical practice and in hospital environments. OBJECTIVE The aim of this study was to characterise the azole resistance mechanism presented by a C. glabrata clinical isolate from a Brazilian university hospital. METHODS Azole susceptibility assays, chemosensitisation, flow cytometry and mass spectrometry were performed. FINDINGS Our study demonstrated extremely high resistance to all azoles tested: fluconazole, voriconazole, posaconazole and itraconazole. This isolate was chemosensitised by FK506, a classical inhibitor of ABC transporters related to azole resistance, and Rhodamine 6G extrusion was observed. A mass spectrometry assay confirmed the ABC protein identification suggesting the probable role of efflux pumps in this resistance phenotype. MAIN CONCLUSIONS This study emphasizes the importance of ABC proteins and their relation to the resistance mechanism in hospital environments and they may be an important target for the development of compounds able to unsettle drug extrusion.

The oxidative stress response of the opportunistic fungal pathogen Candida glabrata / Respuesta al estrés oxidante en el hongo patógeno oportunista 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) (AU)

Los microorganismos han establecido diferentes estrategias para controlar el estrés oxidante generado durante la respiración aeróbica y, por consiguiente, mantener la homeostasia redox en la célula. En particular, los hongos patógenos se exponen a especies reactivas del oxígeno cuando interactúan con las células fagocíticas del huésped que son la primera línea de defensa contra estos agentes infecciosos. Estos patógenos han reclutado sistemas enzimáticos (catalasas, superóxido dismutasas y peroxidasas) y no enzimáticos (glutatión) que normalmente utilizan para mantener la homeostasis redox en la célula, para resistir frente al estrés oxidante y garantizar la supervivencia dentro del huésped. Varios factores de virulencia se han relacionado con la respuesta al estrés oxidante de los hongos patógenos. El hongo patógeno oportunista Candida glabrata (C. glabrata) es la segunda causa más frecuente de candidiasis después de Candida albicans (C. albicans). C. glabrata tiene una respuesta bien definida al estrés oxidante, que incluye sistemas enzimáticos y no enzimáticos y está regulada por los factores de transcripción Yap1, Skn7, Msn2 y Msn4. En esta revisión, describimos los elementos de la respuesta de C. glabrata a dicho estrés, cómo se regula y cómo C. glabrata interacciona con el huésped.Este artículo forma parte de una serie de estudios presentados en el «V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi» (Oaxaca, México, 2012) (AU)

Oxidative stress response to menadione and cumene hydroperoxide in the opportunistic fungal pathogen Candida glabrata

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.