Glabrata Cells Research Articles

Activity and expression of Candida glabrata vacuolar proteases in autophagy-like conditions.

Candida glabrata is an emerging opportunistic pathogen that has intrinsic resistance to azoles. During infection or while living as a commensal, it encounters nutritional stresses such as deficiency of carbon or nitrogen sources. Herein, we investigate the expression and activity of PrA, Ape1, Ape3 and CpY vacuolar proteases during these stressful nutrimental conditions. Our findings demonstrate a differential activity profile depending on the addition or lack of carbon, nitrogen or both. Of the four proteases tested, PrA and Ape3 showed a higher activity in the absence of nitrogen. Steady-state RNA levels for all the proteases were also differentially expressed although not always correlated with its activity, suggesting multiple levels of regulation. Microscopy observations of C. glabrata cells subjected to the different conditions showed an increase in the vacuolar volume. Moreover, the presence of ATG8-PE and an increased expression of ATG8 were observed in the yeast under the tested conditions suggesting that C. glabrata is in autophagy stage. Taken together, our results showed that PrA, Ape1, Ape3 and CpY have varying activities and expression depending on whether nitrogen or carbon is added to the media, and that these vacuolar proteases might have a role in the autophagy process.

A New Determinant of Candida glabrata Virulence: The Acetate Exporter CgDtr1.

Persistence and virulence of Candida glabrata infections are multifactorial phenomena, whose understanding is crucial to design more suitable therapeutic strategies. In this study, the putative multidrug transporter CgDtr1, encoded by ORF CAGL0M06281g, is identified as a determinant of C. glabrata virulence in the infection model Galleria mellonella. CgDTR1 deletion is shown to decrease the ability to kill G. mellonella larvae by decreasing C. glabrata ability to proliferate in G. mellonella hemolymph, and to tolerate the action of hemocytes. The possible role of CgDtr1 in the resistance to several stress factors that underlie death induced by phagocytosis was assessed. CgDTR1 was found to confer resistance to oxidative and acetic acid stress. Consistently, CgDtr1 was found to be a plasma membrane acetic acid exporter, relieving the stress induced upon C. glabrata cells within hemocytes, and thus enabling increased proliferation and virulence against G. mellonella larvae.

Synthetic antimicrobial peptides of the halictines family disturb the membrane integrity of Candida cells

We compared the potency of four derivatives of the antimicrobial peptide halictine-2 against six Candida species. Observed activity was peptide and species specific. Halictines rapidly permeabilized cell membranes and caused the leakage of cytosolic components. Their killing potential was enhanced by the commercial antimicrobial agent octenidine dihydrochloride. The effect on C. glabrata cells did not depend on the activity of Cdr pumps, but was influenced by their lipid composition. The pre-treatment of cells with myriocin, an inhibitor of sphingolipid synthesis, enhanced the peptides' activity, whereas pre-treatment with terbinafine and fluconazole, inhibitors of sterol synthesis, significantly weakened their efficacy. The killing efficacy of peptides increased in combination with amphotericin B. Thus the mode of action of halictines is likely to depend on the plasma-membrane sterols, which might explain the observed differences among the tested Candida species.

Generational distribution of a Candida glabrata population: Resilient old cells prevail, while younger cells dominate in the vulnerable host.

Similar to other yeasts, the human pathogen Candida glabrata ages when it undergoes asymmetric, finite cell divisions, which determines its replicative lifespan. We sought to investigate if and how aging changes resilience of C. glabrata populations in the host environment. Our data demonstrate that old C. glabrata are more resistant to hydrogen peroxide and neutrophil killing, whereas young cells adhere better to epithelial cell layers. Consequently, virulence of old compared to younger C. glabrata cells is enhanced in the Galleria mellonella infection model. Electron microscopy images of old C. glabrata cells indicate a marked increase in cell wall thickness. Comparison of transcriptomes of old and young C. glabrata cells reveals differential regulation of ergosterol and Hog pathway associated genes as well as adhesion proteins, and suggests that aging is accompanied by remodeling of the fungal cell wall. Biochemical analysis supports this conclusion as older cells exhibit a qualitatively different lipid composition, leading to the observed increased emergence of fluconazole resistance when grown in the presence of fluconazole selection pressure. Older C. glabrata cells accumulate during murine and human infection, which is statistically unlikely without very strong selection. Therefore, we tested the hypothesis that neutrophils constitute the predominant selection pressure in vivo. When we altered experimentally the selection pressure by antibody-mediated removal of neutrophils, we observed a significantly younger pathogen population in mice. Mathematical modeling confirmed that differential selection of older cells is sufficient to cause the observed demographic shift in the fungal population. Hence our data support the concept that pathogenesis is affected by the generational age distribution of the infecting C. glabrata population in a host. We conclude that replicative aging constitutes an emerging trait, which is selected by the host and may even play an unanticipated role in the transition from a commensal to a pathogen state.

Mechanistic Insights Underlying Tolerance to Acetic Acid Stress in Vaginal Candida glabrata Clinical Isolates.

During colonization of the vaginal tract Candida glabrata cells are challenged with the presence of acetic acid at a low pH, specially when dysbiosis occurs. To avoid exclusion from this niche C. glabrata cells are expected to evolve efficient adaptive responses to cope with this stress; however, these responses remain largely uncharacterized, especially in vaginal strains. In this work a cohort of 18 vaginal strains and 2 laboratory strains (CBS138 and KUE100) were phenotyped for their tolerance against inhibitory concentrations of acetic acid at pH 4. Despite some heterogeneity has been observed among the vaginal strains tested, in general these strains were considerably more tolerant to acetic acid than the laboratory strains. To tackle the mechanistic insights behind this differential level of tolerance observed, a set of vaginal strains differently tolerant to acetic acid (VG281∼VG49 < VG99 < VG216) and the highly susceptible laboratory strain KUE100 were selected for further studies. When suddenly challenged with acetic acid the more tolerant vaginal strains exhibited a higher activity of the plasma membrane proton pump CgPma1 and a reduced internal accumulation of the acid, these being two essential features to maximize tolerance. Based on the higher level of resistance exhibited by the vaginal strains against the action of a β-1,3-glucanase, it is hypothesized that the reduced internal accumulation of acetic acid inside these strains may originate from them having a different cell wall structure resulting in a reduced porosity to undissociated acetic acid molecules. Both the vaginal and the two laboratory strains were found to consume acetic acid in the presence of glucose indicating that metabolization of the acid is used by C. glabrata species as a detoxification mechanism. The results gathered in this study advance the current knowledge on the mechanisms underlying the increased competitiveness of C. glabrata in the vaginal tract, a knowledge that can be used to guide more suitable strategies to treat infections caused by this pathogenic yeast.

The multidrug resistance transporters CgTpo1_1 and CgTpo1_2 play a role in virulence and biofilm formation in the human pathogen Candida glabrata.

The mechanisms of persistence and virulence associated with Candida glabrata infections are poorly understood, limiting the ability to fight this fungal pathogen. In this study, the multidrug resistance transporters CgTpo1_1 and CgTpo1_2 are shown to play a role in C. glabrata virulence. The survival of the infection model Galleria mellonella, infected with C. glabrata, was found to increase upon the deletion of either CgTPO1_1 or CgTPO1_2. The underlying mechanisms were further explored. In the case of CgTpo1_1, this phenotype was found to be consistent with the observation that it confers resistance to antimicrobial peptides (AMP), such as the human AMP histatin-5. The deletion of CgTPO1_2, on the other hand, was found to limit the survival of C. glabrata cells when exposed to phagocytosis and impair biofilm formation. Interestingly, CgTPO1_2 expression was found to be up-regulated during biofilm formation, but and its deletion leads to a decreased expression of adhesin-encoding genes during biofilm formation, which is consistent with a role in biofilm formation. CgTPO1_2 expression was further seen to decrease plasma membrane potential and affect ergosterol and fatty acid content. Altogether, CgTpo1_1 and CgTpo1_2 appear to play an important role in the virulence of C. glabrata infections, being at the cross-road between multidrug resistance and pathogenesis.

The CgHaa1-Regulon Mediates Response and Tolerance to Acetic Acid Stress in the Human Pathogen Candida glabrata.

To thrive in the acidic vaginal tract, Candida glabrata has to cope with high concentrations of acetic acid. The mechanisms underlying C. glabrata tolerance to acetic acid at low pH remain largely uncharacterized. In this work, the essential role of the CgHaa1 transcription factor (encoded by ORF CAGL0L09339g) in the response and tolerance of C. glabrata to acetic acid is demonstrated. Transcriptomic analysis showed that CgHaa1 regulates, directly or indirectly, the expression of about 75% of the genes activated under acetic acid stress. CgHaa1-activated targets are involved in multiple physiological functions including membrane transport, metabolism of carbohydrates and amino acids, regulation of the activity of the plasma membrane H+-ATPase, and adhesion. Under acetic acid stress, CgHaa1 increased the activity and the expression of the CgPma1 proton pump and contributed to increased colonization of vaginal epithelial cells by C. glabrata. CgHAA1, and two identified CgHaa1-activated targets, CgTPO3 and CgHSP30, are herein demonstrated to be determinants of C. glabrata tolerance to acetic acid. The protective effect of CgTpo3 and of CgHaa1 was linked to a role of these proteins in reducing the accumulation of acetic acid inside C. glabrata cells. In response to acetic acid stress, marked differences were found in the regulons controlled by CgHaa1 and by its S. cerevisiae ScHaa1 ortholog, demonstrating a clear divergent evolution of the two regulatory networks. The results gathered in this study significantly advance the understanding of the molecular mechanisms underlying the success of C. glabrata as a vaginal colonizer.

Replicative Aging in Candida auris Has an Effect on Antifungal Resistance

Background Candida auris can colonize patients for a prolonged time causing life-threatening systemic infections. Replicative aging is the result of asymmetric division, which cause phenotypic changes between mother and daughter cells. We have previously published that older Cryptococcus neoformans and C. glabrata cells exhibit enhanced resistance to antifungals including fluconazole (FLC), five flucytosine (5FC), and sub-therapeutic amphotericin B (AMB). Additionally, they are more resistant to macrophage and neutrophil killing. This is relevant because older fungal cells accumulate in chronic infections. We hypothesized that older C. auris cells would also exhibit enhanced resistance to phagocytic cells and can alter drug resistance.Methods Magnetically labeled cells were grown for 10 generations (Gen) and then were isolated from daughter cells by magnetic column separation. Transmission electron microscopy (TEM) was performed to measure their cell wall thickness. Standard in vitro killing assays were performed with human neutrophils. In vivo virulence was compared in a Galleria injection model. Minimum inhibitory concentration to the antifungals was performed by CSLI methods. Rhodamine 6G (R6G), which is a fluorescent substrate for ABC-transporters, was used to measure efflux.ResultsIn C. auris, 10 Gen old cells are larger than younger cells (0–3 Gen) and exhibit a thicker cell wall on TEM. The older cells are significantly more resistant to neutrophil killing (24.5% vs. 51.6%, P < 0.05 by paired t-test)). Eleven distinct isolates manifest variable virulence in the Galleria infection model (range 1–8 days). In C. auris, 10 Gen cells were significantly more virulent than 0 Gen cells (2 days vs. 4 days). Most (8 of 11) isolates were highly resistant to FLC and showed high efflux of R6G, whereas the three FLC sensitive isolates showed low efflux of R6G. Using these methods, we observed significant (1.5 to 2.0 fold, P < 0.05) increase in efflux activity of older cells in both sensitive and resistant isolates.Conclusion In C. auris replicative aging is associated with enhanced resistance to phagocytic killing, increased cell body size, and cell wall thickness. Aging also enhances efflux activity and thus may contribute even further to antifungal resistance.Disclosures All authors: No reported disclosures.

The Phosphoinositide 3-Kinase Regulates Retrograde Trafficking of the Iron Permease CgFtr1 and Iron Homeostasis in Candida glabrata

The phosphoinositide 3-kinase (PI3K), which phosphorylates phosphatidylinositol and produces PI3P, has been implicated in protein trafficking, intracellular survival, and virulence in the pathogenic yeast Candida glabrata. Here, we demonstrate PI3-kinase (CgVps34) to be essential for maintenance of cellular iron homeostasis. We examine how CgVps34 regulates the fundamental process of iron acquisition, and underscore its function in vesicular trafficking as a central determinant. RNA sequencing analysis revealed iron homeostasis genes to be differentially expressed upon CgVps34 disruption. Consistently, the Cgvps34Δ mutant displayed growth attenuation in low- and high-iron media, increased intracellular iron content, elevated mitochondrial aconitase activity, impaired biofilm formation, and extenuated mouse organ colonization potential. Furthermore, we demonstrate for the first time that C. glabrata cells respond to iron limitation by expressing the iron permease CgFtr1 primarily on the cell membrane, and to iron excess via internalization of the plasma membrane-localized CgFtr1 to the vacuole. Our data show that CgVps34 is essential for the latter process. We also report that macrophage-internalized C. glabrata cells express CgFtr1 on the cell membrane indicative of an iron-restricted macrophage internal milieu, and Cgvps34Δ cells display better survival in iron-enriched medium-cultured macrophages. Overall, our data reveal the centrality of PI3K signaling in iron metabolism and host colonization.

Iron-depletion promotes mitophagy to maintain mitochondrial integrity in pathogenic yeast Candida glabrata

ABSTRACTCandida glabrata, a haploid budding yeast, is the cause of severe systemic infections in immune-compromised hosts. The amount of free iron supplied to C. glabrata cells during systemic infections is severely limited by iron-chelating proteins such as transferrin. Thus, the iron-deficiency response in C. glabrata cells is thought to play important roles in their survival inside the host's body. In this study, we found that mitophagy was induced under iron-depleted conditions, and that the disruption of a gene homologous to ATG32, which is responsible for mitophagy in Saccharomyces cerevisiae, blocked mitophagy in C. glabrata. The mitophagic activity in C. glabrata cells was not detected on short-period exposure to nitrogen-starved conditions, which is a mitophagy-inducing condition used in S. cerevisiae. The mitophagy-deficient atg32Δ mutant of C. glabrata also exhibited decreased longevity under iron-deficient conditions. The mitochondrial membrane potential in Cgatg32Δ cells was significantly lower than that in wild-type cells under iron-depleted conditions. In a mouse model of disseminated infection, the Cgatg32Δ strain resulted in significantly decreased kidney and spleen fungal burdens compared with the wild-type strain. These results indicate that mitophagy in C. glabrata occurs in an iron-poor host tissue environment, and it may contribute to the longevity of cells, mitochondrial quality control, and pathogenesis.

Candida glabrata survives and replicates in human osteoblasts.

Candida glabrata is an opportunistic pathogen that is considered the second most common cause of candidiasis after Candida albicans Many characteristics of its mechanisms of pathogenicity remain unknown. Recent studies have focused on determining the events that underlie interactions between C. glabrata and immune cells, but the relationship between this yeast and osteoblasts has not been studied in detail. The aim of this study was to determine the mechanisms of interaction between human osteoblasts and C. glabrata, and to identify the roles played by some of the molecules that are produced by these cells in response to infection. We show that C. glabrata adheres to and is internalized by human osteoblasts. Adhesion is independent of opsonization, and internalization depends on the rearrangement of the actin cytoskeleton. We show that C. glabrata survives and replicates in osteoblasts and that this intracellular behavior is related to the level of production of nitric oxide and reactive oxygen species. Opsonized C. glabrata stimulates the production of IL-6, IL-8 and MCP-1 cytokines. Adhesion and internalization of the pathogen and the innate immune response of osteoblasts require viable C. glabrata These results suggest that C. glabrata modulates immunological mechanisms in osteoblasts to survive inside the cell.

Potassium Uptake Mediated by Trk1 Is Crucial for Candida glabrata Growth and Fitness.

The maintenance of potassium homeostasis is crucial for all types of cells, including Candida glabrata. Three types of plasma-membrane systems mediating potassium influx with different transport mechanisms have been described in yeasts: the Trk1 uniporter, the Hak cation-proton symporter and the Acu ATPase. The C. glabrata genome contains only one gene encoding putative system for potassium uptake, the Trk1 uniporter. Therefore, its importance in maintaining adequate levels of intracellular potassium appears to be critical for C. glabrata cells. In this study, we first confirmed the potassium-uptake activity of the identified gene’s product by heterologous expression in a suitable S. cerevisiae mutant, further we generated a corresponding deletion mutant in C. glabrata and analysed its phenotype in detail. The obtained results show a pleiotropic effect on the cell physiology when CgTRK1 is deleted, affecting not only the ability of trk1Δ to grow at low potassium concentrations, but also the tolerance to toxic alkali-metal cations and cationic drugs, as well as the membrane potential and intracellular pH. Taken together, our results find the sole potassium uptake system in C. glabrata cells to be a promising target in the search for its specific inhibitors and in developing new antifungal drugs.

Membrane Proteome-Wide Response to the Antifungal Drug Clotrimazole in Candida glabrata: Role of the Transcription Factor CgPdr1 and the Drug:H+ Antiporters CgTpo1_1 and CgTpo1_2

Azoles are widely used antifungal drugs. This family of compounds includes triazoles, mostly used in the treatment of systemic infections, and imidazoles, such as clotrimazole, often used in the case of superficial infections. Candida glabrata is the second most common cause of candidemia worldwide and presents higher levels of intrinsic azole resistance when compared with Candida albicans, thus being an interesting subject for the study of azole resistance mechanisms in fungal pathogens.Since resistance often relies on the action of membrane transporters, including drug efflux pumps from the ATP-binding cassette family or from the Drug:H(+) antiporter (DHA)(1) family, an iTRAQ-based membrane proteomics analysis was performed to identify all the membrane-associated proteins whose abundance changes in C. glabrata cells exposed to the azole drug clotrimazole. Proteins found to have significant expression changes in this context were clustered into functional groups, namely: glucose metabolism, oxidative phosphorylation, mitochondrial import, ribosome components and translation machinery, lipid metabolism, multidrug resistance transporters, cell wall assembly, and stress response, comprising a total of 37 proteins. Among these, the DHA transporter CgTpo1_2 (ORF CAGL0E03674g) was identified as overexpressed in the C. glabrata membrane in response to clotrimazole. Functional characterization of this putative drug:H(+) antiporter, and of its homolog CgTpo1_1 (ORF CAGL0G03927g), allowed the identification of these proteins as localized to the plasma membrane and conferring azole drug resistance in this fungal pathogen by actively extruding the drug to the external medium. The cell wall protein CgGas1 was also shown to confer azole drug resistance through cell wall remodeling. Finally, the transcription factor CgPdr1 in the clotrimazole response was observed to control the expression of 20 of the identified proteins, thus highlighting the existence of additional unforeseen targets of this transcription factor, recognized as a major regulator of azole drug resistance in clinical isolates.

Candida glabrata susceptibility to antifungals and phagocytosis is modulated by acetate.

Candida glabrata is considered a major opportunistic fungal pathogen of humans. The capacity of this yeast species to cause infections is dependent on the ability to grow within the human host environment and to assimilate the carbon sources available. Previous studies have suggested that C. albicans can encounter glucose-poor microenvironments during infection and that the ability to use alternative non-fermentable carbon sources, such as carboxylic acids, contributes to the virulence of this fungus. Transcriptional studies on C. glabrata cells identified a similar response, upon nutrient deprivation. In this work, we aimed at analyzing biofilm formation, antifungal drug resistance, and phagocytosis of C. glabrata cells grown in the presence of acetic acid as an alternative carbon source. C. glabrata planktonic cells grown in media containing acetic acid were more susceptible to fluconazole and were better phagocytosed and killed by macrophages than when compared to media lacking acetic acid. Growth in acetic acid also affected the ability of C. glabrata to form biofilms. The genes ADY2a, ADY2b, FPS1, FPS2, and ATO3, encoding putative carboxylate transporters, were upregulated in C. glabrata planktonic and biofilm cells in the presence of acetic acid. Phagocytosis assays with fps1 and ady2a mutant strains suggested a potential role of FPS1 and ADY2a in the phagocytosis process. These results highlight how acidic pH niches, associated with the presence of acetic acid, can impact in the treatment of C. glabrata infections, in particular in vaginal candidiasis.

New Mechanisms of Flucytosine Resistance in C. glabrata Unveiled by a Chemogenomics Analysis in S. cerevisiae.

5-Flucytosine is currently used as an antifungal drug in combination therapy, but fungal pathogens are rapidly able to develop resistance against this drug, compromising its therapeutic action. The understanding of the underlying resistance mechanisms is crucial to deal with this problem. In this work, the S. cerevisiae deletion mutant collection was screened for increased resistance to flucytosine. Through this chemogenomics analysis, 183 genes were found to confer resistance to this antifungal agent. Consistent with its known effect in DNA, RNA and protein synthesis, the most significant Gene Ontology terms over-represented in the list of 5-flucytosine resistance determinants are related to DNA repair, RNA and protein metabolism. Additional functional classes include carbohydrate and nitrogen—particularly arginine—metabolism, lipid metabolism and cell wall remodeling. Based on the results obtained for S. cerevisiae as a model system, further studies were conducted in the pathogenic yeast Candida glabrata. Arginine supplementation was found to relieve the inhibitory effect exerted by 5-flucytosine in C. glabrata. Lyticase susceptibility was found to increase within the first 30min of 5-flucytosine exposure, suggesting this antifungal drug to act as a cell wall damaging agent. Upon exponential growth resumption in the presence of 5-flucytosine, the cell wall exhibited higher resistance to lyticase, suggesting that cell wall remodeling occurs in response to 5-flucytosine. Additionally, the aquaglyceroporin encoding genes CgFPS1 and CgFPS2, from C. glabrata, were identified as determinants of 5-flucytosine resistance. CgFPS1 and CgFPS2 were found to mediate 5-flucytosine resistance, by decreasing 5-flucytosine accumulation in C. glabrata cells.

Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq.

Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein's distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C. glabrata cells (the calibration genome) with S. cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio – OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this ‘internal standard’ calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using qPCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics.

Exogenous folates stimulate growth and budding of Candida glabrata.

Folate, vitamin B9, is well recognized as being essential for cell growth. The utilization of folate is common to all cells, but the source of it may be quite different. For example, mammalian cells depend on exogenous uptake of folates, while plants and microbes can synthesize them. There has been little consideration of uptake of folate in microbial cells, and studies on the effects of folates in mammalian cells, where conditions are restricted. This study shows that exogenous folates (folic acid or folinic acid), causes Candida glabrata cells suspended in water alone to undergo two cycles of cell division and to form multiple buds. The effect was limited to cells in the stationary phase and more profound in quiescent cells. These data indicate a novel response of yeast to folates that may increase the utility of yeast as a model to study folate transport and signaling.

The mitogen-activated protein kinase CgHog1 is required for iron homeostasis, adherence and virulence in Candida glabrata.

Candida glabrata has emerged as a major fungal pathogen over the last two decades, although our understanding of its survival strategies inside the mammalian host remains rudimentary. An important requirement for survival in vivo is the ability to acquire critical nutrients such as iron from host niches of varied iron content. In the present study, we demonstrate for the first time that C. glabrata cells respond to high external iron levels via activation of two stress-responsive mitogen-activated protein kinases, CgHog1 and CgSlt2, and lack of either kinase results in sensitivity to the high-iron medium. Furthermore, we show that CgHOG1 deletion led to perturbed iron homeostasis (elevated intracellular iron content and high mitochondrial aconitase activity), reduced survival in macrophages and attenuated virulence in the murine model of disseminated candidiasis. Consistently, several genes implicated in iron acquisition and storage displayed deregulated expression in the Cghog1∆ mutant. Genome-wide transcriptional profiling analysis revealed upregulation of genes implicated in DNA repair, RNA processing and autophagy, and downregulation of genes related to cellular respiration and organonitrogen compound metabolism under iron-limiting conditions. In contrast, genes involved in the respiratory electron transport chain were induced under iron-replete conditions. Gene expression microarrays also identified a set of iron-responsive regulon in C. glabrata. Lastly, we present evidence for the iron-regulated expression of the major adhesin-encoding EPA1 gene, decreased histone deacetylase activity in a high-iron environment and increased adherence of iron-surplus-medium-grown C. glabrata cells to epithelial cells. Together, our findings yield novel insights into iron abundance-based regulation of transcriptional and mitogen-activated protein kinase signaling pathways in C. glabrata.

The superoxide dismutases of Candida glabrata protect against oxidative damage and are required for lysine biosynthesis, DNA integrity and chronological life survival.

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.

An essential role for phosphatidylinositol 3-kinase in the inhibition of phagosomal maturation, intracellular survival and virulence in Candida glabrata.

The yeast class III phosphoinositide 3-kinase (PI3K) that catalyses production of the lipid signalling molecule, phosphatidylinositol-3-phosphate, is primarily implicated in vesicle-mediated transport and autophagy. In this study, we identified, through a genetic screen, the Candida glabrata CgVPS15 gene, an orthologue of the Saccharomyces cerevisiae PI3K regulatory subunit-encoding open reading frame (ORF) to be required for impairment of phagosomal maturation in human macrophages. We also disrupted catalytic subunit of the C. glabrata PI3K complex, CgVps34, and found it to be pivotal to arrest mature phagolysosome biogenesis. Further, deletion of either CgVPS15 or CgVPS34 rendered C. glabrata cells hyperadherent to epithelial cells and susceptible to the antimicrobial arsenal of primary murine and cultured human macrophages and diverse stresses. Despite no growth retardation at 37°C, Cgvps15Δ and Cgvps34Δ mutants were severely virulence attenuated in mice. We demonstrate that trafficking and/or processing of the vacuolar lumenal hydrolase, carboxypeptidase Y, and the major adhesin, Epa1, rely on PI3K regulatory mechanisms in C. glabrata. By disrupting autophagy-related PI3K complex genes, we show that C. glabrata PI3K-impeded phagolysosomal acidification is primarily owing to its role in cellular trafficking events. Altogether, our findings underscore the essentiality of PI3K signalling in modulation of host immune response, intracellular survival and virulence in C. glabrata.