Clumping Morphology Influences Virulence Uncoupled from Echinocandin Resistance in Candida glabrata.

Here, we report two paired sets of an index wild-type Candida glabrata bloodstream isolate and subsequent echinocandin-resistant FKS mutant. One paired set exhibited a higher proportion of clumping cells and was more virulent in the invertebrate host Galleria mellonella than the other paired set. No virulence difference between the paired index and FKS strains was observed. These findings imply a potential link of clumping morphology with virulence in C. glabrata that is uncoupled from FKS-mediated echinocandin resistance. IMPORTANCE Candida glabrata is a leading cause of invasive candidiasis. In contrast to other species, it has a high propensity for developing resistance to echinocandins, which are the first-line treatment. Unlike the dimorphic Candida albicans which can grow invasive filamentous hyphae, C. glabrata lacks this ability. Here, we report a link between virulence and clumping cell morphology in two different sets of clinical C. glabrata strains obtained from patients failing echinocandin therapy. One set of paired strains (echinocandin-susceptible and subsequent resistant mutant) had a high proportion of clumping cells in the population and were significantly more virulent than another set which had fewer clumping cells. Additionally, we corroborate that echinocandin resistance does not impart a significant fitness cost. Our findings suggest that clumping morphology may be an important but previously underestimated virulence factor for C. glabrata and also aid our understand for the high prevalence of resistance observed in this species.

Synergistic interaction of fluconazole/sodium bicarbonate on the inhibition of Candida glabrata phospholipase gene

Abstract Candida glabrata infections are responsible for deaths of people globally. Fluconazole is known to be less effective against C. glabrata, which developed many strategies to evade being destroyed by fluconazole. To achieve enhanced efficacy of fluconazole against C. glabrata, the interaction of fluconazole with sodium bicarbonate was investigated using the CLSI guidelines. The efficacy of fluconazole alone and in combination with sodium bicarbonate was evaluated using the time-kill and phospholipase production assays. Eventually, the expression of PLB was assessed using semi-quantitative RT-PCR to investigate the inhibitory properties of fluconazole alone and in combination with sodium bicarbonate against C. glabrata. The fluconazole/sodium bicarbonate combination displayed synergistic and antagonistic effects (FICI= 0.375-4.25). In C. glabrata ATCC, SN 152, and SN 164, the fluconazole/sodium bicarbonate combination exhibited a significant fungicidal activity (p< 0.05) but antagonistic effect in the case of SN 283. With exception of SN 283, a significant reduction was noted in phospholipase production in clinical isolates of C. glabrata treated with fluconazole/sodium bicarbonate combination. The PLB was down-regulated significantly by 0.168-0.515 fold in C. glabrata treated with fluconazole/sodium bicarbonate. The results suggested fluconazole/sodium bicarbonate to have a potential synergistic interaction in C. glabrata, and the underlying mechanism may be associated with phospholipase gene

A novel class of Candida glabrata cell wall proteins with ß-helix fold mediates adhesion in clinical isolates.

Candida glabrata is an opportunistic pathogenic yeast frequently causing infections in humans. Though it lacks typical virulence factors such as hyphal development, C. glabrata contains a remarkably large and diverse set of putative wall adhesins that is crucial for its success as pathogen. Here, we present an analysis of putative adhesins from the homology clusters V and VI. First, sequence similarity network analysis revealed relationships between cluster V and VI adhesins and S. cerevisiae haze protective factors (Hpf). Crystal structures of A-regions from cluster VI adhesins Awp1 and Awp3b reveal a parallel right-handed ß-helix domain that is linked to a C-terminal ß-sandwich. Structure solution of the A-region of Awp3b via single wavelength anomalous diffraction phasing revealed the largest known lanthanide cluster with 21 Gd3+ ions. Awp1-A and Awp3b-A show structural similarity to pectate lyases but binding to neither carbohydrates nor Ca2+ was observed. Phenotypic analysis of awp1Δ, awp3Δ, and awp1,3Δ double mutants did also not confirm their role as adhesins. In contrast, deletion mutants of the cluster V adhesin Awp2 in the hyperadhesive clinical isolate PEU382 demonstrated its importance for adhesion to polystyrene or glass, biofilm formation, cell aggregation and other cell surface-related phenotypes. Together with cluster III and VII adhesins our study shows that C. glabrata CBS138 can rely on a set of 42 Awp1-related adhesins with ß-helix/α-crystallin domain architecture for modifying the surface characteristics of its cell wall.

Human albumin enhances the pathogenic potential of Candida glabrata on vaginal epithelial cells.

The opportunistic pathogen Candida glabrata is the second most frequent causative agent of vulvovaginal candidiasis (VVC), a disease that affects 70-75% of women at least once during their life. However, C. glabrata is almost avirulent in mice and normally incapable of inflicting damage to vaginal epithelial cells in vitro. We thus proposed that host factors present in vivo may influence C. glabrata pathogenicity. We, therefore, analyzed the impact of albumin, one of the most abundant proteins of the vaginal fluid. The presence of human, but not murine, albumin dramatically increased the potential of C. glabrata to damage vaginal epithelial cells. This effect depended on macropinocytosis-mediated epithelial uptake of albumin and subsequent proteolytic processing. The enhanced pathogenicity of C. glabrata can be explained by a combination of beneficial effects for the fungus, which includes an increased access to iron, accelerated growth, and increased adhesion. Screening of C. glabrata deletion mutants revealed that Hap5, a key regulator of iron homeostasis, is essential for the albumin-augmented damage potential. The albumin-augmented pathogenicity was reversed by the addition of iron chelators and a similar increase in pathogenicity was shown by increasing the iron availability, confirming a key role of iron. Accelerated growth not only led to higher cell numbers, but also to increased fungal metabolic activity and oxidative stress resistance. Finally, the albumin-driven enhanced damage potential was associated with the expression of distinct C. glabrata virulence genes. Transcriptional responses of the epithelial cells suggested an unfolded protein response (UPR) and ER-stress responses combined with glucose starvation induced by fast growing C. glabrata cells as potential mechanisms by which cytotoxicity is mediated.Collectively, we demonstrate that albumin augments the pathogenic potential of C. glabrata during interaction with vaginal epithelial cells. This suggests a role for albumin as a key player in the pathogenesis of VVC.

Evaluating the accuracy and diagnostic value of CFW and a new fluorescent reagents, fluorescent brightener 85, for the diagnosis of vulvovaginal candidiasis.

BACKGROUND: Vulvovaginal candidiasis (VVC) is a common vaginitis in females. The commonly used diagnostic method, 10% potassium hydroxide (KOH) smear microscopy, makes it not very easy to recognize fungi. METHODS: Vaginal secretions were collected from clinically suspected VVC patients and divided into four groups and examined using KOH, CFW (Calcofluor White), FB 85(fluorescent brightener 85), and culture. The data were statistically analyzed. RESULTS: In total, 110 patients with suspected VVC were recruited. The positive rates of KOH, CFW, FB 85, and the culture method were 68.2%, 64.5%, 61.8%, and 77%, respectively. According to the McNemar test, there was no statistically significant difference between the KOH, CFW, and the FB 85 methods (p > 0.05). However, CFW had a shorter diagnosis time than the KOH method and had a statistically significant difference (p < 0.001). Moreover, CFW has the highest sensitivity, specificity, and accuracy. In morphological recognition, it was easier to recognize fungal structures with CFW and FB 85 than with the KOH. CONCLUSIONS: The fluorescent method is a good method for the diagnosis of VVC. And the fungi can be found more quickly. Similar to CFW, FB 85 is also a potential good fluorescent reagent for the diagnosis of VVC and has potential value for application in clinical fungal infection diseases.

Transient Mitochondria Dysfunction Confers Fungal Cross-Resistance against Phagocytic Killing and Fluconazole.

Loss or inactivation of antivirulence genes is an adaptive strategy in pathogen evolution. Candida glabrata is an important opportunistic pathogen related to baker's yeast, with the ability to both quickly increase its intrinsic high level of azole resistance and persist within phagocytes. During C. glabrata's evolution as a pathogen, the mitochondrial DNA polymerase CgMip1 has been under positive selection. We show that CgMIP1 deletion not only triggers loss of mitochondrial function and a petite phenotype, but increases C. glabrata's azole and endoplasmic reticulum (ER) stress resistance and, importantly, its survival in phagocytes. The same phenotype is induced by fluconazole and by exposure to macrophages, conferring a cross-resistance between antifungals and immune cells, and can be found in clinical isolates despite a slow growth of petite strains. This suggests that petite constitutes a bet-hedging strategy of C. glabrata and, potentially, a relevant cause of azole resistance. Mitochondrial function may therefore be considered a potential antivirulence factor. IMPORTANCE Candida glabrata is an opportunistic pathogen whose incidence has been increasing in the last 40 years. It has risen to become the most prominent non-Candida albicans Candida (NCAC) species to cause candidemia, constituting about one-third of isolates in the United States, and steadily increasing in European countries and in Australia. Despite its clinical importance, C. glabrata's pathogenicity strategies remain poorly understood. Our research shows that loss of mitochondrial function and the resulting petite phenotype is advantageous for C. glabrata to cope with infection-related stressors, such as antifungals and host immune defenses. The (cross-)resistance against both these factors may have major implications in the clinical outcome of infections with this major fungal pathogen.

Candida glabrata produces a melanin-like pigment that protects against stress conditions encountered during parasitism.

Aim: Melanin has been linked to pathogenesis in several fungi. They often produce melanin-like pigments in the presence of L-dihydroxyphenylalanine (L-DOPA), but this is poorly studied in Candida glabrata. Methods & materials:C. glabrata was grown in minimal medium with or without L-DOPA supplementation and submitted to a chemical treatment with denaturant and hot acid. Results:C. glabrata turned black when grown in the presence of L-DOPA, whereas cells grown without L-DOPA supplementation remained white. Biophysical properties demonstrated that the pigment was melanin. Melanized C. glabrata cells were effectively protected from azoles and amphotericin B, incubation at 42°C and macrophage killing. Conclusion: In the presence of L-DOPA, C. glabrata produces melanin, increases antifungal resistance and enhances host survival.

Aim: Melanin is a pigment that can help fungi to cause disease. Fungi often produce melanin-like pigments in the presence of L-dihydroxyphenylalanine (L-DOPA), but this is poorly studied in Candida glabrata, a yeast species that can cause human disease. Methods & materials:C. glabrata was grown in minimal medium with or without L-DOPA supplementation and submitted to a chemical treatment to isolate melanin. Results:C. glabrata turned black when grown in the presence of L-DOPA, whereas cells grown without L-DOPA supplementation remained white. Several experiments demonstrated that the black pigment was melanin. Melanized C. glabrata cells were effectively protected from antifungal drugs, incubation at 42°C and killing by cells of the immune system. Conclusion: In the presence of L-DOPA, C. glabrata produces melanin, increases antifungal resistance and has enhanced survival in contact with immunologic defense cells.

Comparative assessment of immune evasion mechanisms in human whole-blood infection assays by a systems biology approach.

Computer simulations of mathematical models open up the possibility of assessing hypotheses generated by experiments on pathogen immune evasion in human whole-blood infection assays. We apply an interdisciplinary systems biology approach in which virtual infection models implemented for the dissection of specific immune mechanisms are combined with experimental studies to validate or falsify the respective hypotheses. Focusing on the assessment of mechanisms that enable pathogens to evade the immune response in the early time course of a whole-blood infection, the least-square error (LSE) as a measure for the quantitative agreement between the theoretical and experimental kinetics is combined with the Akaike information criterion (AIC) as a measure for the model quality depending on its complexity. In particular, we compare mathematical models with three different types of pathogen immune evasion as well as all their combinations: (i) spontaneous immune evasion, (ii) evasion mediated by immune cells, and (iii) pre-existence of an immune-evasive pathogen subpopulation. For example, by testing theoretical predictions in subsequent imaging experiments, we demonstrate that the simple hypothesis of having a subpopulation of pre-existing immune-evasive pathogens can be ruled out. Furthermore, in this study we extend our previous whole-blood infection assays for the two fungal pathogens Candida albicans and C. glabrata by the bacterial pathogen Staphylococcus aureus and calibrated the model predictions to the time-resolved experimental data for each pathogen. Our quantitative assessment generally reveals that models with a lower number of parameters are not only scored with better AIC values, but also exhibit lower values for the LSE. Furthermore, we describe in detail model-specific and pathogen-specific patterns in the kinetics of cell populations that may be measured in future experiments to distinguish and pinpoint the underlying immune mechanisms.

DNA damage response of major fungal pathogen Candida glabrata offers clues to explain its genetic diversity.

How cells respond to DNA damage is key to maintaining genome integrity or facilitating genetic change. In fungi, DNA damage responses have been extensively characterized in the model budding yeast Saccharomyces cerevisiae, which is generally not pathogenic. However, it is not clear how closely these responses resemble those in fungal pathogens, in which genetic change plays an important role in the evolutionary arms race between pathogen and host and the evolution of antifungal drug resistance. A close relative of S. cerevisiae, Candida glabrata, is an opportunistic pathogen that displays high variability in chromosome structure among clinical isolates and rapidly evolves antifungal drug resistance. The mechanisms facilitating such genomic flexibility and evolvability in this organism are unknown. Recently we characterized the DNA damage response of C. glabrata and identified several features that distinguish it from the well characterized DNA damage response of S. cerevisiae. First, we discovered that, in contrast to the established paradigm, C. glabrata effector kinase Rad53 is not hyperphosphorylated upon DNA damage. We also uncovered evidence of an attenuated DNA damage checkpoint response, wherein in the presence of DNA damage C. glabrata cells did not accumulate in S-phase and proceeded with cell division, leading to aberrant mitoses and cell death. Finally, we identified evidence of transcriptional rewiring of the DNA damage response of C. glabrata relative to S. cerevisiae, including an upregulation of genes involved in mating and meiosis-processes that have not been reported in C. glabrata. Together, these results open new possibilities and raise tantalizing questions of how this major fungal pathogen facilitates genetic change.

Role of CgTpo4 in Polyamine and Antimicrobial Peptide Resistance: Determining Virulence in Candida glabrata.

Candida glabrata is an emerging fungal pathogen whose success depends on its ability to resist antifungal drugs but also to thrive against host defenses. In this study, the predicted multidrug transporter CgTpo4 (encoded by ORF CAGL0L10912g) is described as a new determinant of virulence in C. glabrata, using the infection model Galleria mellonella. The CgTPO4 gene was found to be required for the C. glabrata ability to kill G. mellonella. The transporter encoded by this gene is also necessary for antimicrobial peptide (AMP) resistance, specifically against histatin-5. Interestingly, G. mellonella's AMP expression was found to be strongly activated in response to C. glabrata infection, suggesting AMPs are a key antifungal defense. CgTpo4 was also found to be a plasma membrane exporter of polyamines, especially spermidine, suggesting that CgTpo4 is able to export polyamines and AMPs, thus conferring resistance to both stress agents. Altogether, this study presents the polyamine exporter CgTpo4 as a determinant of C. glabrata virulence, which acts by protecting the yeast cells from the overexpression of AMPs, deployed as a host defense mechanism.

Revealing Candida glabrata biofilm matrix proteome: global characterization and pH response.

Candida glabrata is a clinically relevant human pathogen with the ability to form high recalcitrant biofilms that contribute to the establishment and persistence of infection. A defining trait of biofilms is the auto-produced matrix, which is suggested to have structural, virulent and protective roles. Thus, elucidation of matrix components, their function and modulation by the host environment is crucial to disclose their role in C. glabrata pathogenesis. As a major step toward this end, this study aimed to reveal, for the first time, the matrix proteome of C. glabrata biofilms, to characterize it with bioinformatic tools and to study its modulation by the environmental pH (acidic and neutral). The results showed the presence of several pH-specific matrix proteins (51 acidic- and 206 neutral-specific) and also proteins commonly found at both pH conditions (236). Of note, several proteins related to mannan and ß-glucan metabolism, which have a potential role in the delivery/organization of carbohydrates in the matrix, were found in both pH conditions but in much higher quantity under the neutral environment. Additionally, several virulence-related proteins, including epithelial adhesins, yapsins and moonlighting enzymes, were found among matrix proteins. Importantly, several proteins seem to have a non-canonical secretion pathway and Pdr1 was found to be a potential regulator of matrix proteome. Overall, this study indicates a relevant impact of environmental cues in the matrix proteome and provides a unique resource for further functional investigation of matrix proteins, contributing to the identification of potential targets for the development of new therapies against C. glabrata biofilms.

Experimental Evolution of Candida by Serial Passaging in Host Cells.

Experimental evolution is an experiment class of its own; instead of requiring an a priori hypothesis, the genetic adaptation of microbes to defined environments tells us about the underlying pathways and mechanisms. Such experiments are often deceptively simple in their design, based on a single abiotic stressor and what is in essence a long-term continuous culture. However, they generally provide a starting point to thorough follow-up analyses (which are specific for the organism at hand and not part of this method chapter). In this chapter, we describe a method to use a biotic stressor which is frequently encountered by pathogenic fungi-macrophage-like cells-in a serial passaging regime. Experimental evolution under such conditions can reveal new virulence attributes and mechanisms by selecting for adaptive mutations against the host cell-induced stress.It is important to note that every evolution experiment is different, and these techniques should be taken as a general guideline to be adapted to different organisms and questions. Then, it is a powerful tool with many potential applications in pathobiology research.

Identification of Candida glabrata Transcriptional Regulators That Govern Stress Resistance and Virulence.

The mechanisms by which Candida glabrata resists host defense peptides and caspofungin are incompletely understood. To identify transcriptional regulators that enable C. glabrata to withstand these classes of stressors, a library of 215 C. glabrata transcriptional regulatory deletion mutants was screened for susceptibility to both protamine and caspofungin. We identified eight mutants that had increased susceptibility to both host defense peptides and caspofungin. Of these mutants, six were deleted for genes that were predicted to specify proteins involved in histone modification. These genes were ADA2, GCN5, SPT8, HOS2, RPD3, and SPP1 Deletion of ADA2, GCN5, and RPD3 also increased susceptibility to mammalian host defense peptides. The Δada2 and Δgcn5 mutants had increased susceptibility to other stressors, such as H2O2 and SDS. In the Galleria mellonella model of disseminated infection, the Δada2 and Δgcn5 mutants had attenuated virulence, whereas in neutropenic mice, the virulence of the Δada2 and Δrpd3 mutants was decreased. Thus, histone modification plays a central role in enabling C. glabrata to survive host defense peptides and caspofungin, and Ada2 and Rpd3 are essential for the maximal virulence of this organism during disseminated infection.

The involvement of the Candida glabrata trehalase enzymes in stress resistance and gut colonization.

Candida glabrata is an opportunistic human fungal pathogen and is frequently present in the human microbiome. It has a high relative resistance to environmental stresses and several antifungal drugs. An important component involved in microbial stress tolerance is trehalose. In this work, we characterized the three C. glabrata trehalase enzymes Ath1, Nth1 and Nth2. Single, double and triple deletion strains were constructed and characterized both in vitro and in vivo to determine the role of these enzymes in virulence. Ath1 was found to be located in the periplasm and was essential for growth on trehalose as sole carbon source, while Nth1 on the other hand was important for oxidative stress resistance, an observation which was consistent by the lower survival rate of the NTH1 deletion strain in human macrophages. No significant phenotype was observed for Nth2. The triple deletion strain was unable to establish a stable colonization of the gastrointestinal (GI) tract in mice indicating the importance of having trehalase activity for colonization in the gut.

Potency of gastrointestinal colonization and virulence of Candida auris in a murine endogenous candidiasis.

BACKGROUND: Candida auris infections have recently emerged worldwide, and this species is highly capable of colonization and is associated with high levels of mortality. However, strain-dependent differences in colonization capabilities and virulence have not yet been reported. OBJECTIVES: In the present study, we aimed to clarify the differences between clinically isolated invasive and non-invasive strains of C. auris. METHODS: We evaluated colonization, dissemination, and survival rates in wild C57BL/6J mice inoculated with invasive or non-invasive strains of C. auris under cortisone acetate immunosuppression, comparing with those of Candida albicans and Candida glabrata infections. We also evaluated the potency of biofilm formation. RESULTS: Stool fungal burdens were significantly higher in mice inoculated with the invasive strains than in those infected with the non-invasive strain. Along with intestinal colonization, liver and kidney fungal burdens were also significantly higher in mice inoculated with the invasive strains. In addition, histopathological findings revealed greater dissemination and colonization of the invasive strains. Regarding biofilm-forming capability, the invasive strain of C. auris exhibited a significantly higher capacity of producing biofilms. Moreover, inoculation with the invasive strains resulted in significantly greater loss of body weight than that noted following infection with the non-invasive strain. CONCLUSIONS: Invasive strains showed higher colonization capability and rates of dissemination from gastrointestinal tracts under cortisone acetate immunosuppression than non-invasive strains, although the mortality rates caused by C. auris were lower than those caused by C. albicans.

Fístula traqueoesofágica por Candida galbrata: una patología infrecuente / Tracheoesophageal fistula due to candida galbrata: a rare pathology

La fístula traqueoesofágica consiste en una comunicación anómala entre el sistema respiratorio y el digestivo, que puede ser a causa de una enfermedad congénita, o bien secundaria a una patología tal como la infecciosa, la tumoral o postraumática. Entre las etiologías infecciosas, Candida o Aspergillus son dos hongos que provocan esofagitis fúngica, la cual podría complicarse con esta entidad, aunque son casos extremadamente raros. El diagnóstico de las fístulas se basa en la clínica, las pruebas de imagen como la TC y la endoscopia, donde se podrá apreciar la solución de continuidad. Este defecto debe ser tratado mediante cirugía o endoscópicamente

The tracheoesophageal fistula consists of an abnormal communication between the respiratory and digestive systems, which may be due to a congenital disease or secondary to a pathology such as infectious, tumorous or post-traumatic. Among the infectious etiologies, Candida or Aspergillus are two fungi that cause fungal esophagitis, which could be complicated by this entity, although they are extremely rare cases. The diagnosis of fistulas is based on the clinic, imaging tests such as CT and endoscopy, where the solution of continuity can be appreciated. This defect must be treated surgically or endoscopically, a technique that is currently on the rise due to its fewer complications

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.

A new regulator in the crossroads of oxidative stress resistance and virulence in Candida glabrata: The transcription factor CgTog1.

Candida glabrata is a prominent pathogenic yeast which exhibits a unique ability to survive the harsh environment of host immune cells. In this study, we describe the role of the transcription factor encoded by the gene CAGL0F09229g, here named CgTog1 after its Saccharomyces cerevisiae ortholog, as a new determinant of C. glabrata virulence. Interestingly, Tog1 is absent in the other clinically relevant Candida species (C. albicans, C. parapsilosis, C. tropicalis, C. auris), being exclusive to C. glabrata. CgTog1 was found to be required for oxidative stress resistance and for the modulation of reactive oxygen species inside C. glabrata cells. Also, CgTog1 was observed to be a nuclear protein, whose activity up-regulates the expression of 147 genes and represses 112 genes in C. glabrata cells exposed to H2O2, as revealed through RNA-seq-based transcriptomics analysis. Given the importance of oxidative stress response in the resistance to host immune cells, the effect of CgTOG1 expression in yeast survival upon phagocytosis by Galleria mellonella hemocytes was evaluated, leading to the identification of CgTog1 as a determinant of yeast survival upon phagocytosis. Interestingly, CgTog1 targets include many whose expression changes in C. glabrata cells after engulfment by macrophages, including those involved in reprogrammed carbon metabolism, glyoxylate cycle and fatty acid degradation. In summary, CgTog1 is a new and specific regulator of virulence in C. glabrata, contributing to oxidative stress resistance and survival upon phagocytosis by host immune cells.

Functional information from clinically-derived drug resistant forms of the Candida glabrata Pdr1 transcription factor.

Azole drugs are the most frequently used antifungal agents. The pathogenic yeast Candida glabrata acquires resistance to azole drugs via single amino acid substitution mutations eliciting a gain-of-function (GOF) hyperactive phenotype in the Pdr1 transcription factor. These GOF mutants constitutively drive high transcription of target genes such as the ATP-binding cassette transporter-encoding CDR1 locus. Previous characterization of Pdr1 has demonstrated that this factor is negatively controlled by the action of a central regulatory domain (CRD) of ~700 amino acids, in which GOF mutations are often found. Our earlier experiments demonstrated that a Pdr1 derivative in which the CRD was deleted gave rise to a transcriptional regulator that could not be maintained as the sole copy of PDR1 in the cell owing to its toxically high activity. Using a set of GOF PDR1 alleles from azole-resistant clinical isolates, we have analyzed the mechanisms acting to repress Pdr1 transcriptional activity. Our data support the view that Pdr1-dependent transactivation is mediated by a complex network of transcriptional coactivators interacting with the extreme C-terminal part of Pdr1. These coactivators include but are not limited to the Mediator component Med15A. Activity of this C-terminal domain is controlled by the CRD and requires multiple regions across the C-terminus for normal function. We also provide genetic evidence for an element within the transactivation domain that mediates the interaction of Pdr1 with coactivators on one hand while restricting Pdr1 activity on the other hand. These data indicate that GOF mutations in PDR1 block nonidentical negative inputs that would otherwise restrain Pdr1 transcriptional activation. The strong C-terminal transactivation domain of Pdr1 uses multiple different protein regions to recruit coactivators.

Host-pathogen interaction in Candida glabrata infection: current knowledge and implications for antifungal therapy.

INTRODUCTION: The opportunistic fungal pathogen Candida glabrata poses a clinical challenge in the successful treatment of invasive Candida infections, owing to its low inherent susceptibility toward azole antifungals and the recent acquisition of coresistance toward azole and echinocandin drugs. Compared to other prevalent Candida bloodstream pathogens, C. glabrata neither exhibits secreted proteolytic activity nor invokes a strong immune response in a variety of host cells and is less virulent. It also does not form true hyphae, and the success of C. glabrata, therefore, as a prevalent human fungal pathogen, appears to be built upon a distinct set of virulence attributes. AREAS COVERED: The focus of this review is to outline, in brief, the interaction of C. glabrata with the host, deduced from the knowledge gained from different in vitro, ex vivo, and in vivo model systems. In addition, we briefly discuss the current antifungals, antifungal resistance mechanisms, and the development of new antifungal therapies, along with the available information on the host response. EXPERT OPINION: A detailed understanding of stresses, selection pressures and differential immune responses in the presence and absence of antifungals that C. glabrata encounters in varied niches of the host, is required to design effective antifungal therapy.