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Background: The prevalence of Candida glabrata healthcare-associated infections is on the rise worldwide and in Lebanon, Candida glabrata infections are difficult to treat as a result of their resistance to azole antifungals and their ability to form biofilms., Objectives: The first objective of this study was to quantify biofilm biomass in the most virulent C. glabrata isolates detected in a Lebanese hospital. In addition, other pathogenicity attributes were evaluated. The second objective was to identify the mechanisms of azole resistance in those isolates., Methods: A mouse model of disseminated systemic infection was developed to evaluate the degree of virulence of 41 azole-resistant C. glabrata collected from a Lebanese hospital. The most virulent isolates were further evaluated alongside an isolate having attenuated virulence and a reference strain for comparative purposes. A DNA-sequencing approach was adopted to detect single nucleotide polymorphisms (SNPs) leading to amino acid changes in proteins involved in azole resistance and biofilm formation. This genomic approach was supported by several phenotypic assays., Results: All chosen virulent isolates exhibited increased adhesion and biofilm biomass compared to the isolate having attenuated virulence. The amino acid substitutions D679E and I739N detected in the subtelomeric silencer Sir3 are potentially involved- in increased adhesion. In all isolates, amino acid substitutions were detected in the ATP-binding cassette transporters Cdr1 and Pdh1 and their transcriptional regulator Pdr1., Conclusions: In summary, increased adhesion led to stable biofilm formation since mutated Sir3 could de-repress adhesins, while decreased azole susceptibility could result from mutations in Cdr1, Pdh1 and Pdr1., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Background: Side effects associated with antimicrobial drugs, as well as their high cost, have prompted a search for low-cost herbal medicinal substances with fewer side effects. These substances can be used as supplements to medicine or to strengthen their effects. The current study investigated the effect of oleuropein on the inhibition of fungal and bacterial biofilm in-vitro and at the molecular level., Materials and Methods: In this experimental study, antimicrobial properties were evaluated using microbroth dilution method. The effect of oleuropein on the formation and eradication of biofilm was assessed on 96-well flat bottom microtiter plates and their effects were observed through scanning electron microscopy (SEM). Its effect on key genes (Hwp1, Als3, Epa1, Epa6, LuxS, Pfs) involved in biofilm formation was investigated using the quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) method., Results: The minimum inhibitory concentration (MIC) and minimum fungicidal/bactericidal concentration (MFC/MBC) for oleuropein were found to be 65 mg/ml and 130 mg/ml, respectively. Oleuropein significantly inhibited biofilm formation at MIC/2 (32.5 mg/ml), MIC/4 (16.25 mg/ml), MIC/8 (8.125 mg/ml) and MIC/16 (4.062 mg/ml) (p < 0.0001). The anti-biofilm effect of oleuropein was confirmed by SEM. RT-qPCR indicated significant down regulation of expression genes involved in biofilm formation in Candida albicans (Hwp1, Als3) and Candida glabrata (Epa1, Epa6) as well as Escherichia coli (LuxS, Pfs) genes after culture with a MIC/2 of oleuropein (p < 0.0001)., Conclusions: The results indicate that oleuropein has antifungal and antibacterial properties that enable it to inhibit or destroy the formation of fungal and bacterial biofilm., (© 2024. The Author(s).)

Objective: In the defense against microorganisms like Candida albicans, macrophages recruit LC3(Microtubule-associated protein 1A/1B-light chain 3) to the periplasm, engaging in the elimination process through the formation of a single-membrane phagosome known as LC3-associated phagocytosis (LAP). Building on this, we propose the hypothesis that glucocorticoids may hinder macrophage phagocytosis of Candida glabrata by suppressing LAP, and rapamycin could potentially reverse this inhibitory effect., Methods: RAW264.7 cells were employed for investigating the immune response to Candida glabrata infection. Various reagents, including dexamethasone, rapamycin, and specific antibodies, were utilized in experimental setups. Assays, such as fluorescence microscopy, flow cytometry, ELISA (Enzyme-Linked Immunosorbent Assay), Western blot, and confocal microscopy, were conducted to assess phagocytosis, cytokine levels, protein expression, viability, and autophagy dynamics., Results: Glucocorticoids significantly inhibited macrophage autophagy, impairing the cells' ability to combat Candida glabrata. Conversely, rapamycin exhibited a dual role, initially inhibiting and subsequently promoting phagocytosis of Candida glabrata by macrophages. Glucocorticoids hinder macrophage autophagy in Candida glabrata infection by suppressing the MTOR pathway(mammalian target of rapamycin pathway), while the activation of MTOR pathway by Candida glabrata diminishes over time., Conclusion: Our study elucidates the intricate interplay between glucocorticoids, rapamycin, and macrophage autophagy during Candida glabrata infection. Understanding the implications of these interactions not only sheds light on the host immune response dynamics but also unveils potential therapeutic avenues for managing fungal infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yang, Wang, Dong, Zhao and Li.)

The protein phosphatase calcineurin is vital for the virulence of the opportunistic fungal pathogen Candida glabrata . The host-induced stresses that activate calcineurin signaling are unknown, as are the targets of calcineurin relevant to virulence. To potentially shed light on these processes, millions of transposon insertion mutants throughout the genome of C. glabrata were profiled en masse for fitness defects in the presence of FK506, a specific inhibitor of calcineurin. Eighty-seven specific gene deficiencies depended on calcineurin signaling for full viability in vitro both in wild-type and pdr1∆ null strains lacking pleiotropic drug resistance. Three genes involved in cell wall biosynthesis ( FKS1 , DCW1 , FLC1 ) possess co-essential paralogs whose expression depended on calcineurin and Crz1 in response to micafungin, a clinical antifungal that interferes with cell wall biogenesis. Interestingly, 80% of the FK506-sensitive mutants were deficient in different aspects of vesicular trafficking, such as endocytosis, exocytosis, sorting, and biogenesis of secretory proteins in the endoplasmic reticulum (ER). In response to the experimental antifungal manogepix that blocks GPI-anchor biosynthesis in the ER, calcineurin signaling increased and strongly prevented cell death independent of Crz1, one of its major targets. Comparisons between manogepix, micafungin, and the ER-stressing tunicamycin reveal a correlation between the degree of calcineurin signaling and the degree of cell survival. These findings suggest that calcineurin plays major roles in mitigating stresses of vesicular trafficking. Such stresses may arise during host infection and in response to antifungal therapies.IMPORTANCECalcineurin plays critical roles in the virulence of most pathogenic fungi. This study sheds light on those roles in the opportunistic pathogen Candida glabrata using a genome-wide analysis in vitro . The findings could lead to antifungal developments that also avoid immunosuppression., Competing Interests: The authors declare no conflict of interest.

Candida glabrata is the second frequent etiologic agent of mucosal and invasive candidiasis. Based on the recent developments in molecular methods, C. glabrata has been introduced as a complex composed of C. glabrata , Candida nivariensis , and Candida bracarensis . The four main classes of antifungal drugs effective against C. glabrata are pyrimidine analogs (flucytosine), azoles, echinocandins, and polyenes. Although the use of antifungal drugs is related to the predictable development of drug resistance, it is not clear why C. glabrata is able to rapidly resist against multiple antifungals in clinics. The enhanced incidence and antifungal resistance of C. glabrata and the high mortality and morbidity need more investigation regarding the resistance mechanisms and virulence associated with C. glabrata ; additional progress concerning the drug resistance of C. glabrata has to be further prevented. The present review highlights the mechanism of resistance to antifungal drugs in C. glabrata .

Candida glabrata is an opportunistic pathogen that adheres to human epithelial mucosa and forms biofilm to cause persistent infections. In this work, Single-cell Force Spectroscopy (SCFS) was used to glimpse at the adhesive properties of C. glabrata as it interacts with clinically relevant surfaces, the first step towards biofilm formation. Following a genetic screening, RNA-sequencing revealed that half of the entire transcriptome of C. glabrata is remodeled upon biofilm formation, around 40% of which under the control of the transcription factors CgEfg1 and CgTec1. Using SCFS, it was possible to observe that CgEfg1, but not CgTec1, is necessary for the initial interaction of C. glabrata cells with both abiotic surfaces and epithelial cells, while both transcription factors orchestrate biofilm maturation. Overall, this study characterizes the network of transcription factors controlling massive transcriptional remodelling occurring from the initial cell-surface interaction to mature biofilm formation., (© 2021. The Author(s).)

After Candida albicans , Candida glabrata is one of the most common fungal species associated with candidemia in nosocomial infections. Rapid acquisition of nutrients from the host is important for the survival of pathogens which possess the metabolic flexibility to assimilate different carbon and nitrogen compounds. In Saccharomyces cerevisiae , nitrogen assimilation is controlled through a mechanism known as Nitrogen Catabolite Repression (NCR). NCR is coordinated by the action of four GATA factors; two positive regulators, Gat1 and Gln3, and two negative regulators, Gzf3 and Dal80. A mechanism in C. glabrata similar to NCR in S. cerevisiae has not been broadly studied. We previously showed that in C. glabrata , Gln3, and not Gat1, has a major role in nitrogen assimilation as opposed to what has been observed in S. cerevisiae in which both factors regulate NCR-sensitive genes. Here, we expand the knowledge about the role of Gln3 from C. glabrata through the transcriptional analysis of BG14 and gln3Δ strains. Approximately, 53.5% of the detected genes were differentially expressed (DEG). From these DEG, amino acid metabolism and ABC transporters were two of the most enriched KEGG categories in our analysis (Up-DEG and Down-DEG, respectively). Furthermore, a positive role of Gln3 in AAA assimilation was described, as was its role in the transcriptional regulation of ARO8 . Finally, an unexpected negative role of Gln3 in the gene regulation of ABC transporters CDR1 and CDR2 and its associated transcriptional regulator PDR1 was found. This observation was confirmed by a decreased susceptibility of the gln3Δ strain to fluconazole.

Recent single-cell studies have revealed that yeast stress response involves transcription factors that are activated in pulses. However, it remains unclear whether and how these dynamic transcription factors temporally interact to regulate stress survival. Here we show that budding yeast cells can exploit the temporal relationship between paralogous general stress regulators, Msn2 and Msn4, during stress response. We find that individual pulses of Msn2 and Msn4 are largely redundant, and cells can enhance the expression of their shared targets by increasing their temporal divergence. Thus, functional redundancy between these two paralogs is modulated in a dynamic manner to confer fitness advantages for yeast cells, which might feed back to promote the preservation of their redundancy. This evolutionary implication is supported by evidence from Msn2/Msn4 orthologs and analyses of other transcription factor paralogs. Together, we show a cell fate control mechanism through temporal redundancy modulation in yeast, which may represent an evolutionarily important strategy for maintaining functional redundancy between gene duplicates.

Candida albicans and Candida glabrata are frequently coisolated from the oral cavity in immunosuppressive or immunocompromised individuals. Their relationship is usually defined as competition as C. glabrata can inhibit growth of C. albicans in cohabitation. In this study, eight C. albicans isolates as well as two C. glabrata strains were used to investigate the effects of culture medium (Roswell Park Memorial Institute [RPMI]-1640, YPD, YND), incubation time (24 h, 48 h, 72 h, 96 h), initial inoculum (C. glabrata: C. albicans = 2:1, 1:1, 1:2), and medium state (static and dynamic states) on viable cell enumeration and relative abundance in both Candida SB and MB. The results showed that in most cases, C. glabrata and C. albicans SB and MB flourished in RPMI-1640 at 24 h under dynamic state compared with other conditions. Except YPD medium, there were high proportions of preponderance of C. albicans over C. glabrata in MB compared with SB. High initial inoculum promoted corresponding Candida number in both SB and MB and its abundance in MB relative to SB. This study revealed an impact of several environmental conditions on the formation of C. albicans and C. glabrata SB and MB and their abundance in MB in comparison with SB, deepening our understanding of both Candida interaction and their resistance mechanism in MB., Lay Summary: This study described the effects of diverse experimental conditions on the numbers of Candida albicans and Candida glabrata single biofilms and mixed biofilms and their abundance., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

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., Competing Interests: The authors have declared that no competing interests exist.

Candida glabrata is a high-performance microbial cell factory for the production of organic acids. To elucidate the role of the C. glabrata Mediator tail subunit Med2 ( Cg Med2) at pH 2.0, we deleted or overexpressed Cg Med2 and used transcriptome analysis to identify genes that are regulated by Cg Med2. At pH 2.0, the deletion of Cg Med2 resulted in a cell growth decrease of 26.1% and a survival decrease of 32.3%. Overexpression of Cg Med2 increased cell growth by 12.4% and cell survival by 5.9% compared to the wild-type strain. Transcriptome and phenotypic analyses identified Cg Yap6 as a transcription factor involved in acid pH stress tolerance. Deletion of Cg Yap6 caused growth defects, whereas its overexpression enhanced cell growth at pH 2.0. Furthermore, total glycerophospholipid content and membrane integrity decreased by 33.4% and 21.8%, respectively, in the Cg Med2Δ strain; however, overexpression of Cg Med2 increased the total glycerophospholipid content and membrane integrity by 24.7% and 12.1%, respectively, compared with those of the wild-type strain at pH 2.0. These results demonstrated that under acid pH stress, Cg Med2 physically interacts with Cg Yap6, which translocates from the cytoplasm to the nucleus after being phosphorylated by the protein kinase Cg Yak1. Once in the nucleus, Cg Yap6 recruits Cg Med2 to express glycerophospholipid-related genes. Our study elucidated the function of Cg Med2 under acid pH stress and provides a potential strategy to equip Candida glabrata with low-pH resistance during organic acid fermentation. IMPORTANCE This study investigated the function of the Mediator tail subunit Cg Med2 in C. glabrata under low-pH stress. The protein kinase Cg Yak1 activates Cg Yap6 for the recruitment of Cg Med2, which in turn increases glycerophospholipid content and membrane integrity to confer low-pH stress tolerance. This study establishes a new link between the Mediator tail subunit and transcription factors. Overall, these findings indicate that Cg Med2 is a novel target to induce the low-pH stress response in C. glabrata ., (Copyright © 2020 Zhou et al.)

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.

Genetic studies have revealed critical roles of transcription factor Pdr1 and the Mediator subunit Gal11A in regulating azole resistance in Candida glabrata. Recently, PDR1 gain-of-function (GOF) mutations have been shown to not only increase azole resistance but also enhance adherence during C. glabrata infection. However, mechanism of how Pdr1 regulates adherence, especially the implication of PDR1 GOF mutations in the regulation of the major adhesin gene EPA1, remains uncharacterized. Initially, we unexpectedly observed that expression of PDR1 harbouring GOF mutation G346D down-regulated EPA1 transcription and attenuated adherence to epithelial cells in different strain backgrounds. Given that PDR1 GOF mutations have been previously regarded as stimulators for adherence of this species, these findings prompted us to explore the regulation of EPA1 by wild-type Pdr1 and Pdr1 harbouring G346D mutation. Epitope tagged version of Pdr1 and Gal11A were utilized to determine the association of Pdr1 and Gal11A with EPA1 promoter. A combination of approaches including deletion, molecular, and biochemical assays showed that EPA1 is a direct target of Pdr1, and demonstrated for the first time that PDR1 G346D mutation decreases EPA1 expression and attenuates adherence to epithelial cells via enhancing recruitment of Gal11A. Taken together, our data propose a critical role of Gal11A in Pdr1-regulated EPA1 expression and adherence to epithelial cells, which could be utilized a novel therapeutic target for the treatment of hyper-adherent C. glabrata infection., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Purpose: Clinical and in vitro studies showed selected oral microorganisms to be related to delayed wound healing and ulcerative oral mucositis. However, it is not known whether this effect is due to reduced metabolism and/or the reduced reproductive capacity of epithelial cells. Therefore, we studied the influence of the oral microorganisms Porphyromonas gingivalis, Candida glabrata, and Candida kefyr on cell metabolism and reproductive capacity of oral epithelial cells, aimed to further unravel the pathogenesis of oral mucositis., Methods: Oral epithelial cells were exposed to different concentrations of P. gingivalis, C. glabrata, and C. kefyr as mono-infections or mixed together. An MTT assay was performed to determine the effect on cell metabolism. A clonogenic assay was used to study the effect on the reproductive capacity of oral epithelial cells., Results: The metabolism of oral epithelial cells was reduced when the microorganisms were present in high concentrations: P. gingivalis at a multiplicity of infection (MOI) of 1000 and the Candida spp. at MOI 100. No statistical difference was observed in the ability of a single epithelial cell to grow into a colony of cells between control and P. gingivalis, C. glabrata, and C. kefyr, independent of the concentrations and combinations used., Conclusion: P. gingivalis, C. glabrata, and C. kefyr lowered the metabolic activity of oral epithelial cells in high concentrations, yet they did not influence the reproductive capacity of epithelial cells. Their impact on ulcerative oral mucositis is likely due to an effect on the migration, proliferation, and metabolism of epithelial cells.

Echinocandins represent the first-line therapy of candidemia. Echinocandin resistance among Candida spp. is mainly due to acquired FKS mutations. In this study, we report the emergence of FKS-mutant Candida albicans/glabrata in Switzerland and provide the microbiological and clinical characteristics of 9 candidemic episodes. All patients were previously exposed to echinocandins (median 26 days; range 15-77). Five patients received initial echinocandin therapy with persistent candidemia in 4 of them. Overall mortality was 33%.

Aim: To compare the pathogenesis of vulvovaginal candidiasis by three Candida species in diabetic mice. Materials & methods: Estrogenized and diabetic mice were challenged with C. albicans , C. tropicalis and C. glabrata . Results: Diabetic animals infected with C. albicans and C. tropicalis maintained the highest fungal burden, despite of high levels of proinflammatory cytokines (IL-6 and TNF-α), respectively. For C. glabrata , the results were similar in diabetic and nondiabetic groups. Conclusion: C. tropicalis was as invasive as C. albicans , and both were more effective than C. glabrata . This ability was attributed to filamentation, which may be stimulated by glucose levels from vaginal fluid. In addition, the high burden may be attributed to the apparent immunological inefficiency of the diabetic host.

In Candida glabrata , the transcription factor Cg Rds2 has been previously characterized as a regulator of glycerophospholipid metabolism, playing a crucial role in the response to osmotic stress. Here, we report that Cg Rds2 is also involved in the response to pH 2.0 stress. At pH 2.0, the deletion of CgRDS2 led to reduced cell growth and survival, by 33% and 57%, respectively, compared with those of the wild-type strain. These adverse phenotypes resulted from the downregulation of genes related to energy metabolism in the Cgrds2 Δ strain at pH 2.0, which led to a 34% reduction of the intracellular ATP content and a 24% decrease in membrane permeability. In contrast, the overexpression of CgRDS2 rescued the growth defect of the Cgrds2 Δ strain and increased cell survival at pH 2.0 by 17% compared with that of the wild-type strain, and this effect was accompanied by significant increases in ATP content and membrane permeability, by 42% and 19%, respectively. Furthermore, we found that the calcium/calmodulin-dependent protein kinase (CaMK) Cg Cmk1 physically interacts with the PAS domain of Cg Rds2, and Cg Cmk1 is required for Cg Rds2 activation and translocation from the cytoplasm to the nucleus under pH 2.0 stress. Moreover, Cg Cmk1 is critical for Cg Rds2 function in resistance to pH 2.0 stress, because cells of the Cgrds2-pas strain with a disrupted Cg Cmk1- Cg Rds2 interaction exhibited impaired energy metabolism and membrane permeability at pH 2.0. Therefore, our results indicate that Cg Cmk1 positively regulates Cg Rds2 and suggest that they promote resistance to low-pH stress by enhancing energy metabolism and membrane permeability in C. glabrata IMPORTANCE An acidic environment is the main problem in the production of organic acids in C. glabrata The present study reports that the calcium/calmodulin-dependent protein kinase Cg Cmk1 positively regulates Cg Rds2 to increase intracellular ATP content, membrane permeability, and resistance to low-pH stress. Hence, the transcription factor Cg Rds2 may be a potential target for improving the acid stress tolerance of C. glabrata during the fermentation of organic acids. The present study also establishes a new link between the calcium signaling pathway and energy metabolism., (Copyright © 2020 American Society for Microbiology.)

Vulvovaginal candidiasis (VVC) is an infection usually caused by Candida albicans and increasingly by Candida glabrata, which has an intrinsically high resistance to commonly used antifungals. Candida species possess virulence factors that contribute to VVC development, as the ability to form biofilms in vaginal walls and intrauterine devices. It is known that VVC is promoted by conditions that increase the hormones levels, during pregnancy, however, the effects of hormones on Candida cells are poorly studied, especially in C. glabrata. Thus, the influence of progesterone and β-estradiol, at normal cycle and pregnancy concentrations, on biofilm formation and resistance of C. albicans and C. glabrata vaginal isolates, was analyzed using acidic conditions (pH 4). Biofilms of C. albicans developed in the presence of hormones presented reduced biomass (up to 65%) and impaired cells ability to produce filamentous forms. On the other hand, C. glabrata presented high adaptation to the presence of hormones, which did not affect its biofilm formation. Additionally, hormones impaired the susceptibility of C. albicans and C. glabrata cells to azoles, with potential clinical significance in the presence of pregnancy hormone levels. A similar result was obtained for the susceptibility to hydrogen peroxide, a biological vaginal barrier against Candida growth. Overall, the results of this study suggest that hormones may act as environmental cues promoting Candida protection from vaginal defenses and harmful conditions, what may have implications in Candida vaginal pathogenicity and treatment of VVC, especially in C. glabrata infections due to its high adaptability to vaginal conditions., (© The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Aim: To assemble, characterize and assess the antifungal effects of a new fluconazole (FLZ)-carrier nanosystem. Materials & methods: The nanosystem was prepared by loading FLZ on chitosan (CS)-coated iron oxide nanoparticles (IONPs). Antifungal effects were evaluated on planktonic cells (by minimum inhibitory concentration determination) and on biofilms (by quantification of cultivable cells, total biomass, metabolism and extracellular matrix) of Candida albicans and Candida glabrata . Results: Characterization results ratified the formation of a nanosystem (<320 nm) with FLZ successfully embedded. IONPs-CS-FLZ nanosystem reduced minimum inhibitory concentration values and, in general, showed similar antibiofilm effects compared with FLZ alone. Conclusion: IONPs-CS-FLZ nanosystem was more effective than FLZ mainly in inhibiting Candida planktonic cells. This nanocarrier has potential to fight fungal infections.

Candida glabrata is an important human fungal pathogen known to trigger serious infections in immune-compromised individuals. Its ability to form biofilms, which exhibit high tolerance to antifungal treatments, has been considered as an important virulence factor. However, the mechanisms involving antifungal resistance in biofilms and the impact of host niche environments on these processes are still poorly defined. In this study, we performed a whole-transcriptome analysis of C. glabrata biofilm cells exposed to different environmental conditions and constraints in order to identify the molecular pathways involved in fluconazole resistance and understand how acidic pH niches, associated with the presence of acetic acid, are able to modulate these responses. We show that fluconazole treatment induces gene expression reprogramming in a carbon source and pH-dependent manner. This is particularly relevant for a set of genes involved in DNA replication, ergosterol, and ubiquinone biosynthesis. We also provide additional evidence that the loss of mitochondrial function is associated with fluconazole resistance, independently of the growth condition. Lastly, we propose that C. glabrata Mge1, a cochaperone involved in iron metabolism and protein import into the mitochondria, is a key regulator of fluconazole susceptibility during carbon and pH adaptation by reducing the metabolic flux towards toxic sterol formation. These new findings suggest that different host microenvironments influence directly the physiology of C. glabrata , with implications on how this pathogen responds to antifungal treatment. Our analyses identify several pathways that can be targeted and will potentially prove to be useful for developing new antifungals to treat biofilm-based infections., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2020.)

Candida glabrata represents the second-most frequent cause of candidiasis infections of the mucosa, bloodstream and genito-urinary tract in immunocompromised individuals. The incidence of C glabrata infection has increased significantly in the last two decades, mainly due to this species' abilities to resist various antifungal drugs and to form biofilms. We focused on the relationship between biofilm formation and the product of QDR2, a C glabrata member of the major facilitator superfamily (MFS) gene family, given that fungal biofilm formation limits drug penetration and is associated with persistent infection. The fungal cells in biofilms were compared between a C glabrata ∆qdr2 mutant and its wild-type strain. Cells were analysed for metabolism activity and drug susceptibility (using tetrazolium assay), adhesion activity, growth assay and intracellular pH (using flow cytometry). Compared to the wild type, the C glabrata ∆qdr2 showed lower adhesion activity and higher fluconazole susceptibility when assessed as a biofilm. The mutant also showed decreased metabolic activity during biofilm formation. Furthermore, the mutant grew more slowly under neutral-basic pH conditions. The qdr2 deletion in C glabrata resulted in an impaired ability to maintain pH homeostasis, which led in turn to a reduction of cell growth and of adherence to an artificial matrix. These results suggested that the Qdr2p function is needed for proper biofilm formation and biofilm maintenance in C glabrata as well as biofilm drug resistance towards fluconazole. Qdr2p may play an important role in C glabrata's ability to form biofilms on implanted medical devices in human bodies., (© 2019 Blackwell Verlag GmbH.)

Candida glabrata is a human commensal and an opportunistic human fungal pathogen. It is more closely related to the model yeast Saccharomyces cerevisiae than other Candida spp. Compared with S. cerevisiae, C. glabrata exhibits higher innate tolerance to various environmental stressors, including hyperthermal stress. Here we investigate the molecular mechanisms of C. glabrata adaptation to heat stress via adaptive laboratory evolution. We show that all parallel evolved populations readily adapt to hyperthermal challenge (from 47 °C to 50 °C) and exhibit convergence in evolved phenotypes with extensive cross-tolerance to various other environmental stressors such as oxidants, acids, and alcohols. Genome resequencing identified fixation of mutations in CgSTE11 in all parallel evolved populations. The CgSTE11 homolog in S. cerevisiae plays crucial roles in various mitogen-activated protein kinase (MAPK) signaling pathways, but its role is less understood in C. glabrata. Subsequent verification confirmed that CgSTE11 is important in hyperthermal tolerance and the observed extensive cross-tolerance to other environmental stressors. These results support the hypothesis that CgSTE11 mediates cross-talks between MAPK signaling pathways in C. glabrata in response to environmental challenges.

Iron is an essential element to most microorganisms, yet an excess of iron is toxic. Hence, living cells have to maintain a tight balance between iron uptake and iron consumption and storage. The control of intracellular iron concentrations is particularly challenging for pathogens because mammalian organisms have evolved sophisticated high-affinity systems to sequester iron from microbes and because iron availability fluctuates among the different host niches. In this review, we present the current understanding of iron homeostasis and its regulation in the fungal pathogen Candida glabrata . This yeast is an emerging pathogen which has become the second leading cause of candidemia, a life-threatening invasive mycosis. C. glabrata is relatively poorly studied compared to the closely related model yeast Saccharomyces cerevisiae or to the pathogenic yeast Candida albicans . Still, several research groups have started to identify the actors of C. glabrata iron homeostasis and its transcriptional and post-transcriptional regulation. These studies have revealed interesting particularities of C. glabrata and have shed new light on the evolution of fungal iron homeostasis.

Flexibility in carbon metabolism is pivotal for the survival and propagation of many human fungal pathogens within host niches. Indeed, flexible carbon assimilation enhances pathogenicity and affects the immunogenicity of Candida albicans . Over the last decade, Candida glabrata has emerged as one of the most common and problematic causes of invasive candidiasis. Despite this, the links between carbon metabolism, fitness, and pathogenicity in C. glabrata are largely unexplored. Therefore, this study has investigated the impact of alternative carbon metabolism on the fitness and pathogenic attributes of C. glabrata . We confirm our previous observation that growth on carbon sources other than glucose, namely acetate, lactate, ethanol, or oleate, attenuates both the planktonic and biofilm growth of C. glabrata , but that biofilms are not significantly affected by growth on glycerol. We extend this by showing that C. glabrata cells grown on these alternative carbon sources undergo cell wall remodeling, which reduces the thickness of their β-glucan and chitin inner layer while increasing their outer mannan layer. Furthermore, alternative carbon sources modulated the oxidative stress resistance of C. glabrata as well as the resistance of C. glabrata to an antifungal drug. In short, key fitness and pathogenic attributes of C. glabrata are shown to be dependent on carbon source. This reaffirms the perspective that the nature of the carbon sources available within specific host niches is crucial for C. glabrata pathogenicity during infection.

Candida albicans and Candida glabrata are the two most prevalent etiologic agents of candidiasis worldwide. Although both are recognized as pathogenic, their choice of virulence traits is highly divergent. Indeed, it appears that these different approaches to fungal virulence may be equally successful in causing human candidiasis. In this review, the virulence mechanisms employed by C. albicans and C. glabrata are analyzed, with emphasis on the differences between the two systems. Pathogenesis features considered in this paper include dimorphic growth, secreted enzymes and signaling molecules, and stress resistance mechanisms. The consequences of these traits in tissue invasion, biofilm formation, immune system evasion, and macrophage escape, in a species dependent manner, are discussed. This review highlights the observation that C. albicans and C. glabrata follow different paths leading to a similar outcome. It also highlights the lack of knowledge on some of the specific mechanisms underlying C. glabrata pathogenesis, which deserve future scrutiny.

The fungal pathogen Candida glabrata can cause both mucosal and disseminated infections. Cell adhesion, a key step in colonization and infection, depends in C. glabrata primarily on the Epa family of cell adhesion proteins. While Epa proteins have been documented to mediate specific adhesion to host glycans, some of them also promote nonspecific adhesion to abiotic surfaces, though this is incompletely understood. Here we address this issue using a combination of genetics and single-cell force measurements. By quantifying the forces driving the attachment of single C. glabrata cells to hydrophobic and hydrophilic substrates, we show that cell adhesion is strongly increased by loss of Sir-mediated silencing. Using a series of mutant strains lacking specific EPA genes, we demonstrate unexpectedly that three major Epa proteins, Epa1, Epa6, and Epa7, primarily contribute to both hydrophilic and hydrophobic interactions, suggesting a broad role for the Epa adhesins in mediating specific and nonspecific adherence and implicating Epa genes in biofilm formation on abiotic surfaces. IMPORTANCE Candida glabrata cell wall proteins mediate the attachment of C. glabrata to abiotic surfaces through molecular interactions that are poorly understood. Here, we study the forces engaged in Epa-dependent adhesion using single-cell techniques. Fungal adhesion to hydrophilic and hydrophobic substrates involves mainly three Epa proteins, suggesting a broad role for the Epa adhesins in mediating adherence. These proteins might represent a potential target for the development of innovative antifungal drugs., (Copyright © 2019 Valotteau et al.)

Denture stomatitis (DS) is a condition characterized by inflammation of the oral mucosa in direct contact with dentures and affects a significant number of otherwise healthy denture wearers. Candida- associated DS is predominantly caused by Candida albicans , a dimorphic fungus that readily colonizes and forms biofilms on denture materials. Previous studies showed a requirement for Candida biofilm formation on both palate and dentures in infection and identified fungal morphogenic transcription factors, Efg1 and Bcr1, as key players in DS pathogenesis. While both C. albicans and Candida glabrata are frequently coisolated in mucosal candidiasis, a pathogenic role for C. glabrata in DS remains unknown. Using an established rat model of DS, we sought to determine whether C. glabrata alone or coinoculation with C. albicans establishes colonization and causes palatal tissue damage and inflammation. Rats fitted with custom dentures were inoculated with C. albicans and/or C. glabrata and monitored over a 4-week period for fungal burden (denture/palate), changes in body weight, and tissue damage via lactate dehydrogenase (LDH) release as well as palatal staining by hematoxylin and eosin (H&E) and immunohistochemistry for myeloperoxidase (MPO) as measures of inflammation. C. glabrata colonized the denture/palate similarly to C. albicans In contrast to C. albicans , colonization by C. glabrata resulted in minimal changes in body weight, palatal LDH release, and MPO expression. Coinoculation with both species had no obvious modulation of C. albicans -mediated pathogenic effects. These data suggest that C. glabrata readily establishes colonization on denture and palate but has no apparent role for inducing/enhancing C. albicans pathogenesis in DS. IMPORTANCE Many denture wearers suffer from Candida -associated denture stomatitis (DS), a fungal infection of the hard palate in contact with dentures. Biofilm formation by Candida albicans on denture/palate surfaces is considered a central process in the infection onset. Although Candida glabrata is frequently coisolated with C. albicans , its role in DS pathogenesis is unknown. We show here, using a contemporary rat model that employed a patented intraoral denture system, that C. glabrata established stable colonization on the denture/palate. However, in contrast to C. albicans inoculated rats, rats inoculated with C. glabrata exhibited minimal changes in weight gain or palatal tissue damage. Likewise, coinoculation with the two Candida species resulted in no exacerbation of C. albicans -induced DS pathology. Together, our findings indicate that C. glabrata has no inducing/enhancing role in DS pathogenesis., (Copyright © 2019 Yano et al.)

Candida glabrata is an opportunistic yeast pathogen, whose incidence has increased over the last decades. Despite its genus name, this species is actually more closely related to the budding yeast Saccharomyces cerevisiae than to other Candida pathogens, such as Candida albicans. Hence, C. glabrata and C. albicans must have acquired the ability to infect humans independently, which is reflected in the use of different mechanism for virulence, and survival in the host. Yet, research on C. glabrata suffers from assumptions carried over from the more studied C. albicans. Regarding the adaptation of C. glabrata to the human host, the prejudice was that, just as C. albicans, C. glabrata is a natural human commensal that turns deadly when immune defenses weaken. It was also considered asexual, as no one has observed mating, diploids, or spores, despite great efforts. However, the recent analysis of whole genomes from globally distributed C. glabrata isolates have shaken these assumptions. C. glabrata seems to be only secondarily associated to humans, as indicated by a lack of co-evolution with its host, and genomic footprints of recombination shows compelling evidence that this yeast is able to have sex. Here, we discuss the implications of this and other recent findings and highlight the new questions opened by this change in paradigm.

Candida glabrata is a common human fungal commensal and opportunistic pathogen. This fungus shows remarkable resilience as it can form recalcitrant biofilms on indwelling catheters, has intrinsic resistance against azole antifungals, and is causing vulvovaginal candidiasis. As a nosocomial pathogen, it can cause life-threatening bloodstream infections in immune-compromised patients. Here, we investigate the potential role of the high osmolarity glycerol response (HOG) MAP kinase pathway for C. glabrata virulence. The C. glabrata MAP kinase CgHog1 becomes activated by a variety of environmental stress conditions such as osmotic stress, low pH, and carboxylic acids and subsequently accumulates in the nucleus. We found that CgHog1 allows C. glabrata to persist within murine macrophages, but it is not required for systemic infection in a mouse model. C. glabrata and Lactobacilli co-colonise mucosal surfaces. Lactic acid at a concentration produced by vaginal Lactobacillus spp. causes CgHog1 phosphorylation and accumulation in the nucleus. In addition, CgHog1 enables C. glabrata to tolerate different Lactobacillus spp. and their metabolites when grown in co-culture. Using a phenotypic diverse set of clinical C. glabrata isolates, we find that the HOG pathway is likely the main quantitative determinant of lactic acid stress resistance. Taken together, our data indicate that CgHog1 has an important role in the confrontation of C. glabrata with the common vaginal flora., (© 2018 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.)

The formation of Candida biofilms on implanted medical devices is crucial to the development of infections and an important clinical problem because of elevated resistance to antifungals. The aim of this study was to compare the in vitro activity of liposomal amphotericin B (L-AMB) and micafungin (MCFG) against four species of Candida biofilms, and the efficacy of systemic plus lock therapy with L-AMB and MCFG in a Candida biofilm-associated catheter infection model. An XTT-reduction assay was used to measure the metabolic activity of the biofilms to evaluation of in vitro antibiofilm activity. MCFG had better in vitro activity than L-AMB against Candida glabrata biofilms, whereas L-AMB had better activity than MCFG against Candida albicans and Candida tropicalis biofilms. L-AMB and MCFG had comparable efficacy against Candida parapsilosis biofilms. In an in vitro lock therapy model, 2 mg/ml L-AMB, unlike 2 mg/ml MCFG, significantly reduced the metabolic activity of all the strains of biofilms by >96%. Systemic and intraluminal lock treatment with L-AMB for 3-days resulted in more than about 2 log 10 reduction of Candida compared with that of systemic treatment and the control group in the C. albicans SP-20012, C. glabrata SP-20040, C. glabrata SP-20131, C. parapsilosis SP-20137, and C. tropicalis SP-20047 infection models. L-AMB was more effective at eradicating Candida biofilms in 3-day course of systemic and lock therapy than MCFG. L-AMB may be useful for the treatment of catheter-related Candida biofilm infections, but this finding will need to be confirmed by further studies including a long treatment duration., (Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Candida spp., especially Candida albicans, is one of the main colonisers of the oral cavity. Due to its ability to form biofilms, it can be implicated in dental caries, periodontal disease and denture stomatitis. Microbial cells in biofilms are minimally impacted by conventional drugs. The aim of this study was to find new substances able to inhibit the adhesion of Candida spp. in order to prevent biofilm formation in the oral cavity. This study focused on the red raspberry (Rubus idaeus) fruit, known for its richness in potentially antimicrobial tannins. Extraction with a polarity gradient was performed on acetone extracts from frozen ripe and unripe fruits, resulting in eight extracts. The antifungal and anti-adhesion effects of the extracts were determined using broth microdilution and XTT methods, respectively, against C. albicans, Candida glabrata and Candida parapsilosis strains. Interestingly, four extracts (hexane and ethyl acetate) displayed anti-adhesion activity against C. albicans at low concentrations [50% inhibitory concentration (IC 50 ) 15.6-62.5 µg/mL]. Bioassay-guided fractionation by chromatographic methods of the most active extract obtained from ripe fruit (ethyl acetate extract) led to two subfractions enriched in anti-adhesion compounds, identified by mass spectrometry analysis as hydrolysable and condensed tannins. Their activities were dose-dependent with maximum inhibition at 80% (IC 50  = 25 µg/mL and 12.5 µg/mL). Regarding antifungal activity, no extract was active against planktonic cells of the tested strains. This work highlights for the first time the potential of raspberries to prevent oral C. albicans biofilms., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In the present study, we have evaluated the antifungal activity of the seed, root and leaf of Paeonia emodi (commonly known as Himalayan peony) in four common solvents (acetone, chloroform, methanol and water) against six fungal strains. The methanolic seed extract (MSE) showed promising antifungal activity against Candida albicans (6.25mg/mL), Candida glabrata (3.12mg/mL) and Candida parapsilosis (12.50mg/mL) among all the fungal strains tested. Combination of the MSE with the well-known commercial antifungal drugs amphotericin B (Amp B), nystatin (NYS) and fluconazole (FLC) resulted in the killing of C. glabrata at non-inhibitory concentrations, i.e., 0.35μg/mL for Amp B, 0.55μg/mL for NYS and 1.19μg/mL for FLC. Notably, MSE caused cell wall damage of C. glabrata cells, as confirmed by confocal microscopy, flowcytometry and scanning electron microscopy (SEM). The MSE was fractionated by thin layer chromatography (TLC). TLC-bioautography was used to determine the active compounds present in the MSE. Column chromatography was used to separate the potential active compounds from the MSE. Furthermore, gas chromatography-mass spectrometry (GC-MS) andfourier-transform infrared spectroscopy (FTIR) were used to identify the phytocomponents of the MSE. These experiments revealed 13-docosenamide/9-octadecenamide/trans-13-docosenamide (89.70%) as being the predominant compound using a chloroform/methanol solvent system for the separation. Interestingly, the MSE also exhibited less significant cytotoxicity at the minimum inhibitory concentration (MIC) against mammalian cells (HeLa and HEK293). This study suggests that the MSE of P. emodi can be used for the treatment of C. glabrata infection., (Copyright © 2018. Published by Elsevier Masson SAS.)

Convergent evolution is often due to selective pressures generating a similar phenotype. We observe relatively recent duplications in a spectrum of Saccharomycetaceae yeast species resulting in multiple phosphatases that are regulated by different nutrient conditions - thiamine and phosphate starvation. This specialization is both transcriptional and at the level of phosphatase substrate specificity. In Candida glabrata , loss of the ancestral phosphatase family was compensated by the co-option of a different histidine phosphatase family with three paralogs. Using RNA-seq and functional assays, we identify one of these paralogs, CgPMU3 , as a thiamine phosphatase. We further determine that the 81% identical paralog CgPMU2 does not encode thiamine phosphatase activity; however, both are capable of cleaving the phosphatase substrate, 1-napthyl-phosphate. We functionally demonstrate that members of this family evolved novel enzymatic functions for phosphate and thiamine starvation, and are regulated transcriptionally by either nutrient condition, and observe similar trends in other yeast species. This independent, parallel evolution involving two different families of histidine phosphatases suggests that there were likely similar selective pressures on multiple yeast species to recycle thiamine and phosphate. In this work, we focused on duplication and specialization, but there is also repeated loss of phosphatases, indicating that the expansion and contraction of the phosphatase family is dynamic in many Ascomycetes. The dynamic evolution of the phosphatase gene families is perhaps just one example of how gene duplication, co-option, and transcriptional and functional specialization together allow species to adapt to their environment with existing genetic resources., (Copyright © 2018 Nahas et al.)

Recently, Candida glabrata has emerged as a health-threatening pathogen and the rising resistance to antifungal agent in C. glabrata often leads to clinical treatment failure. To investigate the evolution of drug resistance and adherence ability in four paired clinical isolates collected before and after antifungal treatment. Sequence analysis, gene disruption, drug-susceptibility, adhesion tests and real-time quantitative PCR were performed. The azole-susceptible strains acquired azole resistance after antifungal therapy. Four gain-of-function (GOF) mutations in CgPDR1 were revealed by sequence analysis, namely G1099D, G346D, L344S and P927S, the last being reported for the first time. CDR1, CDR2 and SNQ2 efflux pump gene expression levels were elevated in strains harbouring GOF mutations in CgPDR1, resulting in decreased azole susceptibility. CgPDR1 alleles with distinct GOF mutations displayed different expression profiles for the drug-related genes. CgPDR1GOF mutations led to increased efflux pumps expression levels in a strain background independent way. Hyperactive Pdr1 G1099D and Pdr1 P927S displayed strain background-dependent increased adherence to host cells via upregulation of EPA1 transcription. Interestingly, the drug transporter gene expression levels did not always correspond with that of the adhesin EPA1 gene. GOF mutations in CgPDR1 conferred drug resistance and increased adherence in the clinical strains, possibly endowing C. glabrata with increased viability and pathogenicity., (© 2018 Blackwell Verlag GmbH.)

Macrophages play a critical role in the elimination of fungal pathogens. They are sensed via cell surface pattern-recognition receptors and are phagocytosed into newly formed organelles called phagosomes. Phagosomes mature through the recruitment of proteins and lysosomes, resulting in addition of proteolytic enzymes and acidification of the microenvironment. Our earlier studies demonstrated an essential role of Dectin-1-dependent activation of spleen tyrosine kinase (Syk) in the maturation of fungal containing phagosomes. The absence of Syk activity interrupted phago-lysosomal fusion resulting in arrest at an early phagosome stage. In this study, we sought to define the contribution of Syk to the control of phagocytosed live Candida glabrata in primary macrophages. To accurately measure intracellular yeast division, we designed a carboxyfluorescein succinimidyl ester (CFSE) yeast division assay in which bright fluorescent parent cells give rise to dim daughter cells. The CFSE-labeling of C. glabrata did not affect the growth rate of the yeast. Following incubation with macrophages, internalized CFSE-labeled C. glabrata were retrieved by cellular lysis, tagged using ConA-647, and the amount of residual CFSE fluorescence was assessed by flow cytometry. C. glabrata remained undivided (CFSE bright) for up to 18 h in co-culture with primary macrophages. Treatment of macrophages with R406, a specific Syk inhibitor, resulted in loss of intracellular control of C. glabrata with initiation of division within 4 h. Delayed Syk inhibition after 8 h was less effective indicating that Syk is critically required at early stages of macrophage-fungal interaction. In conclusion, we demonstrate a new method of tracking division of C. glabrata using CFSE labeling. Our results suggest that early Syk activation is essential for macrophage control of phagocytosed C. glabrata .

Candidiases are the most recurrent fungal infections, especially among immunosuppressed patients. Although Candida albicans is still the most widespread isolated species, non-Candida albicans Candida species have been increasing. The goal of this work was to determine the susceptibility of C. glabrata biofilms to echinocandins and to evaluate their effect on the biofilm matrix composition, comparing the results with other Candida species. Drug susceptibilities were assessed through the determination of minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC) and minimum biofilm eradication concentration (MBEC) of caspofungin (Csf) and micafugin (Mcf). The β-1,3 glucans content of the matrices was assessed after contact with the drugs. The data suggest that, generally, after contact with echinocandins, the concentration of β-1,3 glucans increased. These adjustments in the matrix composition of C. glabrata biofilms and the chemical differences between Csf and Mcf, seem responsible and may determine the effectivity of the drug responses.

The condition of neutropenia, i.e., a reduced absolute neutrophil count in blood, constitutes a major risk factor for severe infections in the affected patients. Candida albicans and Candida glabrata are opportunistic pathogens and the most prevalent fungal species in the human microbiota. In immunocompromised patients, they can become pathogenic and cause infections with high mortality rates. In this study, we use a previously established approach that combines experiments and computational models to investigate the innate immune response during blood stream infections with the two fungal pathogens C. albicans and C. glabrata . First, we determine immune-reaction rates and migration parameters under healthy conditions. Based on these findings, we simulate virtual patients and investigate the impact of neutropenic conditions on the infection outcome with the respective pathogen. Furthermore, we perform in silico treatments of these virtual patients by simulating a medical treatment that enhances neutrophil activity in terms of phagocytosis and migration. We quantify the infection outcome by comparing the response to the two fungal pathogens relative to non-neutropenic individuals. The analysis reveals that these fungal infections in neutropenic patients can be successfully cleared by cytokine treatment of the remaining neutrophils; and that this treatment is more effective for C. glabrata than for C. albicans .

Candida is a member of the normal human microbiota and often resides on mucosal surfaces such as the oral cavity or the gastrointestinal tract. In addition to their commensality, Candida species can opportunistically become pathogenic if the host microbiota is disrupted or if the host immune system becomes compromised. An important factor for Candida pathogenesis is its ability to form biofilm communities. The two most medically important species- Candida albicans and Candida glabrata -are often coisolated from infection sites, suggesting the importance of Candida coculture biofilms. In this work, we report that biofilm formation of the coculture population depends on the relative ratio of starting cell concentrations of C. albicans and C. glabrata When using a starting ratio of C. albicans to C. glabrata of 1:3, ∼6.5- and ∼2.5-fold increases in biofilm biomass were observed relative to those of a C. albicans monoculture and a C. albicans / C. glabrata ratio of 1:1, respectively. Confocal microscopy analysis revealed the heterogeneity and complex structures composed of long C. albicans hyphae and C. glabrata cell clusters in the coculture biofilms, and reverse transcription-quantitative PCR (qRT-PCR) studies showed increases in the relative expression of the HWP1 and ALS3 adhesion genes in the C. albicans / C. glabrata 1:3 biofilm compared to that in the C. albicans monoculture biofilm. Additionally, only the 1:3 C. albicans / C. glabrata biofilm demonstrated an increased resistance to the antifungal drug caspofungin. Overall, the results suggest that interspecific interactions between these two fungal pathogens increase biofilm formation and virulence-related gene expression in a coculture composition-dependent manner. IMPORTANCE Candida albicans and Candida glabrata are often coisolated during infection, and the occurrence of coisolation increases with increasing inflammation, suggesting possible synergistic interactions between the two Candida species in pathogenesis. During the course of an infection, the prevalence of each Candida species may change over time due to differences in metabolism and in the resistance of each species to antifungal therapies. Therefore, it is necessary to understand the dynamics between C. albicans and C. glabrata in coculture to develop better therapeutic strategies against Candida infections. Existing in vitro work has focused on understanding how an equal-part culture of C. albicans and C. glabrata impacts biofilm formation and pathogenesis. What is not understood, and what is investigated in this work, is how the composition of Candida species in coculture impacts overall biofilm formation, virulence gene expression, and the therapeutic treatment of biofilms., (Copyright © 2018 American Society for Microbiology.)

The relationship among Candida species may be influenced by several factors. Thus, this study evaluated the interactions between Candida albicans and Candida glabrata in biofilms, varying the strain type, culture medium and glucose supplementation. Biofilms were formed for 48 hours in Sabouraud dextrose broth (SDB) or RPMI 1640, supplemented with 0%, 1% or 5% glucose. Each strain of C. albicans was combined with two strains of C. glabrata, generating four biofilm associations, which were quantified by colony-forming units (CFUs), total biomass and metabolic activity. Data were analysed by ANOVA and Tukey's HSD test (α = 0.05). For CFUs, all associations were classified as indifferent for biofilms formed in RPMI 1640, while for SDB the interactions were antagonistic for C. albicans and indifferent for C. glabrata. The association of reference strains resulted in a dual-species biofilm with biomass significantly higher than that observed for each single biofilm developed in SDB. The metabolic activity of dual-species biofilms did not significantly differ from that found for single ones, except for co-culture of the reference strains. Glucose supplementation and culture media had a significant influence on all parameters. In conclusion, the strain type, culture medium and glucose supplementation influenced the interactions between C. albicans and C. glabrata., (© 2017 Blackwell Verlag GmbH.)

Bloodstream infections by the human-pathogenic fungi Candida albicans and Candida glabrata increasingly occur in hospitalized patients and are associated with high mortality rates. The early immune response against these fungi in human blood comprises a concerted action of humoral and cellular components of the innate immune system. Upon entering the blood, the majority of fungal cells will be eliminated by innate immune cells, i.e., neutrophils and monocytes. However, recent studies identified a population of fungal cells that can evade the immune response and thereby may disseminate and cause organ dissemination, which is frequently observed during candidemia. In this study, we investigate the so far unresolved mechanism of fungal immune evasion in human whole blood by testing hypotheses with the help of mathematical modeling. We use a previously established state-based virtual infection model for whole-blood infection with C. albicans to quantify the immune response and identified the fungal immune-evasion mechanism. While this process was assumed to be spontaneous in the previous model, we now hypothesize that the immune-evasion process is mediated by host factors and incorporate such a mechanism in the model. In particular, we propose, based on previous studies that the fungal immune-evasion mechanism could possibly arise through modification of the fungal surface by as of yet unknown proteins that are assumed to be secreted by activated neutrophils. To validate or reject any of the immune-evasion mechanisms, we compared the simulation of both immune-evasion models for different infection scenarios, i.e., infection of whole blood with either C. albicans or C. glabrata under non-neutropenic and neutropenic conditions. We found that under non-neutropenic conditions, both immune-evasion models fit the experimental data from whole-blood infection with C. albicans and C. glabrata . However, differences between the immune-evasion models could be observed for the infection outcome under neutropenic conditions with respect to the distribution of fungal cells across the immune cells. Based on these predictions, we suggested specific experimental studies that might allow for the validation or rejection of the proposed immune-evasion mechanism.

Candida spp. is able to form a biofilm, which is considered resistant to the majority of antifungals used in medicine. The aim of this study was to evaluate the in vitro activity of micafungin against Candida spp. biofilms at different stages of their maturation (2, 6, and 24 h). We assessed the inhibitory effect of micafungin against 78 clinical isolates of Candida spp., growing as planktonic or sessile cells, by widely recommended broth microdilution method. The in vitro effect on sessile cells viability was evaluated by colorimetric reduction assay. All examined strains were susceptible or intermediate to micafungin when growing as planktonic cells. At the early stages of biofilm maturation, from 11 (39.3%) to 20 (100%), tested strains, depending on the species, exhibited sessile minimal inhibitory concentrations (SMICs) of micafungin at ≤ 2 mg/L. For 24-h-old Candida spp. biofilms, from 3 (10.7%) to 20 (100%) of the tested strains displayed SMICs of micafungin at ≤ 2 mg/L. Our findings confirm that micafungin exhibits high potential anti-Candida-biofilm activity. However, this effect does not comprise all Candida species and strains. All strains were susceptible or intermediate to micafungin when growing as planktonic cells, but for biofilms, micafungin displays species- and strain-specific activity. Paradoxical growth of C. albicans and C. parapsilosis was observed. Antifungal susceptibility testing of Candida spp. biofilms would be the best solution, but to date, no reference method is available. The strongest antibiofilm activity of micafungin is observed at early stages of biofilm formation. Possibly, micafungin could be considered as an effective agent for prevention of biofilm-associated candidiasis, especially catheter-related candidaemia.

The frequent emergence of azole resistance among Candida glabrata strains contributes to increase the incidence of infections caused by this species. Whole-genome sequencing of a fluconazole and voriconazole-resistant clinical isolate (FFUL887) and subsequent comparison with the genome of the susceptible strain CBS138 revealed prominent differences in several genes documented to promote azole resistance in C. glabrata. Among these was the transcriptional regulator CgPdr1. The CgPdr1 FFUL887 allele included a K274Q modification not documented in other azole-resistant strains. Transcriptomic profiling evidenced the upregulation of 92 documented targets of CgPdr1 in the FFUL887 strain, supporting the idea that the K274Q substitution originates a CgPdr1 gain-of-function mutant. The expression of CgPDR1K274Q in the FFUL887 background sensitised the cells against high concentrations of organic acids at a low pH (4.5), but had no detectable effect in tolerance towards other environmental stressors. Comparison of the genome of FFUL887 and CBS138 also revealed prominent differences in the sequence of adhesin-encoding genes, while comparison of the transcriptome of the two strains showed a significant remodelling of the expression of genes involved in metabolism of carbohydrates, nitrogen and sulphur in the FFUL887 strain; these responses likely reflecting adaptive responses evolved by the clinical strain during colonisation of the host., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Candida glabrata is an opportunistic fungal pathogen that ranks as the second most common cause of systemic candidiasis. Despite its genus name, this yeast is more closely related to the model yeast Saccharomyces cerevisiae than to other Candida pathogens, and hence its ability to infect humans is thought to have emerged independently. Moreover, C. glabrata has all the necessary genes to undergo a sexual cycle but is considered an asexual organism due to the lack of direct evidence of sexual reproduction. To reconstruct the recent evolution of this pathogen and find footprints of sexual reproduction, we assessed genomic and phenotypic variation across 33 globally distributed C. glabrata isolates. We cataloged extensive copy-number variation, which particularly affects genes encoding cell-wall-associated proteins, including adhesins. The observed level of genetic variation in C. glabrata is significantly higher than that found in Candida albicans. This variation is structured into seven deeply divergent clades, which show recent geographical dispersion and large within-clade genomic and phenotypic differences. We show compelling evidence of recent admixture between differentiated lineages and of purifying selection on mating genes, which provides the first evidence for the existence of an active sexual cycle in this yeast. Altogether, our data point to a recent global spread of previously genetically isolated populations and suggest that humans are only a secondary niche for this yeast., (Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Candida glabrata is the second most common source of Candida infections in humans. In this pathogen, the maintenance of cell wall integrity (CWI) frequently precludes effective pharmacological treatment by antifungal agents. In numerous fungi, cell wall modulation is reported to be controlled by endoplasmic reticulum (ER) stress, but how the latter affects CWI maintenance in C. glabrata is not clearly understood. Here, we characterized a C. glabrata strain harboring a mutation in the CNE1 gene, which encodes a molecular chaperone associated with nascent glycoprotein maturation in the ER. Disruption of cne1 induced ER stress and caused changes in the normal cell wall structure, specifically a reduction in the β-1,6-glucan content and accumulation of chitin. Conversely, a treatment with the typical ER stress inducer tunicamycin up-regulated the production of cell wall chitin but did not affect β-1,6-glucan content. Our results also indicated that C. glabrata features a uniquely evolved ER stress-mediated CWI pathway, which differs from that in the closely related species Saccharomyces cerevisiae. Furthermore, we demonstrated that ER stress-mediated CWI pathway in C. glabrata is also induced by the disruption of other genes encoding proteins that function in a correlated manner in the quality control of N-linked glycoproteins in the ER. These results suggest that calcineurin and ER quality control system act as a platform for maintaining CWI in C. glabrata., (Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The Bomanins (Boms) are a family of a dozen secreted peptides that mediate the innate immune response governed by the Drosophila Toll receptor. We recently showed that deleting a cluster of 10 Bom genes blocks Toll-mediated defenses against a range of fungi and gram-positive bacteria. Here, we characterize the activity of individual Bom family members. We provide evidence that the Boms overlap in function and that a single Bom gene encoding a mature peptide of just 16 amino acids can act largely or entirely independent of other family members to provide phenotypic rescue in vivo. We further demonstrate that the Boms function in Drosophila humoral immunity, mediating the killing of the fungal pathogen Candida glabrata in an in vitro assay of cell-free hemolymph. In addition, we find that the level of antifungal activity both in vivo and in vitro is linked to the level of Bom gene expression. Although Toll dictates expression of the antimicrobial peptides (AMPs) drosomycin and metchnikowin, we find no evidence that Boms act by modifying the expression of the mature forms of these antifungal AMPs., (© 2018 S. Karger AG, Basel.)

Candida glabrata is the second most common pathogen of severe candidiasis in immunocompromised hosts, following C. albicans. Although C. glabrata and C. albicans belong to the same genus, they are phylogenetically distinct. C-type lectin receptors (CLRs), acting as pattern-recognition receptors (PRRs), play critical roles in host defense against C. albicans infections. However, our understanding of the specific roles of CLRs in host defense against C. glabrata is limited. Here, we explored the potential roles of the C-type lectins Dectin-1 and Dectin-2 in host defense against C. glabrata. We found that both Dectin-1-deficient mice (Dectin-1 -/- ) and Dectin-2-deficient mice (Dectin-2 -/- ) are more susceptible to C. glabrata infection. Dectin-1confers host higher sensitivity for sensing C. glabrata infections, while the effect of Dectin-2 in the host defense against C. glabrata is infection dose dependent. Dectin-1 is required for host myeloid cells recognition, killing of C. glabrata, and development of subsequent Th1 and Th17 cell-mediated adaptive immune response. Significantly impaired inflammatory responses such as inflammatory cells recruitment and cytokines release that were induced by C. glabrata were manifested in Dectin-1-deficient mice. Together, our study demonstrates that Dectin-1 plays an important role in host defense against systemic Candida glabrata infections, indicating a previous unknown control mechanism for this particular type of infection in host. Our study, therefore, provides new insights into the host defense against C. glabrata.

Candida spp. adhere to medical devices, such as catheters, forming drug-tolerant biofilms that resist killing by the immune system. Little is known about how C. glabrata, an emerging pathogen, resists attack by phagocytes. Here we show that upon encounter with planktonic (non-biofilm) C. glabrata, human neutrophils initially phagocytose the yeast and subsequently release neutrophil extracellular traps (NETs), complexes of DNA, histones, and proteins capable of inhibiting fungal growth and dissemination. When exposed to C. glabrata biofilms, neutrophils also release NETs, but significantly fewer than in response to planktonic cells. Impaired killing of biofilm parallels the decrease in NET production. Compared to biofilm, neutrophils generate higher levels of reactive oxygen species (ROS) when presented with planktonic organisms, and pharmacologic inhibition of NADPH-oxidase partially impairs NET production. In contrast, inhibition of phagocytosis nearly completely blocks NET release to both biofilm and planktonic organisms. Imaging of the host response to C. glabrata in a rat vascular model of infection supports a role for NET release in vivo. Taken together, these findings show that C. glabrata triggers NET release. The diminished NET response to C. glabrata biofilms likely contributes to the resilience of these structured communities to host defenses.

Aim: This study examined the antifungal activity of the combination of tyrosol and farnesol against Candida albicans and Candida glabrata in the planktonic state or forming biofilms., Methods and Results: The effect of drug association against Candida planktonic cells was assessed by the fractional inhibitory concentration index. Mono- and dual-species biofilms were developed during 24 h and then treated with the compounds for 3 days, with two daily treatments of 1 min each. After, the total biomass, metabolic activity and the number of cultivable cells were quantified. Planktonic cells of the two species showed a similar susceptibility to the drug combination, however, a synergistic effect was only verified for C. glabrata. Regarding biofilm susceptibility, significant reductions in C. glabrata biomass, metabolism of C. albicans and mixed biofilms, and cultivable cells of single biofilms were verified for the drug combination, indicating an additive effect. For all other experiments, the effects were classified as indifferent., Conclusion: The combined use of tyrosol and farnesol was advantageous for some of the analysed parameters against Candida species., Significance and Impact of the Study: These findings may contribute to the development of oral care products containing tyrosol and farnesol to combat oral infections caused by Candida species., (© 2017 The Society for Applied Microbiology.)

Candida glabrata is one of most prevalent yeast in fungal infections, especially in immunocompromised patients. Its azole resistance results in a low therapeutic response, particularly when associated with biofilms. The main goal of this work was to study the effectiveness of voriconazole (Vcz) against C. glabrata biofilms oral pathologies, as esophageal or oropharyngeal candidiasis. Antifungal susceptibilities were determined in pre-formed 24-h-biofilms and ERG genes expression was determined by qRT-PCR. Protein quantification was performed using BCA ® Kit, carbohydrate was estimated according to the Dubois assay and β-1,3 glucans concentration were determined using Glucatell ® kit. Finally, ergosterol, Vcz, and fluconazole (Flu) concentrations within the biofilm matrices were determined by RP-HPLC. Results showed that C. glabrata biofilms were more susceptible to Vcz than to Flu and that ERG genes expression evidenced an overexpression of the three ERG genes in the presence of both azoles. The matrix content presented a remarked decrease in proteins and an increase in carbohydrates, namely β-1,3 glucans. Ergosterol was successfully detected and quantified in the biofilm matrices, with no differences in all the considered conditions. Vcz demonstrated better diffusion through the biofilms and better cell penetration capacities, than Flu, indicating that the structure of the drug molecule fully influences its dissemination through the biofilm matrices. This work showed that Vcz is notably more effective than Flu for the treatment of resistant C. glabrata oral biofilms, which demonstrates a clinical relevance in its future use for the treatment of oropharyngeal/esophageal candidiasis caused by this species.