Glabrata Cells Research Articles

Enhancement of ROS Production by Catechin Is a Primary Effect of Increased Azole Efficacy in Nakaseomyces glabratus (Candida glabrata) Cells Lacking the ERG6 Gene.

Fungal infections have become an important public health problem. Currently, there are only three available classes of antifungals for the treatment of invasive infections. Two of them, azoles and polyenes, target the synthesis of ergosterol or bind to sterols. A promising strategy to improve current therapies is the use of natural compounds in combinational therapies with the existing antifungals. In this work, we analyzed the changes in the susceptibility of the mutant strain of Nakaseomyces glabratus (Candida glabrata) lacking the ERG6 gene (encoding the sterol C-24 methyltransferase in ergosterol biosynthesis) in the presence of catechin and antifungal azoles. The reduced content of ergosterol in the Cgerg6Δ mutant resulted in the increased tolerance of the mutant cells to both azoles and polyenes. The combination of catechin with fluconazole or miconazole led to the growth inhibition of the azole-resistant Cgerg6Δ mutant strain. In the presence of catechin and miconazole, the Cgerg6Δ mutant fails to properly activate the expression of genes encoding the transcription factors CgYap1p and CgMsn4p, as well as the gene expression of CgCTA1, which are involved in oxidative stress response and lead to the intracellular accumulation of ROS. Finally, we show that catechin administration reduces mortality in a Galleria mellonella model infected with C. glabrata. Our work thus supports the use of catechin in combination therapies for fungal infections and shows that the CgERG6 gene could be a potential new drug target.

Two promising natural lipopeptides from Bacillus subtilis effectively induced membrane permeabilization in Candida glabrata.

Candida glabrata is an important opportunistic human pathogen well known to develop resistance to antifungal drugs. Due to their numerous desirable qualities, antimicrobial lipopeptides have gained significant attention as promising candidates for antifungal drugs. In the present study, two bioactive lipopeptides (AF4 and AF5 m/z 1071.5 and 1085.5, respectively), coproduced and purified from Bacillus subtilis RLID12.1, consist of seven amino acid residues with lipid moieties. In our previous studies, the reversed phased-HPLC purified lipopeptides demonstrated broad-spectrum of antifungal activities against over 110 Candida albicans, Candida non-albicans and mycelial fungi. Two lipopeptides triggered membrane permeabilization of C. glabrata cells, as confirmed by propidium iodide-based flow cytometry, with PI uptake up to 99% demonstrating fungicidal effects. Metabolic inactivation in treated cells was confirmed by FUN-1-based confocal microscopy. Together, the results indicate that these lipopeptides have potentials to be developed into a new set of antifungals for combating fungal infections.

Overlooked Candida glabrata petites are echinocandin tolerant, induce host inflammatory responses, and display poor in vivo fitness.

Candida glabrata is a major fungal pathogen, which is able to lose mitochondria and form small and slow-growing colonies, called "petite." This attenuated growth rate has created controversies and questioned the clinical importance of petiteness. Herein, we have employed multiple omics technologies and in vivo mouse models to critically assess the clinical importance of petite phenotype. Our WGS identifies multiple genes potentially underpinning petite phenotype. Interestingly, petite C. glabrata cells engulfed by macrophages are dormant and, therefore, are not killed by the frontline antifungal drugs. Interestingly, macrophages infected with petite cells mount distinct transcriptomic responses. Consistent with our ex vivo observations, mitochondrial-proficient parental strains outcompete petites during systemic and gut colonization. Retrospective examination of C. glabrata isolates identified petite prevalence a rare entity, which can significantly vary from country to country. Collectively, our study overcomes the existing controversies and provides novel insights regarding the clinical relevance of petite C. glabrata isolates.

Biochemical and metabolomic insights into antifungal mechanism of berberine against Candida glabrata.

An unprecedented expansion of antifungal therapy failure incidences in healthcare settings of Candida glabrata is the matter of global concern that needs to be addressed efficiently and effectively. In this pursuit, the present study has investigated the antifungal mechanism of benzylisoquinoline alkaloid berberine using biochemical, metabolic, and gene expression analysis, with the aim to delineate its therapeutic activity against C. glabrata and differentially fluconazole-responsive clinical isolates. Interestingly, the clinical isolates were found to be highly susceptible to berberine. Berberine was found to control the surface properties like hydrophobicity and charge of the cells. The cell membrane composition was altered by berberine, where the ergosterol and fatty acids were affected. The efflux pump activity was inhibited, and osmotic stress was generated in C. glabrata cells upon berberine exposure. The berberine has also generated oxidative stress and activated antioxidant system in C. glabrata cells. Furthermore, these observations were supported by the transcriptional expression study of C. glabrata cell genes (CDR1, RLM1, SLT2, SUR4, KRE1) and metabolomics analysis. Based on fold change analysis, the study identified 20 differential metabolites upon berberine treatment, which belong to central carbon, amino acids, and nucleotide pathways. The checkerboard analysis revealed the potentiation of some classically used antifungal drugs by berberine, thus suggesting it as a combinatorial nutraceutical adjuvant for the eradication of fungal infections. KEY POINTS: • Berberine exhibited better potency against azole-resistant clinical isolates • Berberine modulated metabolites of different pathways • Berberine generated oxidative stress and blocked efflux pump activity.

Effect of Escin Alone or in Combination with Antifungal Agents on Resistant Candida glabrata Biofilms: Mechanisms of Action.

Nowadays, the increase in antimicrobial-resistant fungi (AMR) is certainly a major health concern, and the development of alternative therapeutic strategies has become crucial. Natural products have been used to treat various infections, and their chemical properties contribute to the performance of their biological activities, such as antifungal action. The various virulence factors and mechanisms of resistance to antifungals contribute to making Candida glabrata one of the most frequent agents of candidiasis. Here we investigate the in vitro and in vivo activity of β-escin against Candida glabrata. The β-escin MICs were determined for a reference strain and two clinical isolates of C. glabrata. Furthermore, growth kinetics assays and biofilm inhibition/eradication assays (crystal violet) were performed. The differences in the expression of some anti-biofilm-associated genes were analyzed during biofilm inhibition treatment so that reactive oxygen species could be detected. The efficacy of β-escin was evaluated in combination with fluconazole, ketoconazole, and itraconazole. In addition, a Galleria mellonella infection model was used for in vivo treatment assays. Results have shown that β-escin had no toxicity in vitro or in vivo and was able to inhibit or destroy biofilm formation by downregulating some important genes, inducing ROS activity and affecting the membrane integrity of C. glabrata cells. Furthermore, our study suggests that the combination with azoles can have synergistic effects against C. glabrata biofilm. In summary, the discovery of new antifungal drugs against these resistant fungi is crucial and could potentially lead to the development of future treatment strategies.

Macrophage internalization creates a multidrug-tolerant fungal persister reservoir and facilitates the emergence of drug resistance

Candida glabrata is a major fungal pathogen notable for causing recalcitrant infections, rapid emergence of drug-resistant strains, and its ability to survive and proliferate within macrophages. Resembling bacterial persisters, a subset of genetically drug-susceptible C. glabrata cells can survive lethal exposure to the fungicidal echinocandin drugs. Herein, we show that macrophage internalization induces cidal drug tolerance in C. glabrata, expanding the persister reservoir from which echinocandin-resistant mutants emerge. We show that this drug tolerance is associated with non-proliferation and is triggered by macrophage-induced oxidative stress, and that deletion of genes involved in reactive oxygen species detoxification significantly increases the emergence of echinocandin-resistant mutants. Finally, we show that the fungicidal drug amphotericin B can kill intracellular C. glabrata echinocandin persisters, reducing emergence of resistance. Our study supports the hypothesis that intra-macrophage C. glabrata is a reservoir of recalcitrant/drug-resistant infections, and that drug alternating strategies can be developed to eliminate this reservoir.

Synthesis of Benzimidazole-1,2,4-triazole Derivatives as Potential Antifungal Agents Targeting 14α-Demethylase.

Invasive fungal infections (IFIs) are increasing as major infectious diseases around the world, and the limited efficacy of existing medications has resulted in substantial morbidity and death in patients due to the lack of effective antifungal agents and serious drug resistance. In this study, a series of benzimidazole-1,2,4-triazole derivatives (6a-6l) were synthesized and characterized by 1H NMR, 13C NMR, and HR-MS spectral analysis. All the target compounds were screened for their in vitro antifungal activity against four fungal strains, namely, C. albicans, C. glabrata, C. krusei, and C. parapsilopsis. The synthesized compounds exhibited significant antifungal potential, especially against C. glabrata. Three compounds (6b, 6i, and 6j) showed higher antifungal activity with their MIC values (0.97 μg/mL) compared with voriconazole and fluconazole. Molecular docking provided a possible binding mode of compounds 6b, 6i, and 6j in the 14α-demethylase active site. Our studies suggested that the benzimidazole-1,2,4-triazole derivatives can be used as a new fungicidal lead targeting 14α-demethylase for further structural optimization. In addition, their effects on the L929 cell line were also investigated to evaluate the cytotoxic effects of the compounds. SEM analyses were performed to examine the effects of compounds 6a, 6i, and 6j on C. glabrata cells under in vivo experimental conditions.

Influence of menthol on biofilm formation, ergosterol content, and cell surface hydrophobicity of Candida glabrata.

Resistance to synthetic antifungals has become one of the leading public health challenges around the world. Accordingly, novel antifungal products like naturally occurring molecules can be one of the potential ways to reach efficient curative approaches to control candidiasis. This work evaluated the effect of menthol on cell surface hydrophobicity (CSH), biofilm formation, growth and ergosterol content of Candida glabrata (C. glabrata), a yeast with a high resistance against antifungal agents. Disc diffusion method (susceptibility to synthetic antifungals), broth micro-dilution method (Susceptibility to menthol), MTT reduction assay (biofilm formation), HPLC technique (ergosterol content) and adherence to n-hexadecane (CSH) were employed to determine the influence of menthol against C. glabrata isolates. The MIC range of menthol versus C. glabrata was 1250-5000µg/mL (mean±SD: 3375 ± 1375µg/mL). The mean rate of C. glabrata biofilm formation was decreased up to 97.67, 81.15, 71.21, 63.72, 47.53, 26.31 and 0.051% at 625, 1250, 2500, 5000, 10000, 20000 and 40000µg/mL concentrations, respectively. The percentages of CSH were significantly in groups treated with MIC/2 (17.51±5.52%) and MIC/4 (26±5.87%) concentrations of menthol. Also, the percentage changes in membrane ergosterol were 15.97, 45.34 and 73.40% at 0.125, 0.25 and 0.5mg/mL concentrations of menthol, respectively, in comparison with untreated control. The results showed the menthol impact versus sessile and planktonic C. glabrata cells, and the interference with ergosterol content, CSH, and biofilm formation, which made it a potent natural antifungal.

The inhibitory effects of tyrosol on clinical Candida glabrata planktonic and biofilm cells

Biofilm formation is an important problem in the healthcare industry and veterinary medicine and is very common in natural, industrial or hospital environments. Microorganisms can become very resistant to antimicrobials and environmental factors by biofilm forming on biotic or abiotic surfaces. There is a need to develop new, effective and specific antimicrobials that can reduce pathogenicity in biofilm formation that threatens public health due to their role in medical device-related or infectious diseases. Candida species are opportunistic pathogenic yeasts and can cause superficial or disseminated infections. Especially C. glabrata is one of the most common microorganisms causing fungal infections in immunocompromised patients and drug resistance is observed when associated with biofilm. Tyrosol (2-[4-hydroxyphenyl] ethanol) can act as both a quorum sensing molecule and an exogenous agent on Candida species. In this study, the antifungal activity of tyrosol against a clinical C. glabrata isolate was investigated on both planktonic and biofilm forms. Broth microdilution test results demonstrated the inhibitory effect of tyrosol on C. glabrata. Transmission electron microscopic findings showed that tyrosol affected the planktonic C. glabrata cells in a multi targeted manner, and in the groups treated with tyrosol, significant damage was observed in the cell wall, cell membrane, cytoplasm, nucleus and mitochondria. Also, scanning electron microscopic images confirmed biofilm reduction in pre-/post-biofilm applications as a result of tyrosol treatment. In conclusion, tyrosol may be a potential alternative candidate for reducing the C. glabrata biofilm.

Genomic evolution towards azole resistance in Candida glabrata clinical isolates unveils the importance of CgHxt4/6/7 in azole accumulation

The increasing prevalence of candidosis caused by Candida glabrata is related to its ability to acquire azole resistance. Although azole resistance mechanisms are well known, the mechanisms for azole import into fungal cells have remained obscure. In this work, we have characterized two hexose transporters in C. glabrata and further investigate their role as potential azole importers. Three azole susceptible C. glabrata clinical isolates were evolved towards azole resistance and the acquired resistance phenotype was found to be independent of CgPDR1 or CgERG11 mutations. Through whole-genome sequencing, CgHXT4/6/7 was found to be mutated in the three evolved strains, when compared to their susceptible parents. CgHxt4/6/7 and the 96% identical CgHxt6/7 were found to confer azole susceptibility and increase azole accumulation in C. glabrata cells, strikingly rescuing the susceptibility phenotype imposed by CgPDR1 deletion, while the identified loss-of-function mutation in CgHXT4/6/7, leads to increased azole resistance. In silico docking analysis shows that azoles display a strong predicted affinity for the glucose binding site of CgHxt4/6/7. Altogether, we hypothesize that hexose transporters, such as CgHxt4/6/7 and CgHxt6/7, may constitute a family of azole importers, involved in clinical drug resistance in fungal pathogens, and constituting promising targets for improved antifungal therapy.

P029 Synergic effect of deferoxamine combined with fluconazole against fluconazole-resistant Candida Spp. through inhibited Cek1 MAPK signaling pathway

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesThe opportunistic fungal infections represent an increasing threat to humans with the increase of immunocompromised patients, in which Candida albicans is the most common fungal pathogen. Though fluconazole (FCA) is still the first line choice to treat C. albicans infections, several limitations such as an increase in drug resistance compromised its clinical application. This study proposes a combination therapy of deferoxamine (DFO) and FCA to overcome C. albicans resistance.MethodsCheckerboard microdilution assay was used to determine the minimum inhibitory concentration (MIC) of DFO used alone and in combination with FCA against FCA-resistant Candida Spp. Spot assay and time-kill curves were used to investigate the cell viability and dynamic inhibitory effect. Hyphal formation was performed to investigate the underlying mechanism of DFO. Then, a murine model of cutaneous candidiasis was established to explore the in vivo synergistic activity of DFO and FCA.ResultsDFO combined with FCA showed synergistic antifungal activity against FCA-resistant C. albicans, with a fractional inhibitory concentration index (FICI) of 0.25. Moreover, DFO combined with FCA significantly inhibited the activity of C. glabrata cells, which is naturally insensitive to antifungal drugs. The spot assay and time-kill curve assay indicated that DFO can turn the fungistasis activity of FCA into fungicidal activity. Hyphal formation study showed the inhibition of hyphal induction of C. albicans. DFO combined with FCA also significantly inhibited the expression of Cek1 MAPK signaling pathway-related genes (CEK1 and CPH1) and adhesion-related genes (ALS1). In vivo data showed DFO combined with FCA significantly reduced the pustule, CFU numbers, and inflammatory cell infiltration of skin tissue.ConclusionOur results suggest that DFO combined with FCA inhibited the transformation of yeast-hyphae through Cek1 MAPK signaling pathway, resulting in reduced infectivity and resistance of C. albicans in vitro and in vivo, which may provide a new option for the treatment of cutaneous candidiasis.

Erg6p is essential for antifungal drug resistance, plasma membrane properties and cell wall integrity in Candida glabrata.

ERG6 gene encodes C-24 methyltransferase, one of the specific enzymes that differ in mammalian and yeast sterol biosynthesis. To explore the function of CgErg6p in the yeast pathogen Candida glabrata, we have constructed the Cgerg6Δ deletion mutant. We found that C. glabrata cells lacking CgErg6p exhibit reduced susceptibility to both antifungal azoles and polyenes. The reduced content of ergosterol in the Cgerg6 deletion mutant was accompanied by increased expression of genes encoding the last steps of the ergosterol biosynthetic pathway. The absence of CgErg6p leads to plasma membrane hyperpolarization and decrease in its fluidity compared to the parental C. glabrata strain. The absence of sterols containing C-24 alkyls influenced the susceptibility of Cgerg6Δ mutant cells to alkali metal cations and several other metabolic inhibitors. Our results thus show that sterols lacking C-24 alkyls are not sufficient substitutes for maintaining yeast plasma membrane function. The absence of CgErg6p influences also the cell wall integrity and calcineurin signaling in C. glabrata.

Inhibition of Candida glabrata Biofilm by Combined Effect of Dendritic Compounds and Amphotericin.

In the last decade, Candida glabrata has become an important emerging opportunistic pathogen not only because of the increase in nosocomial infections frequency but also because of its ability to form biofilms and its innate resistance to commercial antifungals. These characteristics make this pathogen a major problem in hospital settings, including problems regarding equipment, and in immunosuppressed patients, who are at high risk for candidemia. Therefore, there is an urgent need for the development of and search for new antifungal drugs. In this study, the efficacy of two dendritic wedges with 4-phenyl butyric acid (PBA) at the focal point and cationic charges on the surface ArCO2G2(SNMe3I)4 (1) and ArCO2G3(SNMe3I)8 (2) was studied against C. glabrata strain to inhibit the formation of biofilms and eliminate established biofilm. For this, MBIC (minimum biofilm inhibitory concentration), MBDC (minimum biofilm damaging concentrations), as well as MFCB (minimum fungicidal concentration in biofilm) and MBEC (minimum biofilm eradicating concentration) were determined. In addition, different combinations of dendrons and amphotericin B were tested to study possible synergistic effects. On the other hand, cytotoxicity studies were performed. C. glabrata cells and biofilm structure were visualized by confocal microscopy. ArCO2G2(SNMe3I)4 (1) and ArCO2G3(SNMe3I)8 (2) dendrons showed both an MBIC of 8 mg/L and a MBDC of 32 mg/L and 64 mg/L, respectively. These dendrons managed to eradicate the entirety of an established biofilm. In combination with the antifungal amphotericin, it was possible to prevent the generation of biofilms and eradicate established biofilms at lower concentrations than those required individually for each compound at these conditions.

Elucidating the lactic acid tolerance mechanism in vaginal clinical isolates of Candida glabrata.

Incidence of vulvovaginal candidiasis are strikingly high and treatment options are limited with nearly 50% Candida glabrata cases left untreated or experience treatment failures. The vaginal microenvironment is rich in lactic acid, and the adaptation of C. glabrata to lactic acid (LA) is the main reason for clinical treatment failure. In the present study, C. glabrata and its vaginal clinical isolates were comprehensively investigated for their growth response, metabolic adaptation and altered cellular pathway to LA using different biochemical techniques, metabolic profiling and transcriptional studies. C. glabrata shown considerable variations in its topological and biochemical features without compromising growth in LA media. Chemical profiling data highlighted involvement of cell wall/membrane, ergosterol and oxidative stress related pathways in mediating adaptative response of C. glabrata towards LA. Further, one dimensional proton (1H) NMR spectroscopy based metabolic profiling revealed significant modulation in 19 metabolites of C. glabrata cells upon growth in LA. Interestingly myo-inositol, xylose, putrescine and betaine which are key metabolites for cell growth and viability were found to be differentially expressed by clinical isolates. These observations were supported by the transcriptional expression study of selected genes evidencing cell wall/membrane re-organisation, altered oxidative stress, and reprogramming of carbon metabolic pathways. Collectively, the study advances our understanding on adaptative response of C. glabrata in vaginal microenvironment to lactic acid for survival and virulence.

Histone Acetylation Regulator Gcn5 Mediates Drug Resistance and Virulence of Candida glabrata.

ABSTRACTCandida glabrata is poised to adapt to drug pressure rapidly and acquire antifungal resistance leading to therapeutic failure. Given the limited antifungal armamentarium, there is an unmet need to explore new targets or therapeutic strategies for antifungal treatment. The lysine acetyltransferase Gcn5 has been implicated in the pathogenesis of C. albicans. Yet how Gcn5 functions and impacts antifungal resistance in C. glabrata is unknown. Disrupting GCN5 rendered C. glabrata cells more sensitive to various stressors, partially reverted resistance in drug-resistant mutants, and attenuated the emergence of resistance compared to wild-type cells. RNA sequencing (RNA-seq) analysis revealed transcriptomic changes involving multiple biological processes and different transcriptional responses to antifungal drugs in gcn5Δ cells compared to wild-type cells. GCN5 deletion also resulted in reduced intracellular survival within THP-1 macrophages. In summary, Gcn5 plays a critical role in modulating the virulence of C. glabrata and regulating its response to antifungal pressure and host defense.IMPORTANCE As an important and successful human pathogen, Candida glabrata is known for its swift adaptation and rapid acquisition of resistance to the most commonly used antifungal agents, resulting in therapeutic failure in clinical settings. Here, we describe that the histone acetyltransferase Gcn5 is a key factor in adapting to antifungal pressure and developing resistance in C. glabrata. The results provide new insights into epigenetic control over the drug response in C. glabrata and may be useful for drug target discovery and the development of new therapeutic strategies to combat fungal infections.

Niacin Limitation Promotes Candida glabrata Adhesion to Abiotic Surfaces.

Candida glabrata is a prevalent fungal pathogen in humans, which is able to adhere to host cells and abiotic surfaces. Nicotinic acid (NA) limitation has been shown to promote the adherence of C. glabrata to human epithelial cells. Clinically, the elderly and hospitalized patients who are prone to C. glabrata–related denture stomatitis often suffer from vitamin deficiency. This study aimed to investigate C. glabrata adhesion to abiotic surfaces, including acrylic resin (a denture material) surfaces, cell surface hydrophobicity and adhesion gene expression. C. glabrata CBS138 was grown in media containing decreasing NA concentrations (40, 0.4, 0.04 and 0.004 µM). Adherence of C. glabrata to glass coverslips and acrylic resin was analyzed. C. glabrata adhesion to both surfaces generally increased with decreasing NA concentrations. The highest adhesion was found for the cells grown with 0.004 µM NA. The cell surface hydrophobicity test indicated that NA limitation enhanced hydrophobicity of C. glabrata cells. Quantitative PCR showed that of all adhesion genes tested, EPA1, EPA3 and EPA7 were significantly up-regulated in both 0.004 µM NA and 0.04 µM NA groups compared to those in the 40 µM NA group. No significant up- or down-regulation under NA limitation was observed for the other tested adhesion genes, namely AWP3, AWP4, AWP6 and EPA6. NA limitation resulted in increased expression of some adhesion genes, higher surface hydrophobicity of C. glabrata and enhanced adhesion to abiotic surfaces. NA deficiency is likely a risk factor for C. glabrata–related denture stomatitis in the elderly.

Plausible Mechanistic Insights in Biofilm Eradication Potential against Candida spp. Using In Situ-Synthesized Tyrosol-Functionalized Chitosan Gold Nanoparticles as a Versatile Antifouling Coating on Implant Surfaces.

In the present study, tyrosol-functionalized chitosan gold nanoparticles (Chi-TY-AuNPs) were prepared as an alternative treatment strategy to combat fungal infections. Various biophysical techniques were used to characterize the synthesized Chi-TY-AuNPs. The antifungal and antibiofilm activities of Chi-TY-AuNPs were evaluated against Candida albicans and C. glabrata, and efforts have been made to elucidate the possible mechanism of action. Chi-TY-AuNPs showed a high fungicidal effect against both sessile and planktonic cells of Candida spp. Additionally, Chi-TY-AuNPs completely eradicated (100%) the mature biofilms of both the Candida spp. FESEM analysis highlighted the morphological alterations in Chi-TY-AuNP-treated Candida biofilm cells. The effect of Chi-TY-AuNPs on the ECM components showed significant reduction in protein content in the C. glabrata biofilm and substantial decrease in extracellular DNA content of both the Candida spp. ROS generation analysis using DCFDA-PI staining showed high ROS levels in both the Candida spp., whereas pronounced ROS production was observed in the Chi-TY-AuNP-treated C. glabrata biofilm. Biochemical analysis revealed decreased ergosterol content in Chi-TY-AuNP-treated C. glabrata cells, while inconsequential changes were observed in C. albicans. Furthermore, the transcriptional expression of selected genes (ergosterol biosynthesis, efflux, sterol importer, and glucan biogenesis) was reduced in C. glabrata in response to Chi-TY-AuNPs except ERG11 and CDR1. Conclusively, the result showed the biofilm inhibition and biofilm eradication efficacy of Chi-TY-AuNPs in both the Candida spp. Findings of the present study manifest Chi-TY-AuNPs as a potential therapeutic solution to Candida biofilm-related chronic infections and overcome biofilm antifungal resistance.

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.

From the first touch to biofilm establishment by the human pathogen Candida glabrata: a genome-wide to nanoscale view

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.

Geraniol eradicates Candida glabrata biofilm by targeting multiple cellular pathways.

Global burden of fungal infections and associated health risk has accelerated at an incredible pace and needs to be attended at the earliest with an unbeatable therapeutic intervention. Candida glabrata is clinically the most relevant and least drug susceptible Candida species. In the pursuit of mining alternative novel drug candidates, the antifungal activity of a monoterpene phytoactive molecule geraniol (GR) against C. glabrata biofilm was evaluated. Biofilm inhibitory and eradication ability of GR evaluated against C. glabrata along with its clinical isolates. Impact of GR on various cellular pathways was evaluated to delineate its antifungal mode of action. GR has inhibited both planktonic and sessile growth of all the studied C. glabrata strains and eradicated the mature biofilm. GR reduced the carbohydrate and eDNA content, as well as hydrolytic enzyme activity in extracellular matrix of C. glabrata. The chemical profiling, microscopic, and spectroscopic studies revealed that GR targets chitin and β-glucan in cell wall. Further, results highlighted the reduction of cell membrane ergosterol content, and blocking of ABC drug efflux pump by GR which was also confirmed by RT-PCR where expression of CDR1 and ERG4 was downregulated in GR exposed C. glabrata cells. The fluorescence microscopy and flow cytometry results emphasized the alteration in mitochondrial activity, increased Ca+2 uptake, thus changing the membrane permeability ensuing increased cytochrome C release from mitochondria to cytoplasm. Indeed, GR also has arrested cell cycle in G1/S phase and interfered with DNA replication. These observations suggest GR targets multiple cellular pathways and mediated killing of C. glabrata cells via apoptosis. In conclusion, the present study strengthens the candidacy of GR as novel antifungal therapeutic. Key points • GR inhibits growth and eradicates biofilm of C. glabrata and its clinical isolates. • GR inactivates the hydrolytic enzymes in extracellular matrix. • GR mediates C. glabrata apoptosis by interfering with multiple signaling pathways.