ERG11 Gene Research Articles

Candida albicans Clinical Isolates from a Southwest Brazilian Tertiary Hospital Exhibit MFS-mediated Azole Resistance Profile.

Candida albicans is the most frequent fungal species that causes infections in humans. Fluconazole is the main antifungal used to treat Candida infections, and its prolonged and indiscriminate use for the last decades are the most established causes which originated resistant strains. Fungal drug resistance is associated to alterations in ERG11 gene and overexpression of multidrug resistance (MDR) transporters belonging to two families: ATP-binding cassette (ABC) and Major Facilitator Superfamily (MFS). To evaluate the role of MFS transporters in azoles resistance of C. albicans clinical strains, this study aimed to analyze four Candida albicans clinical isolates from the University Hospital in Juiz de Fora (Minas Gerais/Brazil), selected in our previous study as they were unaffected by FK506, an ABC pumps inhibitor. In a primary investigation on MFS proteins overexpression, the extrusion of fluorescent substrates (rhodamine 6G and nile red) was analyzed by fluorescence microscopy and flow cytometry. Results suggest participation of MFS transporters in azole resistance of C. albicans isolates and indicate the existence of secondary resistance mechanisms. Therefore, this study contributes to the information about Candida albicans infections in Brazil and reinforces the importance of epidemiological studies focusing on an improved understanding of the disease and further resistance reversion.

Biochemical and Molecular Effects of <i>Phoenix dactylifera</i> and <i>Ziziphus spina-christi</i> Extracts on <i>Candida albicans</i>

Our overall knowledge of the medicinal uses of plants suggests that natural compounds could be useful in the treatment of cutaneous fungal infections in tropical regions. Furthermore, the possibilities of treatment using plant extracts may be even broader than is already known when one considers plants that have not been extensively studied in this context, such as the regional species Phoenix dactylifera and Ziziphus spina-christi. This study compared the antimicrobial activity of Phoenix dactylifera and Ziziphus spina-christi extracts in terms of their biochemical and molecular effects on Candida albicans (ATCC CA 10231). These effects included altered levels of intracellular sterols, changes in the permeability of the cell membrane, and changes in the TEF1: QRTTEF1, CaERG1: ERG1, CdERG12: CdERG1, and ERG25: ERG25 genes. Scanning electron microscopy (SEM) was used to identify morphological characteristics, and energy-dispersive X-ray spectroscopy (EDAX) analyses were conducted. In treated samples, the SEM and EDAX analyses showed cell cavities and shrinkage of the cell wall, and the number of cells was reduced to only a few abnormal cells as compared with that in the untreated samples. Yttrium was detected in the cells treated with Z. spina-christi, and high levels of osmium were detected in the cells treated with P. dactylifera. Compared with control cells, cells exposed to the concentration 150 μl/ml of Z. spina-christi extract had an average sterol concentration that was nearly 3 times higher, while the concentration was 5.5 times higher for cells treated with the 150 μl/ml of P. dactylifera extract. The ethanol extracts affected the permeability of C. albicans cell membrane. Gene sequencing showed gaps and mismatches in the ERG1, ERG12, and ERG25 genes after treatment with P. dactylifera and Z. spina-christi extracts compared with that in the controls. The results were highly significant (p ≤ 0.01). We conclude that the ethanol extracts of P. dactylifera and Z. spina-christi have antimicrobial activity through several mechanisms in the yeast cell.

Expression of Lanosterol 14-Demethylase (ERG11) Gene of Three-Drug Combinations in Candida albicans

Patients with impaired immunity are at particular risk of infections with Candida albicans. Antifungal drugs such as azoles commonly used for candidiasis treatment, but drug resistance is one of the most common problems for public health. The aim of this study was to evaluate the expression of lanosterol 14-demethylase (ERG11) gene for three-drug combinations in C. albicans. Disk diffusion and broth microdilution susceptibility tests were employed to evaluate the synergic effects of amphotericin B, fluconazole, voriconazole, ketoconazole and itraconazole. Quantification of ERG11 gene expression was carried out in C. albicans treated with three-drug combinations of fluconazole/ ketoconazole/ voriconazole and fluconazole/ ketoconazole/ itraconazole. Three-drug combinations revealed synergistic and partial synergistic effect for all tested isolates (FIC index range of 0.27-0.77). The expression levels of ERG11 were down-regulated by three-drug combination of fluconazole/ ketoconazole/ voriconazole treatment. Fluconazole synergizes with ketoconazole and voriconazole in three-drug combination against C. albicans by targeting of the ERG11 gene.

Distribution of secreted aspartyl protease (SAP) virulence genes and antifungal resistance genes at vulvovaginal candidiasis isolates

Virulence factors and antifungal resistance features of Candida albicans considering a growing health problem worldwide. This study made to show the expression of both virulence and azole resistance genes at 100 clinical isolates of Candida we used a model of infection of human vaginal epithelial cells with C. albicans strains isolated from Egyptian women with vulvovaginal candidiasis (VVC). The detection and expression of virulence genes and azole resistance genes were performed using PCR technic. All isolates were susceptible to ketoconazole (KTC), 3 isolates (3%) only were resist to both nystatin (NY) and amphotericin B (AMB) ,all isolates found resist to griseofulvin (AGF) 10 µg, eighty five isolates (85%)were resist to flucytosine (AFY), four isolates (4%)were resist to miconazole (MCL), seventy one isolates (71%) were resist to voriconazole (VO), thirty three isolates (33 %) were resist to itraconazole (ITC), forty one isolates and twenty seven isolates (27%)were resist to 100 µg fluconazole (flu). All isolated strains expressed SAP4-SAP6 (100%) and almost all expressed SAP1-SAP3 (91%) In this study, fluconazole resistance was identified in 27% of the strains, whereas (27%) had positive ERG11 gene, (27%) were positive MDR1 gene and (14%) were positive CDR1 gene. The results indicate that the strains that infect Egyptian patients suffering from VVC are highly virulent and virtually all are insensitive to fluconazole.

Ergosterol reduction impairs mitochondrial DNA maintenance in S. cerevisiae

Sterols are essential lipids, involved in many biological processes. In Saccharomyces cerevisiae, the enzymes of the ergosterol biosynthetic pathway (Erg proteins) are localized in different cellular compartments. With the aim of studying organelle interactions, we discovered that Erg27p resides mainly in Lipid Droplets (LDs) in respiratory competent cells, while in absence of respiration, is found mostly in the ER. The results presented in this paper demonstrate an interplay between the mitochondrial respiration and ergosterol production: on the one hand, rho° cells show lower ergosterol content when compared with wild type respiratory competent cells, on the other hand, the ergosterol biosynthetic pathway influences the mitochondrial status, since treatment with ketoconazole, which blocks the ergosterol pathway, or the absence of the ERG27 gene, induced rho° production in S. cerevisiae. The loss of mitochondrial DNA in the ∆erg27 strain is fully suppressed by exogenous addition of ergosterol. These data suggest the notion that ergosterol is essential for maintaining the mitochondrial DNA attached to the inner mitochondrial membrane.

Molecular mechanisms of azole resistance in Candida tropicalis isolates causing invasive candidiasis in China

ObjectiveWe investigated molecular mechanisms responsible for azole resistance in Candida tropicalis isolates. MethodsWe studied 507 C. tropicalis isolates causing invasive candidiasis from ten hospitals over 5 years. Antifungal susceptibility was determined by broth microdilution methods. Point mutations in the C. tropicalis ERG11 gene that may confer azole resistance were explored and verified. The expression levels of ERG11, CYTb, MDR1 and CDR1 genes were compared in 20 fluconazole-susceptible and 20 fluconazole-resistant isolates. ResultsFluconazole-susceptible, -susceptible dose-dependent and -resistant strains accounted for 76.7% (389/507), 10.5% (53/507) and 12.8% (65/507) of C. tropicalis isolates, respectively. The ERG11 mutation A395T/W occurred in 10.7% (54/507) of isolates, all of which were resistant to fluconazole. The nucleotide mutation C461T/Y was the second most common (50/507 isolates, 9.9%), and all isolates carrying C461T/Y also had the mutation A395T/W. However, the presence of C461T did not contribute to the azole-resistant phenotype. Substitutions V125A, Y257H and G464S (<2% of isolates), which were reported for the first time in C. tropicalis, also conferred fluconazole non-susceptible phenotypes. Compared with fluconazole susceptible isolates, fluconazole-resistant isolates had higher ERG11 (fold expression level 1.42 versus 0.79, p < 0.01) but lower CYTb (fold expression level 1.26 versus 2.67, p < 0.01) gene expression levels. Three azole-resistant isolates carrying the wild-type ERG11 gene had higher levels of CDR1 and MDR1 expression. ConclusionsERG11 missense mutations were the major mechanism responsible for azole resistance in C. tropicalis isolates, but overexpression of ERG11, CDR1 and MDR1, as well as reduced expression of CYTb, also contributed to resistance.

Erg6 gene is essential for stress adaptation in Kluyveromyces lactis.

We investigated the effect of Kluyveromyces lactis ERG6 gene deletion on plasma membrane function and showed increased susceptibility of mutant cells to salt stress, cationic drugs and weak organic acids. Contrary to Saccharomyces cerevisiae, Klerg6 mutant cells exhibited increased tolerance to tunicamycin. The content of cell wall polysacharides did not significantly vary between wild-type and mutant cells. Although the expression of the NAD+-dependent glycerol 3-phosphate dehydrogenase (KlGPD1) in the Klerg6 mutant cells was only half of that in the parental strain, it was induced in the presence of calcofluor white. Also, cells exposed to this drug accumulated glycerol. The absence of KlErg6p led to plasma membrane hyperpolarization but had no statistically significant influence on the plasma membrane fluidity. We propose that the phenotype of Klerg6 mutant cells to a large extent was a result of the reduced activity of specific plasma membrane proteins that require proper lipid composition for full activity.

Changes in lipid metabolism convey acid tolerance in Saccharomyces cerevisiae

BackgroundThe yeast Saccharomyces cerevisiae plays an essential role in the fermentation of lignocellulosic hydrolysates. Weak organic acids in lignocellulosic hydrolysate can hamper the use of this renewable resource for fuel and chemical production. Plasma-membrane remodeling has recently been found to be involved in acquiring tolerance to organic acids, but the mechanisms responsible remain largely unknown. Therefore, it is essential to understand the underlying mechanisms of acid tolerance of S. cerevisiae for developing robust industrial strains.ResultsWe have performed a comparative analysis of lipids and fatty acids in S. cerevisiae grown in the presence of four different weak acids. The general response of the yeast to acid stress was found to be the accumulation of triacylglycerols and the degradation of steryl esters. In addition, a decrease in phosphatidic acid, phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine, and an increase in phosphatidylinositol were observed. Loss of cardiolipin in the mitochondria membrane may be responsible for the dysfunction of mitochondria and the dramatic decrease in the rate of respiration of S. cerevisiae under acid stress. Interestingly, the accumulation of ergosterol was found to be a protective mechanism of yeast exposed to organic acids, and the ERG1 gene in ergosterol biosynthesis played a key in ergosterol-mediated acid tolerance, as perturbing the expression of this gene caused rapid loss of viability. Interestingly, overexpressing OLE1 resulted in the increased levels of oleic acid (18:1n-9) and an increase in the unsaturation index of fatty acids in the plasma membrane, resulting in higher tolerance to acetic, formic and levulinic acid, while this change was found to be detrimental to cells exposed to lipophilic cinnamic acid.ConclusionsComparison of lipid profiles revealed different remodeling of lipids, FAs and the unsaturation index of the FAs in the cell membrane in response of S. cerevisiae to acetic, formic, levulinic and cinnamic acid, depending on the properties of the acid. In future work, it will be necessary to combine lipidome and transcriptome analysis to gain a better understanding of the underlying regulation network and interactions between central carbon metabolism (e.g., glycolysis, TCA cycle) and lipid biosynthesis.

Functional characterization of squalene epoxidase genes in the medicinal plant Tripterygium wilfordii

Squalene epoxidase, thought to be one of the rate-limiting enzymes in the biosynthetic pathways of both membrane sterols and triterpenes (e.g., celastrol), catalyses the formation of oxidosqualene as the common precursor of sterols and triterpenoids. In this work, we first found five squalene epoxidase genes (TwSEs) from Tripterygium wilfordii. Tissue expression pattern, consistent with methyl jasmonate induction study, showed that TwSEs1–4 were involved in the production of special metabolites. In contrast, TwSE5 showed a different tissue expression pattern and was not induced by methyl jasmonate. To probe the functions of the TwSEs, we first tried using a prokaryotic system by constructing an engineered bacterium, but we failed to detect their products. Next, we used the CRISPR/Cas9 tool to construct an erg1 mutant yeast by knocking out the ERG1 gene of yeast strain BY4741 and then applied this mutant to identify the function of TwSEs. We found that only TwSEs1–4 can functionally complement the erg1 mutant yeast. This study laid the foundation for the heterologous biosynthesis of special metabolites in Tripterygium wilfordii.

ERG11 couples oxidative stress adaptation, hyphal elongation and virulence in Candida albicans.

Candida albicans is a major fungal opportunistic pathogen for humans. In the treatment of C. albicans, azole drugs target the sterol 14α-demethylase (CYP51) encoded by ERG11 gene. Most studies have focused on the fact that the ERG11 mutant results in drug resistance, but its mechanism of action as a drug target has not been described yet. Our results showed that deletion of ERG11 reduced filamentous and invasive growth, and impaired hyphal elongation in sensing serum. Lack of ERG11 increased susceptibility to H2O2 and was defective in clearing reactive oxygen species. ERG11 may affect oxidative stress adaptation by specifically downregulating CAT1 expression. In addition, C. albicans cells lacking ERG11 were more efficiently killed by macrophages and became avirulent in vivo. This study is the first to indicate that ERG11 plays an essential role in hyphal elongation, oxidative stress adaptation and virulence in C. albicans. We speculated that azole drugs not only inhibit the growth of C. albicans, but also assist the host immune system in clearing the fungal organism. The new understanding of mechanisms of action of antifungal drugs should facilitate the development of treatment strategies for resistant fungal infections.

Multi-azole-resistant strain of Malassezia pachydermatis isolated from a canine Malassezia dermatitis

In the case presented here, we describe the isolation of an azole-resistant strain of M. pachydermatis from a canine Malassezia dermatitis. The isolate (NUBS18001) from this case exhibited a minimum inhibitory concentration (MIC) of 320μg/ml to itraconazole (ITZ) by broth microdilution (BM) assay, >32μg/ml to ITZ by E-test, and >32μg/ml to KTZ by E-test. Synergistic effects between FK506 and ITZ in the azole-resistant strain was evaluated using the microdilution checker-board method. The ITZ-resistant strain exhibited MICs of 320μg/mL of ITZ alone and 5μg/ml of FK506 alone; the addition of FK506 attenuated the ITZ MIC to 2.5μg/ml, yielding an ITZ FICI value of 0.507. This result suggested that the combination of ITZ and FK506 exerted an additive effect against the ITZ-resistant strain. To understand the other mechanism inferred to be present in our multi-azole-resistant strain, we sequenced the ERG11 gene from this isolate, and detected missense mutations (A412G and C905T) in the sequence of the ERG11 open reading frame (ORF). To the best of our knowledge, this work is the first report that a multi-azole-resistant M. pacydermatis strain contains mutations in ERG11.

Lovastatin synergizes with itraconazole against planktonic cells and biofilms of Candida albicans through the regulation on ergosterol biosynthesis pathway

The increase of fungal infectious diseases and lack of safe and efficacious antifungal drugs result in the urgent need of new therapeutic strategies. Here, we repurposed the lovastatin (LOV) as a synergistic antifungal potentiator to itraconazole (ITZ) against Candida albicans planktonic cells and biofilms in vitro for the first time. Mutants from ergosterol biosynthesis pathway were employed and key gene expression profiles of ergosterol pathway were also measured. LOV single treatment was unable to inhibit C. albicans strains except the ERG3 and ERG11 double mutant. LOV and ITZ combination was capable of inhibiting the C. albicans planktonic cells and biofilms synergistically including the ITZ resistant mutants. The synergistic antifungal ability was stronger in either ERG11 or ERG3 dysfunctional mutants compared to wild type. The combination lost the synergistic activities in the ERG11 and ERG3 double mutant, while it was sensitive to LOV single treatment. The expression of HMG1, encoding HMG-CoA the target of LOV, was significantly upregulated in ERG11 and ERG3 double mutant strain by the treatment of the combination at 1.5 and 3h. The combination also significantly increased the HMG1 expression in mutants from ergosterol pathway compared with wild type. The ERG11 and ERG3 gene expressions were upregulated by ITZ and its combination with LOV, but seemingly not by LOV single treatment after 1.5 and 3h. The combination of LOV and ITZ on C. albicans planktonic cells and biofilms highlights its potential clinical practice especially against the azole drug-resistant mutants.

MDR1 overexpression combined with ERG11 mutations induce high-level fluconazole resistance in Candida tropicalis clinical isolates

BackgroundMarked increases in fluconazole resistance in Candida tropicalis have been recently reported. In this study, the molecular mechanisms behind fluconazole resistance were investigated.MethodsTwenty-two C. tropicalis clinical isolates, including 12 fluconazole-resistant isolates and 10 fluconazole-susceptible isolates, were collected from a tertiary care teaching hospital in Beijing between 2013 and 2017. Antifungal susceptibility testing, multilocus sequence typing, ERG11 amplification and sequencing, quantitative real-time reverse transcription-polymerase chain reaction (ERG11, UPC2, MDR1, and CDR1), and clinical data collection were performed for all C. tropicalis isolates.ResultsMultilocus sequence typing revealed that the 10 fluconazole-susceptible isolates and 12 fluconazole-resistant isolates were divided into nine and seven diploid sequence types, respectively. Of the 12 patients with fluconazole-resistant isolates, six had been previously exposed to azole and four had a fatal outcome. Y132F and S154F amino acid substitutions in Erg11p were found in all fluconazole-resistant isolates except one. MDR1 gene overexpression was identified in fluconazole-resistant isolates. In particular, seven high-level fluconazole resistant isolates (minimum inhibitory concentration ≥ 128 mg/L) and three pan-azole resistant isolates were identified. CDR1, ERG11, and UPC2 gene expression levels in fluconazole-resistant isolates were not significantly different from the control isolates (P = 0.262, P = 0.598, P = 0.114, respectively).ConclusionsThis study provides evidence that the combination of MDR1 gene overexpression and ERG11 missense mutations is responsible for high-level fluconazole resistance and pan-azole resistance in C. tropicalis clinical isolates. To the best of our knowledge, this is the first study investigating the relationship between MDR1 gene overexpression and increased fluconazole resistance.

Analysis of ergosterol and gene expression profiles of sterol Δ5,6-desaturase (ERG3) and lanosterol 14α-demethylase (ERG11) in Candida albicans treated with carvacrol

Introduction: Usually, for treatment of fungal infections, antifungals such as azoles are used, but one of the biggest problems faced in clinical practice is the emergence of resistance for most of these drugs. Antifungal drugs derived from plants may alleviate this problem. The aims of this study were to analyse the ergosterol and gene expression profiles of ERG genes in Candida albicans treated with carvacrol. Methods: We used carvacrol and conducted a series of follow-up studies to examine the inhibitors of Candida species isolated from immunocompromised patients. Antifungal susceptibility test, time-kill study, ergosterol binding assay and ergosterol content were investigated. Eventually, the expression of ERG3 and ERG11genes was carried out to investigate the inhibitory properties of antifungal activity against Candida albicans using quantitative real time RT-PCR. Results: Carvacrol was able to inhibit Candida species and reduce time-kill kinetic in C. albicans. This phytoconstituent acted by binding to ergosterol in the fungal membrane and caused a reduction of 52% of the ergosterol content compared to the untreated growth control. Finally, carvacrol displayed significant down-regulation of ERG3 and ERG11genes in C. albicans. Conclusion: These results provide proof of concept for the implementation of carvacrol inhibitors of Candida species. In addition, ERG3 and ERG11 genes could be probable target of carvacrol against C. albicans.

Antifungal Activity of Carvacrol on Ergosterol Synthesis in Multidrug Resistant Candida albicans

Introduction: Greatly increased use of antifungal therapies has resulted in the development of multidrug resistant. The phenolic compound carvacrol have been reported to have anti-Candida activity. This work is an attempt to examine effect of carvacrol on ergosterol synthesis against multidrug resistant Candida albicans. Methods: This cross-sectional study has been conducted on 30 immune-compromised patients from vagina, mouth and skin surfaces during 2016-2017. Colonizing clinical isolates of C. albicans were identified and drug resistant isolates detected using WHONET software. The susceptibility tests for carvacrol were carried out in terms of disk diffusion, broth microdilution and time kill assays against multidrug resistant C. albicans. The ability to change from yeast to hyphal morphology exploited using a light microscopy. Ergosterol quantification has been investigated by spectrophotometric analysis. The expression profile of ERG11 gene was studied using quantitative real time RT-PCR to admit the possibility of further associated ergosterol pathway. Statistical analysis was performed with SPSS 21.0. Comparisons were performed using one-way ANOVA, with Tukeyrs tests. Results: Ten colonizing clinical isolates of C. albicans were identified. Multidrug resistant isolates of C. albicans were detected. Carvacrol was found to have MIC90 of 100-200 mg/ml for multidrug drug resistant isolates of C. albicans. The time kill curve results show that carvacrol could significantly inhibit the growth of C. albicans (Pl0.05). In multidrug resistant C. albicans treated with carvacrol there was a marked reduction of the transition of yeast cells to hyphal cells. Significant decrease of ergosterol content was noted in multidrug resistant C. albicans treated with carvacrol. Furthermore, significant down-regulation was observed on ERG11 gene in multidrug resistant C. albicans treated with carvacrol (Pl0.05). Conclusion: Carvacrol show strong antifungal activity against multidrug resistant C. albicans. These results provide proof of concept for the implementation of carvacrol that may have potential applications in the treatment of drug resistance C. albicans infections. n

ERG3 and ERG11 genes are critical for the pathogenesis of Candida albicans during the oral mucosal infection

The hyphal development of Candida albicans (C. albicans) has been considered as an essential virulent factor for host cell damage. However, the missing link between hyphae and virulence of C. albicans is also been discovered. Here, we identified that the null mutants of ERG3 and ERG11, two key genes in ergosterol biosynthesis pathway, can form typical hyphae but failed to cause the oral mucosal infection in vitro and in vivo for the first time. In particular, the erg3Δ/Δ and erg11Δ/Δ strains co-cultured with epithelial cells significantly reduced the adhesion, damage, and cytokine (interleukin-1α (IL-1α)) production, whereas the invasion was not affected in vitro. Importantly, they were incapable of extensive hyphal invasion, formation of micro-abscesses, and tongue epithelium damage compared to wild type due to the decrease of the colonization and epithelial infection area in a murine oropharyngeal candidiasis model. The fluconazole (FLC), an antifungal targeted at ergosterol biosynthesis, relieved the epithelial infection of C. albicansin vitro and in vivo even under non-growth inhibitory dosage confirming the virulent contribution of ergosterol biosynthesis pathway. The erg3Δ/Δ and erg11Δ/Δ strains were cleared by macrophages similar to wild type, whereas their virulence factors including agglutinin-like sequence 1 (Als1), secreted aspartyl proteinase 6 (Sap6), and hyphal wall protein-1 (Hwp1) were significantly reduced indicated that the non-toxicity might not result from the change on immune tolerance but the defective virulence. The incapacity of erg3Δ/Δ and erg11Δ/Δ in epithelial infection highlights the contribution of ergosterol biosynthesis pathway to C. albicans pathogenesis and fluconazole can not only eliminate the fungal pathogens but also reduced their virulence even at low dosage.

Sterol uptake and sterol biosynthesis act coordinately to mediate antifungal resistance in Candidaglabrata under azole and hypoxic stress.

Pathogenic fungi, including Candida glabrata, develop strategies to grow and survive both in vitro and in vivo under azole stress. However, the mechanisms by which yeast cells counteract the inhibitory effects of azoles are not completely understood. In the current study, it was demonstrated that the expression of the ergosterol biosynthetic genes ERG2, ERG3, ERG4, ERG10, and ERG11 was significantly upregulated in C. glabrata following fluconazole treatment. Inhibiting ergosterol biosynthesis using fluconazole also increased the expression of the sterol influx transporter AUS1 and the sterol metabolism regulators SUT1 and UPC2 in fungal cells. The microarray study quantified 35 genes with elevated mRNA levels, including AUS1, TIR3, UPC2, and 8 ERG genes, in a C. glabrata mutant strain lacking ERG1, indicating that sterol importing activity is increased to compensate for defective sterol biosynthesis in cells. Bioinformatic analyses further revealed that those differentially expressed genes were involved in multiple cellular processes and biological functions, such as sterol biosynthesis, lipid localization, and sterol transport. Finally, to assess whether sterol uptake affects yeast susceptibility to azoles, we generated a C. glabrata aus1∆ mutant strain. It was shown that loss of Aus1p in C. glabrata sensitized the pathogen to azoles and enhanced the efficacy of drug exposure under low oxygen tension. In contrast, the presence of exogenous cholesterol or ergosterol in medium rendered the C. glabrata AUS1 wild-type strain highly resistant to fluconazole and voriconazole, suggesting that the sterol importing mechanism is augmented when ergosterol biosynthesis is suppressed in the cell, thus allowing C. glabrata to survive under azole pressure. On the basis of these results, it was concluded that sterol uptake and sterol biosynthesis may act coordinately and collaboratively to sustain growth and to mediate antifungal resistance in C. glabrata through dynamic gene expression in response to azole stress and environmental challenges.

Invasive Fungal Infections in Patients with Hematological Malignancies: Emergence of Resistant Pathogens and New Antifungal Therapies.

Invasive fungal infections caused by drug-resistant organisms are an emerging threat to heavily immunosuppressed patients with hematological malignancies. Modern early antifungal treatment strategies, such as prophylaxis and empirical and preemptive therapy, result in long-term exposure to antifungal agents, which is a major driving force for the development of resistance. The extended use of central venous catheters, the nonlinear pharmacokinetics of certain antifungal agents, neutropenia, other forms of intense immunosuppression, and drug toxicities are other contributing factors. The widespread use of agricultural and industrial fungicides with similar chemical structures and mechanisms of action has resulted in the development of environmental reservoirs for some drug-resistant fungi, especially azole-resistant Aspergillus species, which have been reported from four continents. The majority of resistant strains have the mutation TR34/L98H, a finding suggesting that the source of resistance is the environment. The global emergence of new fungal pathogens with inherent resistance, such as Candida auris, is a new public health threat. The most common mechanism of antifungal drug resistance is the induction of efflux pumps, which decrease intracellular drug concentrations. Overexpression, depletion, and alteration of the drug target are other mechanisms of resistance. Mutations in the ERG11 gene alter the protein structure of C-demethylase, reducing the efficacy of antifungal triazoles. Candida species become echinocandin-resistant by mutations in FKS genes. A shift in the epidemiology of Candida towards resistant non-albicans Candida spp. has emerged among patients with hematological malignancies. There is no definite association between antifungal resistance, as defined by elevated minimum inhibitory concentrations, and clinical outcomes in this population. Detection of genes or mutations conferring resistance with the use of molecular methods may offer better predictive values in certain cases. Treatment options for resistant fungal infections are limited and new drugs with novel mechanisms of actions are needed. Prevention of resistance through antifungal stewardship programs is of paramount importance.

A multicentre study of antifungal susceptibility patterns among 350 Candida auris isolates (2009-17) in India: role of the ERG11 and FKS1 genes in azole and echinocandin resistance.

Candida auris has emerged globally as an MDR nosocomial pathogen in ICU patients. We studied the antifungal susceptibility of C. auris isolates (n = 350) from 10 hospitals in India collected over a period of 8 years. To investigate azole resistance, ERG11 gene sequencing and expression profiling was conducted. In addition, echinocandin resistance linked to mutations in the C. auris FKS1 gene was analysed. CLSI antifungal susceptibility testing of six azoles, amphotericin B, three echinocandins, terbinafine, 5-flucytosine and nystatin was conducted. Screening for amino acid substitutions in ERG11 and FKS1 was performed. Overall, 90% of C. auris were fluconazole resistant (MICs 32 to ≥64 mg/L) and 2% and 8% were resistant to echinocandins (≥8 mg/L) and amphotericin B (≥2 mg/L), respectively. ERG11 sequences of C. auris exhibited amino acid substitutions Y132 and K143 in 77% (n = 34/44) of strains that were fluconazole resistant whereas WT genotypes, i.e. without substitutions at these positions, were observed in isolates with low fluconazole MICs (1-2 mg/L) suggesting that these substitutions confer a phenotype of resistance to fluconazole similar to that described for Candida albicans. No significant expression of ERG11 was observed, although expression was inducible in vitro with fluconazole exposure. Echinocandin resistance was linked to a novel mutation S639F in FKS1 hot spot region I. Overall, 25% and 13% of isolates were MDR and multi-azole resistant, respectively. The most common resistance combination was azoles and 5-flucytosine in 14% followed by azoles and amphotericin B in 7% and azoles and echinocandins in 2% of isolates.

The impact of farnesol in combination with fluconazole on Candida albicans biofilm: regulation of ERG20, ERG9, and ERG11 genes.

Farnesol (FAR) has already demonstrated an inhibitory effect on Candida albicans biofilm. The aim of this work was to determine the effectiveness of externally added FAR in combination with fluconazole (FLC) on Candida albicans biofilm and on regulation of the ergosterol genes ERG20, ERG9, and ERG11. The effectiveness of compounds was determined by MTT assay and evaluated by the minimal inhibitory concentrations reducing a sessile biofilm to 50% activity (0.5μg/mL and 200μmol/L for FLC and FAR, respectively). These concentrations as well as 30 and 100μmol/L FAR were selected for a study of the effectiveness of the FAR/FLC combination. The reduction in biofilm robustness mainly caused by the presence of 200μmol/L FAR-alone or in combination with FLC-was accompanied by a significant inhibition of the yeast-to-hyphae transition that was observed by light microscopy and CLSM. Results from qRT-PCR indicated that while 30μmol/L FAR only slightly regulated the expression of all 3 genes in the 48-h biofilm, the presence of 200μmol/L FAR downregulated all the tested genes. However, the addition of 0.5μg/mL of FLC to the samples with 200μmol/L FAR restored the downregulation of the ERG20 and ERG11 genes to the control level. Moreover, the gene ERG9 was slightly upregulated. In summary, FAR acted via multiple effects on the C. albicans biofilm, but only a higher concentration of FAR proved to be effective.