P4-ATPase subunit Cdc50 plays a role in yeast budding and cell wall integrity in Candida glabrata.

BACKGROUND: As highly-conserved types of lipid flippases among fungi, P4-ATPases play a significant role in various cellular processes. Cdc50 acts as the regulatory subunit of flippases, forming heterodimers with Drs2 to translocate aminophospholipids. Cdc50 homologs have been reported to be implicated in protein trafficking, drug susceptibility, and virulence in Saccharomyces cerevisiae, Candida albicans and Cryptococcus neoformans. It is likely that Cdc50 has an extensive influence on fungal cellular processes. The present study aimed to determine the function of Cdc50 in Candida glabrata by constructing a Δcdc50 null mutant and its complemented strain. RESULTS: In Candida glabrata, the loss of Cdc50 led to difficulty in yeast budding, probably caused by actin depolarization. The Δcdc50 mutant also showed hypersensitivity to azoles, caspofungin, and cell wall stressors. Further experiments indicated hyperactivation of the cell wall integrity pathway in the Δcdc50 mutant, which elevated the major cell wall contents. An increase in exposure of ß-(1,3)-glucan and chitin on the cell surface was also observed through flow cytometry. Interestingly, we observed a decrease in the phagocytosis rate when the Δcdc50 mutant was co-incubated with THP-1 macrophages. The Δcdc50 mutant also exhibited weakened virulence in nematode survival tests. CONCLUSION: The results suggested that the lipid flippase subunit Cdc50 is implicated in yeast budding and cell wall integrity in C. glabrata, and thus have a broad influence on drug susceptibility and virulence. This work highlights the importance of lipid flippase, and offers potential targets for new drug research.

Erg11 mutations associated with azole resistance in clinical isolates of Candida albicans.

The widespread use of azoles has led to increasing azole resistance among Candida albicans strains. One mechanism of azole resistance involves point mutations in the ERG11 gene, which encodes the target enzyme (cytochrome P450 lanosterol 14α-demethylase). In the present study, we amplified and sequenced the ERG11 gene of 23 C. albicans clinical isolates. Seventeen mutations encoding distinct amino acid substitutions were found, of which seven (K143Q, Y205E, A255V, E260V, N435V, G472R, and D502E) were novel. We further verified the contribution of the amino acid substitutions to azole resistance using site-directed mutagenesis of the ERG11 gene to recreate these mutations for heterologous expression in Saccharomyces cerevisiae. We observed that substitutions A114S, Y132H, Y132F, K143R, Y257H, and a new K143Q substitution contributed to significant increases (≧fourfold) in fluconazole and voriconazole resistance; changes in itraconazole resistance were not significant (≦twofold).

Regulatory role of Mss11 in Candida glabrata virulence: adhesion and biofilm formation.

Introduction: Candida glabrata has emerged as a fungal pathogen with high infection and mortality rates, and its primary virulence factors are related to adhesion and biofilm formation. These virulence factors in C.glabrata are primarily mediated by epithelial adhesins (Epas), most of which are encoded in subtelomeric regions and regulated by subtelomeric silencing mechanisms. The transcription factor Mss11, known for its regulatory role in adhesion, biofilm formation, and filamentous growth in Saccharomyces cerevisiae and Candida albicans, has also been implicated in the expression of EPA6, suggesting its potential influence on C.glabrata virulence. The present study aims to determine the regulatory role of Mss11 in the virulence of C. glabrata. Methods: In this work, a Δmss11 null mutant and its complemented strain were constructed from a C.glabrata standard strain. The impact of the transcription factor Mss11 on the virulence of C.glabrata was investigated through a series of phenotypic experiments, including the microbial adhesion to hydrocarbons (MATH) test, adherence assay, biofilm assay, scanning electron microscopy and Galleria mellonella virulence assay. Furthermore, transcriptome sequencing, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and chromatin immunoprecipitation sequencing (ChIP-seq) were employed to investigate the molecular mechanisms behind the regulation of Mss11. Results: In C.glabrata, the loss of MSS11 led to a significant reduction in several virulence factors including cell surface hydrophobicity, epithelial cell adhesion, and biofilm formation. These observations were consistent with the decreased virulence of the Δmss11 mutant observed in the Galleria mellonella infection model. Further exploration demonstrated that Mss11 modulates C. glabrata virulence by regulating EPA1 and EPA6 expression. It binds to the upstream regions of EPA1 and EPA6, as well as the promoter regions of the subtelomeric silencing-related genes SIR4, RIF1, and RAP1, indicating the dual regulatory role of Mss11. Conclusion: Mss11 plays a crucial role in C. glabrata adhesion and biofilm formation, and thus has a broad influence on virulence. This regulation is achieved by regulating the expression of EPA1 and EPA6 through both promoter-specific regulation and subtelomeric silencing.

FLO8 deletion leads to decreased adhesion and virulence with downregulated expression of EPA1, EPA6, and EPA7 in Candida glabrata.

BACKGROUND: The Candida glabrata does not develop into a pathogenic hiphal form; however, it has become the second most common pathogen of fungal infections in humans, partly because of its adhesion ability and virulence. OBJECTIVES: The present study aimed to determine whether Flo8, a transcription factor that plays an important role in the virulence and drug resistance in Candida albicans, has a similar role in C. glabrata. METHODS: We constructed FLO8 null strains of a C. glabrata standard strain and eight clinical strains from different sources, and a FLO8 complemented strain. Real-time quantitative PCR, biofilm formation assays, hydrophobicity tests, adhesion tests, Caenorhabditis elegans survival assay, and drug-susceptibility were then performed. RESULTS: Compared with the wild-type strains, the biofilm formation, hydrophobicity, adhesion, and virulence of the FLO8-deficient strains decreased, accompanied by decreased expression of EPA1, EPA6, and EPA7. On the other hand, it showed no changes in antifungal drug resistance, although the expression levels of CDR1, CDR2, and SNQ2 increased after FLO8 deletion. CONCLUSIONS: These results indicated that Flo8 is involved in the adhesion and virulence of C. glabrata, with FLO8 deletion leading to decreased expression of EPA1, EPA6, and EPA7 and decreased biofilm formation, hydrophobicity, adhesion, and virulence.

Correction to: The H741D mutation in Tac1p contributes to the upregulation of CDR1 and CDR2 expression in Candida albicans.

Unfortunately, an error occurred in the author affiliations.

The H741D mutation in Tac1p contributes to the upregulation of CDR1 and CDR2 expression in Candida albicans.

The wide use of antifungal agents has led to the development of resistance in the pathogenic yeast strain Candida albicans. Gain-of-function mutations in transcription factors such as Tac1p demonstrated their ability to control expression of the ABC transporter genes CDR1 and CDR2, and mediation of azole resistance. Previously, we obtained a series of azole-resistant isolates with high-level expression of CDR1 or/and CDR2, and identified the novel H741D mutation in Tac1p. In the present study, the TAC1 alleles from isolate C13 were introduced into tac1Δ/Δ mutant. The H741D change was seen in TAC1C13 in addition to several other amino acid differences. Hyperactive alleles TAC1C13 exhibited higher minimum inhibitory concentrations (MICs) of fluconazole and itraconazole than that observed in SN152 containing the wild-type TAC1 allele. And alleles TAC1C13 conferred constitutively high levels of Cdr1p and Cdr2p. Moreover, the importance of H741D in conferring hyperactivity to TAC1 was also confirmed by site-directed mutagenesis. Compared with SN152, the presence of H741D resulted in > 2-fold increase in CDR1 and CDR2 gene and protein expression, > 4-fold increase in fluconazole and itraconazole MICs and higher rates of Rhodamine 6G efflux by 43.24%.

The discovery of potential phosphopantetheinyl transferase Ppt2 inhibitors against drug-resistant Candida albicans.

With the high-frequency use or abuse of antifungal drugs, the crisis of drug-resistant fungi continues to increase worldwide; in particular, the infection of drug-resistant Candida albicans brings the great challenge to the clinical treatment. Therefore, to decelerate the spread of this resistance, it is extremely urgent to facilitate the new antifungal targets with novel drugs. Phosphopantetheinyl transferases PPTases (Ppt2 in Candida albicans) had been identified in bacterium and fungi and mammals, effects as a vital enzyme in the metabolism of organisms in C. albicans. Ppt2 transfers the phosphopantetheinyl group of coenzyme A to the acyl carrier protein Acp1 in mitochondria for the synthesis of lipoic acid that is essential for fungal respiration, so making Ppt2 an ideal target for antifungal drugs. In this study, 110 FDA-approved drugs were utilized to investigate the Ppt2 inhibition against drug-resistant Candida albicans by the improved fluorescence polarization experiments, which have enough druggability and structural variety under the novel strategy of drug repurposing. Thereinto, eight agents revealed the favourable Ppt2 inhibitory activities. Further, broth microdilution assay of incubating C. albicans with these eight drugs showed that pterostilbene, procyanidine, dichlorophen and tea polyphenol had the superior MIC values. In summary, these findings provide more valuable insight into the treatment of drug-resistant C. albicans.

FLO8 deletion leads to azole resistance by upregulating CDR1 and CDR2 in Candida albicans.

Candida albicans has the ability to switch reversibly between budding yeast, filamentous, pseudohypha, and hyphal forms, a process in which the transcription factor Flo8 plays an important role. This ability is important for the virulence and pathogenicity of C. albicans. To determine whether Flo8 plays a role in the regulation of drug sensitivity, we constructed a FLO8 null mutant flo8/flo8 from the parental strain SN152 and a Flo8-overexpressing strain, flo8/flo8::FLO8. The susceptibility of the isolates to antifungal agents was then evaluated using the agar dilution and broth microdilution methods. Expression of drug resistance-related genes by the isolates was investigated by real-time PCR. The flo8/flo8 mutation isolates exhibited increased resistance to fluconazole, voriconazole, and itraconazole compared with the wild-type and drug sensitivity was restored by FLO8 overexpression (flo8/flo8∷FLO8). Of seven drug resistance-related genes, the FLO8 null mutation resulted in increased CDR1 and CDR2 expression (1.60-fold and 5.27-fold, respectively) compared with SN152, while FLO8 overexpression resulted in decreased CDR1 expression (0.63-fold). These results suggest that Flo8 is involved in the susceptibility of C. albicans to antifungal azoles, with FLO8 deletion leading to constitutive overexpression of CDR1 and CDR2 and resistance to antifungal azoles.

Multilocus sequence typing of Candida tropicalis shows clonal cluster enrichment in azole-resistant isolates from patients in Shanghai, China.

To explore the putative correlation between the multilocus sequence types (MLST) and antifungal susceptibility of clinical Candida tropicalis isolates in Mainland China. Eighty-two clinical C. tropicalis isolates were collected from sixty-nine patients at Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China, from July 2012 to February 2015, and antifungal susceptibility tests were performed. Genetic profiles of those 82 isolates (30 azole-resistant and 52 azole-susceptible) were characterised by multilocus sequence typing. Phylogenetic analysis of the data was conducted with the clustering method, using UPGMA (unweighted pair group method with arithmetic averages) and the minimal spanning tree algorithm. MLST clonal clusters were analysed using the eBURST V3 package. Of the six gene fragments identified in multilocus sequence typing, SAPT4 presented the highest typing efficiency, whereas SAPT2 was the least efficient. Of the 44 diploid sequence types (DSTs) differentiated, 32 DSTs and 12 genotypes were identified as new to the C. tropicalis DST database. Twenty (45.45%) of the 44 DSTs were assigned to seven major groups based on eBURST analysis. Of these, Group 6, which contained DST 376, DST 505, DST 506 and DST 507, accounted for 76.7% of the 30 azole-resistant isolates. However, the genetic relationships among the azole-susceptible isolates were relatively decentralised. This MLST analysis of the putative correlation between the MLST types and antifungal susceptibility of clinical C. tropicalis isolates in Mainland China shows that DSTs 376, 505, 506 and 507 are closely related azole-resistant C. tropicalis clones.

Mutations in the Flo8 transcription factor contribute to virulence and phenotypic traits in Candida albicans strains.

The ability of Candida albicans to switch between multiple morphological states, including yeast (blastospores), fliamentous, pseudohyphal and hyphal forms, has been shown to be important for its pathogenicity and virulence. The transcription factor Flo8, which contains the LisH domain, is a downstream regulator of the cAMP/PKA pathway. Four clinical strains from adult women with recurrent vaginitis were isolated, and their morphology was observed. The results showed that two strains presented longer hyphal threads, stronger adherence to plastic and invasion into agar medium, and one strain was defective in filament and biofilm growth. Interestingly, mutations in the FLO8 gene were identified in these strains. We analyzed the contribution of these mutants to filamentous growth by constructing mutant strains and investigating their morphological and ultrastructural characteristics, including putative virulence traits, in vitro and in vivo. The results showed that the G723R and T751D Flo8 mutants enhanced activation of the Flo8C terminus, thereby promoting filamentous growth and increasing virulence.

Mechanisms of azole resistance in Candida albicans clinical isolates from Shanghai, China.

This study was undertaken to characterize the mechanism(s) of azole resistance in clinical isolates of Candida albicans collected in Shanghai, China, focusing on the role of efflux pumps, target enzymes of fluconazole (Erg11), respiratory status and the ergosterol biosynthetic pathway. Clinical isolates of C. albicans (n = 30) were collected from 30 different non-HIV-infected patients in four hospitals in Shanghai. All 30 C. albicans isolates were susceptible to amphotericin B and 5-fluorocytosine. Twelve C. albicans isolates showed resistance to at least one type of triazole antifungal. Flow cytometry analysis of rhodamine 6G efflux showed that azole-resistant isolates had greater efflux pump activity, which was consistent with elevated levels of CDR1 and CDR2 genes that code for ABC efflux pumps. However, we did not observe increased expression of ERG11 and MDR1 or respiratory deficiency. Several mutations of ERG11 and TAC1 genes were detected. The F964Y mutation in the TAC1 gene was identified for the first time. Two main sterols, ergosterol and lanosterol, were identified by GC-MS chromatogram, and no missense mutations were found in ERG3. Furthermore, seven amino acid substitutions in ERG11, A114S, Y132H, Y132F, K143Q, K143R, Y257H and G448E were found, by Type II spectral quantitative analysis, to contribute to low affinity binding between Erg11 and fluconazole.

Mutations in transcription factor Mrr2p contribute to fluconazole resistance in clinical isolates of Candida albicans.

The Candida albicans zinc cluster proteins are a family of transcription factors (TFs) that play essential roles in the development of antifungal drug resistance. Gain-of-function mutations in several TFs, such as Tac1p, Mrr1p and Upc2p, have been previously well documented in azole-resistant clinical C. albicans isolates. Mrr2p (multidrug resistance regulator 2) is a novel TF controlling expression of the ABC transporter gene CDR1 and mediating fluconazole resistance. In this study, the relationship between naturally occurring mutations in MRR2 and fluconazole resistance in clinical C. albicans isolates was investigated. Among a group of 20 fluconazole-resistant clinical C. albicans and 10 fluconazole-susceptible C. albicans, 12 fluconazole-resistant isolates overexpressed CDR1 by at least two-fold compared with the fluconazole-susceptible isolates. Of these 12 resistant isolates, three (C7, C9, C15) contained 11 identical missense mutations, 6 of which occurred only in the azole-resistant isolates. The contribution of these mutations to CDR1 overexpression and therefore to fluconazole resistance was further verified by generating recombinant strains containing the mutated MRR2 gene. The mutated MRR2 alleles from isolate C9 contributed to an almost six-fold increase in CDR1 expression and an eight-fold increase in fluconazole resistance; the missense mutations S466L and T470N resulted in an increase in CDR1 expression of more than two-fold and a four-fold increase in fluconazole resistance. In contrast, the other four missense mutations conferred only two- to four-fold increases in fluconazole resistance, with no significant increase in CDR1 expression. These findings provide some insight into the mechanism by which MRR2 regulates C. albicans multidrug resistance.