Dataset Information

Antifungal Activity of the Noncytotoxic Human Peptide Hepcidin 20 against Fluconazole-Resistant Candida glabrata in Human Vaginal Fluid.

ABSTRACT: Vaginal infections caused by Candida glabrata are difficult to eradicate due to this species' scarce susceptibility to azoles. Previous studies have shown that the human cationic peptide hepcidin 20 (Hep-20) exerts fungicidal activity in sodium phosphate buffer against a panel of C. glabrata clinical isolates with different levels of susceptibility to fluconazole. In addition, the activity of the peptide was potentiated under acidic conditions, suggesting an application in the topical treatment of vaginal infections. To investigate whether the peptide activity could be maintained in biological fluids, in this study the antifungal activity of Hep-20 was evaluated by a killing assay in (i) a vaginal fluid simulant (VFS) and in (ii) human vaginal fluid (HVF) collected from three healthy donors. The results obtained indicated that the activity of the peptide was maintained in VFS and HVF supplemented with EDTA. Interestingly, the fungicidal activity of Hep-20 was enhanced in HVF compared to that observed in VFS, with a minimal fungicidal concentration of 25 ?M for all donors. No cytotoxic effect on human cells was exerted by Hep-20 at concentrations ranging from 6.25 to 100 ?M, as shown by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide tetrazolium salt (XTT) reduction assay and propidium iodide staining. A piece of indirect evidence of Hep-20 stability was also obtained from coincubation experiments of the peptide with HVF at 37°C for 90 min and for 24 h. Collectively, these results indicate that this peptide should be further studied as a novel therapeutic agent for the topical treatment of vaginal C. glabrata infections.

SUBMITTER: Del Gaudio G

PROVIDER: S-EPMC3754342 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Antifungal Activity of the Noncytotoxic Human Peptide Hepcidin 20 against Fluconazole-Resistant Candida glabrata in Human Vaginal Fluid.

Del Gaudio Gaetano G Lombardi Lisa L Maisetta Giuseppantonio G Esin Semih S Batoni Giovanna G Sanguinetti Maurizio M Senesi Sonia S Tavanti Arianna A

Antimicrobial agents and chemotherapy 20130624 9

Vaginal infections caused by <i>Candida glabrata</i> are difficult to eradicate due to this species' scarce susceptibility to azoles. Previous studies have shown that the human cationic peptide hepcidin 20 (Hep-20) exerts fungicidal activity in sodium phosphate buffer against a panel of <i>C. glabrata</i> clinical isolates with different levels of susceptibility to fluconazole. In addition, the activity of the peptide was potentiated under acidic conditions, suggesting an application in the topi ...[more]

PMID: 23796919

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Project description: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.

Project description:BackgroundIncreasing numbers of immunocompromised patients have resulted in greater incidence of invasive fungal infections with high mortality. Candida albicans infections dominate, but during the last decade, Candida glabrata has become the second highest cause of candidemia in the United States and Northern Europe. Reliable and early diagnosis, together with appropriate choice of antifungal treatment, is needed to combat these challenging infections.ObjectivesTo confirm the identity of 183 Candida glabrata isolates from different human body sites using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and VITEK(®)2, and to analyze isolate protein profiles and antifungal susceptibility. The minimum inhibitory concentration (MIC) of seven antifungal drugs was determined for the isolates to elucidate susceptibility.DesignA total of 183 C. glabrata isolates obtained between 2002 and 2012 from Norwegian health-care units were analyzed. For species verification and differentiation, biochemical characterization (VITEK(®)2) and mass spectrometry (MALDI-TOF) were used. MIC determination for seven antifungal drugs was undertaken using E-tests(®).ResultsUsing VITEK(®)2, 92.9% of isolates were identified as C. glabrata, while all isolates (100%) were identified as C. glabrata using MALDI-TOF. Variation in protein spectra occurred for all identified C. glabrata isolates. The majority of isolates had low MICs to amphotericin B (≤1 mg/L for 99.5%) and anidulafungin (≤0.06 mg/L for 98.9%). For fluconazole, 18% of isolates had MICs >32 mg/L and 82% had MICs in the range ≥0.016 mg/L to ≤32 mg/L.ConclusionsProtein profiles and antifungal susceptibility characteristics of the C. glabrata isolates were diverse. Clustering of protein profiles indicated that many azole resistant isolates were closely related. In most cases, isolates had highest susceptibility to amphotericin B and anidulafungin. The results confirmed previous observations of high MICs to fluconazole and flucytosine. MALDI-TOF was more definitive than VITEK(®)2 for C. glabrata identification.

Project description:The high prevalence of fluconazole resistance among clinical isolates of Candida glabrata has greatly hampered the utility of fluconazole for the treatment of invasive candidiasis. Fluconazole resistance in this yeast is almost exclusively due to activating mutations in the transcription factor Pdr1, which result in upregulation of the ABC transporter genes CDR1, PDH1, and SNQ2 and therefore increased fluconazole efflux. However, the regulation of Pdr1 is poorly understood. In order to identify genes that interact with the Pdr1 transcriptional pathway and influence the susceptibility of C. glabrata to fluconazole, we screened a collection of deletion mutants for those exhibiting increased resistance to fluconazole. Deletion of the gene coding for a protein homologous to the Saccharomyces cerevisiae J protein Jjj1 resulted in decreased fluconazole susceptibility. We used the SAT1 flipper method to generate independent deletion mutants for JJJ1 in an SDD clinical isolate. Expression of both CDR1 and PDR1 was increased in the absence of JJJ1. In the absence of CDR1 or PDR1, deletion of JJJ1 has only a modest effect on fluconazole susceptibility. Transcriptional profiling using transcriptome sequencing (RNA-seq) revealed upregulation of genes of the Pdr1 regulon in the absence of JJJ1. Jjj1 appears to be a negative regulator of fluconazole resistance in C. glabrata and acts primarily through upregulation of the ABC transporter gene CDR1 via activation of the Pdr1 transcriptional pathway. IMPORTANCECandida glabrata is the second most common species of Candida recovered from patients with invasive candidiasis. The increasing number of infections due to C. glabrata, combined with its high rates of resistance to the commonly used, well-tolerated azole class of antifungal agents, has limited the use of this antifungal class. This has led to the preferential use of echinocandins as empirical treatment for serious Candida infections. The primary mechanism of resistance found in clinical isolates is the presence of an activating mutation in the gene encoding the transcription factor Pdr1 that results in upregulation of one or more of the efflux pumps Cdr1, Pdh1, and Snq2. By developing a better understanding of this mechanism of resistance to the azoles, it will be possible to develop strategies for reclaiming the utility of the azole antifungals against this important fungal pathogen.

Dataset Information

Antifungal Activity of the Noncytotoxic Human Peptide Hepcidin 20 against Fluconazole-Resistant Candida glabrata in Human Vaginal Fluid.

Publications

Antifungal Activity of the Noncytotoxic Human Peptide Hepcidin 20 against Fluconazole-Resistant Candida glabrata in Human Vaginal Fluid.

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