Unexpected effects of azole transporter inhibitors on antifungal susceptibility in Candida glabrata and other pathogenic Candida species

The pathogenic fungus Candida glabrata is often resistant to azole antifungal agents. Drug efflux through azole transporters, such as Cdr1 and Cdr2, is a key mechanism of azole resistance and these genes are under the control of the transcription factor Pdr1. Recently, the monoamine oxidase A (MAO-A) inhibitor clorgyline was shown to inhibit the azole efflux pumps, leading to increased azole susceptibility in C. glabrata. In the present study, we have evaluated the effects of clorgyline on susceptibility of C. glabrata to not only azoles, but also to micafungin and amphotericin B, using wild-type and several mutant strains. The addition of clorgyline to the culture media increased fluconazole susceptibility of a C. glabrata wild-type strain, whereas micafungin and amphotericin B susceptibilities were markedly decreased. These phenomena were also observed in other medically important Candida species, including Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida krusei. Expression levels of CDR1, CDR2 and PDR1 mRNAs and an amount of Cdr1 protein in the C. glabrata wild-type strain were highly increased in response to the treatment with clorgyline. However, loss of Cdr1, Cdr2, Pdr1, and a putative clorgyline target (Fms1), which is an ortholog of human MAO-A, or overexpression of CDR1 did not affect the decreased susceptibility to micafungin and amphotericin B in the presence of clorgyline. The presence of other azole efflux pump inhibitors including milbemycin A4 oxime and carbonyl cyanide 3-chlorophenylhydrazone also decreased micafungin susceptibility in C. glabrata wild-type, Δcdr1, Δcdr2, and Δpdr1 strains. These findings suggest that azole efflux pump inhibitors increase azole susceptibility but concurrently induce decreased susceptibility to other classes of antifungals independent of azole transporter functions

Vacuolar proton-translocating ATPase is required for antifungal resistance and virulence of Candida glabrata

Vacuolar proton-translocating ATPase (V-ATPase) is located in fungal vacuolar membranes.It is involved in multiple cellular processes, including the maintenance of intracellular ion homeostasis by maintaining acidic pH within the cell. The importance of V-ATPase in virulence has been demonstrated in several pathogenic fungi, including Candida albicans. However, it remains to be determined in the clinically important fungal pathogen Candida glabrata.Increasing multidrug resistance of C. glabrata is becoming a critical issue in the clinical setting. In the current study, we demonstrated that the plecomacrolide V-ATPase inhibitor bafilomycin B1 exerts a synergistic effect with azole antifungal agents, including fluconazole and voriconazole, against a C. glabrata wild-type strain. Furthermore,the deletion of the VPH2 gene encoding an assembly factor of V-ATPase was sufficient to interfere with V-ATPase function in C. glabrata, resulting in impaired pH homeostasis in the vacuole and increased sensitivity to a variety of environmental stresses, such as alkaline conditions (pH 7.4), ion stress (Na+, Ca2+, Mn2+, and Zn2+ stress),exposure to the calcineurin inhibitor FK506 and antifungal agents (azoles and amphotericin B), and iron limitation. In addition, virulence of C. glabrata Δvph2 mutant in a mouse model of disseminated candidiasis was reduced in comparison with that of the wild-type and VPH2-reconstituted strains.These findings support the notion that V-ATPase is a potential attractive target for the development of effective antifungal strategies

Effects of two known azole transporter inhibitors, carbonyl cyanide 3-chlorophenylhydrazone and milbemycin A4 oxime, on micafungin susceptibility in <i>C</i>. <i>glabrata</i> wild-type strain and <i>CDR1</i>, <i>CDR2</i>, and <i>PDR1</i> mutant strains.

<p>Spot dilution tests were performed as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180990#pone.0180990.g001" target="_blank">Fig 1B</a>. Final drug concentrations: carbonyl cyanide 3-chlorophenylhydrazone (CCCP), 40 μM; milbemycin A4 oxime (Milbemycin), 4 μM; micafungin (MCFG), 0.03 μg/ml. <i>C</i>. <i>glabrata</i> strains: Wild type, CBS138; Δ<i>cdr1</i>, TG-C1; Δ<i>cdr2</i>, TG-C2; and Δ<i>pdr1</i>, TG-C3.</p

Strains used in this study.

<p>Strains used in this study.</p

Interaction of micafungin and clorgyline against <i>Candida</i> species determined by fractional inhibitory concentration index.

<p>Interaction of micafungin and clorgyline against <i>Candida</i> species determined by fractional inhibitory concentration index.</p

Unexpected effects of azole transporter inhibitors on antifungal susceptibility in <i>Candida glabrata</i> and other pathogenic <i>Candida</i> species - Fig 4

<p>(A) Time-course analysis of <i>FMS1</i> expression in <i>C</i>. <i>glabrata</i> wild-type strain. Cell culture, RNA extraction, real-time qRT-PCR, and data presentation were performed as described in the Materials and Methods section and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180990#pone.0180990.g003" target="_blank">Fig 3A</a> legend. (B) Effects of clorgyline on susceptibility to micafungin and amphotericin B in the <i>C</i>. <i>glabrata</i> wild-type and <i>FMS1</i> mutant strains. Spot dilution tests were performed as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180990#pone.0180990.g001" target="_blank">Fig 1B</a>. Final drug concentrations: clorgyline, 80 μg/ml; micafungin (MCFG), 0.03 μg/ml; and amphotericin B (AMPH-B), 1.25 μg/ml. <i>C</i>. <i>glabrata</i> strains: Wild type, CBS138; and Δ<i>fms1</i>, TG-C4.</p

Effects of clorgyline on susceptibility to micafungin and amphotericin B in <i>Candida</i> wild-type strains.

<p>Spot dilution tests were performed using MIN plates as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180990#pone.0180990.g001" target="_blank">Fig 1B</a>. Clorgyline was added at a final concentration of 0 μg/ml or 80 μg/ml. Micafungin (MCFG) final concentrations: <i>C</i>. <i>glabrata</i>, 0.06 μg/ml; <i>C</i>. <i>albicans</i>, 0.08 μg/ml; <i>C</i>. <i>parapsilosis</i>, 0.25 μg/ml; <i>C</i>. <i>tropicalis</i>, 0.06 μg/ml; and <i>C</i>. <i>krusei</i>, 0.08 μg/ml. Amphotericin B (AMPH-B) final concentrations: <i>C</i>. <i>glabrata</i>, 0.5 μg/ml; <i>C</i>. <i>albicans</i>, 0.5 μg/ml; <i>C</i>. <i>parapsilosis</i>, 1 μg/ml; <i>C</i>. <i>tropicalis</i>, 0.5 μg/ml; and <i>C</i>. <i>krusei</i>, 2 μg/ml. <i>Candida</i> wild-type strains: <i>C</i>. <i>glabrata</i>, CBS138; <i>C</i>. <i>albicans</i>, SC5314; <i>C</i>. <i>parapsilosis</i>, ATCC 90018; <i>C</i>. <i>tropicalis</i>, ATCC 750; and <i>C</i>. <i>krusei</i>, ATCC 6258.</p

Antifungal Susceptibilities of Aspergillus fumigatus Clinical Isolates Obtained in Nagasaki, Japan

We investigated the triazole, amphotericin B, and micafungin susceptibilities of 196 A. fumigatus clinical isolates in Nagasaki, Japan. The percentages of non-wild-type (non-WT) isolates for which MICs of itraconazole, posaconazole, and voriconazole were above the ECV were 7.1%, 2.6%, and 4.1%, respectively. A G54 mutation in cyp51A was detected in 64.2% (9/14 isolates) and 100% (5/5 isolates) of non-WT isolates for itraconazole and posaconazole, respectively. Amphotericin B MICs of ≥2 μg/ml and micafungin minimum effective concentrations (MECs) of ≥16 μg/ml were recorded for two and one isolates, respectively

How to prevent contamination with Candida albicans during the fabrication of transplantable oral mucosal epithelial cell sheets

We have utilized patients' own oral mucosa as a cell source for the fabrication of transplantable epithelial cell sheets to treat limbal stem cell deficiency and mucosal defects after endoscopic submucosal dissection of esophageal cancer. Because there are abundant microbiotas in the human oral cavity, the oral mucosa was sterilized and 40 μg/mL gentamicin and 0.27 μg/mL amphotericin B were added to the culture medium in our protocol. Although an oral surgeon carefully checked each patient's oral cavity and although candidiasis was not observed before taking the biopsy, contamination with Candida albicans (C. albicans) was detected in the conditioned medium during cell sheet fabrication. After adding 1 μg/mL amphotericin B to the transportation medium during transport from Nagasaki University Hospital to Tokyo Women's Medical University, which are 1200 km apart, no proliferation of C. albicans was observed. These results indicated that the supplementation of transportation medium with antimycotics would be useful for preventing contamination with C. albicans derived from the oral mucosa without hampering cell proliferation