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 g/ml and micafungin minimum effective concentrations (MECs) of >16 g/ml were recorded for two and one isolates, respectively.

The pathogenic fungus Candida glabrata is relatively resistant to azole antifungals, which target lanosterol 14α-demethylase (Erg11p) in the ergosterol biosynthesis pathway. Our study revealed that C. glabrata exhibits increased azole susceptibility under low-iron conditions. To investigate the molecular basis of this phenomenon, we generated a strain lacking the heme (iron protoporphyrin IX)-binding protein Dap1 in C. glabrata. The Δdap1 mutant displayed growth defects under iron-limited conditions, decreased azole tolerance, decreased production of ergosterol, and increased accumulation of 14α-methylated sterols lanosterol and squalene. All the Δdap1 phenotypes were complemented by wild-type DAP1, but not by DAP1(D91G) , in which a heme-binding site is mutated. Furthermore, azole tolerance of the Δdap1 mutant was rescued by exogenous ergosterol but not by iron supplementation alone. These results suggest that heme binding by Dap1 is crucial for Erg11 activity and ergosterol biosynthesis, thereby being required for azole tolerance. A Dap1-GFP fusion protein predominantly localized to vacuolar membranes and endosomes, and the Δdap1 cells exhibited aberrant vacuole morphologies, suggesting that Dap1 is also involved in the regulation of vacuole structures that could be important for iron storage. Our study demonstrates that Dap1 mediates a functional link between iron homeostasis and azole resistance in C. glabrata.

Vacuolar H(+)-ATPase (V-ATPase) is responsible for the acidification of eukaryotic intracellular compartments and plays an important role in oxidative stress response (OSR), but its molecular bases are largely unknown. Here, we investigated how V-ATPase is involved in the OSR by using a strain lacking VPH2, which encodes an assembly factor of V-ATPase, in the pathogenic fungus Candida glabrata The loss of Vph2 resulted in increased H2O2 sensitivity and intracellular reactive oxygen species (ROS) level independently of mitochondrial functions. The Δvph2 mutant also displayed growth defects under alkaline conditions accompanied by the accumulation of intracellular ROS and these phenotypes were recovered in the presence of the ROS scavenger N-acetyl-l-cysteine. Both expression and activity levels of mitochondrial manganese superoxide dismutase (Sod2) and catalase (Cta1) were decreased in the Δvph2 mutant. Phenotypic analyses of strains lacking and overexpressing these genes revealed that Sod2 and Cta1 play a predominant role in endogenous and exogenous OSR, respectively. Furthermore, supplementation of copper and iron restored the expression of SOD2 specifically in the Δvph2 mutant, suggesting that the homeostasis of intracellular cupper and iron levels maintained by V-ATPase was important for the Sod2-mediated OSR. This report demonstrates novel roles of V-ATPase in the OSR in C. glabrata.

a b s t r a c tWe 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 mg/mL gentamicin and 0.27 mg/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 mg/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.

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.