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Most previous studies on fungal NADPH oxidases (Nox) focused on multicellular fungi and highlighted the important roles of Nox-derived reactive oxygen species (ROS) in cellular differentiation and signaling communication. However, there are few reports about Nox in unicellular fungi. A novel NOX ortholog, CAGL0K05863g (named CgNOX1), in Candida glabrata was investigated in this study. Deletion of CgNOX1 led to a decrease in both intracellular and extracellular ROS production. In addition, the Cgnox1∆ mutant exhibited hypersensitivity to hydrogen peroxide and menadione. Also, the wild-type strain showed higher levels of both CgNOX1 mRNA expression and ROS production under oxidative stress. Moreover, the absence of CgNOX1 resulted in impaired ferric reductase activity. Although there was no effect on in vitro biofilm formation, the CgNOX1 mutant did not produce hepatic apoptosis, which might be mediated by fungal Nox-derived ROS during co-incubation. Together, these results indicated that the novel NOX gene plays important roles in unicellular pathogenic C. glabrata and its interaction with host cells.

With only four classes of antifungal drugs available for the treatment of invasive systemic fungal infections, the number of resistant fungi is increasing, highlighting the urgent need for novel antifungal drugs. Ergosterol, an essential component of cell membranes, and its synthetic pathway have been targeted for antifungal drug development. Sterol-C4-methyl monooxygenase (Erg25p), which is a greater essential target than that of existing drugs, represents a promising drug target. However, the development of antifungal drugs must consider potential side effects, emphasizing the importance of evaluating their selective toxicity against fungi. In this study, we knocked in ERG25 of Candida glabrata and its human ortholog, SC4MOL, in ERG25-deleted Saccharomyces cerevisiae. Utilizing these strains, we evaluated 1181-0519, an Erg25p inhibitor, that exhibited selective toxicity against the C. glabrata ERG25 knock-in strain. Furthermore, 1181-0519 demonstrated broad-spectrum antifungal activity against pathogenic Candida species, including Candida auris. The approach of utilizing a gene that is functionally conserved between yeast and humans and subsequently screening for molecular target drugs enables the identification of selective inhibitors for both species.

Outbreaks of invasive infections, with high mortality rates, caused by multidrug-resistant Candida auris have been reported worldwide. Although hotspot mutations in FKS1 are an established cause of echinocandin resistance, the actual contribution of these mutations to echinocandin resistance remains unknown. Here, we sequenced the FKS1 gene of a caspofungin-resistant clinical isolate (clade I) and identified a novel resistance mutation (G4061A inducing R1354H). We applied the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to generate a recovered strain (H1354R) in which only this single nucleotide mutation was reverted to its wild-type sequence. We also generated mutant strains with only the R1354H mutation introduced into C. auris wild-type strains (clade I and II) and analyzed their antifungal susceptibility. Compared to their parental strains, the R1354H mutants exhibited a 4- to 16-fold increase in caspofungin minimum inhibitory concentration (MIC) while the H1354R reverted strain exhibited a 4-fold decrease in caspofungin MIC. In a mouse model of disseminated candidiasis, the in vivo therapeutic effect of caspofungin was more closely related to the FKS1 R1354H mutation and the virulence of the strain than its in vitro MIC. The CRISPR-Cas9 system could thus aid in elucidating the mechanism underlying drug resistance in C. auris.

Mitochondrial dysfunction or morphological abnormalities in human pathogenic fungi are known to contribute to azole resistance; however, the underlying molecular mechanisms are unknown. In this study, we investigated the link between mitochondrial morphology and azole resistance in Candida glabrata, which is the second most common cause of human candidiasis worldwide. The ER-mitochondrial encounter structure (ERMES) complex is thought to play an important role in the mitochondrial dynamics necessary for mitochondria to maintain their function. Of the five components of the ERMES complex, deletion of GEM1 increased azole resistance. Gem1 is a GTPase that regulates the ERMES complex activity. Point mutations in GEM1 GTPase domains were sufficient to confer azole resistance. The cells lacking GEM1 displayed abnormalities in mitochondrial morphology, increased mtROS levels, and increased expression of azole drug efflux pumps encoded by CDR1 and CDR2. Interestingly, treatment with N-acetylcysteine (NAC), an antioxidant, reduced ROS production and the expression of CDR1 in Δgem1 cells. Altogether, the absence of Gem1 activity caused an increase in mitochondrial ROS concentration, leading to Pdr1-dependent upregulation of the drug efflux pump Cdr1, resulting in azole resistance.

The prevalence of antifungal resistance in Candida glabrata, especially against azole drugs, results in difficult-to-treat and potentially life-threatening infections. Understanding the molecular basis of azole resistance in C. glabrata is crucial to designing more suitable therapeutic strategies. In this study, the role of the transcription factor encoded by ORF CAGL0B03421g, here denominated as CgMar1 (Multiple Azole Resistance 1), in azole susceptibility was explored. Using RNA-sequencing, CgMar1 was found to regulate 337 genes under fluconazole stress, including several related to lipid biosynthesis pathways. In this context, CgMar1 and its target CgRSB1, encoding a predicted sphingoid long-chain base efflux transporter, were found to contribute to plasma membrane sphingolipid incorporation and membrane permeability, decreasing fluconazole accumulation. CgMar1 was found to associate with the promoter of CgRSB1, which contains two instances of the CCCCTCC consensus, found to be required for CgRSB1 activation during fluconazole stress. Altogether, a regulatory pathway modulating azole susceptibility in C. glabrata is proposed, resulting from what appears to be a neofunctionalization of a Hap1-like transcription factor.