Evaluation of Antifungal Selective Toxicity Using Candida glabrata ERG25 and Human SC4MOL Knock-In Strains.

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.

Activation of CWI pathway through high hydrostatic pressure, enhancing glycerol efflux via the aquaglyceroporin Fps1 in Saccharomyces cerevisiae.

The fungal cell wall is the initial barrier for the fungi against diverse external stresses, such as osmolarity changes, harmful drugs, and mechanical injuries. This study explores the roles of osmoregulation and the cell-wall integrity (CWI) pathway in response to high hydrostatic pressure in the yeast Saccharomyces cerevisiae. We demonstrate the roles of the transmembrane mechanosensor Wsc1 and aquaglyceroporin Fps1 in a general mechanism to maintain cell growth under high-pressure regimes. The promotion of water influx into cells at 25 MPa, as evident by an increase in cell volume and a loss of the plasma membrane eisosome structure, activates the CWI pathway through the function of Wsc1. Phosphorylation of Slt2, the downstream mitogen-activated protein kinase, was increased at 25 MPa. Glycerol efflux increases via Fps1 phosphorylation, which is initiated by downstream components of the CWI pathway, and contributes to the reduction in intracellular osmolarity under high pressure. The elucidation of the mechanisms underlying adaptation to high pressure through the well-established CWI pathway could potentially translate to mammalian cells and provide novel insights into cellular mechanosensation.

Evaluation of a Novel FKS1 R1354H Mutation Associated with Caspofungin Resistance in Candida auris Using the CRISPR-Cas9 System.

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.

In Candida glabrata, ERMES Component GEM1 Controls Mitochondrial Morphology, mtROS, and Drug Efflux Pump Expression, Resulting in Azole Susceptibility.

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.

A Putative NADPH Oxidase Gene in Unicellular Pathogenic Candida glabrata Is Required for Fungal ROS Production and Oxidative Stress Response.

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.

Multiple genome analysis of Candida glabrata clinical isolates renders new insights into genetic diversity and drug resistance determinants.

The emergence of drug resistance significantly hampers the treatment of human infections, including those caused by fungal pathogens such as Candida species. Candida glabrata ranks as the second most common cause of candidiasis worldwide, supported by rapid acquisition of resistance to azole and echinocandin antifungals frequently prompted by single nucleotide polymorphisms (SNPs) in resistance associated genes, such as PDR1 (azole resistance) or FKS1/2 (echinocandin resistance). To determine the frequency of polymorphisms and genome rearrangements as the possible genetic basis of C. glabrata drug resistance, we assessed genomic variation across 94 globally distributed isolates with distinct resistance phenotypes, whose sequence is deposited in GenBank. The genomes of three additional clinical isolates were sequenced, in this study, including two azole resistant strains that did not display Gain-Of-Function (GOF) mutations in the transcription factor encoding gene PDR1. Genomic variations in susceptible isolates were used to screen out variants arising from genome diversity and to identify variants exclusive to resistant isolates. More than half of the azole or echinocandin resistant isolates do not possess exclusive polymorphisms in PDR1 or FKS1/2, respectively, providing evidence of alternative genetic basis of antifungal resistance. We also identified copy number variations consistently affecting a subset of chromosomes. Overall, our analysis of the genomic and phenotypic variation across isolates allowed to pinpoint, in a genome-wide scale, genetic changes enriched specifically in antifungal resistant strains, which provides a first step to identify additional determinants of antifungal resistance. Specifically, regarding the newly sequenced strains, a set of mutations/genes are proposed to underlie the observed unconventional azole resistance phenotype.

Ultrastructural examination of mouse kidney glomerular capillary loop by sandwich freezing and freeze-substitution.

Sandwich freezing is a method of rapid freezing by sandwiching specimens between two metal disks and has been used for observing exquisite the close-to-native ultrastructure of living yeast and bacteria. Recently, this method has been found to be useful for preserving cell images of glutaraldehyde-fixed animal and human tissues. In the present study, this method was applied to observe the fine structure of mouse glomerular capillary loops. Morphometry was then performed, and the results were compared with the data obtained by an in vivo cryotechnique, which may provide the closest ultrastructure to the native state of living tissue. The results show that the ultrastructure of glomerular capillary loops obtained by sandwich freezing-freeze-substitution after glutaraldehyde fixation was close to that of the ultrastructure obtained by in vivo cryotechnique not only in the quality of cell image but also in quantitative morphometry. They indicate that the ultrastructure obtained by sandwich freezing-freeze-substitution after glutaraldehyde fixation may more closely reflect the living state of cells and tissues than conventional chemical fixation and dehydration at room temperature and conventional rapid freezing-freeze-substitution of excised tissues without glutaraldehyde fixation. Sandwich freezing-freeze-substitution techniques should be used routinely as a standard method for observing the close-to-native ultrastructure of biological specimens.

Erg25 Controls Host-Cholesterol Uptake Mediated by Aus1p-Associated Sterol-Rich Membrane Domains in Candida glabrata.

The uptake of cholesterol from the host is closely linked to the proliferation of pathogenic fungi and protozoa during infection. For some pathogenic fungi, cholesterol uptake is an important strategy for decreasing susceptibility to antifungals that inhibit ergosterol biosynthesis. In this study, we show that Candida glabrata ERG25, which encodes an enzyme that demethylates 4,4-dimethylzymosterol, is required for cholesterol uptake from host serum. Based on the screening of C. glabrata conditional knockdown mutants for each gene involved in ergosterol biosynthesis, ERG25 knockdown was found to decrease lethality of infected mice. ERG25 knockdown impairs the plasma membrane localization of the sterol importer Aus1p, suggesting that the accumulated 4,4-dimethylzymosterol destabilizes the lipid domain with which Aus1p functionally associates. ERG25 knockdown further influences the structure of the membrane compartment of Can1p (MCC)/eisosomes (ergosterol-rich lipid domains), but not the localization of the membrane proteins Pma1p and Hxt1p, which localize to sterol-poor domains. In the sterol-rich lipid domain, Aus1p-contining domain was mostly independent of MCC/eisosomes, and the nature of these domains was also different: Ausp1-contining domain was a dynamic network-like domain, whereas the MCC/eisosomes was a static dot-like domain. However, deletion of MCC/eisosomes was observed to influence the localization of Aus1p after Aus1p was transported from the endoplasmic reticulum (ER) through the Golgi apparatus to the plasma membrane. These findings suggest that ERG25 plays a key role in stabilizing sterol-rich lipid domains, constituting a promising candidate target for antifungal therapy.

Characterization of the Candida glabrata Transcription Factor CgMar1: Role in Azole Susceptibility.

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.

Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy.

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ultrastructure of cells; artifacts and extraction of cell contents are usually observed. Rapid freezing is a better alternative for the preservation of cell structure. Sandwich freezing of living yeast or bacteria followed by freeze-substitution has been used for observing the exquisite natural ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells or human tissues has also been used to reveal the ultrastructure of cells and tissues. These studies have thus far been carried out with a handmade sandwich freezing device, and applications to studies in other laboratories have been limited. A new sandwich freezing device has recently been fabricated and is now commercially available. The present paper shows how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. Also shown is the preparation of specimens for ultrathin sectioning after rapid freezing and procedures for freeze-substitution, resin embedding, trimming of blocks, cutting of ultrathin sections, recovering of sections, staining, and covering of grids with support films.

From the first touch to biofilm establishment by the human pathogen Candida glabrata: a genome-wide to nanoscale view.

Candida glabrata is an opportunistic pathogen that adheres to human epithelial mucosa and forms biofilm to cause persistent infections. In this work, Single-cell Force Spectroscopy (SCFS) was used to glimpse at the adhesive properties of C. glabrata as it interacts with clinically relevant surfaces, the first step towards biofilm formation. Following a genetic screening, RNA-sequencing revealed that half of the entire transcriptome of C. glabrata is remodeled upon biofilm formation, around 40% of which under the control of the transcription factors CgEfg1 and CgTec1. Using SCFS, it was possible to observe that CgEfg1, but not CgTec1, is necessary for the initial interaction of C. glabrata cells with both abiotic surfaces and epithelial cells, while both transcription factors orchestrate biofilm maturation. Overall, this study characterizes the network of transcription factors controlling massive transcriptional remodelling occurring from the initial cell-surface interaction to mature biofilm formation.

The Lack of SNARE Protein Homolog Syn8 Influences Biofilm Formation of Candida glabrata.

Biofilm formation of Candida species is considered to be a pathogenic factor of host infection. Since biofilm formation of Candida glabrata has not been as well studied as that of Candida albicans, we performed genetic screening of C. glabrata, and three candidate genes associated with biofilm formation were identified. Candida glabrata SYN8 (CAGL0H06325g) was selected as the most induced gene in biofilm cells for further research. Our results indicated that the syn8Δ mutant was defective not only in biofilm metabolic activity but also in biofilm morphological structure and biomass. Deletion of SYN8 seemed to have no effect on extracellular matrix production, but it led to a notable decrease in adhesion ability during biofilm formation, which may be linked to the repression of two adhesin genes, EPA10 and EPA22. Furthermore, hypersensitivity to hygromycin B and various ions in addition to the abnormal vacuolar morphology in the syn8Δ mutant suggested that active vacuolar function is required for biofilm formation of C. glabrata. These findings enhance our understanding of biofilm formation in this fungus and provide information for the development of future clinical treatments.

Role of CgTpo4 in Polyamine and Antimicrobial Peptide Resistance: Determining Virulence in Candida glabrata.

Candida glabrata is an emerging fungal pathogen whose success depends on its ability to resist antifungal drugs but also to thrive against host defenses. In this study, the predicted multidrug transporter CgTpo4 (encoded by ORF CAGL0L10912g) is described as a new determinant of virulence in C. glabrata, using the infection model Galleria mellonella. The CgTPO4 gene was found to be required for the C. glabrata ability to kill G. mellonella. The transporter encoded by this gene is also necessary for antimicrobial peptide (AMP) resistance, specifically against histatin-5. Interestingly, G. mellonella's AMP expression was found to be strongly activated in response to C. glabrata infection, suggesting AMPs are a key antifungal defense. CgTpo4 was also found to be a plasma membrane exporter of polyamines, especially spermidine, suggesting that CgTpo4 is able to export polyamines and AMPs, thus conferring resistance to both stress agents. Altogether, this study presents the polyamine exporter CgTpo4 as a determinant of C. glabrata virulence, which acts by protecting the yeast cells from the overexpression of AMPs, deployed as a host defense mechanism.

Sandwich freezing device for rapid freezing of viruses, bacteria, yeast, cultured cells and animal and human tissues in electron microscopy.

We have been using sandwich freezing of living yeast and bacteria followed by freeze-substitution for observing close-to-native ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells and human tissues have been found to give excellent preservation of ultrastructure of cells and tissues. These studies, however, have been conducted using a handmade sandwich freezing device and have been limited in a few laboratories. To spread the use of this method to other laboratories, we fabricated and commercialized a new sandwich freezing device. The new device is inexpensive, portable and sterilizable. It can be used to rapid-freeze viruses, bacteria, yeast, cultured cells and animal and human tissues to a depth of 0.2 mm if tissues are prefixed with glutaraldehyde. The commercial availability of this device will expand application of rapid freezing to wide range of biological materials.

A new regulator in the crossroads of oxidative stress resistance and virulence in Candida glabrata: The transcription factor CgTog1.

Candida glabrata is a prominent pathogenic yeast which exhibits a unique ability to survive the harsh environment of host immune cells. In this study, we describe the role of the transcription factor encoded by the gene CAGL0F09229g, here named CgTog1 after its Saccharomyces cerevisiae ortholog, as a new determinant of C. glabrata virulence. Interestingly, Tog1 is absent in the other clinically relevant Candida species (C. albicans, C. parapsilosis, C. tropicalis, C. auris), being exclusive to C. glabrata. CgTog1 was found to be required for oxidative stress resistance and for the modulation of reactive oxygen species inside C. glabrata cells. Also, CgTog1 was observed to be a nuclear protein, whose activity up-regulates the expression of 147 genes and represses 112 genes in C. glabrata cells exposed to H2O2, as revealed through RNA-seq-based transcriptomics analysis. Given the importance of oxidative stress response in the resistance to host immune cells, the effect of CgTOG1 expression in yeast survival upon phagocytosis by Galleria mellonella hemocytes was evaluated, leading to the identification of CgTog1 as a determinant of yeast survival upon phagocytosis. Interestingly, CgTog1 targets include many whose expression changes in C. glabrata cells after engulfment by macrophages, including those involved in reprogrammed carbon metabolism, glyoxylate cycle and fatty acid degradation. In summary, CgTog1 is a new and specific regulator of virulence in C. glabrata, contributing to oxidative stress resistance and survival upon phagocytosis by host immune cells.

Functional characteristics of Svl3 and Pam1 that are required for proper cell wall formation in yeast cells.

In the budding yeast Saccharomyces cerevisiae, Svl3 and Pam1 proteins work as functional homologues. Loss of their function causes increased levels of chitin deposition in the cell wall and temperature sensitivity, suggesting their involvement in cell wall formation. We found that the N- and C-termini of these proteins have distinctive and critical functions. They contain an N-terminal part that has a probable 2-dehydropantoate 2-reductase domain. In Svl3, this part can be replaced with the yeast 2-dehydropantoate 2-reductase, Pan5, suggesting that Svl3 and its homologues may be able to mediate 2-dehydropantoate 2-reductase function. On the other hand, Svl3 is recruited to the bud tip and bud neck via multiple localization signals in the C-terminal part. One of such signals is the lysine-rich region located in the C-terminal end. The function and localization of Svl3 are significantly disrupted by the loss of this lysine-rich region; however, its localization is not completely abolished by the mutation because another localization signal enables appropriate transport. Svl3 and Pam1 orthologues are found in cells across fungal species. The Svl3 orthologues of Candida glabrata can complement the loss of Svl3 and Pam1 in S. cerevisiae. C. glabrata cells lacking the SVL3 and PAM1 orthologue genes exhibit phenotypes similar to those observed in svl3∆pam1∆ S. cerevisiae cells. Thus, Svl3 homologues may be generally required for the assembly of the cell wall in fungal cells.

Candida glabrata Transcription Factor Rpn4 Mediates Fluconazole Resistance through Regulation of Ergosterol Biosynthesis and Plasma Membrane Permeability.

The ability to acquire azole resistance is an emblematic trait of the fungal pathogen Candida glabrata Understanding the molecular basis of azole resistance in this pathogen is crucial for designing more suitable therapeutic strategies. This study shows that the C. glabrata transcription factor (TF) CgRpn4 is a determinant of azole drug resistance. RNA sequencing during fluconazole exposure revealed that CgRpn4 regulates the expression of 212 genes, activating 80 genes and repressing, likely in an indirect fashion, 132 genes. Targets comprise several proteasome and ergosterol biosynthesis genes, including ERG1, ERG2, ERG3, and ERG11 The localization of CgRpn4 to the nucleus increases upon fluconazole stress. Consistent with a role in ergosterol and plasma membrane homeostasis, CgRpn4 is required for the maintenance of ergosterol levels upon fluconazole stress, which is associated with a role in the upkeep of cell permeability and decreased intracellular fluconazole accumulation. We provide evidence that CgRpn4 directly regulates ERG11 expression through the TTGCAAA binding motif, reinforcing the relevance of this regulatory network in azole resistance. In summary, CgRpn4 is a new regulator of the ergosterol biosynthesis pathway in C. glabrata, contributing to plasma membrane homeostasis and, thus, decreasing azole drug accumulation.

Screening the Drug:H+ Antiporter Family for a Role in Biofilm Formation in Candida glabrata.

Biofilm formation and drug resistance are two key pathogenesis traits exhibited by Candida glabrata as a human pathogen. Interestingly, specific pathways appear to be in the crossroad between the two phenomena, making them promising targets for drug development. In this study, the 10 multidrug resistance transporters of the Drug:H+ Antiporter family of C. glabrata were screened for a role in biofilm formation. Besides previously identified players in this process, namely CgTpo1_2 and CgQdr2, two others are shown to contribute to biofilm formation: CgDtr1 and CgTpo4. The deletion of each of these genes was found to lead to lower biofilm formation, in both SDB and RPMI media, while their expression was found to increase during biofilm development and to be controlled by the transcription factor CgTec1, a predicted key regulator of biofilm formation. Additionally, the deletion of CgDTR1, CgTPO4, or even CgQDR2 was found to increase plasma membrane potential and lead to decreased expression of adhesin encoding genes, particularly CgALS1 and CgEPA1, during biofilm formation. Although the exact role of these drug transporters in biofilm formation remains elusive, our current model suggests that their control over membrane potential by the transport of charged molecules, may affect the perception of nutrient availability, which in turn may delay the triggering of adhesion and biofilm formation.

Phenotypic and Molecular Biological Analysis of Polymycovirus AfuPmV-1M From Aspergillus fumigatus: Reduced Fungal Virulence in a Mouse Infection Model.

The filamentous fungal pathogen Aspergillus fumigatus is one of the most common causal agents of invasive fungal infection in humans; the infection is associated with an alarmingly high mortality rate. In this study, we investigated whether a mycovirus, named AfuPmV-1M, can reduce the virulence of A. fumigatus in a mouse infection model. AfuPmV-1M has high sequence similarity to AfuPmV-1, one of the polymycovirus that is a capsidless four-segment double-stranded RNA (dsRNA) virus, previously isolated from the genome reference strain of A. fumigatus, Af293. However, we found the isolate had an additional fifth dsRNA segment, referred to as open reading frame 5 (ORF5), which has not been reported in AfuPmV-1. We then established isogenic lines of virus-infected and virus-free A. fumigatus strains. Mycovirus infection had apparent influences on fungal phenotypes, with the virus-infected strain producing a reduced mycelial mass and reduced conidial number in comparison with these features of the virus-free strain. Also, resting conidia of the infected strain showed reduced adherence to pulmonary epithelial cells and reduced tolerance to macrophage phagocytosis. In an immunosuppressed mouse infection model, the virus-infected strain showed reduced mortality in comparison with mortality due to the virus-free strain. RNA sequencing and high-performance liquid chromatography (HPLC) analysis showed that the virus suppressed the expression of genes for gliotoxin synthesis and its production at the mycelial stage. Conversely, the virus enhanced gene expression and biosynthesis of fumagillin. Viral RNA expression was enhanced during conidial maturation, conidial germination, and the mycelial stage. We presume that the RNA or translation products of the virus affected fungal phenotypes, including spore formation and toxin synthesis. To identify the mycovirus genes responsible for attenuation of fungal virulence, each viral ORF was ectopically expressed in the virus-free KU strain. We found that the expression of ORF2 and ORF5 reduced fungal virulence in the mouse model. In addition, ORF3 affected the stress tolerance of host A. fumigatus in culture. We hypothesize that the respective viral genes work cooperatively to suppress the pathogenicity of the fungal host.