Correction: Jiménez-Ortigosa et al. Cryo-Electron Tomography of Candida glabrata Plasma Membrane Proteins. J. Fungi 2021, 7, 120.

The authors wish to update the article title to "Cryo-Electron Tomography of Candida glabrata Plasma Membrane Proteins" [...].

Heterologous Expression of Candida Antifungal Target Genes in the Model Organism Saccharomyces cerevisiae.

Understanding how a gene variant may influence antifungal resistance, or other phenotypic characteristics, is an important step in determining or dissecting resistance mechanisms. The influence of specific genes or gene alleles on a phenotype can initially be assessed within the model organism, Saccharomyces cerevisiae. S. cerevisiae exhibits efficient rates of homologous recombination making it amendable for heterologous expression and represents a susceptible organism that can be used to determine changes in antifungal susceptibilities. Many groups have developed different methodologies for the cloning, expression, and screening processes. In this chapter, we present straightforward methodology that utilizes gap-repair cloning to express a plasmid-borne copy of Candida auris ERG11 within S. cerevisiae. Multiple alleles can be compared in order to determine how specific alterations influence triazole susceptibility. Primer design, gap-repair co-transformation, and colony PCR screening are detailed.

Impact of Erg11 Amino Acid Substitutions Identified in Candida auris Clade III Isolates on Triazole Drug Susceptibility.

ERG11 sequencing of 28 Candida auris clade III isolates revealed the presence of concomitant V125A and F126L substitutions. Heterologous expression of Erg11-V125A/F126L in Saccharomyces cerevisiae led to reduced fluconazole and voriconazole susceptibilities. Generation of single substitution gene variants through site-directed mutagenesis uncovered that F126L primarily contributes to the elevated triazole MICs. A similar yet diminished pattern of reduced susceptibility was observed with the long-tailed triazoles posaconazole and itraconazole for the V125A/F126L, F126L, Y132F, and K143R alleles.

Critical Assessment of Cell Wall Integrity Factors Contributing to in vivo Echinocandin Tolerance and Resistance in Candida glabrata.

Fungal infections are on the rise, and emergence of drug-resistant Candida strains refractory to treatment is particularly alarming. Resistance to azole class antifungals, which have been extensively used worldwide for several decades, is so high in several prevalent fungal pathogens, that another drug class, the echinocandins, is now recommended as a first line antifungal treatment. However, resistance to echinocandins is also prominent, particularly in certain species, such as Candida glabrata. The echinocandins target 1,3-ß-glucan synthase (GS), the enzyme responsible for producing 1,3-ß-glucans, a major component of the fungal cell wall. Although echinocandins are considered fungicidal, C. glabrata exhibits echinocandin tolerance both in vitro and in vivo, where a subset of the cells survives and facilitates the emergence of echinocandin-resistant mutants, which are responsible for clinical failure. Despite this critical role of echinocandin tolerance, its mechanisms are still not well understood. Additionally, most studies of tolerance are conducted in vitro and are thus not able to recapitulate the fungal-host interaction. In this study, we focused on the role of cell wall integrity factors in echinocandin tolerance in C. glabrata. We identified three genes involved in the maintenance of cell wall integrity - YPS1, YPK2, and SLT2 - that promote echinocandin tolerance both in vitro and in a mouse model of gastrointestinal (GI) colonization. In particular, we show that mice colonized with strains carrying deletions of these genes were more effectively sterilized by daily caspofungin treatment relative to mice colonized with the wild-type parental strain. Furthermore, consistent with a role of tolerant cells serving as a reservoir for generating resistant mutations, a reduction in tolerance was associated with a reduction in the emergence of resistant strains. Finally, reduced susceptibility in these strains was due both to the well described FKS-dependent mechanisms and as yet unknown, FKS-independent mechanisms. Together, these results shed light on the importance of cell wall integrity maintenance in echinocandin tolerance and emergence of resistance and lay the foundation for future studies of the factors described herein.

Preliminary Structural Elucidation of ß-(1,3)-glucan Synthase from Candida glabrata Using Cryo-Electron Tomography.

Echinocandin drugs have become a front-line therapy against Candida spp. infections due to the increased incidence of infections by species with elevated azole resistance, such as Candida glabrata. Echinocandins target the fungal-specific enzyme ß-(1,3)-glucan synthase (GS), which is located in the plasma membrane and catalyzes the biosynthesis of ß-(1,3)-glucan, the major component of the fungal cell wall. However, resistance to echinocandin drugs, which results from hotspot mutations in the catalytic subunits of GS, is an emerging problem. Little structural information on GS is currently available because, thus far, the GS enzyme complex has resisted homogenous purification, limiting our understanding of GS as a major biosynthetic apparatus for cell wall assembly and an important therapeutic drug target. Here, by applying cryo-electron tomography (cryo-ET) and subtomogram analysis, we provide a preliminary structure of the putative C. glabrata GS complex as clusters of hexamers, each subunit with two notable cytosolic domains, the N-terminal and central catalytic domains. This study lays the foundation for structural and functional studies of this elusive protein complex, which will provide insight into fungal cell wall synthesis and the development of more efficacious antifungal therapeutics.

Differential Regulation of Echinocandin Targets Fks1 and Fks2 in Candida glabrata by the Post-Transcriptional Regulator Ssd1.

Invasive infections caused by the opportunistic pathogen Candida glabrata are treated with echinocandin antifungals that target ß-1,3-glucan synthase, an enzyme critical for fungal cell wall biosynthesis. Echinocandin resistance develops upon mutation of genes (FKS1 or FKS2) that encode the glucan synthase catalytic subunits. We have analyzed cellular factors that influence echinocandin susceptibility and here describe effects of the post-transcriptional regulator Ssd1, which in S. cerevisiae, can bind cell wall related gene transcripts. The SSD1 homolog in C. glabrata was disrupted in isogenic wild type and equivalent FKS1 and FKS2 mutant strains that demonstrate echinocandin resistance (MICs ˃ 0.5 µg/mL). A reversal of resistance (8- to 128-fold decrease in MICs) was observed in FKS1 mutants, but not in FKS2 mutants, following SSD1 deletion. Additionally, this phenotype was complemented upon expression of SSD1 from plasmid (pSSD1). All SSD1 disruptants displayed susceptibility to the calcineurin inhibitor FK506, similar to fks1∆. Decreases in relative gene expression ratios of FKS1 to FKS2 (2.6- to 4.5-fold) and in protein ratios of Fks1 to Fks2 (2.7- and 8.4-fold) were observed in FKS mutants upon SSD1 disruption. Additionally, a complementary increase in protein ratio was observed in the pSSD1 expressing strain. Overall, we describe a cellular factor that influences Fks1-specific mediated resistance and demonstrates further differential regulation of FKS1 and FKS2 in C. glabrata.

Novel FKS1 and FKS2 modifications in a high-level echinocandin resistant clinical isolate of Candida glabrata.

Echinocandin resistance in Candida glabrata poses a serious clinical challenge. The underlying resistance mechanism of a pan-echinocandin-resistant C. glabrata isolate (strain L74) was investigated in this study. FKS mutants carrying specific mutations found in L74 were reconstructed by the Alt-R CRISPR-Cas9 system (Fks1 WT/Fks2-E655K, strain CRISPR 31) and site-directed mutagenesis (strain fks1Δ/Fks2-E655K). Sequence analysis of strain L74 revealed a premature stop codon W508stop in FKS1 and an E655K mutation preceding the hotspot 1 region in FKS2. Introduction of the Fks2-E655K mutation in ATCC 2001 (strain CRISPR 31) conferred a modest reduction in susceptibility. However, the same FKS2 mutation in the fks1Δ background (strain fks1Δ/Fks2-E655K) resulted in high levels of resistance to echinocandins. Glucan synthase isolated from L74 was dramatically less sensitive to micafungin (MCF) relative to ATCC 2001. Both FKS1/FKS2 transcript ratios and Fks1/Fks2 protein ratios were significantly lower in L74 and fks1Δ/Fks2-E655K compared to ATCC 2001 and CRISPR 31 (P <0.05). Mice challenged with CRISPR 31 and fks1Δ/Fks2-E655K mutants failed to respond to MCF. In conclusion, the high-level of echinocandin resistance in the clinical isolate of C. glabrata L74 was concluded to result from the combination of null function of Fks1 and the point mutation E655K in Fks2.

Spontaneous Mutational Frequency and FKS Mutation Rates Vary by Echinocandin Agent against Candida glabrata.

Echinocandins are front-line agents for treatment of invasive candidiasis. There are no reported agent-specific differences in Candida mutational frequency of resistance or propensity to develop FKS mutations. The objective of this study was to measure spontaneous and FKS mutation rates among Candida glabrata strains. Twenty bloodstream isolates from patients with or without prior echinocandin exposure were included. Minimum inhibitory concentrations (MICs), minimum fungicidal concentrations (MFCs), and mutation prevention concentrations were higher for caspofungin than for anidulafungin (P < 0.0001) and micafungin (P < 0.0001). Mutational frequencies of resistance at 3× the baseline MIC were highest for caspofungin and lowest for micafungin. A total of 247 isolates were recovered at or above the MFC for caspofungin (n = 159), anidulafungin (n = 74), or micafungin (n = 14). Agent-specific MIC increases were noted for anidulafungin and caspofungin, but not micafungin. Thirty-three percent of isolates harbored hot spot mutations in FKS1 (n = 6) or FKS2 (n = 76). Mutations at the Ser629 (Fks1) or Ser663 (Fks2) loci were more common after selection with anidulafungin or micafungin than with caspofungin (P = 0.003). Four isolates demonstrated >4-fold increases in MICs without FKS hot spot mutations; three of these harbored Fks2 mutations upstream of hot spot 1. The final isolate was FKS1 and FKS2 wild-type, but the 50% inhibitory concentrations of caspofungin and micafungin were increased 2.7- and 8-fold, respectively. In conclusion, micafungin may be superior in vitro to the other agents in limiting the emergence of resistance among C. glabrata Caspofungin exposure may be most likely to promote resistance development. These data provide a foundation for future investigations of newly developed echinocandin agents.

Fungal Resistance to Echinocandins and the MDR Phenomenon in Candida glabrata.

Candida glabrata has thoroughly adapted to successfully colonize human mucosal membranes and survive in vivo pressures. prior to and during antifungal treatment. Out of all the medically relevant Candida species, C. glabrata has emerged as a leading cause of azole, echinocandin, and multidrug (MDR: azole + echinocandin) adaptive resistance. Neither mechanism of resistance is intrinsic to C. glabrata, since stable genetic resistance depends on mutation of drug target genes, FKS1 and FKS2 (echinocandin resistance), and a transcription factor, PDR1, which controls expression of major drug transporters, such as CDR1 (azole resistance). However, another hallmark of C. glabrata is the ability to withstand drug pressure both in vitro and in vivo prior to stable "genetic escape". Additionally, these resistance events can arise within individual patients, which underscores the importance of understanding how this fungus is adapting to its environment and to drug exposure in vivo. Here, we explore the evolution of echinocandin resistance as a multistep model that includes general cell stress, drug adaptation (tolerance), and genetic escape. The extensive genetic diversity reported in C. glabrata is highlighted.

Limited ERG11 Mutations Identified in Isolates of Candida auris Directly Contribute to Reduced Azole Susceptibility.

Multiple Erg11 amino acid substitutions were identified in clinical isolates of Candida auris originating from India and Colombia. Elevated azole MICs were detected in Saccharomyces cerevisiae upon heterologous expression of C. aurisERG11 alleles that encoded for Y132F or K143R substitutions; however, expression of alleles encoding I466M, Y501H, or other clade-defined amino acid differences yielded susceptible MICs. Similar to other Candida species, specific C. aurisERG11 mutations resulted directly in reduced azole susceptibility.

Multilocus Sequence Typing (MLST) Genotypes of Candida glabrata Bloodstream Isolates in Korea: Association With Antifungal Resistance, Mutations in Mismatch Repair Gene (Msh2), and Clinical Outcomes.

Candida glabrata bloodstream infection (BSI) isolates from a particular geographic area have been reported to comprise a relatively small number of the major sequence types (STs) by multilocus sequence typing (MLST) analysis. Yet little is known about the characteristics of major ST strains of C. glabrata. To address this question in Korea, we investigated antifungal resistance and non-synonymous mutations of the mismatch repair gene (msh2 mutations) in C. glabrata BSI isolates, as well as associated clinical characteristics, and compared the results according to MLST genotype. We assessed a total of 209 C. glabrata BSI isolates from seven hospitals in Korea for 2 years (2009 and 2014). Clinical features of candidemia and their outcomes were analyzed for 185 available cases. According to MLST, ST7 (47.8%) was the most common type, followed by ST3 (22.5%); the remainder represented 28 types of minor STs (29.7%). Fluconazole-resistance (FR) rates for ST7, ST3, and other strains were 9.0% (9/100), 8.5% (4/47), and 4.8% (3/62), respectively, and all were susceptible to amphotericin B and micafungin. All ST7 isolates harbored the V239L mutation in msh2, known to confer hypermutability, while 91.5% of ST3 isolates did not harbor the msh2 mutation. Overall, isolates of the same ST had identical msh2 mutations, with the exception of nine isolates. The msh2 mutations were identified in 68.8% (11/16) of the FR isolates and 67.4% (130/193) of the fluconazole susceptible-dose dependent isolates. There was no significant difference in all clinical characteristics between ST3 and ST7. However, the 30-day mortality of C. glabrata candidemia due to the two major ST (ST3 or ST7) strains was significantly higher than that of candidemia due to other minor ST strains (45.1 vs. 25.0%, p < 0.05). Multivariate logistic regression analysis also showed that two major STs (ST3 and ST7) were independent predictors of 30-day mortality. This study showed for the first time that two STs (ST7 and ST3) were predominant among BSI isolates in Korea, and that C. glabrata BSI isolates belonging to two major MLST genotypes are characterized by higher mortality. In addition, most msh2 mutations align with MLST genotype, irrespective of FR.

Molecular Analysis of Resistance and Detection of Non-Wild-Type Strains Using Etest Epidemiological Cutoff Values for Amphotericin B and Echinocandins for Bloodstream Candida Infections from a Tertiary Hospital in Qatar.

A total of 301 Candida bloodstream isolates collected from 289 patients over 5 years at a tertiary hospital in Qatar were evaluated. Out of all Candida infections, 53% were diagnosed in patients admitted to the intensive care units. Steady increases in non-albicans Candida species were reported from 2009 to 2014 (30.2% for Candida albicans versus 69.8% for the other Candida species). Etest antifungal susceptibility testing was performed on all recovered clinical isolates to determine echinocandin (micafungin and anidulafungin) and amphotericin B susceptibilities and assess non-wild-type (non-WT) strains (strains for which MICs were above the epidemiological cutoff values). DNA sequence analysis was performed on all isolates to assess the presence of FKS mutations, which confer echinocandin resistance in Candida species. A total of 3.9% of isolates (12/301) among strains of C. albicans and C. orthopsilosis contained FKS hot spot mutations, including heterozygous mutations in FKS1 For C. tropicalis, the Etest appeared to overestimate strains non-WT for micafungin, anidulafungin, and amphotericin B, as 14%, 11%, and 35% of strains, respectively, had values above the epidemiological cutoff value. However, no FKS mutations were identified in this species. For all other species, micafungin best reported the echinocandin non-WT strains relative to the FKS genotype, as anidulafungin tended to overestimate non-wild-type strains. Besides C. tropicalis, few strains were classified as non-WT for amphotericin B.

VT-1598 inhibits the in vitro growth of mucosal Candida strains and protects against fluconazole-susceptible and -resistant oral candidiasis in IL-17 signalling-deficient mice.

Background: Chronic mucocutaneous candidiasis (CMC) treatment often induces drug resistance, posing long-term challenges. A novel broad-spectrum fungal CYP51 inhibitor, VT-1598, specifically targets fungal CYP51, but not human CYP enzymes. Objectives: To determine the efficacy of VT-1598 in the treatment of oral Candida infection caused by fluconazole-susceptible and -resistant clinical isolates. Methods: The MICs of VT-1598 and fluconazole for 28 Candida isolates recovered from patients with inherited CMC were determined using CLSI M27-A3 and M27-S4 guidelines. Plasma and tongue VT-1598 or fluconazole concentrations were measured in mice following oral administration to determine tissue distribution. Tongue fungal load was determined in IL-17 signalling-deficient Act1-/- mice following sublingual Candida albicans infection and oral treatment with fluconazole or VT-1598. Results: Among the 28 Candida isolates, 10 (36%) had fluconazole MICs of ≥4 mg/L, whereas VT-1598 demonstrated potent in vitro activity against all isolates (MIC90, 0.125 mg/L). After oral administration, VT-1598 levels in mouse plasma and tongue were significantly greater than those of fluconazole. In vivo, VT-1598 exhibited significant efficacy against fluconazole-susceptible and -resistant C. albicans, even at low drug doses. Furthermore, after a 10 day washout period, tongue fungal burdens in fluconazole-treated mice returned to vehicle control levels, whereas, in contrast, they were undetectable in mice treated with VT-1598. Conclusions: VT-1598 effectively controls in vitro growth of mucosally derived Candida clinical isolates, including fluconazole-resistant strains. In vivo, VT-1598 eliminates C. albicans, even after a long washout period or at low doses. Therefore, VT-1598 is a promising drug candidate that may significantly improve treatment options for CMC patients.

Absence of Azole or Echinocandin Resistance in Candida glabrata Isolates in India despite Background Prevalence of Strains with Defects in the DNA Mismatch Repair Pathway.

Candida glabrata infections are increasing worldwide and exhibit greater rates of antifungal resistance than those with other species. DNA mismatch repair (MMR) gene deletions, such as msh2Δ, in C. glabrata resulting in a mutator phenotype have recently been reported to facilitate rapid acquisition of antifungal resistance. This study determined the antifungal susceptibility profiles of 210 C. glabrata isolates in 10 hospitals in India and investigated the impact of novel MSH2 polymorphisms on mutation potential. No echinocandin- or azole-resistant strains and no mutations in FKS hot spot regions were detected among the C. glabrata isolates, supporting our in vitro susceptibility testing results. CLSI antifungal susceptibility data showed that the MICs of anidulafungin (geometric mean [GM], 0.12 µg/ml) and micafungin (GM, 0.01 µg/ml) were lower and below the susceptibility breakpoint compared to that of caspofungin (CAS) (GM, 1.31 µg/ml). Interestingly, 69% of the C. glabrata strains sequenced contained six nonsynonymous mutations in MSH2, i.e., V239L and the novel mutations E459K, R847C, Q386K, T772S, and V239/D946E. Functional analysis of MSH2 mutations revealed that 49% of the tested strains (40/81) contained a partial loss-of-function MSH2 mutation. The novel MSH2 substitution Q386K produced higher frequencies of CAS-resistant colonies upon expression in the msh2Δ mutant. However, expression of two other novel MSH2 alleles, i.e., E459K or R847C, did not confer selection of resistant colonies, confirming that not all mutations in the MSH2 MMR pathway affect its function or generate a phenotype of resistance to antifungal drugs. The lack of drug resistance prevented any correlations from being drawn with respect to MSH2 genotype.

The Gastrointestinal Tract Is a Major Source of Echinocandin Drug Resistance in a Murine Model of Candida glabrata Colonization and Systemic Dissemination.

Candida species are a part of the human microbiome and can cause systemic infection upon immune suppression. Candida glabrata infections are increasing and have greater rates of antifungal resistance than other species. Here, we present a C. glabrata gastrointestinal (GI) colonization model to explore whether colonized yeast exposed to caspofungin, an echinocandin antifungal, develop characteristic resistance mutations and, upon immunosuppression, breakthrough causing systemic infection. Daily therapeutic dosing (5 mg/kg of body weight) of caspofungin resulted in no reduction in fecal burdens, organ breakthrough rates similar to control groups, and resistance rates (0 to 10%) similar to those reported clinically. Treatment with 20 mg/kg caspofungin initially reduced burdens, but a rebound following 5 to 9 days of treatment was accompanied by high levels of resistance (FKS1/FKS2 mutants). Although breakthrough rates decreased in this group, the same FKS mutants were recovered from organs. In an attempt to negate drug tolerance that is critical for resistance development, we cotreated mice with daily caspofungin and the chitin synthase inhibitor nikkomycin Z. The largest reduction (3 log) in GI burdens was obtained within 3 to 5 days of 20 mg/kg caspofungin plus nikkomycin treatment. Yet, echinocandin resistance, characterized by a novel Fks1-L630R substitution, was identified following 5 to 7 days of treatment. Therapeutic caspofungin plus nikkomycin treatment left GI burdens unchanged but significantly reduced organ breakthrough rates (20%; P < 0.05). Single-dose pharmacokinetics demonstrated low levels of drug penetration into the GI lumen posttreatment with caspofungin. Overall, we show that C. glabrata echinocandin resistance can arise within the GI tract and that resistant mutants can readily disseminate upon immunosuppression.

Genetic Drivers of Multidrug Resistance in Candida glabrata.

Both the incidence of invasive fungal infections and rates of multidrug resistance associated with fungal pathogen Candida glabrata have increased in recent years. In this perspective, we will discuss the mechanisms underlying the capacity of C. glabrata to rapidly develop resistance to multiple drug classes, including triazoles and echinocandins. We will focus on the extensive genetic diversity among clinical isolates of C. glabrata, which likely enables this yeast to survive multiple stressors, such as immune pressure and antifungal exposure. In particular, over half of C. glabrata clinical strains collected from U.S. and non-U.S. sites have mutations in the DNA mismatch repair gene MSH2, leading to a mutator phenotype and increased frequencies of drug-resistant mutants in vitro. Furthermore, recent studies and data presented here document extensive chromosomal rearrangements among C. glabrata strains, resulting in a large number of distinct karyotypes within a single species. By analyzing clonal, serial isolates derived from individual patients treated with antifungal drugs, we were able to document chromosomal changes occurring in C. glabrata in vivo during the course of antifungal treatment. Interestingly, we also show that both MSH2 genotypes and chromosomal patterns cluster consistently into specific strain types, indicating that C. glabrata has a complex population structure where genomic variants arise, perhaps during the process of adaptation to environmental changes, and persist over time.

Prevalent mutator genotype identified in fungal pathogen Candida glabrata promotes multi-drug resistance.

The fungal pathogen Candida glabrata has emerged as a major health threat since it readily acquires resistance to multiple drug classes, including triazoles and/or echinocandins. Thus far, cellular mechanisms promoting the emergence of resistance to multiple drug classes have not been described in this organism. Here we demonstrate that a mutator phenotype caused by a mismatch repair defect is prevalent in C. glabrata clinical isolates. Strains carrying alterations in mismatch repair gene MSH2 exhibit a higher propensity to breakthrough antifungal treatment in vitro and in mouse models of colonization, and are recovered at a high rate (55% of all C. glabrata recovered) from patients. This genetic mechanism promotes the acquisition of resistance to multiple antifungals, at least partially explaining the elevated rates of triazole and multi-drug resistance associated with C. glabrata. We anticipate that identifying MSH2 defects in infecting strains may influence the management of patients on antifungal drug therapy.

Sphingolipids mediate differential echinocandin susceptibility in Candida albicans and Aspergillus nidulans.

The cell wall synthesis-inhibiting echinocandins, including caspofungin and micafungin, play important roles in the treatment of candidiasis and aspergillosis. Previous studies revealed that, in the haploid yeast Candida glabrata, sphingolipid biosynthesis pathway mutations confer caspofungin reduced susceptibility (CRS) but micafungin increased susceptibility (MIS). Here, we describe one Candida albicans strain (of 10 tested) that similarly yields CRS-MIS mutants at relatively high frequency. Mutants demonstrated increased levels of long-chain bases (sphingolipid pathway intermediates) and, unique to this strain, loss of His104/Pro104 heterozygosity in the TSC13-encoded enoyl reductase. CRS-MIS was similarly observed in a C. albicans homozygous fen1Δ fen12Δ laboratory strain and in diverse wild-type strains following exogenous long-chain-base treatment. Analogous to these results, CRS-MIS was demonstrated in an Aspergillus nidulans basA mutant encoding defective sphingolipid C4-hydroxylase and in its wild-type parent exposed to long-chain bases. Sphingolipids likely modulate echinocandin interaction with their Fks membrane target in all susceptible fungi, with potential implications for optimizing therapy with existing antifungals and the development of novel agents.

Fks1 and Fks2 are functionally redundant but differentially regulated in Candida glabrata: implications for echinocandin resistance.

The echinocandins caspofungin, micafungin, and anidulafungin, inhibitors of cell wall ß-1,3-glucan synthesis, were recently elevated to first-line agents for treating infections due to the azole-refractory yeast Candida glabrata. In Candida albicans, echinocandin resistance is strictly associated with mutations in Fks1, a large integral membrane protein and putative ß-1,3-glucan synthase, while mutations in both Fks1 and its paralog Fks2 (but not Fks3) have been associated with resistance in C. glabrata. To further explore their function, regulation, and role in resistance, C. glabrata fks genes were disrupted and subjected to mutational analysis, and their differential regulation was explored. An fks1Δ fks2Δ double disruptant was not able to be generated; otherwise, all three single and remaining two double disruptants displayed normal growth and echinocandin susceptibility, indicating Fks1-Fks2 redundancy. Selection on echinocandin-containing medium for resistant mutants was dependent on strain background: only fks1Δ and fks1Δ fks3Δ strains consistently yielded mutants exhibiting high-level resistance, all with Fks2 hot spot 1 mutations. Thus, Fks1-Fks2 redundancy attenuates the rate of resistance; further analysis showed that it also attenuates the impact of resistance-conferring mutations. Growth of the fks1Δ and, especially, fks1Δ fks3Δ strains was specifically susceptible to the calcineurin inhibitor FK506. Relatedly, FK506 addition or calcineurin gene CMP2 disruption specifically reversed Fks2-mediated resistance of laboratory mutants and clinical isolates. RNA analysis suggests that transcriptional control is not the sole mechanism by which calcineurin modulates Fks2 activity.

CRS-MIS in Candida glabrata: sphingolipids modulate echinocandin-Fks interaction.

Infections with the azole-refractory yeast Candida glabrata are now commonly treated with the echinocandins caspofungin (CSF) or micafungin (MCF). True resistance (> 32-fold decreased susceptibility) to these lipopeptide inhibitors of cell wall synthesis is rare and strictly associated with mutations in integral membrane proteins Fks1 or Fks2. In contrast, mutants exhibiting 4- to 32-fold CSF reduced susceptibility (CRS) were readily selected in vitro, and surprisingly demonstrated 4- to 32-fold MCF increased susceptibility (MIS). Sequencing and gene deletion demonstrated that CRS-MIS is Fks-independent. To explore alternative mechanisms, we initially employed Saccharomyces cerevisiae, and observed that CRS was conferred by multiple mutations (fen1Δ, sur4Δ, cka2Δ and tsc10-ts) disrupting sphingolipid biosynthesis. Following this lead, C. glabrata fen1Δ and cka2Δ deletants were constructed, and shown to exhibit CRS-MIS. Sphingolipid analysis of CRS-MIS laboratory mutants and clinical isolates demonstrated elevated dihydrosphingosine (DHS) and phytosphingosine (PHS) levels, and consistent with this sequencing revealed fen1, sur4, ifa38 and sur2 mutations. Moreover, exogenous DHS or PHS conferred a CRS-MIS phenotype on wild-type C. glabrata. Exogenous PHS failed, however, to suppress CRS-MIS in a sur2 mutant blocked in conversion of DHS to PHS, implying that accumulation of these intermediates confers CRS-MIS. We conclude that membrane sphingolipids modulate echinocandin-Fks interaction.