OBJECTIVES:Fluconazole resistance has been documented in Candida albicans and Candida parapsilosis, both leading causes of invasive candidiasis, driven in part by mutations in MRR1 that encodes a transcription factor that regulates drug efflux pump expression. A correlation has been observed between susceptibilities to fluconazole and manogepix, the active moiety of fosmanogepix, a promising new antifungal with activity against most Candida species. We aimed to determine whether MRR1-activating mutations driving fluconazole resistance in these species conferred cross-resistance to manogepix. METHODS:C. albicans and C. parapsilosis clinical isolates and strains engineered to express wild-type or mutant MRR1 alleles, and their derivatives deleted for target drug transporters, were assessed for fluconazole and manogepix susceptibility. RESULTS:Introducing Mrr1 I283R and A854V substitutions into C. parapsilosis clinical isolate Cp13 increased fluconazole MIC from 0.25 to 16-32 mg/L and increased manogepix MIC from 0.008 to 0.25 mg/L, whereas correcting to MRR1 wild-type sequence in fluconazole-resistant clinical isolates reduced fluconazole MICs from 64, 128, and 32 mg/L to 2, 0.5, and 2 mg/L, respectively, and manogepix MICs from 0.125, 0.25, and 0.125 mg/L to 0.015 mg/L for all. This was dependent upon genes encoding Mdr1B and Cdr1B transporters. In C. albicans, introducing different MRR1-activating mutations into SC5314 increased fluconazole MICs from 0.5 mg/L to 4-16 mg/L and increased manogepix MICs from 0.015 mg/L to 0.06 mg/L. Activating mutations in clinical isolates increased fluconazole MICs from 1-2 mg/L to 32-64 mg/L and manogepix MICs by 2- to 4-fold. MRR1 deletion in resistant isolates restored susceptibility to both drugs. CONCLUSIONS:Our findings demonstrate that fluconazole and manogepix cross-resistance in C. parapsilosis conferred by MRR1-activating mutations is mediated by Cdr1B and Mdr1B transporters. MRR1-activating mutations conferred cross-resistance, to a lesser degree, in C. albicans, but independent of drug transporters known to be regulated by Mrr1.

The fungal pathogen Candida auris is of global concern due to high levels of multidrug resistance and its propensity to cause infectious outbreaks. Over 90% of isolates are resistant to fluconazole, the most commonly prescribed antifungal worldwide. Fluconazole resistance is multifactorial with many isolates carrying mutations in the gene encoding the transcriptional regulator Tac1B, leading to increased expression of the gene encoding the ATP-binding cassette (ABC) transporter Cdr1. Recently, a study in C. auris examining mechanisms of in vitro-evolved reduced susceptibility to manogepix, a promising antifungal agent currently in clinical trials, found a TAC1B mutation that confers reduced manogepix and fluconazole susceptibility. We hypothesized that mutations in C. auris TAC1B and similar transcription factors in other Candida species that confer fluconazole resistance might also confer reduced susceptibility to manogepix. We measured manogepix susceptibilities for selected isolates and strains and found that mutations in C. auris TAC1B, C. albicans and C. parapsilosis TAC1, and C. glabrata PDR1 confer reduced manogepix susceptibility in a manner dependent on ABC transporters, such as Cdr1. Our findings raise the possibility of fluconazole and manogepix cross-resistance for clinical isolates harboring mutations in these genes.

OBJECTIVES:Candidozyma (Candida) auris is an emerging fungal pathogen of global concern that often exhibits multidrug resistance. Of the six described C. auris clades, Clade III has been found to be nearly universally fluconazole resistant, and almost every Clade III isolate described carries a mutation leading to a VF125AL substitution in ERG11, the gene encoding the fluconazole target lanosterol 14α-demethylase, and a mutation leading to a N647T substitution in the gene encoding Mrr1a, a transcriptional regulator of the Mdr1 transporter. In this study, we aimed to determine the specific contributions of these mutations to triazole antifungal susceptibility in Clade III C. auris isolates. METHODS:We introduced the Clade III MRR1A mutation into a Clade I background using CRISPR-Cas9 gene editing. In two Clade III clinical isolates, we corrected the native MRR1A and ERG11 mutations to their wild-type sequences, as well as disrupted MDR1. Triazole susceptibilities and MDR1 gene expression were measured in all strains. RESULTS:Introduction of the N647T substitution in a Clade I background increases fluconazole MIC from 2 to 4 μg/mL. Correction of MRR1A or disruption of MDR1 in each Clade III background resulted in a one-dilution decrease in fluconazole MIC (64-32 μg/mL), whereas the ERG11 correction resulted in a three-to four-dilution decrease (64 to 4-8 μg/mL) in fluconazole MIC. Correction of both mutations together resulted in a further decrease to 4 μg/mL. Similar changes were observed for voriconazole. CONCLUSIONS:Our findings show that although the MRR1A mutation makes a modest contribution, the ERG11 mutation is responsible for most of the fluconazole resistance observed in Clade III isolates. We also show that although these mutations likewise affect voriconazole susceptibility, they have no effect on itraconazole, isavuconazole, or posaconazole susceptibility, suggesting the therapeutic utility of these antifungals for infections due to Clade III C. auris isolates.

Candida auris has emerged as a fungal pathogen of particular concern owing in part to its propensity to exhibit antifungal resistance, especially to the commonly prescribed antifungal fluconazole. A mutation in TAC1B, which encodes a zinc cluster transcription factor, has been shown to confer increased resistance to fluconazole. In this work, we aimed to determine how mutations in TAC1B exert this effect. TAC1B mutations leading to A640V, A657V, and F862_N866del, found in fluconazole-resistant clinical isolates, were introduced into two susceptible Clade I backgrounds using CRISPR-Cas9 gene editing. These TAC1B mutations conferred increased fluconazole resistance, as well as increased resistance to other triazoles as measured by broth microdilution. RNA-seq revealed that the ATP-binding cassette (ABC) transporter gene CDR1 as well as the major facilitator superfamily (MFS) transporter gene MDR1 were both upregulated in the presence of these TAC1B mutations. Disruption of CDR1 increased susceptibility in strains with TAC1B mutations, whereas disruption of MDR1 had little to no effect. However, disruption of both CDR1 and MDR1 resulted in an additional increase in susceptibility as compared with disruption of CDR1 alone. TAC1B mutations, leading to A640V, A657V, and F862_N866del all result in increased resistance to fluconazole and other triazole antifungals and increased expression of both CDR1 and MDR1 in C. auris. Together, these data suggest CDR1 is the primary driver of resistance conferred by these TAC1B mutations.

OBJECTIVE:Candida auris has emerged as a fungal pathogen of particular concern owing in part to its propensity to exhibit antifungal resistance, especially to the commonly prescribed antifungal fluconazole. In this work we aimed to determine how mutations in the transcription factor gene TAC1B, which are common among resistant isolates and confer fluconazole resistance, exert this effect. METHODS:Selected TAC1B mutations from clinical isolates were introduced into a susceptible isolate and reverted to the wild-type sequence in select clinical isolates using CRISPR Cas9 gene editing. Disruption mutants were likewise generated for select genes of interest. TAC1B mutants were subjected to transcriptional profiling by RNA-seq, and relative expression of specific genes of interest was determined by qRT-PCR. Antifungal susceptibilities were determined by modified CLSI broth microdilution. RESULTS:TAC1B mutations leading to A640V, A657V, and F862_N866del conferred fluconazole resistance, as well as increased resistance to other triazoles, when introduced into a susceptible isolate. RNA-seq revealed that the ATP-Binding Cassette (ABC) transporter gene CDR1 as well as the Major Facilitator Superfamily (MFS) transporter gene MDR1 were both upregulated by these TAC1B mutations. Disruption of CDR1 greatly abrogated resistance in strains with TAC1B mutations whereas disruption of MDR1 had little to no effect. However, disruption of both CDR1 and MDR1 resulted in an additional reduction in resistance as compared to disruption of either gene alone. CONCLUSION:TAC1B mutations leading to A640V, A657V, and F862_N866del all result in increased resistance to fluconazole and other triazole antifungals, and increased expression of both CDR1 and MDR1 in C. auris. CDR1 is the primary driver of resistance conferred by these TAC1B mutations.

The adaptation of gene deletion methods based on the CRISPR-Cas9 system has facilitated the genetic manipulation of the pathogenic yeast Candida albicans, because homozygous mutants of this diploid fungus can now be generated in a single step, allowing the rapid screening of candidate genes for their involvement in a phenotype of interest. However, the Cas9-mediated double-strand breaks at the target site may result in an undesired loss of heterozygosity (LOH) on the affected chromosome and cause phenotypic alterations that are not related to the function of the investigated gene. In our present study, we harnessed Cas9-facilitated gene deletion to probe a set of genes that are constitutively overexpressed in strains containing hyperactive forms of the transcription factor Mrr1 for a possible contribution to the fluconazole resistance of such strains. To this aim, we used gene deletion cassettes containing two different dominant selection markers, caSAT1 and HygB, which confer resistance to nourseothricin and hygromycin, respectively, for simultaneous genomic integration in a single step, hypothesizing that this would minimize undesired LOH events at the target locus. We found that selection for resistance to both nourseothricin and hygromycin strongly increased the proportion of homozygous deletion mutants among the transformants compared with selection on media containing only one of the antibiotics, but it did not avoid undesired LOH events. Our results demonstrate that LOH on the target chromosome is a significant problem when using Cas9 for the generation of C. albicans gene deletion mutants, which demands a thorough examination of recombination events at the target site. IMPORTANCE Candida albicans is one of the medically most important fungi and a model organism to study fungal pathogenicity. Investigating gene function in this diploid yeast has been facilitated by the adaptation of gene deletion methods based on the bacterial CRISPR-Cas9 system, because they enable the generation of homozygous mutants in a single step. We found that, in addition to increasing the efficiency of gene replacement by selection markers, the Cas9-mediated double-strand breaks also result in frequent loss of heterozygosity on the same chromosome, even when two different selection markers were independently integrated into the two alleles of the target gene. Since loss of heterozygosity for other genes can result in phenotypic alterations that are not caused by the absence of the target gene, these findings show that it is important to thoroughly analyze recombination events at the target locus when using Cas9 to generate gene deletion mutants in C. albicans.

ABSTRACT Zinc cluster transcription factors (ZCFs) are a family of transcription regulators that are almost exclusively found in the fungal kingdom. Activating mutations in the ZCFs Mrr1, Tac1, and Upc2 frequently cause acquired resistance to the widely used antifungal drug fluconazole in the pathogenic yeast Candida albicans . Similar to a hyperactive Tac1, a constitutively active form of the ZCF Znc1 causes increased fluconazole resistance by upregulating the multidrug efflux pump-encoding gene CDR1 . Hyperactive forms of both Tac1 and Znc1 also cause overexpression of RTA3 , which encodes a seven-transmembrane receptor protein involved in the regulation of asymmetric lipid distribution in the plasma membrane. RTA3 expression is also upregulated by miltefosine, an antiparasitic drug that is active against fungal pathogens and considered for treatment of invasive candidiasis, and rta3 Δ mutants are hypersensitive to miltefosine. We found that activated forms of both Tac1 and Znc1 confer increased miltefosine resistance, which was dependent on RTA3 whereas CDR1 was dispensable. Intriguingly, the induction of RTA3 expression by miltefosine depended on Znc1, but not Tac1, in contrast to the known Tac1-dependent RTA3 upregulation by fluphenazine. In line with this observation, znc1 Δ mutants were hypersensitive to miltefosine, whereas tac1 Δ mutants showed wild-type tolerance. Forced expression of RTA3 reverted the hypersensitivity of znc1 Δ mutants, demonstrating that the hypersensitivity was caused by the inability of the mutants to upregulate RTA3 in response to the drug. These findings establish Znc1 as a key regulator of miltefosine-induced RTA3 expression that is important for wild-type miltefosine tolerance. IMPORTANCE Transcription factors are central regulators of gene expression, and knowledge about which transcription factor regulates specific genes in response to a certain signal is important to understand the behavior of organisms. In the pathogenic yeast Candida albicans , the RTA3 gene is required for wild-type tolerance of miltefosine, an antiparasitic drug that is considered for treatment of invasive candidiasis. Activated forms of the transcription factors Tac1 and Znc1 cause constitutive overexpression of RTA3 and thereby increased miltefosine resistance, but only Tac1 mediates upregulation of RTA3 in response to the known inducer fluphenazine. RTA3 expression is also induced by miltefosine, and we found that this response depends on Znc1, whereas Tac1 is dispensable. Consequently, znc1 Δ mutants were hypersensitive to miltefosine, whereas tac1 Δ mutants showed wild-type tolerance. These findings demonstrate that Znc1 is the key regulator of RTA3 expression in response to miltefosine that is important for wild-type miltefosine tolerance.

Objectives: The aim of this study was to determine how mutations in CpERG11 and CpTAC1 contribute to fluconazole resistance in a collection of clinical isolates.Methods: Sequences of CpERG11 and CpTAC1 were determined for 35 resistant Candida parapsilosis clinical isolates. A plasmid-based CRISPR-Cas9 system was used to introduce mutations leading to amino acid substitution in CpTac1 and CpErg11. Triazole susceptibility was determined by broth microdilution and E-test. Differential expression of genes mediated by CpTAC1 mutation was determined by RNA sequencing, and relative expression of individual transporter genes was assessed with RT-qPCR.Results: Six isolates carried a mutation in CpTAC1 in combination with the CpERG11 mutation, leading to the CpErg11Y132F substitution. When introduced into susceptible isolates, this CpERG11 mutation led to a 4- to 8-fold increase in fluconazole minimum inhibitory concentrations (MIC; 0.125 pg/mL vs. 0.5 pg/mL, 0.125 pg/mL vs. 0.5 pg/mL, and 0.5 pg/mL vs. 4 pg/mL). When introduced into a susceptible isolate, the CpTAC1 mutation leading to the G650E substitution resulted in an 8-fold increase in fluconazole MIC (0.25 pg/mL vs. 2 pg/mL), whereas correction of this mutation in resistant isolates led to a 16-fold reduction in MIC (32 pg/mL vs. 2 pg/mL). CpCDR1, CpCDR1B, and CpCDR1C were overexpressed in the presence CpTac1G650E. Disruption of these genes in combination resulted in a 4-fold reduction in fluconazole MIC (32 pg/mL vs. 8 pg/mL).Discussion: These results define the specific contribution made by the Y132F substitution in CpERG11 and demonstrate a role for activating mutations in CpTAC1 in triazole resistance in C. parapsilosis. Laura A. Doorley, Clin Microbiol Infect 2023;29:1602.e1-1602.e7 (c) 2023 Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.

ObjectiveCandida auris has emerged as a health-care-associated and multidrug-resistant fungal pathogen of great clinical concern. As many as 50% of C. auris clinical isolates are reported to be resistant to amphotericin B, but no mechanisms contributing to this resistance have been identified. Here we describe a clinical case in which high-level amphotericin B resistance was acquired in vivo during therapy and undertake molecular and genetic studies to identify and characterize the genetic determinant of resistance.MethodsWhole-genome sequencing was performed on four C. auris isolates obtained from a single patient case. Cas9-mediated genetic manipulations were then used to generate mutant strains harbouring mutations of interest, and these strains were subsequently subjected to amphotericin B susceptibility testing and comprehensive sterol profiling.ResultsA novel mutation in the C. auris sterol-methyltransferase gene ERG6 was found to be associated with amphotericin B resistance, and this mutation alone conferred a >32-fold increase in amphotericin B resistance. Comprehensive sterol profiling revealed an abrogation of ergosterol biosynthesis and a corresponding accumulation of cholesta-type sterols in isolates and strains harbouring the clinically derived ERG6 mutation.ConclusionsTogether these findings definitively demonstrate mutations in C. auris ERG6 as the first identified mechanism of clinical amphotericin B resistance in C. auris and represent a significant step forward in the understanding of antifungal resistance in this emerging public health threat.

Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus.

Resistance to fluconazole is one of clinical characteristics most frequently challenging the treatment of invasive Candida auris infections, and is observed among >90% of all characterized clinical isolates. In this work, the native C. auris ERG11 allele in a previously characterized fluconazole-susceptible clinical isolate was replaced with the ERG11 alleles from three highly fluconazole-resistant clinical isolates (MIC ≥256 mg/L), encoding the amino acid substitutions VF125AL, Y132F, and K143R, using Cas9-ribonucleoprotein (RNP) mediated transformation system. Reciprocally, the ERG11WT allele from the same fluconazole-susceptible clinical isolate, lacking any resistance-associated mutation, was introduced into a previously characterized fluconazole-resistant clinical isolate, replacing the native ERG11K143R allele, using the same methods. The resulting collection of strains was subjected to comprehensive triazole susceptibility testing, and the direct impact each of these clinically-derived ERG11 mutations on triazole MIC was determined. Introduction of each of the three mutant ERG11 alleles was observed to increase fluconazole and voriconazole MIC by 8- to 16-fold. The MIC for the other clinically available triazoles were not significantly impacted by any ERG11 mutation. In the fluconazole-resistant clinical isolate background, correction of the K143R encoding mutation led to a similar 16-fold decrease in fluconazole MIC, and 8-fold decrease in voriconazole MIC, while the MIC of other triazoles were minimally changed. Taken together, these findings demonstrate that mutations in C. auris ERG11 significantly contribute to fluconazole and voriconazole resistance, but alone cannot explain the substantially elevated MIC observed among clinical isolates of C. auris. IMPORTANCE Candida auris is an emerging multidrug-resistant and health care-associated pathogen of urgent clinical concern. The triazoles are the most widely prescribed antifungal agents worldwide and are commonly utilized for the treatment of invasive Candida infections. Greater than 90% of all C. auris clinical isolates are observed to be resistant to fluconazole, and nearly all fluconazole-resistant isolates of C. auris are found to have one of three mutations (encoding VF125AL, Y132F, or K143R) in the gene encoding the target of the triazoles, ERG11. However, the direct contribution of these mutations in ERG11 to fluconazole resistance and the impact these mutations may have the susceptibility of the other triazoles remains unknown. The present study seeks to address this knowledge gap and potentially inform the future application the triazole antifungals for the treatment of infections caused by C. auris.

Background: Candida albicans is the primary etiological agent of vulvovaginal candidiasis (VVC) and exerts its pathogenicity through secretion of the peptide toxin candidalysin encoded by the ECE1 gene. A highly conserved variant ECE1 sequence exists across a diverse set of clinical isolates. Thus, we sought to determine the relative pathogenicity and mechanism(s) associated with this alternative ECE1 allele. Methods: Isogenic strains harboring WT or variant ECE1 sequences were engineered in an Δ/Δece1 background. After confirmation of equivalent expression by qPCR, pathogenicity of strains were tested using in vitro epithelial cell and in vivo VVC models of infection and LDH, IL-1β, neutrophil levels monitored. Follow up studies using synthetic candidalysin peptide were also performed. Lastly, a panel of ECE1 chimeras were constructed to assess potential processing defects and detected by a novel HiBiT-tagging approach. Results: Strains transformed with either the variant full length ECE1 or candidalysin allele, as compared to the WT sequence, demonstrated significantly reduced immunopathogenicity during in vitro or in vivo infection despite equivalent fungal burden. Interestingly, epithelial challenge with WT or variant synthetic peptide revealed similar capacity to elicit damage and IL-1β. Allele profiling and ECE1 chimera experiments demonstrated that defects in pathogenicity are at least partly due to inefficient ECE1 processing at the peptide 2-peptide 3 junction. Discussion: The ECE1 gene displays conserved polymorphisms that alter candidalysin secretion and strain pathogenicity. Future work is focused on determining specific amino acid sequences that contribute to these affects across clinical isolates and disease states.

One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus . Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance. Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus . Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.

Candida albicans is a ubiquitous commensal fungus that colonizes human mucosal tissues. C. albicans can become pathogenic, clinically manifesting most commonly as oropharyngeal, dermal or vulvovaginal candidiasis (VVC). Studies in mice and humans convincingly show that Th17/IL-17-driven immunity is essential to controlling oral and dermal candidiasis. In contrast, the role of the IL-17 pathway during VVC remains controversial, with conflicting reports from human data and mouse models. It has been observed that a strong IL-17-related gene signature is induced in the vagina during estrogen (E2)-dependent murine VVC. IL-17R deficient mice are resistant to VVC in this standard model of E2-induced disease. Since estrogen increases susceptibility to vaginal colonization and subsequent immunopathology, first we asked whether its use in the standard VVC model masks a role for the Th17/IL-17 axis. Here, we demonstrate that mice lacking IL-17 receptor signaling showed no evidence for altered VVC susceptibility or immunopathology, regardless of estrogen administration, supporting the emerging consensus that Th17/IL-17 axis signaling is dispensable for the immunopathogenesis of VVC. We further considered the epithelial dynamics during murine VVC. C. albicans colonizes mucosal epithelial tissues, particularly those expressing Keratin 13 (K13). When estrogen levels are high, this epithelium becomes increasingly keratinized, characterized by a thick layer of thin, flat pieces of tissue. In order to visualize the expression patterns in the vaginal epithelium, we developed a reporter mouse where LACZ is expressed in all tissues containing K13. We present data using these mice to assess epithelial dynamics during VVC.

Candida albicans, a ubiquitous commensal fungus that colonizes human mucosal tissues and skin, can become pathogenic, clinically manifesting most commonly as oropharyngeal candidiasis and vulvovaginal candidiasis (VVC). Studies in mice and humans convincingly show that T-helper 17 (Th17)/interleukin 17 (IL-17)-driven immunity is essential to control oral and dermal candidiasis. However, the role of the IL-17 pathway during VVC remains controversial, with conflicting reports from human data and mouse models. Like others, we observed induction of a strong IL-17-related gene signature in the vagina during estrogen-dependent murine VVC. As estrogen increases susceptibility to vaginal colonization and resulting immunopathology, we asked whether estrogen use in the standard VVC model masks a role for the Th17/IL-17 axis. We demonstrate that mice lacking IL-17RA, Act1, or interleukin 22 showed no evidence for altered VVC susceptibility or immunopathology, regardless of estrogen administration. Hence, these data support the emerging consensus that Th17/IL-17 axis signaling is dispensable for the immunopathogenesis of VVC.

ABSTRACTCandida aurishas emerged as a multidrug-resistant pathogen of great clinical concern. Approximately 90% of clinicalC. aurisisolates are resistant to fluconazole, the most commonly prescribed antifungal agent, yet it remains unknown what mechanisms underpin this fluconazole resistance. To identify novel mechanisms contributing to fluconazole resistance inC. auris, the fluconazole-susceptibleC. aurisclinical isolate AR0387 was passaged in media supplemented with fluconazole to generate derivative strains which had acquired increased fluconazole resistancein vitro. Comparative analysis of comprehensive sterol profiles, [3H]-fluconazole uptake, sequencing ofC. aurisgenes homologous to genes known to contribute to fluconazole resistance in other species ofCandida, and the relative expression ofC. auris ERG11, CDR1, andMDR1were performed. All fluconazole-evolved derivative strains were found to have acquired mutations in the zinc-cluster transcription factor-encoding gene,TAC1B, and a corresponding increase inCDR1expression relative to the parental clinical isolate, AR0387. Mutations inTAC1Bwere also identified in a set of 304 globally distributedC. aurisclinical isolates representing each of the four major clades. Introduction of the most common mutation found among fluconazole-resistant clinical isolates ofC. aurisinto the fluconazole-susceptible isolate AR0387, was confirmed to increase fluconazole resistance by 8-fold, and the correction of the same mutation in a fluconazole-resistant isolate, AR0390, decreased fluconazole MIC by 16-fold. Taken together, these data demonstrate thatC. auriscan rapidly acquire resistance to fluconazolein-vitro, and that mutations inTAC1Bsignificantly contribute to clinical fluconazole resistance.IMPORTANCECandida aurisis an emerging multidrug-resistant pathogen of global concern, known to be responsible for outbreaks on six continents and commonly resistant to antifungals. While the vast majority of clinicalC. aurisisolates are highly resistant to fluconazole, an essential part of the available antifungal arsenal, very little is known about the mechanisms contributing to resistance. In this work, we show that mutations in the transcription factorTAC1Bsignificantly contribute to clinical fluconazole resistance. These studies demonstrate that mutations inTAC1Bcan arise rapidly in vitro upon exposure to fluconazole, and that a multitude of resistance-associatedTAC1Bmutations are present among the majority of fluconazole-resistantC. aurisisolates from a global collection and appear specific to a subset of lineages or clades. Thus, identification of this novel genetic determinant of resistance significantly adds to the understanding of clinical antifungal resistance inC. auris.

Candida auris has rapidly emerged as a health care-associated and multidrug-resistant pathogen of global concern. In this work, we examined the relative expression of the four C. auris genes with the highest degree of homology to Candida albicans CDR1 and MDR1 among three triazole-resistant clinical isolates as compared to the triazole-susceptible genome reference clinical isolate. We subsequently utilized a novel Cas9-mediated system for genetic manipulations to delete C. auris CDR1 and MDR1 in both a triazole-resistant clinical isolate and a susceptible reference strain and observed that MICs for all clinically available triazoles decreased as much as 128-fold in the CDR1 deletion strains. The findings of this work reveal for the first time that C. auris CDR1 and MDR1 are more highly expressed among triazole-resistant clinical isolates of C. auris and that the overexpression of CDR1 is a significant contributor to clinical triazole resistance.

ABSTRACT Inactivation of sterol Δ5,6-desaturase (Erg3p) in the prevalent fungal pathogen Candida albicans is one of several mechanisms that can confer resistance to the azole antifungal drugs. However, loss of Erg3p activity is also associated with deficiencies in stress tolerance, invasive hyphal growth, and attenuated virulence in a mouse model of disseminated infection. This may explain why relatively few erg3-deficient strains have been reported among azole-resistant clinical isolates. In this study, we examined the consequences of Erg3p inactivation upon C. albicans pathogenicity and azole susceptibility in mouse models of mucosal and disseminated infection. While a C. albicans erg3Δ/Δ mutant was unable to cause lethality in the disseminated model, it induced pathology in a mouse model of vaginal infection. The erg3Δ/Δ mutant was also more resistant to fluconazole treatment than the wild type in both models of infection. Thus, complete loss of Erg3p activity confers azole resistance but also niche-specific virulence deficiencies. Serendipitously, we discovered that loss of azole-inducible ERG3 transcription (rather than complete inactivation) is sufficient to confer in vitro fluconazole resistance, without compromising C. albicans stress tolerance, hyphal growth, or pathogenicity in either mouse model. It is also sufficient to confer fluconazole resistance in the mouse vaginal model, but not in the disseminated model of infection, and thus confers niche-specific azole resistance without compromising C. albicans pathogenicity at either site. Collectively, these results establish that modulating Erg3p expression or activity can have niche-specific consequences on both C. albicans pathogenicity and azole resistance. IMPORTANCE While conferring resistance to the azole antifungals in vitro, loss of sterol Δ5,6-desaturase (Erg3p) activity has also been shown to reduce C. albicans pathogenicity. Accordingly, it has been presumed that this mechanism may not be significant in the clinical setting. The results presented here challenge this assumption, revealing a more complex relationship between Erg3p activity, azole resistance, C. albicans pathogenicity, and the specific site of infection. Most importantly, we have shown that even modest changes in ERG3 transcription are sufficient to confer azole resistance without compromising C. albicans fitness or pathogenicity. Given that previous efforts to assess the importance of ERG3 as a determinant of clinical azole resistance have focused almost exclusively on detecting null mutants, its role may have been grossly underestimated. On the basis of our results, a more thorough investigation of the contribution of the ERG3 gene to azole resistance in the clinical setting is warranted.

The human fungal pathogen Candida albicans is the major etiological agent of vulvovaginal candidiasis (VVC). Despite this fact, other non-albicans Candida (NAC) species have frequently been reported, as well. Despite their presence in the vaginal environment, little is known about their capacities to elicit immune responses classically associated with C. albicans-mediated immunopathology, including neutrophil recruitment and proinflammatory cytokine signaling. Therefore, using a combination of in vitro and in vivo approaches, we undertook a comparative analysis to determine whether a representative panel of NAC species could colonize, induce immunopathological markers, or cause damage at the vaginal mucosa. Using a murine model of VVC, C. albicans was found to induce robust immunopathology (neutrophils and interleukin 1β [IL-1β]) and elicit mucosal damage. However, all the NAC species tested (including C. dubliniensis, C. tropicalis, C. parapsilosis, C. krusei, C. glabrata, and C. auris) induced significantly less damage and neutrophil recruitment than C. albicans, despite achieving similar early colonization levels. These results largely correlated with a notable lack of ability by the NAC species (including C. dubliniensis and C. tropicalis) to form hyphae both in vitro and in vivo Furthermore, both C. dubliniensis and C. tropicalis induced significantly less expression of the ECE1 gene encoding candidalysin, a key fungal virulence determinant driving VVC immunopathology. In order to determine the relative capacities of these species to elicit inflammasome-dependent IL-1β release, both wild-type and NLRP3-/- THP-1 cells were challenged in vitro While most species tested elicited only modest amounts of IL-1β, challenge with C. albicans led to significantly elevated levels that were largely NLRP3 dependent. Collectively, our findings demonstrate that although NAC species are increasingly reported as causative agents of VVC, C. albicans appears to be exceedingly vaginopathogenic, exhibiting robust immunopathology, hypha formation, and candidalysin expression. Thus, this study provides mechanistic insight into why C. albicans is overwhelmingly the major pathogen reported during VVC.