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Experimental Evolution Identifies Adaptive Aneuploidy as a Mechanism of Fluconazole Resistance in Candida auris.

ABSTRACT: Candida auris is a newly emerging fungal pathogen of humans and has attracted considerable attention from both the clinical and basic research communities. Clinical isolates of C. auris are often resistant to one or more antifungal agents. To explore how antifungal resistance develops, we performed experimental evolution assays using a fluconazole-susceptible isolate of C. auris (BJCA001). After a series of passages through medium containing increasing concentrations of fluconazole, fungal cells acquired resistance. By sequencing and comparing the genomes of the parental fluconazole-susceptible strain and 26 experimentally evolved strains of C. auris, we found that a portion of fluconazole-resistant strains carried one extra copy of chromosome V. In the absence of fluconazole, C. auris cells rapidly became susceptible and lost the extra copy of chromosome V. Genomic and transcriptome sequencing (RNA-Seq) analyses indicate that this chromosome carries a number of drug resistance-related genes, which were transcriptionally upregulated in the resistant, aneuploid strains. Moreover, missense mutations were identified in the genes TAC1B, RRP6, and SFT2 in all experimentally evolved strains. Our findings suggest that the gain of an extra copy of chromosome V is associated with the rapid acquisition of fluconazole resistance and may represent an important evolutionary mechanism of antifungal resistance in C. auris.

SUBMITTER: Bing J

PROVIDER: S-EPMC7927865 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The recently emerged pathogenic yeast Candida auris is a major concern for human health, because it is easily transmissible, difficult to eradicate from hospitals, and highly drug resistant. Most C. auris isolates are resistant to the widely used antifungal drug fluconazole due to mutations in the target enzyme Erg11 and high activity of efflux pumps, such as Cdr1. In the well-studied, distantly related yeast Candida albicans, overexpression of drug efflux pumps also is a major mechanism of acquired fluconazole resistance and caused by gain-of-function mutations in the zinc cluster transcription factors Mrr1 and Tac1. In this study, we investigated a possible involvement of related transcription factors in efflux pump expression and fluconazole resistance of C. auris The C. auris genome contains three genes encoding Mrr1 homologs and two genes encoding Tac1 homologs, and we generated deletion mutants lacking these genes in two fluconazole-resistant strains from clade III and clade IV. Deletion of TAC1b decreased the resistance to fluconazole and voriconazole in both strain backgrounds, demonstrating that the encoded transcription factor contributes to azole resistance in C. auris strains from different clades. CDR1 expression was not or only minimally affected in the mutants, indicating that Tac1b can confer increased azole resistance by a CDR1-independent mechanism.IMPORTANCE Candida auris is a recently emerged pathogenic yeast that within a few years after its initial description has spread all over the globe. C. auris is a major concern for human health, because it can cause life-threatening systemic infections, is easily transmissible, and is difficult to eradicate from hospital environments. Furthermore, C. auris is highly drug resistant, especially against the widely used antifungal drug fluconazole. Mutations in the drug target and high activity of efflux pumps are associated with azole resistance, but it is not known how drug resistance genes are regulated in C. auris We have investigated the potential role of several candidate transcriptional regulators in the intrinsic fluconazole resistance of C. auris and identified a transcription factor that contributes to the high resistance to fluconazole and voriconazole of two C. auris strains from different genetic clades, thereby providing insight into the molecular basis of drug resistance of this medically important yeast.

Project description:Candida auris has emerged as a multidrug-resistant pathogen of great clinical concern. Approximately 90% of clinical C. auris isolates are resistant to fluconazole, the most commonly prescribed antifungal agent, and yet it remains unknown what mechanisms underpin this fluconazole resistance. To identify novel mechanisms contributing to fluconazole resistance in C. auris, fluconazole-susceptible C. auris clinical isolate AR0387 was passaged in media supplemented with fluconazole to generate derivative strains which had acquired increased fluconazole resistance in vitro Comparative analyses of comprehensive sterol profiles, [3H]fluconazole uptake, sequencing of C. auris genes homologous to genes known to contribute to fluconazole resistance in other species of Candida, and relative expression levels of C. auris ERG11, CDR1, and MDR1 were performed. All fluconazole-evolved derivative strains were found to have acquired mutations in the zinc-cluster transcription factor-encoding gene TAC1B and to show a corresponding increase in CDR1 expression relative to the parental clinical isolate, AR0387. Mutations in TAC1B were also identified in a set of 304 globally distributed C. auris clinical isolates representing each of the four major clades. Introduction of the most common mutation found among fluconazole-resistant clinical isolates of C. auris into 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 that C. auris can rapidly acquire resistance to fluconazole in vitro and that mutations in TAC1B significantly contribute to clinical fluconazole resistance.IMPORTANCE Candida auris is an emerging multidrug-resistant pathogen of global concern, known to be responsible for outbreaks on six continents and to be commonly resistant to antifungals. While the vast majority of clinical C. auris isolates 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 factor TAC1B significantly contribute to clinical fluconazole resistance. These studies demonstrated that mutations in TAC1B can arise rapidly in vitro upon exposure to fluconazole and that a multitude of resistance-associated TAC1B mutations are present among the majority of fluconazole-resistant C. auris isolates 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 in C. auris.

Project description:Candida tropicalis is less commonly isolated from clinical specimens than Candida albicans. Unlike C. albicans, which can be occasionally found as a commensal, C. tropicalis is almost always associated with the development of fungal infections. In addition, C. tropicalis has been reported to be resistant to fluconazole (FLC). To analyze the development of FLC resistance in C. tropicalis, an FLC-susceptible strain (ATCC 750) (MIC = 1.0 microg/ml) was cultured in liquid medium containing increasing FLC concentrations from 8.0 to 128 microg/ml. The strain developed variable degrees of FLC resistance which paralleled the concentrations of FLC used in the medium. The highest MICs of FLC were 16, 256, and 512 microg/ml for strains grown in medium with 8.0, 32, and 128 microg of FLC per ml, respectively. Development of resistance was rapid and could be observed already after a single subculture in azole-containing medium. The resistant strains were cross-resistant to itraconazole (MIC > 1.0 microg/ml) and terbinafine (MIC > 512 microg/ml) but not to amphotericin B. Isolates grown in FLC at concentrations of 8.0 and 32 microg/ml reverted to low MICs (1.0 microg/ml) after 12 and 11 passages in FLC-free medium, respectively. The MIC for one isolate grown in FLC (128 microg/ml) (128 R) reverted to 16 microg/ml but remained stable over 60 passages in FLC-free medium. Azole-resistant isolates revealed upregulation of two different multidrug efflux transporter genes: the major facilitators gene MDR1 and the ATP-binding cassette transporter CDR1. The development of FLC resistance in vitro correlated well with the results obtained in an experimental model of disseminated candidiasis. While FLC given at 10 mg/kg of body weight/day was effective in reducing the fungal burden of mice infected with the parent strain, the same dosing regimen was ineffective in mice infected with strain 128 R. Finally, the acquisition of in vitro FLC resistance in strain 128 R was related to a loss of virulence. The results of our study elucidate important characteristics and potential mechanisms of FLC resistance in C. tropicalis.

Project description:Regulated erroneous protein translation (adaptive mistranslation) increases proteome diversity and produces advantageous phenotypic variability in the human pathogen Candida albicans. It also increases fitness in the presence of fluconazole, but the underlying molecular mechanism is not understood. To address this question, we evolved hypermistranslating and wild-type strains in the absence and presence of fluconazole and compared their fluconazole tolerance and resistance trajectories during evolution. The data show that mistranslation increases tolerance and accelerates the acquisition of resistance to fluconazole. Genome sequencing, array-based comparative genome analysis, and gene expression profiling revealed that during the course of evolution in fluconazole, the range of mutational and gene deregulation differences was distinctively different and broader in the hypermistranslating strain, including multiple chromosome duplications, partial chromosome deletions, and polyploidy. Especially, the increased accumulation of loss-of-heterozygosity events, aneuploidy, translational and cell surface modifications, and differences in drug efflux seem to mediate more rapid drug resistance acquisition under mistranslation. Our observations support a pivotal role for adaptive mistranslation in the evolution of drug resistance in C. albicans. IMPORTANCE Infectious diseases caused by drug-resistant fungi are an increasing threat to public health because of the high mortality rates and high costs associated with treatment. Thus, understanding of the molecular mechanisms of drug resistance is of crucial interest for the medical community. Here we investigated the role of regulated protein mistranslation, a characteristic mechanism used by C. albicans to diversify its proteome, in the evolution of fluconazole resistance. Such codon ambiguity is usually considered highly deleterious, yet recent studies found that mistranslation can boost adaptation in stressful environments. Our data reveal that CUG ambiguity diversifies the genome in multiple ways and that the full spectrum of drug resistance mechanisms in C. albicans goes beyond the traditional pathways that either regulate drug efflux or alter the interactions of drugs with their targets. The present work opens new avenues to understand the molecular and genetic basis of microbial drug resistance.

Project description:Candida auris exhibits resistance to multiple antifungal drug classes and sterilization agents, posing threats to the immunocompromised worldwide. Among the four major geographical clades, the East Asian clade 2 isolates of C. auris are mostly drug susceptible. In this study, we experimentally evolved one such drug-susceptible isolate for multiple generations in the presence of the antifungal compound fluconazole and analyzed changes in the karyotype, DNA sequence, and gene expression profiles in three evolved drug-resistant isolates. Next-generation sequencing and electrophoretic karyotyping confirm the presence of segmental aneuploidy as supernumerary chromosomes originating from centromere-inclusive chromosomal duplication events in two such cases. A 638-kb region and a 675-kb region, both of which originated from chromosome 5 and contained its centromere region, are instances of supernumerary chromosome formation identified in two evolved fluconazole-resistant isolates. Loss of the supernumerary chromosomes from the drug-resistant isolates results in a complete reversal of fluconazole susceptibility. Transcriptome analysis of the third isolate identified overexpression of drug efflux pumps as a possible non-aneuploidy-driven mechanism of drug resistance. Together, this study reveals how both aneuploidy-driven and aneuploidy-independent mechanisms may operate in parallel in an evolving population of C. auris in the presence of an antifungal drug, in spite of starting from the same strain grown under similar conditions, to attain various levels of fluconazole resistance. IMPORTANCE Fungal pathogens develop drug resistance through multiple pathways by acquiring gene mutations, increasing the copy number of genes, or altering gene expression. In this study, we attempt to understand the mechanisms of drug resistance in the recently emerged superbug, C. auris. One approach to studying this aspect is identifying various mechanisms operating in drug-resistant clinical isolates. An alternative approach is to evolve a drug-susceptible isolate in the presence of an antifungal compound and trace the changes that result in drug resistance. Here, we evolve a drug-susceptible isolate of C. auris in the laboratory in the presence of a widely used antifungal compound, fluconazole. In addition to the already known changes like overexpression of drug efflux pumps, this study identifies a novel mechanism of azole resistance by the emergence of additional chromosomes through segmental duplication of chromosomal regions, including centromeres. The centric supernumerary chromosome helps stable amplification of a set of genes with an extra copy to confer fluconazole resistance.

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Experimental Evolution Identifies Adaptive Aneuploidy as a Mechanism of Fluconazole Resistance in Candida auris.

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