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Doorley, L. A.

Publications and source records attributed to Doorley, L. A..

5 recordsLinked to original sources

Emergence and spread of outbreak-adapted Candidozyma auris harboring mutations in RBA1

Candidozyma auris is an emerging healthcare-associated fungal pathogen, increasingly isolated from clinical outbreaks, with a high propensity to colonize patients and the medical environment. Here, we leveraged C. auris isolates from multi-facility clinical outbreaks to identify genomic patho-adaptations promoting persistence and dissemination in the healthcare environment. Genomic and phylogenetic analyses revealed loss-of-function mutations in the uncharacterized C. auris transcription factor gene RBA1 to have independently emerged multiple times within these outbreaks. We demonstrate loss of RBA1 increases C. auris adhesion to plastic and human keratinocytes, enhances biofilm formation, and exacerbates fungal burden in a mouse model of catheter-associated urinary tract infection. Finally, we uncover mutations in RBA1 have emerged during multiple C. auris outbreaks across the globe, and that RBA1 mutant lineages are present among large ongoing clinical outbreaks. These results reveal loss-of-function mutations in C. auris RBA1 as a novel and clinically relevant genetic determinant of enhanced outbreak characteristics.

microbiology↗

Acquired Amphotericin B Resistance Attributed to a Mutated ERG3 in Candidozyma auris

First identified in 2009, Candidozyma auris (formerly Candida auris) is an emerging multidrug resistant fungus that can cause invasive infections with a crude mortality rate ranging from 30-60%. Currently, 30-50% of C. auris isolates are intrinsically resistant to amphotericin B. In this work, we characterized a clinical case of acquired amphotericin B resistance using whole genome sequencing, a large-scale phenotypic screen, comprehensive sterol profiling, and genotypic reversion using CRISPR. Data obtained in this work provides evidence that a deletion resulting in a frameshift in ERG3 contributes to the observed resistant phenotype. Characterization of this isolate also revealed a fitness cost is associated with the abrogation of ergosterol production and its replacement with other late-stage sterols. This article presents a clinical case description of amphotericin B resistance from a frameshift mutation in ERG3 in C. auris and marks an advancement in the understanding of antifungal resistance in this fungal pathogen.

genetics↗

A Candidozyma (Candida) auris-optimized Episomal Plasmid Induced Cas9-editing system reveals the direct impact of the S639F encoding FKS1 mutation

ObjectivesMutations in the Candidozyma (Candida) auris {beta}-glucan synthase gene (FKS1) altering S639 are frequently associated with clinical echinocandin resistance. However, the direct impact of these mutations remains uncharacterized. We have developed a novel C. auris-optimized Episomal Plasmid-Induced Cas9 (EPIC) gene-editing system capable of recyclable precision genome editing and demonstrate the contribution of FKS1S639F mutation to echinocandin resistance. MethodsThe EPIC gene-editing system was generated for optimized use in C. auris, and ADE2 modification was evaluated in 5 C. auris clades. Mutations leading to Fks1S639F and Fks1WT were placed into echinocandin-susceptible and echinocandin-resistant isolates from Clade-III and -I, respectively, using the EPIC system. Echinocandin susceptibility was determined by CLSI broth microdilution, and cell wall abundance of chitin and {beta}-glucan was assessed by staining with Calcofluor White (CFW) and Aniline Blue (AB). ResultsThe EPIC system was capable of targeted ADE2 editing in C. auris isolates from 5 genetic clades and shown to be precise by confirmatory sequencing of 50 transformants. A single nucleotide change in FKS1 resulting in either the S639F substitution or a silent synonymous mutation was introduced in an echinocandin-susceptible Clade-III isolate. Precision FKS1 editing by the EPIC system was confirmed by whole genome sequencing. Subsequent susceptibility testing demonstrated introduction of the FKS1 S639F mutation to increase resistance to echinocandins. Moreover, introduction of a wildtype Fks1 sequence to an echinocandin-resistant Clade-I clinical isolate, correcting the FKS1S639F mutation, resulted in a restoration of echinocandin sensitivity. Evaluation of cell wall composition showed isolates or strains harboring FKS1S639F to contain significantly elevated {beta}-glucan and chitin content relative to isogenic comparators. ConclusionsThese data demonstrate the potential of our EPIC system in its ability to introduce single nucleotide substitutions in multiple C. auris clade backgrounds while revealing the direct impact of the S639F encoding FKS1 mutation on echinocandin resistance.

microbiology↗

Analysis of clinical Candida parapsilosis isolates reveals copy number variation in key fluconazole resistance genes

We used whole-genome sequencing to analyse a collection of 35 fluconazole resistant and 7 susceptible Candida parapsilosis isolates together with coverage analysis and GWAS techniques to identify new mechanisms of fluconazole resistance. Phylogenetic analysis shows that although the collection is diverse, two probable outbreak groups were identified. We identified copy number variation of two genes, ERG11 and CDR1B, in resistant isolates. Two strains have a CNV at the ERG11 locus; the entire ORF is amplified in one, and only the promoter region is amplified in the other. We show the annotated telomeric gene CDR1B is actually an artefactual in silico fusion of two highly similar neighbouring CDR genes due to an assembly error in the C. parapsilosis CDC317 reference genome. We report highly variable copy numbers of the CDR1B region across the collection. Several strains have increased expansion of the two genes into a tandem array of new chimeric genes. Other strains have experienced a deletion between the two genes creating a single gene with a reciprocal chimerism. We find translocations, duplications, and gene conversion across the CDR gene family in the C. parapsilosis species complex, showing that it is a highly dynamic family.

genomics↗

Loss-of-function ROX1 mutations suppress the fluconazole susceptibility of upc2AΔ mutation in Candida glabrata, implicating additional positive regulators of ergosterol biosynthesis

Two of the major classes of antifungal drugs in clinical use target ergosterol biosynthesis. Despite its importance, our understanding of the transcriptional regulation of ergosterol biosynthesis genes in pathogenic fungi is essentially limited to the role of hypoxia and sterol-stress induced transcription factors such as Upc2 and Upc2A as well as homologs of Sterol Response Element Binding (SREB) factors. To identify additional regulators of ergosterol biosynthesis in Candida glabrata, an important human fungal pathogen with reduced susceptibility to ergosterol biosynthesis inhibitors relative to other Candida spp., we used a serial passaging strategy to isolate suppressors of the fluconazole hypersusceptibility of a upc2A{Delta} deletion mutant. This led to the identification of loss of function mutants in two genes: ROX1, the homolog of a hypoxia gene transcriptional suppressor in Saccharomyces cerevisiae, and CST6, a transcription factor that is involved in the regulation of carbon dioxide response in C. glabrata. Here, we describe a detailed analysis of the genetic interaction of ROX1 and UPC2A. In the presence of fluconazole, loss of Rox1 function restores ERG11 expression to the upc2A{Delta} mutant and inhibits the expression of ERG3 and ERG6, leading to increased levels or ergosterol and decreased levels of the toxic sterol, 14 methyl-ergosta-8,24(28)-dien-3{beta}, 6-diol, relative to upc2A{Delta}. Our observations establish that Rox1 is a negative regulator of ERG gene biosynthesis and indicate that a least one additional positive transcriptional regulator of ERG gene biosynthesis must be present in C. glabrata. ImportanceCandida glabrata is one of the most important human fungal pathogens and has reduced susceptibility to azole class inhibitors of ergosterol biosynthesis. Although ergosterol is the target of two of the three classes of antifungal drugs, relatively little is known about the regulation of this critical cellular pathway. Sterols are both essential components of the eukaryotic plasma membrane and potential toxins; therefore, sterol homeostasis is critical for cell function. Here, we identified two new negative regulators of C. glabrata of ergosterol (ERG) biosynthesis gene expression. Our results also indicate that in addition to Upc2A, the only known activator of ERG genes, additional positive regulators of this pathway must exist.s

microbiology↗