Abstract
Candida auris is an emerging healthcare-associated pathogen of global concern. Recent reports have identified C. auris isolates that grow in cellular aggregates or filaments, often without a clear genetic explanation. To investigate the regulation of C. auris morphogenesis, we applied an Agrobacterium-mediated transformation system to all four C. auris clades. We identified aggregating mutants associated with disruption of chitin regulation, while disruption of ELM1 produced a polarized, filamentous growth morphology. We developed a transiently expressed Cas9 and sgRNA system for C. auris that significantly increased targeted transformation efficiency across the four C. auris clades. Using this system, we confirmed the roles of C. auris morphogenesis regulators. Morphogenic mutants showed dysregulated chitinase expression, attenuated virulence, and altered antifungal susceptibility. Our findings provide insights into the genetic regulation of aggregating and filamentous morphogenesis in C. auris. Furthermore, the genetic tools described here will allow for efficient manipulation of the C. auris genome.
Highlights
Candida auris is an emerging healthcare-associated pathogen of global concern
To assess whether the regulation of cell wall maintenance genes was conserved in C. auris, we investigated the transcriptional change in the chitinase gene CTS1 (B9J08_002761), which is homologous to a key enzyme regulated by Ace[2] and responsible for the degradation of the primary septum during daughter cell separation in S. cerevisiae[38]
We observed no significant difference in the expression of CHS2 in Δace[2] or Δtao[3] mutants compared to the wild type (Fig. 4c). These findings demonstrate that ACE2 and TAO3 are key regulators of C. auris morphogenesis and deletion of either leads to an aggregating phenotype associated with decreased expression of the chitinase gene CTS1
Summary
Candida auris is an emerging healthcare-associated pathogen of global concern. Recent reports have identified C. auris isolates that grow in cellular aggregates or filaments, often without a clear genetic explanation. We developed a transiently expressed Cas[9] and sgRNA system for C. auris that significantly increased targeted transformation efficiency across the four C. auris clades. Using this system, we confirmed the roles of C. auris morphogenesis regulators. Genotoxic stress induced by hydroxyurea or deletion of the DNA damage responsive long non-coding RNA DINOR result in pseudohyphal elongated cells[7,8]. Strains exhibiting filamentous, elongated, or aggregating morphologies have been isolated from populations of C. auris cells following murine infection[10,11]. We hypothesized that specific adaptation of the transiently expressed CRISPR-Cas[9] system to use C. aurisrecognized promoters would increase the rates of targeted transformation efficiency
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