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.

Candida albicans is a common mucosal colonizer, as well as a cause of lethal invasive fungal infections. The major predisposing factor for invasive fungal disease is a compromised immune system. One component of the host immune response to fungal infection is the activation of the inflammasome, a multimeric protein complex that is critical for regulating host pro-inflammatory responses. Here, we describe methods for investigating the interactions between C. albicans and host macrophages, with a focus on the inflammasome. C. albicans isolates differ in the degree to which they activate the inflammasome due to differences in internalization, morphogenic switching, and inflammasome priming. Therefore, we include protocols for identifying these factors. This simple in vitro model can be used to elucidate the contributions of specific C. albicans strains or mutants to different aspects of interactions with macrophages.

Candida auris is an emerging healthcare-associated pathogen of global concern. Although this organism does not display the same morphological plasticity as the related fungal pathogen Candida albicans, recent reports have identified numerous C. auris isolates that grow in cellular aggregates or filaments. However, the genetic circuitry governing C. auris morphology remains largely uncharacterized. Here, we developed an Agrobacterium-mediated transformation system to generate mutants exhibiting aggregating or filamentous cell morphologies. Aggregating strains were associated with disruption of homologs of Saccharomyces cerevisiae chitinase and chitin synthase regulatory proteins, including components of the Regulation of ACE2 Morphogenesis (RAM) pathway, while disruption of a homolog of the S. cerevisiae ELM1 gene resulted in a novel filamentous strain of C. auris. To facilitate targeted genetic manipulation, we developed a transiently expressed Cas9 and sgRNA expression system for use in C. auris. Transformation using this system significantly increased the efficiency of homologous recombination and targeted integration of a reporter cassette in all four clades of C. auris. Using this system, we generated targeted deletion mutants to confirm the roles of RAM and Elm1 proteins in regulating C. auris morphogenesis. Overall, our findings provide novel insights into the genetic regulation of aggregating and filamentous morphogenesis in C. auris. Furthermore, the genetic manipulation tools described here will allow for inexpensive and efficient manipulation of the C. auris genome.ImportanceCandida auris is an emerging and often multi-drug resistant fungal pathogen responsible for outbreaks globally. Current difficulties in performing genetic manipulation in this organism remain a barrier to understanding C. auris biology. Homologous recombination approaches can result in less than 1% targeted integration of a reporter cassette, emphasizing the need for new genetic tools specific for manipulating C. auris. Here, we adapted Agrobacterium-mediated transformation and a transient Cas9 and sgRNA expression system for use in forward and reverse genetic manipulation of C. auris. We demonstrated the efficacy of each system by uncovering genes underlying cellular morphogenesis in C. auris. We identified a novel filamentous mutant of C. auris, demonstrating that this organism has maintained the capacity for filamentous growth. Our findings provide additional options for improving the genetic tractability of C. auris, which will allow for further characterization of this emerging pathogen.

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