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Shaker, H.

Publications and source records attributed to Shaker, H..

2 recordsLinked to original sources

A role for the putative error-prone polymerase REV1 in DNA damage and antifungal drug resistance in Candida albicans

Antimicrobial-induced DNA damage, and subsequent repair via upregulation of DNA repair factors, including error-prone translesion polymerases, can lead to the increased accumulation of mutations in the microbial genome, and ultimately increased risk of acquired mutations associated with antimicrobial resistance. While this phenotype is well described in bacterial species, it is less thoroughly investigated amongst microbial fungi. Here, we monitor DNA damage induced by antifungal agents in the fungal pathogen Candida albicans, and find that commonly used antifungal drugs are able to induce DNA damage, leading to the upregulation of transcripts encoding predicted error-prone polymerases and related factors. We focus on REV1, encoding a putative error-prone polymerase, and find that while deleting this gene in C. albicans leads to increased sensitivity to DNA damage, it also unexpectedly renders cells more likely to incur mutations and evolve resistance to antifungal agents. We further find that deletion of REV1 leads to a significant depletion in the uncharacterized protein Shm1, which itself plays a role in fungal mutagenesis. Together, this work lends new insight into previously uncharacterized factors with important roles in the DNA damage response, mutagenesis, and the evolution of antifungal drug resistance.

microbiology↗

Functional genetic characterization of stress tolerance and biofilm formation in Nakaseomyces glabrata via a novel CRISPR activation system

The overexpression of genes frequently arises in Nakaseomyces (formerly Candida) glabrata via gain-of-function mutations, gene duplication or aneuploidies, with important consequences on pathogenesis traits and antifungal drug resistance. This highlights the need to develop specific genetic tools to mimic and study genetic amplification in this important fungal pathogen. Here, we report the development, validation, and applications of the first CRISPR activation (CRISPRa) system in N. glabrata for targeted genetic overexpression. Using this system, we demonstrate the ability of CRISPRa to drive high levels of gene expression in N. glabrata, and further assess optimal guide RNA targeting for robust overexpression. We demonstrate the applications of CRISPRa to overexpress genes involved in fungal pathogenesis and drug resistance, and detect corresponding phenotypic alterations in these key traits, including the characterization of novel phenotypes. Finally, we capture strain variation using our CRISPRa system in two commonly used N. glabrata genetic backgrounds. Together, this tool will expand our capacity for functional genetic overexpression in this pathogen, with numerous possibilities for future applications.

microbiology↗