Utilizing a Microbial Natural Product to Investigate Cellular Circuitry Governing Fungal Drug Resistance and Morphogenesis
Year: 2016
Type: dissertation
Abstract: Fungal pathogens cause life-threatening infections in immunocompetent and immunocompromised individuals. The three most prevalent fungal pathogens are Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. There is a limited repertoire of antifungals in clinical use, with treatment of invasive infections restricted to polyenes, azoles, and echinocandins. The emergence of drug resistance now threatens the utility of antifungals, necessitating the development of novel strategies to block the emergence of drug resistance and restore antifungal activity against resistant pathogens. My research explores the mechanisms by which the natural product beauvericin enhances azole efficacy, blocks the emergence of azole resistance, inhibits virulence traits, and renders resistant pathogens responsive to treatment in mammalian infection models. My results demonstrate that beauvericin increases azole activity against Saccharomyces cerevisiae and pathogenic fungi such as C. albicans, C. neoformans, and A. fumigatus. Harnessing genome sequencing of S. cerevisiae beauvericin-resistant mutants, affinity purification of a biotinylated beauvericin analog, and biochemical and genetic assays reveals that beauvericin blocks multidrug efflux and inhibits the global regulator TORC1 kinase, thereby activating protein kinase CK2 and inhibiting the molecular chaperone Hsp90. Substitutions in the multidrug transporter Pdr5 that enable beauvericin efflux impair antifungal efflux, thereby impeding resistance to the drug combination. Genome sequence analysis of beauvericin-resistant mutants in C. albicans reveals mutations in genes encoding the transcription factors Zcf29 and Tac1. Transcriptional profiling and chromatin immunoprecipitation coupled to microarray analyses reveals that Zcf29 binds to and regulates the expression of multidrug transporter genes. Beyond drug resistance, I also discovered that beauvericin blocks the C. albicans morphogenetic transition from yeast to filamentous growth, a key virulence trait, in response to diverse host-relevant cues. Thus, my work establishes that beauvericin abrogates antifungal drug resistance and blocks C. albicans morphogenesis, providing a powerful strategy for the development of combination therapy to treat life-threatening fungal infections.
Source: TSpace (University of Toronto)
Author: Tanvi Shekhar‐Guturja
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2618143525