Abstract
Candida auris is an emerging pathogenic fungus causing outbreaks of invasive disease in healthcare facilities worldwide. Isolates frequently display resistance to multiple drug classes, owing to the upregulation of drug transporter genes. The mortality rate associated with invasive disease is high, approaching 60%, posing an increased risk for immunocompromised individuals. Therefore, there is a pressing need for new antifungalsWork presented in this thesis indicates that the deletion of CDR1 and MDR1 in C. auris, respectively encoding an ATP-binding Cassette and a Major Facilitator Superfamily efflux pump, generates a strain that is hypersensitised to drugs and can act as a platform for antifungal drug screening. Targeted homologous recombination of a nourseothricin resistance cassette was used to disrupt CDR1 followed by excision of the drug resistance marker via Cre-LoxP mediated recombination, to enable its re-use to delete MDR1.
The cdr1Dmdr1D mutant was 64- and 8- times more sensitive to fluconazole and voriconazole, respectively, compared with the wild-type strain. When assessing the capacity to form biofilms, a statistically significant difference was observed between the wild type and cdr1Dmdr1D mutant when using a stain (crystal violet) that binds to cells, but there was little to no statistically significant difference observed when using a colourimetric dye (XTT) that measures metabolic activity. Little to no statistically significant difference was observed between wild type and the transporter pump mutant in virulence, as judged by the larval Galleria mellonella infectivity assay. Accordingly, the cdr1Dmdr1D mutant will be useful when screening for drugs that limit formation of biofilms or limit fungal virulence, as these two properties are unaffected when drug efflux is compromised.
A pilot study was completed that screened the cdr1Dmdr1D mutant against a library of 2000 diverse drug- like compounds, identifying twice as many compounds with antifungal activity in comparison to the same screen using the wildtype C. auris strain. These compounds can act as development candidates for medicinal chemistry projects to generate clinically useful drugs that would inhibit the growth of wild type C. auris strains. Structure activity relationship (SAR) analysis of hit compounds and additional panels of structurally related analogues identified compounds with elevated antifungal activity. To identify its mechanism of action, one of these compounds was tested against a panel of Saccharomyces cerevisiae deletes that is diagnostic for different aspects of cellular physiology. The resulting chemogenomic profile suggested a mechanism of action involving compromise of lipid homeostasis.
| Date of Award | 1 Aug 2024 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Barry Panaretou (Supervisor), Charlotte K. Hind (Supervisor), John Sutton (Supervisor) & James Mason (Supervisor) |