Investigation the roles of AZR1 and CDR1 genes of candida glabrata in antifungal drug resistance

Fungal infections are on the rise around the world every day. There are approximately 300 fungi known to be pathogenic to humans. One of the most prevalent fungal diseases is candidiasis, caused by infection with Candida species. In the last several decades Candida glabrata, which is phylogenetically more closely related to the non-pathogenic yeast Saccharomyces cerevisiae than to any other pathogenic Candida species, and it has become the second cause of candidiasis globally. Candida glabrata causes systemic infections that are high in mortality, and are difficult to treat due to reduced antifungal susceptibility. Due to the extensive use of antifungal drugs, multidrug resistance has been clearly documented in many pathogenic fungi. This poses a severe challenge to treatment (Control, and Prevention, 2019, Marquez, and Quave, 2020, Lamoth et al., 2018, Gow et al., 2022). Moreover, the phenomenon of multiple drug resistance is characterized by the simultaneous acquisition of resistance to a large variety of structurally, and functionally non-related cytotoxic chemicals. The effectiveness of treatment is reduced due to the action of transmembrane protein pumps which efflux the drug out of the fungal cell. It is crucial to gain extensive knowledge of the mechanisms underlying the perseverance of antimicrobial resistance to reduce the number of pathogenic agents resistant to multiple drugs. In addition, several mechanisms have been documented in human fungal pathogens, including C. glabrata, that contribute to multidrug resistance (MDR) development. That is including overexpression of or mutations in the gene encoding the target enzyme of an antifungal, lanosterol 14α demethylase, and transcriptional activation of genes encoding drug efflux pump proteins within the ATP-binding cassette (ABC family), as well as within the Major Facilitator Superfamily (MFS family). This research investigated the effect of exposure of C. glabrata cells to the antifungals fluconazole, acetic acid, and simvastatin, with disruption of the genes encoding an ABC transporter (CDR1), and MFS transporter (AZR1). These genes were chosen because they are predicted to have roles in antifungal resistance due to their overexpression in azole-resistant clinical isolates (CDR1), the role of the homolog in S. cerevisiae (AZR1), and as AZR1 has not been deleted previously in C. glabrata. By analysing the deletion mutants of these genes, the relative contribution of each gene to drug resistance can be determined. Bioinformatic analysis was performed to identify all members of the ABC, and MDR gene families in S. cerevisiae, C. glabrata, and C. albicans homologs. This analysis identified the homologs of the CDR1, and AZR1 genes in these species, and showed that C. glabrata Cdr1p was more closely related to S cerevisiae Pdr5p, then with C. albicans Cdr1p. For more, it was found that C. glabrata Cdr1p is very similar to C. glabrata Cdr2/Pdh1p when compared with other genes in C. glabrata. Likewise, C. glabrata Azr1p was most similar to S. cerevisiae Azr1p, whereas C. albicans Seg11p was the least similar. To research gene function, the PRODIGE method was used to successfully generate a CDR1 deletion mutant by homologous integration of the knockout construct. Some adjustment of the method, including redesigning the primers to have higher G+C content in the homology region, was necessary to obtain a deletion mutant of the AZR1 gene. Compared with wildtype, the CDR1 mutant, and AZR1 mutant showed increased susceptibility to antifungals suggesting these genes play a significant role in azole particularly fluconazole, and acetic acid resistance. Based on the results of this study, AZR1 is vital in protecting the plasma membrane against acetic acid, hence the name. It is noteworthy that, statins were initially used to control cholesterol levels in humans, but studies have demonstrated that they possess antifungal activity in vitro. In this project, simvastatin was used as chemicals with antifungal activity. Neither the CDR1 nor AZR1 knockout mutant showed significantly different growth in the presence of simvastatin compared to wildtype C. glabrata. Therefore, the cholesterol lowering drug simvastatin has also been demonstrated to be effective as an antifungal agent and inhibits the growth of C. glabrata, as well as other pathogenic yeasts. Overall, this research shows that C. glabrata is a promising model for studying the genetics of resistance to multiple antifungals. This project has characterized the role of a previously uncharacterized gene, AZR1 in C. glabrata, in resistance to antifungals. Furthermore, this study has uncovered the potential for the repurposing of drugs from the statins such as simvastatin as potential antifungals for the treatment of candidiasis.