There is a significant gap in knowledge concerning the importance of protein kinases (PKs) in drug resistance mechanisms of the pathogenic fungus Candida glabrata. Our long-term goal is to identify key kinases in drug resistance and exploit them for novel Candida drug development. Our overall objectives in the present application are to (i) construct a C. glabrata PK deletion mutant library, (ii) systemically screen mutants in the library for their roles in antifungal drug resistance, (iii) analyze their regulation networks, and (iv) examine their functions in C. glabrata drug resistance isolates. Cna1, the catalytic subunit of the phosphatase calcineurin, is essential in C. glabrata echinocandin drug tolerance. Through direct dephosphorylation, Cna1 activates the transcription factor Crz1 which subsequently induces the expression of Fks2 (the target of echinocandins). In addition, our preliminary data have indicated that loss of CNA1 also enhances C. glabrata susceptibility to triazoles (a different class of antifungal drug). This works through a reduction in expression and function of Pdr1, the key transcription factor in C. glabrata triazole drug resistance. Further analysis indicate that Pdr1 can, in fact, be phosphorylated and its phosphorylation level increases with triazole treatment. Together, these findings indicate that protein phosphorylation is important in C. glabrata mechanisms of resistance against both echinocandin and triazole drugs, and PKs present as potential therapeutic targets for enhancing the efficacy of these two antifungal agents against this inherently resistant species. In Aim 1, we will construct and validate a genome-wide barcoded set of C. glabrata PK deletion mutants using a transient CRISPR/Cas9 strategy. In Aim 2, we will examine the characteristics of PK deletion mutants in C. glabrata antifungal drug resistance. PK candidates that have important roles in drug resistance will be identified by competitive fitness and minimum inhibitory concentration (MIC) assays. With transcriptional profiling (RNA-seq), we will also explore the genomic networks of these PKs. Finally, their roles in drug resistance will also be tested among C. glabrata drug resistance isolates. The proposed studies are innovative as they uniquely focus on the roles of C. glabrata PKs in antifungal drug resistance. Furthermore, our approach is innovative as we will for the first time generate a barcoded C. glabrata PK deletion library in a clinical isolate of the fungal pathogen C. glabrata. This library will be a significant contribution to the fungal research community, since it is suitable for both in vitro and in vivo future in-depth analyses of PK functions in C. glabrata biology.

The proposed research is relevant to the public health because characterizing the roles of protein kinases in Candida glabrata drug resistance mechanism represents ways in which C. glabrata kinase activity might be therapeutically targeted to overcome antifungal resistance. Therefore, this research is relevant to the National Institute of Allergy and Infectious Diseases’ mission that pertains to supporting basic and applied research to better understand, treat, and ultimately prevent infectious diseases, particularly regarding the emphasis area of antimicrobial resistance.