Research

Candida glabrata is the second most common cause of Candida species hospital-acquired bloodstream infections. Both the incidence of C. glabrata infections and the percentage of infections resistant to frontline antifungal drugs have been increasing over the past several decades. The increasing prevalence of drug resistant C. glabrata infections is a growing public health concern, particularly among immunocompromised populations (e.g. transplant recipients, HIV/AIDS patients, premature infants, and cancer patients receiving cytotoxic chemotherapy). Combating anti-fungal resistance will require a better understanding of how drug resistance pathways are regulated and the development of new treatment strategies.

Phosphoregulation of Drug Resistance Pathways

One way fungi develop drug resistance is through increased transcription of genes for xenobiotic efflux pump proteins. These proteins expel antifungal drugs from the cell which prevents drugs from accumulating to toxic levels. In C. glabrata, the transcription factor Pdr1 initiates the production of xenobiotic efflux pumps implicated in resistance to the azole class of antifungal agents. Knockout of the PDR1 gene results in decreased levels of drug efflux pumps and increased susceptibility to antifungal agents, underscoring the critical role of Pdr1 in drug resistance.

The Breen lab is characterizing how post-translational modifications such as phosphorylation regulate Pdr1 and drug resistance in C. glabrata. We have generated Pdr1 variants that lack putative phosphosites and revert C. glabrata to a drug sensitive phenotype. Pdr1 variants are being further investigated to probe how specific phosphorylation at specific sites regulates Pdr1 localization and interactions.

Genetic Code Expansion Tools

Genetic code expansion enables the site-specific incorporation of a non-canonical amino acid into a protein in a living cell. This technique uses a bioorthogonal amino acid tRNA synthetase and tRNA pair to incorporate the non-canonical amino acid at an amber stop codon (UAG). By altering a gene sequence to contain an amber stop codon, a protein can be synthesized with a non-canonical amino acid at the desired location. The incorporation of non-canonical amino acids can give proteins new chemical properties and enable new experiments to probe protein function.

While genetic code expansion has been used in other species, including the closely related organism Saccharomyces cerevisiae, this technique has never been applied in C. glabrata. The Breen lab is developing genetic code expansion systems to produce proteins containing non-canonical amino acids in C. glabrata. The incorporation of non-canonical amino acids will enable us to probe weak and transient protein-protein interactions regulating drug resistance in their native environment.

Funding