The Ca-loop in thymidylate kinase is critical for growth and contributes to pyrimidine drug sensitivity of Candida albicans.

The yeast Candida albicans is the most prevalent opportunistic fungal pathogen in humans. Drug resistance among C. albicans isolates poses a common challenge, and overcoming this resistance represents an unmet need in managing this common pathogen. Here, we investigated CDC8, encoding thymidylate kinase (TMPK), as a potential drug target for the management of C. albicans infections. We found that the region spanning amino acids 106-123, namely the Ca-loop of C. albicans TMPK (CaTMPK), contributes to the hyperactivity of this enzyme compared with the human enzyme (hTMPK) and to the utilization of deoxyuridine monophosphate (dUMP)/deoxy-5-fluorouridine monophosphate (5-FdUMP) as a substrate. Notably, expression of CaTMPK, but not of hTMPK, produced dUTP/5-FdUTP-mediated DNA toxicity in budding yeast (Saccharomyces cerevisiae). CRISPR-mediated deletion of this Ca-loop in C. albicans revealed that the Ca-loop is critical for fungal growth and susceptibility to 5-fluorouridine (5-FUrd). Of note, pathogenic and drug-resistant C. albicans clones were similarly sensitive to 5-FUrd, and we also found that CaTMPK is essential for the growth of C. albicans In conclusion, these findings not only identified a target site for the development of CaTMPK-selective drugs, but also revealed that 5-FUrd may have potential utility as drug for managing C. albicans infections.