FN5-3 | Generation of Viable Candida albicans Mutants Lacking the 'Essential' Protein Kinase Snf1

摘要

Candida albicans is a typical member of the human microbiome, where it generally exists as a commensal. It is also, however, an opportunistic pathogen, posing a particular threat to patients who are immunocompromised. Treatments are limited due to its eukaryotic nature and the evolution of drug resistance under selective pressure; therefore, greater understanding of its mechanisms of virulence and host survival are essential to the development of novel therapies. One particularly attractive class of potential drug targets are protein kinases, which are often vital regulators of cellular processes. In the course of this dual lifestyle, C. albicans encounters a wide variety of carbon sources, though rarely the preferred carbon source, glucose. Therefore, it must have a sophisticated set of tools to efficiently detect, take up, and utilize this multitude of compounds. Several regulatory mechanisms control the response to changes in carbon source, including in particular the Glucose Repression Pathway, which is critically regulated by the protein kinase Snf1. While homologous pathways have been well studied in other organisms, notably the environmental yeast Saccharomyces cerevisiae, it has largely been neglected in C. albicans. This may be in part due to the lack of success in generating snflA mutants, leading to the theory that SNF1 is an essential gene in C. albicans, in contrast with the S. cerevisiae homo-logue. Here we show that utilization of an inducible, recombination-based gene deletion system allows for the successful deletion of SNF1 in C. albicans. Furthermore, phenotypic comparison of the null mutant with a kinase-dead form of Snf1 suggests that kinase activity accounts for the full extent of Snf1 function. These results as well as further phenotypic assays finally elucidate functions of Snf1 in C. albicans and furthermore represent a powerful system for probing the essentiality of genes previously characterized as such.