A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens

All evolutionary processes are underpinned by a cellular capacity to mutate DNA. To identify factors affecting mutagenesis, it is necessary to compare mutation rates between different strains and conditions. Drug resistance-based mutation reporters are used extensively to measure mutation rates, but they are suitable only when the compared strains have identical drug tolerance levels—a condition that is not satisfied under many “real-world” circumstances, e.g., when comparing mutation rates among a series of environmental or clinical isolates. Candida glabrata is a fungal pathogen that shows a high degree of genetic diversity and fast emergence of antifungal drug resistance. To enable meaningful comparisons of mutation rates among C. glabrata clinical isolates, we developed a novel fluorescence-activated cell sorting-based approach to measure the mutation rate of a chromosomally integrated GFP gene. We found that in Saccharomyces cerevisiae this approach recapitulated the reported mutation rate of a wild-type strain and the mutator phenotype of a shu1Δ mutant. In C. glabrata, the GFP reporter captured the mutation rate increases caused either by a genotoxic agent or by deletion of DNA mismatch repair gene MSH2, as well as the specific mutational signature associated with msh2Δ. Finally, the reporter was used to measure the mutation rates of C. glabrata clinical isolates carrying different alleles of MSH2. Together, these results show that fluorescence-based mutation reporters can be used to measure mutation rates in microbes under conditions of unequal drug susceptibility to reveal new insights about drivers of mutagenesis.