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ABSTRACT Although mutation drives evolution over long and short terms, measuring and comparing mutation rates accurately have been particularly difficult. This is especially true when mutations lead to an alteration in fitness. E. Shor, J. Schuyler, and D. S. Perlin (https://doi.org/10.1128/mBio.00120-19) present a new method to compare mutation rates across fungal strains and under different growth conditions: they employ the green fluorescent protein (GFP) as the reporter and count mutations using fluorescence-activated cell sorting (FACS). The estimates of mutation rates using the GFP-FACS approach are similar to those calculated with other reporters, and the method was used to assess if different alleles of the mismatch repair pathway gene MSH2 impact the mutation rates in the human pathogen Candida glabrata. The approach could be extended to other microbes and applications, opening the way for a better understanding of how mutation rates have impacted speciation and the emergence of antimicrobial resistance.

ABSTRACT The ability to adapt to a changing environment provides a selective advantage to microorganisms. In the case of many pathogens, a large change in their environment occurs when they move from a natural setting to a setting within a human host and then during the course of disease development to various locations within that host. Two clinical isolates of the human fungal pathogen Cryptococcus neoformans were identified from a collection of environmental and clinical strains that exhibited a mutator phenotype, which is a phenotype which provides the ability to change rapidly due to the accumulation of DNA mutations at high frequency. Whole-genome analysis of these strains revealed mutations in MSH2 of the mismatch repair pathway, and complementation confirmed that these mutations are responsible for the mutator phenotype. Comparison of mutation frequencies in deletion strains of eight mismatch repair pathway genes in C. neoformans showed that the loss of three of them, MSH2, MLH1, and PMS1, results in an increase in mutation rates. Increased mutation rates enable rapid microevolution to occur in these strains, generating phenotypic variations in traits associated with the ability to grow in vivo, in addition to allowing rapid generation of resistance to antifungal agents. Mutation of PMS1 reduced virulence, whereas mutation of MSH2 or MLH1 had no effect on the level of virulence. These findings thus support the hypothesis that this pathogenic fungus can take advantage of a mutator phenotype in order to cause disease but that it can do so only in specific pathways that lead to a mutator trait without a significant tradeoff in fitness. IMPORTANCE Fungi account for a large number of infections that are extremely difficult to treat; superficial fungal infections affect approximately 1.7 billion (25%) of the general population worldwide, and systemic fungal diseases result in an unacceptably high mortality rate. How fungi adapt to their hosts is not fully understood. This research investigated the role of changes to DNA sequences in adaption to the host environment and the ability to cause disease in Cryptococcus neoformans, one of the world’s most common and most deadly fungal pathogens. The study results showed that microevolutionary rates are enhanced in either clinical isolates or in gene deletion strains with msh2 mutations. This gene has similar functions in regulating the rapid emergence of antifungal drug resistance in a distant fungal relative of C. neoformans, the pathogen Candida glabrata. Thus, microevolution resulting from enhanced mutation rates may be a common contributor to fungal pathogenesis.