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

ABSTRACT 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 r...

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Autores principales:	Erika Shor, Jessica Schuyler, David S. Perlin
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	Candida glabrata GFP MSH2 mutation rate Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/8a2ade67478c48e6ae6767b3d22fe616
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spelling	oai:doaj.org-article:8a2ade67478c48e6ae6767b3d22fe6162021-11-15T15:55:14ZA Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens10.1128/mBio.00120-192150-7511https://doaj.org/article/8a2ade67478c48e6ae6767b3d22fe6162019-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00120-19https://doaj.org/toc/2150-7511ABSTRACT 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. IMPORTANCE Measurements of mutation rates—i.e., how often proliferating cells acquire mutations in their DNA—are essential for understanding cellular processes that maintain genome stability. Many traditional mutation rate measurement assays are based on detecting mutations that cause resistance to a particular drug. Such assays typically work well for laboratory strains but have significant limitations when comparing clinical or environmental isolates that have various intrinsic levels of drug tolerance, which confounds the interpretation of results. Here we report the development and validation of a novel method of measuring mutation rates, which detects mutations that cause loss of fluorescence rather than acquisition of drug resistance. Using this method, we measured the mutation rates of clinical isolates of fungal pathogen Candida glabrata. This assay can be adapted to other organisms and used to compare mutation rates in contexts where unequal drug sensitivity is anticipated.Erika ShorJessica SchuylerDavid S. PerlinAmerican Society for MicrobiologyarticleCandida glabrataGFPMSH2mutation rateMicrobiologyQR1-502ENmBio, Vol 10, Iss 1 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Candida glabrata GFP MSH2 mutation rate Microbiology QR1-502
spellingShingle	Candida glabrata GFP MSH2 mutation rate Microbiology QR1-502 Erika Shor Jessica Schuyler David S. Perlin A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens
description	ABSTRACT 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. IMPORTANCE Measurements of mutation rates—i.e., how often proliferating cells acquire mutations in their DNA—are essential for understanding cellular processes that maintain genome stability. Many traditional mutation rate measurement assays are based on detecting mutations that cause resistance to a particular drug. Such assays typically work well for laboratory strains but have significant limitations when comparing clinical or environmental isolates that have various intrinsic levels of drug tolerance, which confounds the interpretation of results. Here we report the development and validation of a novel method of measuring mutation rates, which detects mutations that cause loss of fluorescence rather than acquisition of drug resistance. Using this method, we measured the mutation rates of clinical isolates of fungal pathogen Candida glabrata. This assay can be adapted to other organisms and used to compare mutation rates in contexts where unequal drug sensitivity is anticipated.
format	article
author	Erika Shor Jessica Schuyler David S. Perlin
author_facet	Erika Shor Jessica Schuyler David S. Perlin
author_sort	Erika Shor
title	A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens
title_short	A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens
title_full	A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens
title_fullStr	A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens
title_full_unstemmed	A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens
title_sort	novel, drug resistance-independent, fluorescence-based approach to measure mutation rates in microbial pathogens
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/8a2ade67478c48e6ae6767b3d22fe616
work_keys_str_mv	AT erikashor anoveldrugresistanceindependentfluorescencebasedapproachtomeasuremutationratesinmicrobialpathogens AT jessicaschuyler anoveldrugresistanceindependentfluorescencebasedapproachtomeasuremutationratesinmicrobialpathogens AT davidsperlin anoveldrugresistanceindependentfluorescencebasedapproachtomeasuremutationratesinmicrobialpathogens AT erikashor noveldrugresistanceindependentfluorescencebasedapproachtomeasuremutationratesinmicrobialpathogens AT jessicaschuyler noveldrugresistanceindependentfluorescencebasedapproachtomeasuremutationratesinmicrobialpathogens AT davidsperlin noveldrugresistanceindependentfluorescencebasedapproachtomeasuremutationratesinmicrobialpathogens
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A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens

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