A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens.
A Novel, Drug Resistance-Independent, Fluorescence-Based Approach To Measure Mutation Rates in Microbial Pathogens.
复制标题
一种新颖的、独立于耐药性、基于荧光的方法来测量微生物病原体的突变率。
DOI：
10.1128/mbio.00120-19
复制
发表时间：
2019
期刊：
影响因子：
6.4
通讯作者：
Perlin,DavidS
中科院分区：
文献类型：
--
作者：
Shor,Erika;Schuyler,Jessica;Perlin,DavidS
所有的进化过程都以细胞突变DNA的能力为基础。为了确定影响诱变的因素，有必要比较不同菌株和条件下的突变率。基于抗药性的突变报告器被广泛用于测量突变率，但只有当比较的菌株具有相同的药物耐受性水平时才适用--这一条件在许多“现实世界”情况下是不满足的，例如，当比较一系列环境或临床分离株的突变率时。光滑假丝酵母菌是一种具有高度遗传多样性和快速产生抗药性的真菌病原菌。为了能够对光滑毛滴虫临床分离株之间的突变率进行有意义的比较，我们开发了一种基于荧光激活细胞分类的新方法来测量染色体整合的GFP基因的突变率。我们发现，在酿酒酵母中，这种方法概括了已报道的野生型菌株的突变率和Assh1Δ突变体的突变子表型。在光肩星天牛中，GFP报告捕捉到了由遗传毒剂或错配修复基因MSH2缺失引起的突变率增加，以及与msh2Δ相关的特定突变特征。最后，用记者检测了携带不同MSH2等位基因的光滑毛滴虫临床分离株的突变率。总之，这些结果表明，基于荧光的突变报告器可以用来测量不同药物敏感性条件下微生物的突变率，以揭示关于突变驱动因素的新见解。重要的是，突变率的测量-即增殖细胞获得DNA突变的频率-对于理解维持基因组稳定的细胞过程至关重要。许多传统的突变率测量分析都是基于检测导致对特定药物产生耐药性的突变。这种分析方法通常对实验室菌株很有效，但在比较具有不同内在药物耐受性水平的临床或环境分离株时有重大限制，这混淆了对结果的解释。在这里，我们报告了一种测量突变率的新方法的开发和验证，该方法检测导致荧光丧失的突变，而不是获得耐药性。用该方法检测了临床分离的光滑念珠菌的突变率。这种检测方法可以适用于其他生物体，并用于比较不同药物敏感性的环境中的突变率。
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 integratedGFPgene. We found that in Saccharomyces cerevisiae this approach recapitulated the reported mutation rate of a wild-type strain and the mutator phenotype of ashu1Δmutant. In C. glabrata, theGFPreporter captured the mutation rate increases caused either by a genotoxic agent or by deletion of DNA mismatch repair geneMSH2, as well as the specific mutational signature associated withmsh2Δ. Finally, the reporter was used to measure the mutation rates of C. glabrata clinical isolates carrying different alleles ofMSH2. 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.IMPORTANCEMeasurements 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.