How eukaryotic pathogens explore the fitness landscape by mitotic recombination

真核病原体如何通过有丝分裂重组探索适应性景观

• 批准号: 8604684
• 负责人: Tim James
• 金额: $ 19.44万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-15 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8604684
• 关键词: Address; Alleles; Animal Model; Antifungal Therapy; Biocompatible Materials; Cell Separation; Chromosomes; Data; Development; Diploidy; Environment; Eukaryota; Event; Evolution; Gene Mutation; Genes; Genetic; Genetic Drift; Genetic Load; Genetic Recombination; Genetic Variation; Genome; Genomics; Genotype; Goals; Green Fluorescent Proteins; Growth; Habitats; Haploidy; Health; Human; Infection; Larva; Life; Life Cycle Stages; Loss of Heterozygosity; Malignant Neoplasms; Maps; Meiosis; Methods; Microbe; Mitosis; Mitotic Recombination; Modeling; Molecular Epidemiology; Moths; Mutation; Natural Selections; Nucleotides; Organism; Pathogenesis; Pathogenicity Island; Phenotype; Play; Population; Population Genetics; Population Sizes; Process; Reactive Oxygen Species; Relative (related person); Research; Role; Saccharomyces cerevisiae; Speed; Staging; System; Temperature; Testing; Variant; Virulence; Virulence Factors; Waxes; Work; Yeast Model System; Yeasts; asexual; base; empowered; epidemiology study; fitness; in vivo; interest; next generation sequencing; novel; pandemic disease; pathogen; positional cloning; public health relevance; research study; sample fixation; sex; stressor; theories; tumorigenesis

DESCRIPTION (provided by applicant): Mitotic recombination occurs in all diploid organisms, but its evolutionary significance has largely been ignored. Mitotic recombination causes loss of heterozygosity (LOH), making it counterintuitive that it could be adaptive. In large multicellular organisms, LOH is considered primarily maladaptive and is frequently associated with tumorigenesis. Yet in organisms with a free-living haploid stage that lack a high genetic load, such as many unicellular fungal and protozoan pathogens, LOH it is likely to be an important mechanism speeding the fixation of beneficial recessive alleles. This assumption is reinforced by studies of natural populations of diploid pathogens that consistently show evidence for polymorphic LOH genomic regions. These observations indicate that LOH is pervasive, however there is a critical need to address whether, under what conditions, and by what mechanisms LOH is an essential component of how diploid pathogens explore their fitness landscapes. Our central hypothesis is that LOH is an important component of evolution by positive selection, but the relative importance of LOH as an adaptive force will be positively correlated with the heterozygosity of the initial genotype or population. We are particularly interested in applying this to the fitness landscape of pathogens because they are notoriously clonal, at least 1,500 described pathogenic protozoon species are diploid, and pathogens appear to have increased rates of LOH in vivo. Here we propose a novel method to test whether heterozygosity speeds the rate of adaptation by mitotic recombination using a yeast-wax worm (Saccharomyces cerevisiae-Galleria mellonella) pathogenesis model. Aim 1 will clonally evolve replicate populations growing inside waxworm larvae initiated from single parental genotypes differing over a 32-fold range of heterozygosity. Using fluorescent cell sorting based on a green fluorescent protein tagged yeast, we will be able to extract pure yeast populations from the infected larvae after 48 hrs of in vivo growth, and this process will be repeated for 100 serial transfers. Cell sorting also allows pathogen fitness to be estimated at each transfer, allowing us to test whether the rate of adaptation is correlated with initial heterozygosity. Aim 2 will use next generation sequencing to genotype the evolved lines to identify parallel LOH events among replicate populations that indicate the action of positive selection and identify virulence genes. Using this experimental evolution approach will allow us to avoid the problems associated genetic drift and mutation accumulation due to small population sizes. Development of the yeast-Galleria infection model into an experimental evolution system will provide a means to map the relationship between pathogen genotype, virulence, and fitness in more powerful way than standard reverse genetic approaches.

描述(由申请人提供)： 有丝分裂重组存在于所有二倍体生物中，但其进化意义在很大程度上被忽视了。有丝分裂重组会导致杂合性丢失(LOH)，这使得它可能是适应性的这一点与直觉相反。在大型多细胞生物体中，杂合性缺失主要被认为是不适应的，并且经常与肿瘤的发生有关。然而，在缺乏高遗传负荷的自由生活单倍体阶段的生物体中，如许多单细胞真菌和原生动物病原体，LOH可能是加速有益隐性等位基因固定的重要机制。对二倍体病原体自然种群的研究证实了这一假设，这些研究一直表明存在多态的LOH基因组区域。这些观察表明杂合性缺失是普遍存在的，然而，迫切需要解决的问题是，杂合性缺失是否是二倍体病原体探索其适应环境的重要组成部分，在什么条件下，以及通过什么机制。我们的中心假设是LOH是正向选择进化的重要组成部分，但LOH作为一种适应力量的相对重要性将与初始基因或群体的杂合性正相关。我们对将这一理论应用于病原体的适应状况特别感兴趣，因为它们是众所周知的克隆，至少1,500种所描述的致病原虫是二倍体，而且病原体在体内的LOH率似乎有所增加。在这里，我们提出了一种新的方法来测试杂合性是否加快了有丝分裂重组的适应速度，使用酵母-蜡虫(Saccharmyces cerevisiae-Galleria mellonella)致病模型。目的1将克隆进化生长在蜡虫幼虫体内的复制群体，这些群体起源于不同杂合度32倍的单亲基因。使用基于绿色荧光蛋白标记的酵母的荧光细胞分选，我们将能够在体内生长48小时后从感染的幼虫中提取纯酵母菌群，并且这个过程将重复100次连续转移。细胞分类还可以在每次转移时估计病原体的适合度，使我们能够测试适应速度是否与初始杂合性相关。目标2将使用下一代测序来对进化的品系进行基因分型，以在复制群体中识别平行的LOH事件，表明正选择的作用，并识别毒力基因。使用这种实验性的进化方法将使我们能够避免由于种群规模较小而导致的遗传漂移和突变积累的问题。酵母-盖氏杆菌感染模型的发展成为一个实验性的进化系统，将提供一种比标准反向遗传方法更强大的方法来绘制病原体基因、毒力和适合度之间的关系图。