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

基本信息

批准号：
8489735
负责人：
Tim James
金额：
$ 23.33万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-01-15 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8489735
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

项目摘要

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）：有丝分裂重组发生在所有二倍体生物中，但其进化意义在很大程度上被忽视。有丝分裂重组会导致杂合性丧失（LOH），使其具有适应性是违反直觉的。在大型多细胞生物体中，LOH 被认为主要是适应不良，并且经常与肿瘤发生相关。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，例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群体的研究进一步证实了这一假设，这些研究一致显示了多态性 LOH 基因组区域的证据。这些观察结果表明 LOH 普遍存在，但迫切需要解决 LOH 是否、在什么条件下以及通过什么机制成为二倍体病原体探索其适应环境的重要组成部分。我们的中心假设是，LOH 是正选择进化的重要组成部分，但 LOH 作为适应性力量的相对重要性将与初始基因型或群体的杂合性正相关。我们对将其应用于病原体的适应性景观特别感兴趣，因为众所周知，它们是克隆性的，至少 1,500 种描述的致病原虫物种是二倍体，并且病原体体内的 LOH 率似乎有所增加。在这里，我们提出了一种新方法，使用酵母蜡虫（酿酒酵母-大蜡螟）发病机制模型来测试杂合性是否加速有丝分裂重组的适应速度。目标 1 将克隆进化出在蜡虫幼虫体内生长的复制种群，这些种群是从杂合度范围超过 32 倍的单一亲本基因型开始的。使用基于绿色荧光蛋白标记酵母的荧光细胞分选，我们将能够在体内生长 48 小时后从受感染的幼虫中提取纯酵母群体，并且该过程将重复 100 次连续转移。细胞分选还可以在每次转移时估计病原体的适应性，从而使我们能够测试适应率是否与初始杂合性相关。目标 2 将使用下一代测序对进化品系进行基因分型，以识别重复群体中的平行 LOH 事件，这些事件表明正向选择的作用并识别毒力基因。使用这种实验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变和突变积累相关的问题。将酵母-Galleria感染模型发展成实验进化系统将提供一种比标准反向遗传方法更强大的方式来绘制病原体基因型、毒力和适应性之间关系的方法。