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）：有丝分裂的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略了。有丝分裂重组会导致杂合性丧失（LOH），使其可以适应性违反直觉。在大型多细胞生物中，LOH被认为主要是适应不良，并且经常与肿瘤发生有关。然而，在缺乏高遗传负荷（例如许多单细胞真菌和原生动物病原体）的自由生活单倍体阶段的生物体中，LOH可能是一个重要的机制，可以超速固定有益的隐性等位基因。通过对多态LOH基因组区域的证据的二倍体病原体的自然种群的研究来加强这种假设。这些观察结果表明LOH无处不在，但是在什么条件下，通过哪种机制来解决二倍体病原体如何探索其健身景观的重要组成部分。我们的中心假设是LOH是阳性选择进化的重要组成部分，但是LOH作为自适应力的相对重要性将与初始基因型或种群的杂合性正相关。我们特别有兴趣将其应用于病原体的适应性景观，因为它们是众所周知的克隆，至少有1,500个描述的病原性原生动物物种是二倍体，而病原体似乎在体内的LOH速率增加。在这里，我们提出了一种新方法，以测试杂合性是否使用酵母蜡蠕虫（酿酒酵母 - 加利利亚梅洛尼亚氏菌）的发病机理模型来促进有丝分裂重组的适应速率。 AIM 1将在蜡虫幼虫内生长的重复种群从单个父母基因型中引发的重复种群，这些基因型在32倍的杂合性范围内不同。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种群，并将重复100个连续传输的过程。细胞分选还可以在每次转移时估计病原体适应性，从而使我们能够测试适应率是否与初始杂合性相关。 AIM 2将使用下一代测序来基因型进化线，以识别复制人群之间的平行LOH事件，这些事件表明阳性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人口量较小而导致的遗传漂移和突变积累的问题。与标准反向遗传学方法相比，酵母 - 加利亚感染模型将酵母 - 加利亚感染模型以实验进化系统的形式提供了一种方法，以更强大的方式绘制病原体基因型，毒力和适应性之间的关系。