Analysis of P. aeruginosa genome diversity and evolution

描述（由申请人提供）：铜绿假单胞菌是水和土壤中的常见居民，也是一系列严重人类感染的原因，包括囊性纤维化（CF）患者的慢性呼吸道疾病。本提案的目标是应用分子工具来定义各种铜绿假单胞菌分离株基因组内的遗传变异。该项目将测试一个假设，即与大多数铜绿假单胞菌菌株不同，能够定植CF患者的菌株的灵活基因库由水平获得的基因内编码的特定基因库组成，通常存在于基因组岛中。此外，在感染的慢性阶段，这些菌株在铜绿假单胞菌基因组中积累了病理适应性突变，这使得细菌在呼吸道中持续多年。首先，使用DNA微阵列，通过PCR分析DNA的可变片段，应用靶向DNA捕获方法和差异杂交策略，我们将鉴定环境和临床菌株中不属于核心基因组的基因，因此可能存在于基因组岛中。将构建完全由来自该灵活基因库的基因组成的DNA微阵列，并将其用于进一步分析大量CF分离株的基因组变化，包括来自个体CF患者的早期和晚期分离株的已建立谱系。该阵列还将用于生成CF特异性岛内的体内表达基因的转录组，其将用于指导相关基因的后续优先级排序。在第二个目标中，将删除（i）存在于绝大多数CF分离株中和（ii）在感染动物模型中表达的那些含有基因组岛的基因，或者将诱变单个基因。将在两种呼吸道感染动物模型中评估这些突变对铜绿假单胞菌毒力的影响。在第三个目标中，还将在从个体CF患者分离的铜绿假单胞菌克隆中监测基因组中点突变的出现。这些突变可能是铜绿假单胞菌引起CF呼吸道慢性持久感染所必需的。这一假设将在大鼠慢性感染模型中进行测试，其中突变的积累将与人类感染中观察到的突变进行比较。本申请中提出的研究结果应该为非常成功的机会致病菌的进化提供新的见解，并阐明在慢性呼吸道感染期间发挥作用的一些意想不到的毒力机制。此外，这项工作的发现将大大补充研究界目前可用的基因组资源，特别是那些使用不同铜绿假单胞菌分离株的研究人员。基于完整或部分完整的基因组序列以及水平获得的岛屿序列的“虚拟基因组”的可用性，应导致对菌株特异性和核心毒力决定因素之间相互作用的更好理解。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa, a common inhabitant of water and soil, is also responsible for a range of serious human infections including the chronic respiratory disease of patients with cystic fibrosis (CF). The goal of this proposal is to apply molecular tools to define genetic variations within the genomes of various P. aeruginosa isolates. The project will test the hypothesis that unlike most strains of P. aeruginosa, the flexible gene pool of strains that are capable of colonizing patients with CF, consists of a specific repertoire of genes encoded within horizontally-acquired genes, often present in genomic islands. Moreover, during the chronic phase of infection these strains accumulate pathoadaptive mutations in the P. aeruginosa genome, which allow the bacteria to persist for many years in the respiratory tract. First, using DNA microarrays, analyzing variable segments of DNA by PCR, applying a targeted DNA capture method, and a differential hybridization strategy, we will identify genes in environmental and clinical strains which are not part of the core gene set and therefore may reside in genomic islands. A DNA microarray will be constructed which consist entirely of the genes from this flexible gene pool and it will be used to further analyze the changes in the genomes of a large set of CF isolates, including established lineages of early and late isolates from individual CF patients. The array will be also used to generate a transcriptome of in vivo expressed genes within the CF-specific islands, which will be used to guide the subsequent prioritization of relevant genes. In the second aim, those genomic island-containing genes that are (i) present in great majority of CF isolates, and (ii) expressed in animal models of infection, will be deleted or individual genes will be mutagenized. The effect of these mutations on the virulence of P. aeruginosa will be assessed in two animal models of respiratory infection. In the third aim, the appearance of point mutations in the genome will be also monitored in the clones of P. aeruginosa isolated over several years from individual CF patients. These mutations may be a required for P. aeruginosa to cause a chronic, long-lasting infection in the CF respiratory tract. This hypothesis will be tested in a rat chronic infection model, where accumulation of mutations will be compared to those seen in human infections. The results of the studies proposed in this application should provide new insights into the evolution of a highly successful opportunistic pathogen and shed light on some unexpected virulence mechanisms that function during chronic respiratory tract infections. Moreover, findings from this work should greatly supplement the currently available genomic resources for the research community, particularly those who work with different P. aeruginosa isolates. The availability of a "virtual genome", based on complete or partially-completed genome sequences as well as sequences of horizontally-acquired islands, should result in an improved understanding of the interplay between strain specific and core virulence determinants.