Analysis of P. aeruginosa genome diversity and evolution

描述(申请人提供)：铜绿假单胞菌，一种常见的水和土壤居民，也是一系列严重的人类感染的罪魁祸首，包括囊性纤维化(CF)患者的慢性呼吸道疾病。这项建议的目标是应用分子工具来定义不同铜绿假单胞菌分离株基因组内的遗传变异。该项目将检验这样一个假设，即与大多数铜绿假单胞菌菌株不同，能够定植CF患者的灵活的菌株基因库由一组特定的基因组成，这些基因编码在水平获得的基因中，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，这使得细菌能够在呼吸道中持续多年。首先，使用DNA微阵列，通过聚合酶链式反应分析DNA的可变片段，应用靶向DNA捕获方法和差异杂交策略，我们将在环境和临床菌株中识别不属于核心基因集的基因，因此可能存在于基因组岛上。将构建一个DNA微阵列，它完全由这个灵活的基因库中的基因组成，并将被用于进一步分析一大组CF分离株的基因组变化，包括已建立的来自单个CF患者的早期和晚期分离株的谱系。该阵列还将用于生成在体内表达的CF特异性岛基因的转录组，该转录组将用于指导后续相关基因的优先顺序。在第二个目标中，那些包含基因组岛的基因(I)存在于绝大多数CF分离株中，并且(Ii)在动物感染模型中表达，将被删除或个别基因被突变。这些突变对铜绿假单胞菌毒力的影响将在两种呼吸道感染动物模型中进行评估。在第三个目标中，还将监测几年来从单个CF患者中分离的铜绿假单胞菌克隆中基因组中点突变的出现。这些突变可能是铜绿假单胞菌在CF呼吸道引起慢性、持久感染所必需的。这一假设将在大鼠慢性感染模型中得到验证，在该模型中，突变的累积将与在人类感染中看到的突变进行比较。本申请中提出的研究结果将为高度成功的机会性病原体的进化提供新的见解，并阐明一些在慢性呼吸道感染中发挥作用的意想不到的毒力机制。此外，这项工作的发现将极大地补充研究界目前可用的基因组资源，特别是那些与不同的铜绿假单胞菌分离株合作的人。基于完整或部分完成的基因组序列以及水平获得的岛的序列的“虚拟基因组”的可获得性应有助于更好地理解毒株特异性决定因素和核心毒力决定因素之间的相互作用。

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.