Network Analysis of Pathogenicity in Pseudomonas aeruginosa

描述（由申请人提供）：高通量技术正在产生关于人类病原体细胞功能的大量数据。然而，尽管对数百种病原体进行了基因组测序，基因组学方法在确定可行的药物靶点方面取得了有限的成功。一个关键的障碍是，测序分析工作确定的目标没有考虑到细胞内的相互作用网络；例如，考虑到系统中通路的冗余性，抑制一种蛋白质的功能可能没有效果。我们建议重建和验证铜绿假单胞菌的代谢和调控网络，特别关注先前发现的已知对其毒力至关重要的突变体。为了在这种人类病原体的细胞内网络背景下制定治疗策略，迫切需要一个定量框架，该框架可用于背景化高通量数据，生成表型预测，并提出有关其生理学的可测试假设。具体来说，我们提出的目标是：(1)重建铜绿假单胞菌的代谢和调控网络，以解释1500个基因的功能，这将导致迄今为止最大的病原体网络重建；(2)在最小培养基条件下，对先前在标记诱变筛选中鉴定出的无毒铜绿假单基因突变体的代谢表型进行表征；(3)分析P. aeruginosa和无毒突变菌株在囊性纤维化特异性培养基中的代谢表型，以制定基于条件必需基因的可能治疗策略。该计划的结果将是一个具有良好特征的、经过良好验证的铜绿假单胞菌模型，该模型可用于系统地识别药物靶点和其致病性的关键特征，以及stm鉴定的突变体的关键代谢表型（例如，生长速率、副产物分泌物）的描述，这些突变体可用于潜在的治疗策略开发。拟议的计划将导致迄今为止最全面的重建具有重大疾病负担的病原体。

DESCRIPTION (provided by applicant): High-throughput technologies are generating a tremendous amount of data about cellular functions of human pathogens. However, despite the genome sequencing of hundreds of pathogens, the genomics approach has had limited success at identifying viable drug targets. One key hurdle has been that targets identified by sequencing analysis efforts do not take into account the network of interactions inside the cell; for instance, inhibiting the function of one protein may have no effect given the redundancy of pathways in the system. We propose to reconstruct and validate the metabolic and regulatory networks of Pseudomonas aeruginosa, with particular attention to previously identified mutants known to be critical for its virulence. In order to develop therapeutic strategies in the context of the intracellular networks in this human pathogen, there exists a significant need for a quantitative framework that can be used to contextualize high-throughput data, generate phenotypic predictions, and propose testable hypotheses regarding its physiology. Specifically, our proposed aims are to: (1) Reconstruct the metabolic and regulatory networks of P. aeruginosa to account for the function of 1500 genes, which will result in the largest network reconstruction of a pathogen to date; (2) Characterize the metabolic phenotypes under minimal media conditions of avirulent P. aeruginosa single-gene mutants identified previously in a signature-tagged mutagenesis screen; and (3) Analyze metabolic phenotypes of P. aeruginosa and avirulent mutant strains in cystic fibrosis-specific medium to develop possible therapeutic strategies based on conditionally essential genes. The outcome of this proposed program will be a well-characterized, well-validated model of P. aeruginosa that can be used for systematically identifying drug targets and key features of its pathogenicity, as well as a delineation of the key metabolic phenotypes (e.g., growth rate, byproduct secretions) of the STM-identified mutants that can be used potentially for the development of therapeutic strategies. The proposed program will lead to the most comprehensive reconstruction to date of a pathogen with a significant disease burden. PUBLIC HEALTH RELEVANCE: The proposed program will lead to the most comprehensive reconstruction to date of a human pathogen. Aside from its significant disease burden in hospital-acquired infections, Pseudomonas aeruginosa poses a significant problem in burn patients, individuals with cystic fibrosis, cancer patients on chemotherapy regimens, and other immuno-compromised individuals. Furthermore, drug resistance is already an important problem in P. aeruginosa infections and will certainly be an even greater challenge in the future. The outcome of this proposed program will be a well-characterized, well-validated model of P. aeruginosa that can be used for systematically identifying drug targets and key features of its pathogenicity.