Validating metabolic pathways in the intracellular pathogen Chlamydia trachomatis

描述（由申请人提供）：沙眼衣原体是可预防失明（全球约800万视力受损人群）和细菌相关性传播疾病（STD）的主要原因，仅在美国每年就有超过90万例STD报告病例。虽然男性的衣原体性传播疾病感染通常有症状，但女性通常无症状感染，导致不孕或危及生命的异位妊娠。临床治疗失败和对选定抗生素的体外耐药性的证明表明，对当前疗法的耐药性是可能的，这使得研究既可以确定新的药物靶点，也可以导致对公共卫生至关重要的疫苗。细菌中的必需代谢途径是常见的药物靶标，并且代谢途径突变体也显示出作为减毒活疫苗株的前景。莽草酸途径（在本申请中称为分支酸途径[CP]）产生分支酸，其是必需芳香族化合物的从头合成所需的。由于CP存在于细菌中，但不存在于哺乳动物中，CP的酶已被用作抗生素开发的靶标，并且CP突变体已被构建用作减毒活疫苗。虽然预测CP途径的基因存在于衣原体基因组中，但它们的功能尚未得到验证，并且在衣原体中不存在利用分支产物的基因同源物。沙眼随后，本申请将测试假设，C。沙眼衣原体使用CP产生用于芳香族化合物合成的分支酸，通过：1）使用遗传和生物化学方法功能性分析关键途径酶（AroG、AroDE和AroA），包括大肠杆菌无效突变体中的互补研究、使用纯化的重组蛋白的酶测定和抑制剂测定; 2）检测来自沙眼衣原体的分支酸。沙眼衣原体细菌提取物;和，3）确定分支酸盐如何被C.使用放射性标记追踪实验和基因组文库筛选检测沙眼。这项申请的成功完成将填补我们对衣原体代谢的理解的空白，确定新的药物靶点以抑制衣原体生长，并将确定基因敲除候选人以创建减毒菌株。衣原体是美国细菌感染的头号原因，也是全球失明和细菌性性病的主要原因。这项研究申请旨在确定治疗和预防衣原体感染的新方法，以及阐明衣原体代谢的未知特征。

DESCRIPTION (provided by applicant): Chlamydia trachomatis is the leading cause of preventable blindness (~8 million visually impaired people worldwide) and bacteria-associated sexually transmitted diseases (STD) with over 900,000 reported cases of STD occurring each year in the United States alone. While chlamydial STD infections in males are typically symptomatic, women often have asymptomatic infections resulting in infertility or life threatening ectopic pregnancies. Clinical treatment failure and demonstration of in vitro resistance to select antibiotics suggests that drug resistance to current therapeutics is possible, making research that could both identify new drug targets and lead to vaccines crucial to public health. Essential metabolic pathways in bacteria are common drug targets and metabolic pathway mutants also have shown promise as live-attenuated vaccine strains. The shikimate pathway (referred to in this application as the chorismate pathway [CP]) produces chorismate, which is required for the de novo synthesis of essential aromatic compounds. Because the CP is present in bacteria, but absent in mammals, enzymes of the CP have been used as targets for antibiotic development and CP mutants have been constructed for use as live-attenuated vaccines. While the genes comprising the CP pathway are predicted to be present in the Chlamydia genomes, they have yet to be functionally validated and chorismate-utilizing gene homologs are absent in C. trachomatis. Subsequently, this application will test the hypothesis that C. trachomatis uses the CP to produce chorismate for aromatic compound synthesis by: 1) functionally analyzing key pathway enzymes (AroG, AroDE, and AroA) using genetic and biochemical approaches, including complementation studies in Escherichia coli null mutants, enzyme assays using purified recombinant proteins, and inhibitor assays; 2) detecting chorismate from C. trachomatis bacterial extracts using mass spectroscopy; and, 3) determining how chorismate is utilized by C. trachomatis using radio-label tracing experiments and genome library screens. Successful completion of this application will fill in gaps in our understanding of chlamydial metabolism, identify new drug targets to inhibit chlamydial growth, and will identify gene knockout candidates to create attenuated strains. Chlamydia is the number one cause of bacterial infections in the United States and the leading cause of blindness and bacterial STD worldwide. This research application seeks to identify new ways to treat and prevent chlamydial infections, as well as to elucidate unknown features of the chlamydial metabolism.