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性失明（全世界约有800万视力受损者）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90万性病病例报告。男性衣原体性传播感染通常是有症状的，而女性通常是无症状感染，导致不孕或危及生命的异位妊娠。临床治疗失败和对某些抗生素的体外耐药性表明，对当前治疗方法的耐药性是可能的，这使得既可以确定新的药物靶点又可以开发疫苗的研究对公共卫生至关重要。细菌的基本代谢途径是常见的药物靶点，代谢途径突变体也显示出作为减毒活疫苗菌株的希望。莽草酸途径（在本应用中称为chorismate途径[CP]）产生chorismate，这是必需的芳香族化合物的从头合成所必需的。由于CP存在于细菌中，但在哺乳动物中不存在，因此CP酶已被用作抗生素开发的靶点，CP突变体已被构建用于减毒活疫苗。虽然预计构成CP途径的基因存在于衣原体基因组中，但它们尚未得到功能验证，并且沙眼衣原体中没有利用chorisate的基因同源物。随后，本申请将测试沙眼衣原体使用CP生产用于芳香族化合物合成的choris酸盐的假设：1)使用遗传和生化方法对关键途径酶（AroG， AroDE和AroA）进行功能分析，包括大肠杆菌零突变体的互补研究，使用纯化重组蛋白的酶分析和抑制剂分析；2)利用质谱法检测沙眼衣原体细菌提取物中的choris酸；3)利用放射性标记追踪实验和基因组文库筛选确定沙眼衣原体如何利用choris酸盐。该应用程序的成功完成将填补我们对衣原体代谢的理解空白，确定抑制衣原体生长的新药物靶点，并将确定基因敲除候选物以创建减毒菌株。衣原体是美国细菌感染的头号原因，也是全球致盲和细菌性性病的主要原因。本研究应用程序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.