Validating metabolic pathways in the intracellular pathogen Chlamydia trachomatis

描述(申请人提供)：沙眼衣原体是可预防的失明(全世界约800万视障人士)和与细菌相关的性传播疾病(STD)的主要原因，仅在美国每年就有超过90万例报告的STD病例。虽然男性的衣原体性病感染通常是有症状的，但女性通常会有无症状的感染，导致不孕不育或危及生命的宫外孕。临床治疗失败和对选定抗生素的体外耐药性表明，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，又可以导致对公共健康至关重要的疫苗。细菌中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，这是必需的芳香族化合物从头合成所必需的。由于CP存在于细菌中，而不存在于哺乳动物中，因此CP的酶已被用作抗生素开发的靶点，并且CP突变体已被构建用于活疫苗。虽然组成CP途径的基因被预测存在于衣原体基因组中，但它们的功能尚未得到验证，沙眼衣原体中缺乏利用分支酸的基因同源物。随后，这一应用将检验沙眼衣原体利用CP产生分支酸用于芳香化合物合成的假设，方法包括：1)使用遗传和生化方法对关键途径酶(Arog、AroDE和ARRA)进行功能分析，包括在大肠杆菌零突变体中的互补性研究、使用纯化的重组蛋白进行酶分析和抑制剂分析；2)使用质谱学检测沙眼衣原体细菌提取物中的分支酸；以及3)使用放射性标记跟踪实验和基因组文库筛选来确定沙眼衣原体如何利用分支酸。这项申请的成功完成将填补我们对衣原体新陈代谢的了解空白，识别抑制衣原体生长的新药物靶点，并将识别候选基因敲除以创造减毒株。衣原体是美国细菌感染的头号原因，也是全世界失明和细菌性STD的主要原因。这项研究应用寻求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.