Validating metabolic pathways in the intracellular pathogen Chlamydia trachomatis

验证细胞内病原体沙眼衣原体的代谢途径

• 批准号: 7483372
• 负责人: Derek James Fisher
• 金额: $ 4.68万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7483372
• 关键词: Antibiotics; Aromatic Compounds; Attenuated; Attenuated Live Virus Vaccine; Bacteria; Bacterial Infections; Bacterial Sexually Transmitted Diseases; Biochemical Genetics; Biological Assay; Blindness; Case Study; Chlamydia; Chlamydia Infections; Chlamydia trachomatis; Clinical Treatment; Development; Drug Delivery Systems; Drug resistance; Ectopic Pregnancy; Enzymes; Escherichia coli; Genes; Genome; Genomic Library; Growth; Homologous Gene; In Vitro; Infection; Infertility; Label; Lead; Life; Mammals; Mass Spectrum Analysis; Metabolic Pathway; Metabolism; Numbers; Pathway interactions; Public Health; Radio; Recombinant Proteins; Research; Resistance; Sexually Transmitted Diseases; Testing; Therapeutic; Treatment Failure; United States; Vaccines; Visually Impaired Persons; Woman; enzyme pathway; inhibitor/antagonist; knockout gene; male; mutant; pathogen; prevent; research study; shikimate; Antibiotics; Aromatic Compounds; Attenuated; Attenuated Live Virus Vaccine; Bacteria; Bacterial Infections; Bacterial Sexually Transmitted Diseases; Biochemical Genetics; Biological Assay; Blindness; Case Study; Chlamydia; Chlamydia Infections; Chlamydia trachomatis; Clinical Treatment; Development; Drug Delivery Systems; Drug resistance; Ectopic Pregnancy; Enzymes; Escherichia coli; Genes; Genome; Genomic Library; Growth; Homologous Gene; In Vitro; Infection; Infertility; Label; Lead; Life; Mammals; Mass Spectrum Analysis; Metabolic Pathway; Metabolism; Numbers; Pathway interactions; Public Health; Radio; Recombinant Proteins; Research; Resistance; Sexually Transmitted Diseases; Testing; Therapeutic; Treatment Failure; United States; Vaccines; Visually Impaired Persons; Woman; enzyme pathway; inhibitor/antagonist; knockout gene; male; mutant; pathogen; prevent; research study; shikimate

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.

描述（由申请人提供）：沙眼衣原体是可预防失明的主要原因（全球范围内约800万视障人士）和与细菌相关的性传播疾病（STD），仅在美国，每年都有900,000例报告的性病病例超过900,000例。虽然男性的衣原体性病感染通常是症状的，但女性通常有无症状的感染导致不孕或威胁生命的异位妊娠。临床治疗失败和对选择抗生素的体外耐药性的证明表明，对当前治疗剂的耐药性是可能的，这使得可以识别新药物靶标并导致疫苗对公共卫生至关重要的研究。细菌中必需的代谢途径是常见的药物靶标，代谢途径突变体也显示出有望为实时销售的疫苗菌株。 Shikimate途径（在本应用中称为依恋途径[CP]）会产生绒毛膜，这是必需芳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的酶已被用作抗生素发育的靶标，并且已构建了CP突变体作为实时衰减疫苗。虽然预计包含CP途径的基因存在于衣原体基因组中，但尚未在功能上进行验证，并且在沙眼梭状芽孢杆菌中不存在酸性利用基因同源物。 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）使用质谱法检测从沙眼梭状芽胞庭细菌提取物中检测绒毛膜； 3）确定如何使用无线电标签示踪实验和基因组库筛选来确定如何利用沙丘菌。成功完成此应用程序将填补我们对衣原体代谢的理解，确定新药靶向抑制衣原体生长的差异，并将确定基因敲除候选者以产生​​衰减菌株。衣原体是美国细菌感染的第一大原因，也是世界范围内失明和细菌性病的主要原因。该研究申请旨在确定治疗和预防衣原体感染的新方法，并阐明衣原体代谢的未知特征。