Molecular Genetic Analysis of Chlamydia Pathogenicity

描述（由申请人提供）：衣原体属细菌是动物和人类的重要病原体。衣原体在男性中引起的疾病包括肺炎、心内膜炎、多发性关节炎、失明，以及多种性传播疾病，包括女性的宫颈炎、输卵管炎、盆腔炎和不育症，以及男性的非淋球菌性尿道炎和急性附睾炎。尽管经过多年的努力，衣原体由于其固有的细胞内生活方式和复杂的发育周期，仍然难以进行遗传分析。我们的长期目标是应用包括遗传学在内的各种分子工具来研究衣原体的致病机制和细胞内代谢。该提案的目标包括一个技术开发目标（遗传工具）和两个假设驱动的目标，以解决衣原体生物学的重要问题。具体目标是：1 -衣原体遗传分析工具的开发。2 -衣原体肽聚糖合成的生化和遗传表征。3 -衣原体生长必需成分摄取的运输系统的鉴定。我们将采用新的和创新的方法，以我们过去的成功为基础，开发目标1的遗传工具。成功实现这一目标将产生重大影响，并通过提供用于衣原体遗传分析的新工具来推进衣原体研究领域。目的2将确定参与肽聚糖（PG）合成的两个关键细胞质步骤的酶，并应用新技术分离和证明衣原体中PG成分的存在。我们还将筛选通过PG片段向宿主发送信号有缺陷的衣原体突变体，作为剖析PG在宿主反应中的作用的第一步。aim 2的成功将最终解决“衣原体异常”问题，并揭示抗菌药物开发的潜在新靶点。目的3将描述衣原体从宿主细胞质中获取铁和生物素的运输系统。衣原体获取铁的机制是完全未知的，我们的创新方法将使用志贺氏菌突变体来筛选衣原体库。由于沙眼衣原体缺乏合成生物素的基因，这种必需维生素的运输对生长至关重要。在这两种情况下，这一目标的成功将阐明作为治疗干预潜在目标的营养获取过程。从更广泛的角度来看，

DESCRIPTION (provided by applicant): Bacteria of the genus Chlamydia are significant pathogens of animals and man. The diseases caused by Chlamydia spp. in man include pneumonia, endocarditis, polyarthritis, blindness, and a wide range of sexually transmitted diseases including cervicitis, salpingitis, pelvic inflammatory disease, and infertility in females and non-gonococcal urethritis and acute epididymitis in males. Despite many years of effort, the Chlamydia remain intractable to genetic analysis due to their obligate intracellular lifestyle and complex developmental cycle. Our long-term goal is to apply the full range of molecular tools including the power of genetics to study the pathogenic mechanisms and intracellular metabolism of Chlamydia. The aims of this proposal include a technology development aim (genetic tools) and two hypothesis-driven aims to address significant questions of Chlamydia biology. The specific aims are: 1 - Development of tools for the genetic analysis of Chlamydia. 2 - Biochemical and genetic characterization of peptidoglycan synthesis in Chlamydia. 3 - Identification of transport systems for uptake of essential constituents for Chlamydia growth. We will employ new and innovative approaches to build on our past success and develop genetic tools in aim 1. Success in achieving this aim will have a significant impact and advance the field of Chlamydia research by making new tools available for genetic analysis of Chlamydia. Aim 2 will identify the enzymes involved in two key cytoplasmic steps in peptidoglycan (PG) synthesis and also apply new techniques to isolate and demonstrate the presence of PG components in Chlamydia. We will also screen for Chlamydia mutants defective in signaling to the host via PG fragments as a first step in dissecting the role of PG in host response. Success in aim 2 will finally resolve the "Chlamydia anomaly" and reveal potential new targets for antimicrobial development. Aim 3 will characterize transport systems used by Chlamydia to acquire iron and biotin from the host cytosol. Mechanisms of iron acquisition by Chlamydia are completely unknown and our innovative approach will use a Shigella mutant to screen a Chlamydia library. Since the genes for biotin synthesis are absent in C. trachomatis, transport of this essential vitamin is critical for growth. In both cases, success in this aim will shed light on processes of nutrient acquisition that are potential targets for therapeutic intervention. In broader terms, the significance of this aim is that it will provide insight into metabolic processes of obligate intracellular pathogens.