Molecular Genetic Analysis of Chlamydia Pathogenicity

描述(申请人提供)：衣原体属细菌是动物和人类的重要病原体。衣原体引起的疾病。男性包括肺炎、心内膜炎、多发性关节炎、失明和广泛的性传播疾病，女性包括宫颈癌、输卵管炎、盆腔炎和不孕症，男性包括非淋菌性尿道炎和急性附睾炎。尽管多年来的努力，衣原体仍然难以进行遗传分析，因为他们专属的细胞内生活方式和复杂的发育周期。我们的长期目标是应用全方位的分子工具，包括遗传学的力量来研究衣原体的致病机制和细胞内代谢。该提案的目的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大问题。具体目标是：1-开发衣原体基因分析工具。衣原体合成肽聚糖的生化和遗传学特征。3-识别吸收衣原体生长必需成分的运输系统。我们将采用新的和创新的方法，在过去成功的基础上，在AIM 1中开发基因工具。成功实现这一目标将产生重大影响，并通过提供新的衣原体基因分析工具，推动衣原体研究领域的发展。目的2将鉴定参与合成肽聚糖(PG)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组分的存在。我们还将筛选在通过PG片段向宿主发送信号方面存在缺陷的衣原体突变株，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”，并揭示抗菌药物开发的潜在新目标。目标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完全未知，我们的创新方法将使用志贺氏菌突变株来筛选衣原体文库。由于沙眼衣原体缺乏生物素合成基因，这种必需维生素的运输对其生长至关重要。在这两种情况下，这一目标的成功将揭示营养获取过程，这些过程是治疗干预的潜在目标。从更广泛的角度来看， 这一目标的意义在于，它将提供对专性细胞内病原体代谢过程的洞察。

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.