Molecular Genetic Analysis of Chlamydia Pathogenicity

性状（由申请人提供）：衣原体属细菌是动物和人类的重要病原体。包括肺炎、心内膜炎、多关节炎、失明和广泛性传播疾病，包括宫颈炎、输卵管炎、盆腔炎和女性不孕症，以及男性的非淋菌性关节炎和急性附睾炎。尽管多年的努力，衣原体仍然难以遗传分析，由于其专性细胞内的生活方式， 复杂的发展周期。我们的长期目标是应用包括遗传学力量在内的全方位分子工具来研究衣原体的致病机制和细胞内代谢。该应用程序的目标包括技术开发目标（遗传工具）和三个假设驱动的目标，以解决衣原体生物学的重大问题。具体目标是： 1 -衣原体基因分析工具的开发 2 -衣原体肽聚糖合成的生化和遗传特征 3 -衣原体生长必需成分摄取的运输系统的鉴定 4 -定义衣原体中的Ser/Thr/Tyr蛋白磷酸化网络 我们将采用新的和创新的方法，在我们过去的成功基础上，开发目标1中的遗传工具。成功实现这一目标将产生重大影响，并通过为衣原体的遗传分析提供新的工具来推进衣原体研究领域。目的2将确定参与两个关键的细胞质步骤肽聚糖（PG）合成的酶，并应用新技术来分离和证明衣原体PG组分的存在。我们还将筛选衣原体突变体缺陷的信号通过PG片段的主机作为解剖PG在主机响应中的作用的第一步。目标2的成功将最终解决“衣原体异常”，并揭示抗菌剂开发的潜在新靶点。目的3将描述衣原体从宿主细胞质中获取铁和生物素的转运系统。衣原体获得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文库。由于C.在沙眼衣原体中，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 阐明了营养获取的过程，这些过程是治疗干预的潜在目标。从更广泛的意义上说，这一目标的意义在于，它将提供洞察专性细胞内病原体的代谢过程。目标4是数据生成目标。通过定义衣原体磷酸化蛋白质组，它将揭示候选蛋白质来测试磷酸化对蛋白质活性的影响，并导致关于这些蛋白质如何调节衣原体发育周期的二态阶段之间的分化的新假设。

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 arthritis and acute epididymitis in males. Despite many years of effort, the Chlamydia remains 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 application include a technology development aim (genetic tools) and three 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 4 - Define the Ser/Thr/Tyr protein phosphorylation network in Chlamydia 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. Aim 4 is a data generating aim. By defining the Chlamydia phosphoproteome, it will reveal candidate proteins to test for the effect of phosphorylation on protein activity and lead to new hypotheses about how these proteins may regulate differentiation between the dimorphic stages of the Chlamydia developmental cycle.