Enzymology of Chlamydia Pathogenesis

衣原体发病机制的酶学

基本信息

批准号：
8707691
负责人：
Dewey G McCafferty
金额：
$ 28.2万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8707691
关键词：
Actins Acute Animal Model Animals Apoptosis Bacteria Bacterial Infections Bacterial Proteins Biological Factors Biology Blindness Caspase-1 Cell Culture Techniques Cell Death Cell model Cell physiology Cells Cellular Morphology Cessation of life Chemicals Chlamydia Chlamydia Infections Chlamydia trachomatis Cytokine Activation Cytoplasm Cytoskeleton Defect Detection Development Disease Drug Formulations Ectopic Pregnancy Effectiveness Endothelial Cells Enzymatic Biochemistry Enzymes Epithelial Cells Epitopes Figs - dietary Genital system Genitourinary system Goals Host Defense Human Immune Immunologic Markers In Vitro Infection Infection prevention Infertility Inflammation Inflammatory Intermediate Filaments Laboratories MEKs Mass Spectrum Analysis Mediating Methods Microtubules Modeling Molecular Motor Mus Pathogenesis Pathway interactions Pelvic Inflammatory Disease Peptide Hydrolases Peptide Library Persons Process Proteins Proteolysis Proteome Recombinants Research Project Grants Resistance Role S Phase Scanning Serine Protease Signal Pathway Signal Transduction Small Interfering RNA Solid Specificity Structure Transmembrane Transport Vaccinated Vaccines Vacuole Virulence Factors acquired immunity adaptive immunity analog antimicrobial base chemical genetics clinically relevant combat conjunctiva cytokine defense response design genetic manipulation in vivo inhibitor/antagonist insight killings lipid transport microbial multicatalytic endopeptidase complex novel overexpression pathogen prevent response restoration salinosporamide A scaffold small molecule tandem mass spectrometry tool

项目摘要

DESCRIPTION (provided by applicant): The obligate intracellular bacterium Chlamydia trachomatis is the most common sexually-transmitted pathogen in the developed world, >100 million persons are infected worldwide annually. C. trachomatis infections cause numerous disease-related complications including pelvic inflammatory disease, ectopic pregnancy, infertility, and blindness. Infecting the single columnar layer of epithelial cells of the urogenital tract and conjunctiva, C. trachomatis persists within parasitophorous inclusion vacuoles. During infection, C. trachomatis releases numerous effector molecules into the host cell cytoplasm to interfere with host signaling, which aids in chlamydial cooption of key host cell functions that benefit the infection. Of these effectors, the serine protease chlamydial protease-like activity factor (CPAF) is an enzyme that has emerged as a central virulence factor due to its multivariable roles in modifying host or bacterial proteins involved in lipid and membrane transport, the actin cytoskeleton, microtubule-based motors, lysosomal recognition of the inclusion, ERK/MEK signaling pathways, and the onset of programmed cell death and inflammation signaling. We have developed the first selective and potent cell permeable inhibitors for CPAF, including a newly discovered nonpeptidic small molecule inhibitor, and we have used CPAF inhibitors to establish a critical role for this enzyme as both an antimicrobial and antivirulence target in cell culture models of human chlamydial infections. Inhibition of CPAF activity leads to decreased bacterial replication, destruction of the inclusion vacuole, and bacterial cell death. In addition, CPAF inhibitors reprogrammed infected cells to initiate host immune defense responses such as secretion of pro-inflammatory cytokines and activation of an inflammasome-dependent programmed cell death pathway in the host cell. Based on these results, we hypothesize that CPAF inhibitors may form the basis for a new class of small molecule 'antimicrobial vaccines' that not only kill bacteria, but may promote the development of adaptive and acquired immunity. Since Chlamydiae sp. are resistant to genetic manipulation, we lack an appropriate understanding of the identity of CPAF targets and substrate processing mechanisms in vivo, an appreciation for the scope of the involvement of CPAF in chlamydial pathogenesis, and an understanding of the type and magnitude of antimicrobial and immune defensive responses triggered by CPAF inhibition in animal models of infection. Our long-term goals for this proposal are to understand the role CPAF in C. trachomatis pathogenesis, to gain insight into the molecular level details of CPAF protease function, inhibition and identification o targets, and to determine the extent to which CPAF inhibitors can clear infections, rescue host immune defense activity, and protect against reinfection in a clinically relevant murine animal model of chlamydial genital tract infection.

描述（由申请人提供）：专性细胞内细菌沙眼衣原体是发达国家最常见的性传播病原体，全世界每年有超过 1 亿人被感染。沙眼衣原体感染会导致许多与疾病相关的并发症，包括盆腔炎、宫外孕、不孕和失明。沙眼衣原体感染泌尿生殖道和结膜的单柱状上皮细胞层，在寄生包涵体液泡中持续存在。在感染过程中，沙眼衣原体释放大量效应分子到宿主细胞细胞质中以干扰宿主信号传导，这有助于衣原体共同选择有利于感染的关键宿主细胞功能。在这些效应子中，丝氨酸蛋白酶衣原体蛋白酶样活性因子（CPAF）是一种酶，由于其在修饰参与脂质和膜运输的宿主或细菌蛋白质、肌动蛋白细胞骨架、微管中的多变量作用，已成为中心毒力因子。基于马达、溶酶体识别内含物、ERK/MEK 信号传导途径以及程序性细胞死亡和炎症信号传导的发生。我们开发了第一个选择性且有效的 CPAF 细胞渗透性抑制剂，包括新发现的非肽类小分子抑制剂，并且我们使用 CPAF 抑制剂确立了这种酶作为人类细胞培养模型中抗菌和抗毒靶点的关键作用衣原体感染。抑制 CPAF 活性会导致细菌复制减少、包涵液泡破坏和细菌细胞死亡。此外，CPAF抑制剂对受感染的细胞进行重新编程，以启动宿主免疫防御反应，例如促炎细胞因子的分泌和宿主细胞中炎症小体依赖性程序性细胞死亡途径的激活。基于这些结果，我们假设 CPAF 抑制剂可能构成一类新型小分子“抗菌疫苗”的基础，这种疫苗不仅可以杀死细菌，还可以促进适应性和获得性免疫的发展。由于衣原体 sp。我们对 CPAF 靶标的身份和体内底物加工机制缺乏适当的了解，对 CPAF 在衣原体发病机制中的参与范围缺乏认识，对抗菌和免疫的类型和程度缺乏了解感染动物模型中 CPAF 抑制引发的防御反应。我们本提案的长期目标是了解 CPAF 在沙眼衣原体发病机制中的作用，深入了解 CPAF 蛋白酶功能、抑制和靶点识别的分子水平细节，并确定 CPAF 抑制剂清除的程度在衣原体生殖道感染的临床相关小鼠动物模型中，挽救宿主免疫防御活性并防止再次感染。