Enzymology of Chlamydia Pathogenesis

衣原体发病机制的酶学

• 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
• 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

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(申请人提供)：专性细胞内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1亿人感染。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、异位妊娠、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，持续存在于寄生虫包涵体空泡内。在感染期间，沙眼衣原体向宿主细胞胞质释放大量的效应分子来干扰宿主信号，从而帮助衣原体选择有利于感染的关键宿主细胞功能。在这些效应物中，丝氨酸蛋白酶、衣原体蛋白酶样活性因子(CPAF)是一种重要的毒力因子，因为它在调节宿主或细菌的脂质和膜运输蛋白、肌动蛋白细胞骨架、基于微管的马达、溶酶体对包涵体的识别、ERK/MEK信号通路以及启动细胞程序性死亡和炎症信号等方面发挥着重要的作用。我们已经开发了第一个选择性和有效的CPAF细胞通透性抑制剂，包括一个新发现的非肽类小分子抑制剂，我们已经使用CPAF抑制剂来确定这种酶在人类衣原体感染的细胞培养模型中作为抗菌和抗毒力靶点的关键作用。抑制CPAF活性会导致细菌复制减少，包涵体空泡破坏，细菌细胞死亡。此外，CPAF抑制剂对感染细胞重新编程，以启动宿主免疫防御反应，如分泌促炎细胞因子和激活宿主细胞中依赖炎症体的程序性细胞死亡途径。基于这些结果，我们推测CPAF抑制剂可能形成一类新的小分子抗菌疫苗的基础，这种疫苗不仅可以杀死细菌，而且可以促进获得性免疫和获得性免疫的发展。自衣原体属(Chlamydiae sp.)在对基因操作具有抵抗力的情况下，我们缺乏对CPAF靶标和体内底物处理机制的适当了解，缺乏对CPAF参与衣原体致病的范围的认识，也缺乏对CPAF抑制在动物感染模型中触发的抗微生物和免疫防御反应的类型和规模的了解。我们这项建议的长期目标是了解CPAF在沙眼衣原体发病机制中的作用，深入了解CPAF蛋白酶功能的分子水平细节，抑制和识别靶点，并确定CPAF抑制剂在多大程度上可以清除感染，挽救宿主免疫防御活性，并在临床相关的衣原体生殖道感染的小鼠动物模型中预防再次感染。