Enzymology of Chlamydia Pathogenesis

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1亿人感染。C.沙眼感染引起许多疾病相关的并发症，包括盆腔炎、异位妊娠、不孕症和失明。感染泌尿生殖道和结膜的单层柱状上皮细胞。沙眼持续存在于寄生虫包涵体空泡内。在感染过程中，C.沙眼衣原体释放许多效应分子到宿主细胞质中，干扰宿主信号传导，这有助于衣原体选择有益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）是一种酶，由于其在修饰参与脂质和膜转运、肌动蛋白细胞骨架、基于微管的马达、包涵体的溶酶体识别、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启动的宿主或细菌蛋白中的多变量作用，其已成为中心毒力因子。我们已经开发了第一个选择性和有效的细胞渗透性抑制剂CPAF，包括一个新发现的非肽小分子抑制剂，我们已经使用CPAF抑制剂，以建立一个关键的作用，这种酶作为一个抗菌剂和抗病力的目标，在细胞培养模型的人衣原体感染。CPAF活性的抑制导致细菌复制减少、包涵体液泡破坏和细菌细胞死亡。此外，CPAF抑制剂重编程感染的细胞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性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抑制剂可能形成一类新的小分子“抗微生物疫苗”的基础，不仅可以杀死细菌，而且可以促进适应性和获得性免疫的发展。由于衣原体属的耐遗传操作，我们缺乏适当的了解的身份CPAF目标和底物处理机制在体内，一个升值的范围内的参与CPAF衣原体发病机制，并了解的类型和规模的抗菌和免疫防御反应引发的CPAF抑制动物模型的感染。我们这个提案的长期目标是了解CPAF在C中的作用。本发明的目的在于研究沙眼衣原体的发病机制，以深入了解CPAF蛋白酶功能、抑制和靶标鉴定的分子水平细节，并确定CPAF抑制剂在衣原体生殖道感染的临床相关鼠动物模型中清除感染、拯救宿主免疫防御活性和防止再感染的程度。

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.