Enzymology of Chlamydial Pathogenesis

描述(由申请人提供)：尽管采取了积极的抗菌治疗方案，但全世界每年仍有超过1亿人感染了性传播的专性细胞内细菌病原体沙眼衣原体。在感染期间，沙眼衣原体将效应蛋白分泌到宿主细胞质中，干扰宿主信号网络，从而抑制宿主的免疫防御。不幸的是，在大多数男性和女性中，感染是无症状的，针对再次感染的长期保护性免疫的发展受到抑制，因此许多感染成为慢性并刺激继发性病理，如传染性失明(沙眼)、盆腔炎、宫外孕和不孕。丝氨酸蛋白酶、衣原体蛋白酶样活性因子(CPAF)在脂质和膜转运、肌动蛋白细胞骨架结构和动力学、微管驱动、溶酶体识别包涵体、ERK/MEK信号通路、细胞程序性死亡和炎症信号转导等方面具有重要作用，已成为衣原体的核心毒力因子。利用基于抑制剂的化学生物学方法，我们最近开发了两类CPAF抑制剂，观察到抑制CPAF活性抑制了细菌的复制，破坏了包含复制衣原体的寄生虫包涵体空泡的完整性，并导致细菌细胞死亡。我们还确定，CPAF抑制通过分泌促炎细胞因子和激活炎症体依赖的程序化宿主细胞死亡途径，对感染细胞重新编程，重新启动对感染的宿主免疫防御反应。基于这些集体数据，我们假设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，还可以增强对衣原体感染的适应性和获得性免疫反应。我们的目标是了解CPAF在沙眼衣原体致病机制中的作用，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。

DESCRIPTION (provided by applicant): Despite aggressive antimicrobial treatment regimens, over 100 million persons are infected annually worldwide with the sexually-transmitted obligate intracellular bacterial pathogen, Chlamydia trachomatis. During infection, C. trachomatis secretes effector proteins into the host cytosol that interfere with host signaling networks to dampen host immune defenses. Unfortunately infections are asymptomatic in the majority of men and women, development of long-term protective immunity against reinfection is inhibited, and therefore many infections become chronic and spur secondary pathologies like infectious blindness (trachoma), pelvic inflammatory disorder, ectopic pregnancy, and infertility. The serine protease chlamydial protease-like activity factor (CPAF) has emerged as a central chlamydial virulence factor due to its importance in lipid and membrane transport, actin cytoskeleton structure and dynamics, microtubule-based motors, lysosomal recognition of the inclusion, ERK/MEK signaling pathways, and the onset of programmed cell death and inflammation signaling. Using an inhibitor-based chemical biology approach, we recently developed two classes of CPAF inhibitors, and observed that inhibition of CPAF activity inhibited bacterial replication, destabilized the integrity of the parasitophorous inclusion vacuole housing replicating chlamydia, and resulted in bacterial cell death. We also determined that CPAF inhibition reprogrammed infected cells to re-initiate host immune defense responses to infection by secreting pro-inflammatory cytokines and activating an inflammasome-dependent programmed host cell death pathway. Based on these collective data, we hypothesize that CPAF inhibitors may form the basis for a new class of small molecule 'antimicrobial vaccines' that not only kill bacteria, but also may enhance adaptive and acquired immunity responses to chlamydial infections. Our 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 of 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.