沙眼衣原体感染过程中的免疫识别和逃逸机理研究

Chlamydia trachomatis infection is a common cause of sexually transmitted disease, leading to male urethritis, female pelvic inflammatory disease (PID), epitopic pregnancy, and infertility. Chlamydia STD has been considered less significant in China, but a recent population based survey has identified Chlamydia STD as a "hidden epidemic" in China. Accordingly, the Ministry of Health lists Chlamydia infection for STD surveillance starting from the beginning of 2013, indicating the severity of the disease. Chlamydia sp. is an obligate intracellular bacterial pathogen that infects mucosal epithelia of the eye and genital tract. A hallmark of chlamydial STD is the persistent and asymptomatic nature of the infection. Up to 70% individuals are unaware of an infection. As an obligate intracellular bacterium, Chlamydia resides in membrane bound vacuoles (also known as inclusions), shielding the organism from host antimicrobial response. The host utilizes pattern-recognition receptors (PRRs) to recognize pathogen associated molecular patterns (PAMPs) for innate immune response. The systems for chlamydial PAMPs recognition as well as the PAMPs remain not well investigated. The bacterium also produces and secretes proteases that target the host proteins of immune response. The chlamydial protease activity factor (CPAF) degrades transcriptional factor RFX5 and upstream stimulation factor-1 of MHC I and MHC II expression. We have recently showed that a tail-specific protease (CT441) selectively cleaves p65 of NF-κB pathway, potentially to down regulate host proinflammatory response. In preliminary studies, we found that Chlamydia trachomatis infection causes STING degradation and promotes MAVS cleavage. In vitro assays showed that the protease activity was associated with Chlamydia, not a host factor. MAVS, also known as VISA, CARDIF and ISP-1, is an adaptor protein that mediates RIG-I pathway activity and host antimicrobial response. We hypothesize that cleavage of MAVS is responsible for Chlamydia evasion of interferon mediated anti-chlamydial response. Two specific AIMS are designed to address host recognition of intracellular Chlamydia and the mechanisms that Chlamydia applies to evade host antimicrobial immunity. Specific AIM 1: characterizations of Chlamydia produced cyclic dinucleotides as innate immune stimulator. Specific AIM 2: Characterization of chlamydial protease that cleaves MAVS. The study of host-pathogen interaction has made significant progress during the past two decades. Most of the progress and breakthrough discoveries are devised from studies of viral pathogens and several important bacterial pathogens. Chlamydia trachomatis is one of the most common pathogens of human diseases. There is very limited knowledge regarding the biology and host antimicrobial response to obligate intracellular bacteria. Mechanistic studies of Chlamydia immune evasion of host cells will uncover novel pathways and mechanisms.

沙眼衣原体（CT）是引起性病的最常见病原体，2013年起卫生部将其与梅毒、HIV等列入重点监控性病。CT是严格胞内寄生性细菌，感染具有隐蔽性和持续性，但机理不清。前期工作鉴定了CT切断NF-κB/p65的蛋白酶，近期发现CT具有作用于干扰素调控通路的能力，包括将MAVS蛋白切割成50和20 kDa片段（不是降解）。课题拟（1）鉴定CT切割MAVS蛋白的蛋白酶（2）研究感染过程的天然免疫识别。内容包括用蛋白质化学和分子生物学方法鉴定CT蛋白酶，确定酶活性中心以及底物切割位点；研究其在感染过程中的表达、释放和再分布等，诱导MAVS切割的条件；用基因敲除以及过表达MAVS片段研究MAVS切割与CT抗凋亡和持续感染的关系；用基因敲除小鼠模型验证MAVS缺失与持续和潜伏感染的关系等。课题实施可以揭示CT致病机理、丰富对严格胞内细菌的免疫识别和逃逸的理论知识，为治疗提供思路。

沙眼衣原体（CT）是严格胞内寄生性细菌，感染具有隐蔽性和持续性，但机理不清。前期工作发现干扰NF-κB的蛋白酶，该项目主要研究影响干扰素通路蛋白酶，目标包括（1）鉴定CT 切割MAVS 蛋白的蛋白酶（2）研究感染过程的天然免疫识别。主要成果包括1）鉴定衣原体蛋白酶特点及切割底物位点都已经在体外及重组蛋白层面验证/证实，该工作由王晶晶博士论文阐述；后期工作利用其底物切割位点制备了荧光探针，希望能够用于检测。该部分工作有一个挑战性问题，即衣原体为严格胞内细菌，蛋白酶在感染状态的释放、如何穿过由双层脂质组成的包涵体膜而作用于胞浆蛋白是本课题的疑难点，我们正在利用该荧光探针，在活细胞检测蛋白切割的特点及意义。收尾工作正在进行中，希望能产生高水平研究论文。2）感染过程中小分子代谢与宿主免疫应答。项目执行期间，我们利用组学进展，开展了点写组学研究，鉴定了两个特异通路，其中鞘脂代谢与衣原体包涵体膜融合以及衣原体成熟密切相关。利用小分子化合物为探针，证明抑制鞘脂从头合成过程可以阻断包涵体融合，阻滞衣原体于非感染性 RB 阶段。3）从核苷酸代谢研究宿主抗微生物应答。衣原体基因组编码一个环二核苷酸合成酶，证明其具有催化 ATP 环合至环二腺苷酸的能力，虽然环二核苷酸具有激活干扰素通路的能力，但是衣原体感染合成的环二核苷酸在时间点上严重滞后干扰素通路激活，表明宿主识别其它模式分子。有关工作在衣原体国际和国内会议交流。4）正在进行的工作：结合人沙眼衣原体工作进展，我们2017年申报了“科技支宁（夏）”计划，并且得到项目资助，拟将基础研究成果用于牛羊流产衣原体致病机理和分布特点研究，为西部开发出力。在中国与工业发达国家一样，沙眼衣原体感染形势严峻，是女性不孕的主要病原体。但是严格胞内细菌研究在国内是小众领域，整体来讲缺少人才和资源。项目执行期间培养研究衣原体的博士研究生3名（一名在读）、硕士研究生3名，希望他们能继续相关领域研究。另外，发表研究论文6篇(总 IF>25），3篇投稿中。