半胱氨酸脱硫酶样蛋白MG_336在生殖支原体致病作用中的机制探索

Mycoplasma genitalium is a sexually transmissible pathogen that can cause a range of urogenital tract infections. The established mechanisms of M. genitalium pathogenesis have yet to fully elucidate its pathogenic ability and persistent infection phenomenon. Previous studies indicated that pathogenic proteins of Mycoplasma play a pivotal role in its infection. This project focuses on the key scientific issue of virulence factors related to M. genitalium infection，and explores function of cysteine desulfurase-like protein MG_336 which is the possible potential virulence factors of M. genitalium. To reveal its role in promoting Mg to establish stable intracellular niche and inducing host cell damage, this study intends to determine subcellular localization of MG_336 by corresponding methods including 2-D electrophoresis and mass spectrometry, and detect catalytic enzyme activity of MG_336 obtained through gene cloning and other corresponding approaches, and then determine antioxidant activity of MG_336 by quantitative RT-PCR and oxidative stress sensitivity test. In order to analyze the function of MG_336, we plan to construct MG_336 functionally deficient strains and complement strains through homologous recombination, and then employ these constructed strains to establish infected SV-HUC-1 cell model and murine model, and detect the cytotoxicity and pathogenicity of MG_336. This project is conducive to further explore the pathogenicity and immune escape mechanisms of M. genitalium, and provide a new potential therapeutic target for M. genitalium infection.

生殖支原体（Mg）是一种可引起泌尿生殖道感染的性传播病原体。目前已明确的Mg致病机制不能完全阐明Mg感染后的致病能力及其持续性感染现象。前期研究表明，支原体致病相关蛋白在其感染中发挥着重要作用。本项目着眼于Mg感染相关的毒力因子这一关键科学问题，对Mg的潜在毒力因子半胱氨酸脱硫酶样蛋白MG_336进行研究。为了揭示其在促进Mg建立稳定的胞内生态位及诱发宿主细胞损伤中的作用，本项目拟采用2-D电泳、质谱等方法分析其亚细胞定位；体外克隆表达MG_336蛋白，鉴定其催化活性；应用qRT-PCR和氧化胁迫敏感性试验检测其抗氧化活性；利用同源重组法构建MG_336功能缺失株及回补株，并构建功能缺失株及回补株的人尿道上皮细胞（SV-HUC-1细胞）模型和小鼠模型，分析MG_336的细胞毒性及其致病作用。本项目的开展有利于进一步探讨Mg的致病性与免疫逃逸机制，为治疗Mg感染提供新的潜在靶点。

生殖支原体作为一种促进性传播疾病流行的超级细菌而受到普遍关注。越来越多的研究表明，生殖支原体代谢相关的酶分子在其感染所致的持续性炎性损伤中发挥着重要作用。本项目聚焦含硫生物分子合成途径的关键酶进行深入探究，成功鉴定了具有潜在半胱氨酸脱硫酶活性的生殖支原体MG_336蛋白。研究结果显示，MG_336蛋白为mg336基因所编码，由408个氨基酸组成，具有保守的参与氨基酸转运和代谢的CsdA结构域，含有多个优势抗原表位，具有良好的免疫原性；该蛋白与其它已知的半胱氨酸脱硫酶同源性较高，其定位于胞浆中，具有磷酸吡哆醛依赖的半胱氨酸脱硫酶活性，位于C端结构域的保守cys残基为其活性位点；MG_336亦可诱导宿主细胞凋亡。这些结果揭示新鉴定的生殖支原体MG_336蛋白可催化宿主来源的代谢生长因子维持自身生存并诱发宿主细胞损伤。本项目的完成有助于进一步阐释Mg 感染所致的泌尿生殖道炎症的机理，并可为Mg 感染的治疗提供新的潜在治疗靶点。