肺炎支原体CARDS Toxin基因缺失对其毒力影响的研究

Unlike many bacterial pathogens, Mycoplasma pneumoniae (Mp) is not known to produce classical toxins, and precisely how Mp injures the respiratory epithelium has remained a mystery. CARDS（Community Acquired Respiratory Distress Syndrome）toxin was a new found virulence factor (MPN372) that possesses cell vacuolating and ADP-ribosyltransferase (ART) activity. The earlier study showed that recombinant CARDS toxin would elicit a robust inflammatory response and lung injury in mice, but its pathogenic mechanism in Mp genome to host is not known, the deletion of this gene whether or not lead to virulence change of Mp? Based on our previous studies on Mp adhensin proteins, in this study, we will construct the Mp strain without CARDS toxin, by observing the strain morphology and cell toxicity, to identify its impact on Mp biological character. Using CARDS toxin gene knockout Mp strain to infect mice, by analyzing the pathology change in mice lung tissue, the cytokines change of bronchoalveolar lavage fluid and the T lymphocytes changes of in whole blood, to identify the effects of CARDS toxin knockout on virulence of Mycoplasma pneumoniae, then further research the CARDS toxin pathogenic mechanism in Mp.

肺炎支原体（Mp）损伤宿主呼吸道上皮细胞作用机制尚不清楚，CARDS toxin是一种新发现的具有细胞空泡毒素作用的Mp毒力因子。重组的CARDS toxin会引起小鼠肺组织病理损害和机体强烈炎症反应。但是CARDS toxin缺失后是否会导致Mp的毒力下降甚至消失？它在Mp导致宿主细胞损伤中的作用机制如何？为了解决这一问题，本研究拟在前期对Mp粘附蛋白研究基础上，构建CARDS toxin基因敲除Mp菌株，通过观察菌株的形态学改变及其对细胞的毒性作用，明确CARDS toxin基因敲除后对Mp菌株生物学性状的影响；采用构建的CARDS toxin基因敲除株建立感染小鼠模型，通过分析感染小鼠肺组织病理学改变、肺泡灌洗液细胞因子的变化以及血液中T淋巴细胞的变化，明确CARDS toxin基因敲除对Mp菌株毒力的影响，进一步阐明CARDS toxin造成宿主细胞损伤的作用机制。

肺炎支原体（M.pneumoniae）是人类社区获得性肺炎中重要病原体之一，CARDS毒素（社区获得性呼吸窘迫综合症，Community Acquired Respiratory Distress Syndrome）是2005年新发现的一种毒力蛋白，与百日咳毒素S1亚单位有27％同源性，可造成细胞的空泡化损伤，是目前唯一发现的肺炎支原体的毒力因子。本研究构建了CARDS基因功能缺失突变株，表达并纯化了重组的CARDS毒素蛋白，对突变株及重组蛋白的功能进行了研究。本项目共获得了CARDS毒素不同位点突变株3株，插入位点分别位于N末端和C末端。研究显示（1）在转座子插入后，CARDS基因在RNA转录水平未发生中断而继续进行，转录序列为插入的转座子和CARDS基因组。蛋白免疫印迹实验证实在转座子位于N端的突变株其原始CARDS毒素未表达，而转座子位于C端的菌株仍可发现部分CARDS毒素蛋白的表达。（2）与标准株相比，CARDS毒素突变株的菌落形态及生长速度无差异，说明CARDS毒素不是肺炎支原体生长必须基因。（3）与标准株相比，CARDS突变株对体外培养的人呼吸道上皮细胞的空泡化作用减弱；动物体内实验中，与标准株相比，CARDS毒素突变株感染组小鼠肺部的病理损伤减轻，肺泡灌洗液中各种细胞因子的表达水平与标准株有明显差异；CARDS基因突变株的毒力未完全缺失，推测除CARDS毒素外，存在其他毒力因子。（4）CARDS毒素基因在不同菌株间的序列较为保守，很少发生点突变。其导致细胞损伤功能的部位主要在C端，同时 C端在血清学检测中有较好的应用价值。