ERS/VTS介导问号钩端螺旋体跨细胞转运及肾细胞内繁殖与出胞的分子机制

Leptospirosis is a globally-spreading zoonotic disease. Animals after infection with Leptospira will discharge leptospires from their urine to contaminate natural water, while humans will be infected by contact with the Leptospira-containing water and the invaded leptospires in patient’s bloodstream diffuse into internal organs to cause the aggravation of patient’s condition. The recent literatures reported that bacteria can utilize cellular endocytic recycling system / vesicular transport system (ERS/VTS) to diffuse in vivo through transcytosis and propagate in vesicles. Our previous study found that leptospires pass through the vascular endothelial cell monolayers by transcytosis and their phagocytotic vesicles did not fuse with lysosomes, while leptospires could also pass through the renal tubule epithelial cells and fibroblasts and the leptospiral number was increased, but their molecular machanims are unknown. In this project, we will study from molecular, cellular and animal levels and use advanced and reliable techniques to determine the mechanism of vascular endothelial cells, renal tubule epithelial cells and fibroblasts phagocytosing the leptospires through ITG-FAK- or CAV1-PI3K-mediated pathway. Subsequently, we will determine the mechanisms of extracellular leptospiral transcytosis through Rab5-Rab4-Rab11 or CAV1-EEA1-Rab11 vesicle transport route, formation of exocyst complex and membrane fusion complex, activation of calcium channels, and FAK or PI3K signaling pathway-mediated Ca2+ / microfilament or microtubule-dependent cytoskeleton rearragment as well as the the mechanisms of leptospiral propagation in phagocytotic vesicles of renal cells and exocytosis of the propagated leptospires. The results of this project have important significance in control and treatment of leptospirosis.

钩端螺旋体（简称钩体）病是全球性人兽共患病。动物感染后尿液排菌形成疫水、人接触疫水感染后从血流扩散至内脏分别是钩体病流行和病情恶化关键。近年发现细菌利用细胞内吞循环系统／囊泡运输系统（ERS/VTS）跨细胞扩散及泡内繁殖。我们前期研究发现钩体通过跨细胞途径穿越血管内皮细胞且吞噬泡不与溶酶体融合，也能穿越肾小管上皮和成纤维细胞且胞内钩体数量增加，但机制不明。本项目拟从分子／细胞和动物不同层次并采用先进可靠技术，先确定ITG-FAK或CAV1-PI3K通路介导血管内皮、肾小管上皮和成纤维细胞内吞钩体分子机制，再确定钩体吞噬泡经Rab5-Rab4-Rab11或CAV1-EEA1-Rab11囊泡转运途径形成出胞及膜融合复合体、激活钙通道并经FAK或PI3K通路介导Ca2+及微丝或微管依赖细胞骨架重排实现胞外钩体跨细胞转运以及肾细胞吞噬泡内钩体繁殖与出胞分子机制，对钩体病防控及治疗均具有重要意义。

主要由问号钩端螺旋体（简称钩体）引起的钩体病是全球性人兽共患传染病。动物感染后从尿液排出问号钩体形成疫水，人接触疫水感染的问号钩体进入血流并扩散至内脏引发疾病，但其跨内皮和上皮屏障播散的分子机制迄今未见报道。此外，问号钩体跨细胞转运过程中钩体与细胞相互作用也不清楚。本项目研究发现，优势流行的问号钩体黄疸出血群赖型赖株利用细胞内吞循环系统／囊泡运输系统（endocytic recycling system / vesicular transport system，ERS/VTS）跨人和小鼠血管内皮细胞、肾小管上皮细胞和成纤维细胞转运和扩散，但仅在小鼠成纤维细胞中增殖。问号钩体赖株均通过细胞小窝／β1整合素-磷脂酰肌醇-3激酶-细胞微丝途径（CAV/ITGB1-PI3K-MF）进入血管内皮和肾小管上皮细胞、细胞小窝／β1整合素-黏附斑激酶-细胞微丝途径（CAV/ITGB1-FAK-MF）进入成纤维细胞形成钩体吞噬泡。问号钩体吞噬泡招募ERS/VTS中Rab5/11形成钩体循环内体（recycling endosome，RE），但不与溶酶体融合，钩体和细胞均存活。问号钩体RE招募VTS中Sec15、VAMP2和SYN1形成钩体RE-SNARE出胞复合体（RE-SNARE exocyst，RSE），然后均通过FAK-MF/MT途径出胞。研究中我们还发现，问号钩体赖株vWA基因产物可分别与人1、3、4和6型胶原（hCOL1/3/4/6）强结合，具有黏附因子作用；M16金属蛋白酶1和3（Lep-MP1和Lep-MP2）介导问号钩体赖株侵入金地鼠血流引起中毒性败血症以及体内播散；vwa-I/II基因产物能与人血小板GPIb-α受体强结合抑制vWF/GPIb介导的凝血而导致肺出血；LA_2144 基因产物具有较强的血小板活化因子乙酰水解酶（PAF-AH）活性和较弱的炎症相关磷脂酶A2（PLA2）活性，参与问号钩体赖株致病过程。本项目预定的研究目标拟发表论文5篇，其中SCI论文3篇，争取有2篇SCI论文影响因子＞5，培养硕、博士生3-5名。本项目已发表SCI研究论文或综述4篇、待发表SCI研究论文1篇，其中2篇SCI论文IF=5.022和6.680，发表中文研究论文或综述5篇，1篇我国最早的新冠肺炎中文综述为疫情防控作出了专业贡献，培养了博士3名硕士3名，全面完成了预定的研究任务。