维生素D对幽门螺杆菌清除的新机制：靶向溶酶体酸化通路

The molecular pathogenesis of chronic Helicobacter pylori infection is unclear. Emerging evidence demonstrated that some bacterial species could devise strategy to promote their intracellular survival through interfering with host cells’ lysosomal acidification after being sequestered by autophagosomes, leading to chronic infection. Our preliminary data showed that H. pylori promoted autophagosome formation with concomitant inhibition of lysosomal acidification so as to sustain their intracellular survival. In this connection, vitamin D reduced the intracellular survival and colonization of H. pylori in vitro and in vivo as well as restored lysosomal acidification. Nevertheless, the molecular mechanism underlying the protective action of vitamin D is unknown. Herein, we proposed (1) to study the protective role of vitamin D on the lysosomes acidification in H. pylori-infection; (2) using gene silence or gene knockout methods to elucidate TRPML3 functional role in vitamin D on H. pylori infection; (3) combining transcriptomics with proteomic analysis of lysosomes isolated from H. pylori-infected gastric epithelial cells to identify novel lysosome-regulatory pathway of vitamin D. The proposed project will not only elucidate the mechanism underlying the rectifying effect of vitamin D on lysosomal acidification in H. pylori infection, but may also open up novel therapeutic avenues for modulating host immune response to achieve infection clearance. The results from this study will also help formulate regimens for more effective H. pylori eradication.

幽门螺杆菌（Hp）慢性感染的分子机制尚不明确。最新研究发现：顽固性病原菌感染细胞后会进入自噬体，并扰乱基于溶酶体酸化的细胞自主防御系统，抑制溶酶体的降解功能，造成慢性持续性感染。申请者前期研究结果证明：Hp慢性感染与其抑制溶酶体酸化功能相关，维生素D能有效降低Hp在细胞内和小鼠胃内定植，这一过程可能与恢复溶酶体酸化功能相关，但具体分子机制不明确。故本项目拟开展：(1)维生素D对细胞溶酶体酸化通路靶向修复的分子机制研究；(2)采用基因沉默或基因敲除技术，揭示TRPML3在维生素D清除Hp中的意义；(3)利用基因表达谱及转录因子网络分析技术，从源头寻找维生素D调控TRPML3基因降解Hp的新的靶基因，并进行功能性验证。本研究成果将阐明维生素D通过靶向溶酶体酸化通路清除Hp的新机制，为我们如何激活机体自主防御系统清除Hp的认知带来新的突破，同时对开发新的根除Hp感染方案有着深远意义。

幽门螺杆菌(Hp)是常见的人类致病菌，一旦感染宿主细胞，就很难被人体免疫系统所清除，临床上发生免疫逃逸的机制尚不明确。另外Hp耐药也呈逐年上升趋势，克服Hp耐药及寻找根除Hp感染的新方案是消化系统疾病研究领域的重点和热点。我们的研究发现Hp慢性感染与溶酶体酸化功能受损相关，Hp能够藏匿于胃上皮细胞自噬体内，破坏自噬-溶酶体通路。维生素D能诱导溶酶体酸化，使溶酶体功能得到修复，从而有效降低Hp包括耐药Hp在永生化胃上皮细胞HFE145内和小鼠胃内的定植。我们的研究证实Hp感染导致溶酶体Ca2+释放受阻，溶酶体内pH值显著增高，其酸性磷酸酶ACP2，β-N乙酰葡萄糖苷酶（NAGLU）活性降低，组织蛋白酶Cathepsin D非成熟蛋白累积；而维生素D处理修复了溶酶体的酸性环境及其酶活性和其对细菌的降解功能。进一步的分子机制研究表明维生素D通过PDIA3/STAT3调控溶酶体Ca2+通道MCOLN3的表达，促进了溶酶体Ca2+释放，最终修复了溶酶体酸性环境，清除了藏匿于自噬体的Hp。本研究成果阐明维生素D通过靶向溶酶体酸化通路清除Hp的新机制，为我们如何激活机体自主防御系统清除Hp带来新的突破，同时对开发新的根除Hp感染方案有着深远意义。