调控因子hilA在安石榴苷调节食源性沙门氏菌致病性中的作用及机制研究

Salmonella is the one of the most common foodborne pathogens in the world. Emergence of antibiotic resistant Salmonella strains poses a great challenge to the control of Salmonella. Therefore, it is imperative to search for effective, safe anti-infective substances which will cause little or no resistance in bacteria. HilA is located in virulence island (SPI) – 1 and play a key role as the regulation factor for SPI-1, hilA is important for Ш secretion system. In our previous studies, the hilA gene expression was significantly inhibited by punicalagin, and the ability of invade the intestinal epithelial cells was significantly affected by punicalagin. In order to explore the role and mechanism of hilA on the virulence of Salmonella regulated by punicalagin, in our experiment, we first constructed hilA deficient mutant , hila-overexpression strain as well as hila-complemented  strains using Salmonella SL1344. On this basis, its effect on the expression of virulence genes, the ability of Salmonella to adhere to and invade epithelial cells and mechanism in vivo were researched through gene chip technology, RT-PCR, Western-blot and Caco-2 tissue culture. It is expected that the findings from this project will provide theoretical bases for food safety system which was interfere with salmonella virulence island as the targets.

沙门氏菌是一种最常见的食源性致病菌。耐药菌株在全球的流行，给预防和控制沙门氏菌带来严峻挑战。探索有效、安全同时又不易引起细菌耐药的抗菌物质具有重要意义。hilA位于毒力岛（SPI）-1上，是SPI-1的正调控因子，调控与侵袭力有关的III型分泌系统。前期研究初步发现安石榴苷对沙门氏菌具有抑制作用，在亚致死浓度下能够极显著抑制hilA基因的表达，并显著影响沙门氏菌侵入肠上皮细胞。本研究拟通过基因重组技术构建hilA基因缺失菌株、回复补偿菌株及过表达菌株；在此基础上，通过基因芯片、RT-PCR、Western-blot及细胞培养等技术手段从影响毒力基因表达、抗粘附和侵入上皮细胞能力、全基因表达谱及体内实验等角度对hilA在安石榴苷调节沙门氏菌致病性中的可能机制进行探讨。本研究将为建立以干扰沙门氏菌毒力岛调控因子为靶点的新型的食品安全控制技术体系提供理论基础。

沙门氏菌是一种最常见的食源性致病菌，易污染食品及引起食物中毒。研究表明安石榴苷对沙门氏菌具有抑制作用，在亚致死浓度下能够显著抑制hilA基因的表达。基于此，本项目研究了安石榴苷对沙门氏菌细胞膜的影响，发现安石榴苷破坏沙门氏菌细胞膜的完整性。研究了安石榴苷抗沙门氏菌感染的体内机制，发现安石榴苷可提高沙门氏菌感染小鼠的生存率，减轻沙门氏菌对小鼠肝和脾的破坏。结合基因组学和蛋白组学技术识别了沙门氏菌在安石榴苷胁迫下的关键性代谢物和基因，通过对关键性基因进行KEGG途径富集分析，发现主要的代谢途径包括：Carbohydrate metabolism、Signal transduction、Amino acid metabolism、Energy metabolism、Membrane transport。基因组学和蛋白组学实验证实安石榴苷改变了hilA基因的表达，从而影响沙门氏菌的致病性。构建了沙门氏菌hilA 基因缺失菌株和过表达菌株，并用RT-PCR技术验证了hilA基因及相关致病基因的表达量变化。本研究将为建立以干扰沙门氏菌毒力岛调控因子为靶点的新型的食品安全控制技术体系提供理论基础。