中文摘要
梦稿本
sopB基因在鼠伤寒沙门菌引起的肠炎中的作用 鼠伤寒沙门菌是一种革兰氏阴性、兼性细胞内寄生菌，能够引起人和多种动物广泛的疾病，是重要的食源性病原菌之一。在全球范围内，鼠伤寒沙门菌每年引起超过2000万人的感染，严重威胁人类健康。鼠伤寒沙门菌病临床上主要表现为食欲减退、呕吐和腹泻等，甚至导致败血症的发生。研究鼠伤寒沙门菌致病机制，对于该病的防治具有重要指导意义。作为重要的的食源性病原菌，鼠伤寒沙门菌主要经胃肠道感染。肠上皮屏障功能的完整性在防御病原感染过程中起重要作用，其中，肠上皮稳态是屏障功能完整性的组成部分，肠上皮细胞的增殖分化和细胞死亡的动态平衡维持着肠上皮稳态。已知SopB作为鼠伤寒沙门菌毒力岛SPI-1编码的一个重要的效应分子，在沙门菌侵袭过程中能够诱导AKT磷酸化，但SopB在肠黏膜感染中的具体作用和机制还不清楚。因此，本实验以鼠伤寒沙门菌株SL1344、sopB基因缺失菌株△sopB SL1344口服感染小鼠，研究SopB在沙门菌侵袭小鼠肠黏膜过程中的作用和机制。小鼠感染存活率分析结果表明，相对于SL1344感染组，△sopB 感染组死亡率显著升高。通过盲肠H&E染以研究组织病理变化情况，结果表明△sopB感染组黏膜下水肿和杯状细胞缺失更为严重，并且肠上皮完整性损伤加剧，其中杯状细胞数量减少尤其明显。进而通过盲肠组织PAS染和Mucin-2免疫组化实验，研究盲肠组织杯状细胞数量及粘蛋白分泌情况，结果表明，△sopB感染组小鼠盲肠组织黏液和Mucin-2的分布均明显减少。综上，相对于亲本菌株，△sopB菌株的强致病性可能与其诱导 更多的杯状细胞死亡相关。
我们以杯状细胞LS174T细胞株作为体外研究模型，探讨SopB在沙门菌诱导其死亡中的作用。乳酸脱氢酶释放（LDH）分析结果显示，△sopB能显著促进LS174T细胞的死亡，并且其诱导死亡效应能被MLKL蛋白（Mixed Lineage Kinase Domain-Like Protein）特异性抑制剂necrosulfonamide（NSA）抑制，而
Receptor Interacting Serine/threonine Kinase 1（RIPK1）抑制剂necrostatin-1（Nec-1）对其无明显抑制作用。盲肠组织免疫组化显示，相对于亲本SL1344菌株，△sopB菌株诱导MLKL磷酸化作用增强；并且MLKL免疫组化与PAS 阳性细胞共定位，表明△sopB菌株明显促进肠道杯状细胞的坏死性凋亡。
目前已知，MLKL是坏死性凋亡（necroptosis）信号的最终执行者，为了进一步证明△sopB是否促进杯状细胞的坏死性凋亡，我们以MLKL基因缺失（MLKL-/-）小鼠作为研究模型。感染小鼠盲肠眼观病理变化结果表明，相比于亲本SL1344菌株，△sopB菌株能够诱导更为严重的肠道病理损伤，具体表现为盲肠体积缩小、内容物减少。在炎症反应、组织荷菌方面，表现为相比于亲本SL1344菌株，△sopB菌株感染WT小鼠诱导明显增多的细胞因子和趋化因子的分泌、组织荷菌数增多，而感染的MLKL-/-小鼠的情况则相反。盲肠组织Mucin-2免疫组化结果显示，相比于亲本SL1344菌株，△sopB菌株能够引
dwg起WT小鼠Mucin-2表达减少，且△sopB菌株感染的MLKL-/-小鼠盲肠Mucin-2表达趋于正常水平。上述结果表明，△sopB能够通过MLKL信号通路诱导杯状细胞坏死性凋亡。
最后，我们通过体外实验分析了sopB在沙门菌感染肠上皮细胞过程中的时间动态表达，发现随着感染时间延长sopB基因的表达水平逐渐降低，之前结果表明了sopB基因缺失能够诱导更为严重的肠道病理损伤，故sopB的动态表达和沙门菌的致病机制相关。
综上所述，SopB在沙门菌感染过程中通过抑制杯状细胞发生坏死性凋亡，维持肠道粘液屏障，抑制细菌易位，进而增强机体对病原菌的抵抗能力。
关键词：
SopB，鼠伤寒沙门菌，杯状细胞，坏死性凋亡
Abstract军事情报分析
The role of Salmonella sopB gene in bacterial colitis Salmonella typhimurium is a facultative intracellular bacterium implicated in a variety of illnesses, and is the leading cause of food borne illnesses in humans. Salmonella typhimurium infects over 20 million people worldwid annually and threatens the human health heavily. Salmonellosis is characterized by anorexia, vomiting and diarrh
ea, even leading to septicemia. The research of the pathogenesis of Salmonella typhimurium has important guiding significance for the prevention and treatment of the disease. As an important foodborne pathogen, Salmonella typhimurium caused diease mainly via gastrointestinal tract infection. The integrity of the intestinal epithelial barrier plays an important role in the defense of pathogen infection. Among them, the intestinal epithelium is a component of the integrity of the barrier function. The dynamic balance of intestinal epithelial cell proliferation, differentiation and cell death maintains the intestinal epithelium barrier.
时间散
Although SopB is known as an important effector molecule encoded by Salmonella typhimurium virulence island SPI-1, and inducing AKT phosphorylation during the invasion of Salmonella, but the role and mechanism of SopB in intestinal mucosal infection is not yet clearly understood. Therefore, we investigated the role and mechanism of SopB on the intestinal mucosa infection. The results of survival experiment showed that the mortality of △sopB infection group was significantly higher than that of SL1344 infection group. Histopathological changes were observed by cecal H&E staining. The results showed that mucosal edema and goblet cell loss were more serious in the △sopB infection group and the intestinal epithelium disorder was exacerbated, especially the decrease of the number of goblet cells. The distribution of mucus and Mucin-2 in cecal tissue of mice with △sopB
荆中秀infection were significantly decreased by PAS staining and Mucin-2 immunohistochemistry in cecal tissue. In summary, the strong pathogenicity of the
strain △sopB may be associated with goblet cell death compared to SL1344 counterparts.pass系统是什么意思
We used the LS174T cell line as an in vitro model to explore the role of SopB in the induction of cell death by Salmonella. Lactate dehydrogenase release (LDH) analysis showed that △sopB significantly promoted the death of LS174T cell, and its induced death effect was inhibited by MLKL specific inhibitor necrosulfonamide (NSA) , but receptor Interacting Serine / threonine Kinase 1 (RIPK1) inhibitor necrostatin-1 (Nec-1) had no significant inhibitory effect. The immunohistochemistry of cecal tissue showed that MLKL phosphorylation was marked induced by △sopB strain compared to SL1344 counterparts, and MLKL immunohistochemistry was co-located with PAS staining indicating that △sopB strain significantly promoted necroptosis of goblet cells.
It is known that MLKL is the ultimate performer of the necroptosis signal. We use MLKL-/- mice as a study model to further demonstrate whether △sopB promotes necroptosis of goblet cells. Our study showed that △sopB led to more sereve gross appearance of cecum compared to SL1344 counterparts, mainly contains obvious shrinkage of the cecum. In the inflammatory response and tiss
ue bacteria,WT mice infected with △sopB strain displayed a significant increase in the secretion of cytokines and chemokines and the number of tissue bacteria load compared to SL1344 counterparts, while MLKL-/- mice ware not. The results of immunohistochemistry showed that WT mice infected with △sopB displayed decreased expression of Mucin-2 and MLKL-/- mice ware not compared to SL1344 counterparts.In conclusion, △sopB was able to induce necroptosis of goblet cells through the MLKL signaling pathway.
Finally, we analyzed the dynamic expression of sopB in the process of intestinal epithelial cell infected with Salmonella and found that the expression level of sopB gene decreased with the prolongation of infection time. Previous results shows sopB gene mutation leads to more serious Intestinal pathological damage, Thus sopB
expression decreasement may be related to the pathogenesis of Salmonella typhimurium.
In summary, SopB maintained intestinal mucosal barrier, inhibited of bacterial translocation by inhibiting the necroptosis of goblet cells to enhance the resistance to pathogens.
Key words：
SopB, Salmonella typhimurium,Goblet cell, necroptosis
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