Role of SPI-1 Secreted Effectors in Acute Bovine Response to Salmonella enterica Serovar Typhimurium: A Systems Biology Analysis Approach

Salmonella enterica Serovar Typhimurium (S. Typhimurium) causes enterocolitis with diarrhea and polymorphonuclear cell (PMN) influx into the intestinal mucosa in humans and calves. The Salmonella Type III Secretion System (T3SS) encoded at Pathogenicity Island I translocates Salmonella effector proteins SipA, SopA, SopB, SopD, and SopE2 into epithelial cells and is required for induction of diarrhea. These effector proteins act together to induce intestinal fluid secretion and transcription of C-X-C chemokines, recruiting PMNs to the infection site. While individual molecular interactions of the effectors with cultured host cells have been characterized, their combined role in intestinal fluid secretion and inflammation is less understood. We hypothesized that comparison of the bovine intestinal mucosal response to wild type Salmonella and a SipA, SopABDE2 effector mutant relative to uninfected bovine ileum would reveal heretofore unidentified diarrhea-associated host cellular pathways. To determine the coordinated effects of these virulence factors, a bovine ligated ileal loop model was used to measure responses to wild type S. Typhimurium (WT) and a ΔsipA, sopABDE2 mutant (MUT) across 12 hours of infection using a bovine microarray. Data were analyzed using standard microarray analysis and a dynamic Bayesian network modeling approach (DBN). Both analytical methods confirmed increased expression of immune response genes to Salmonella infection and novel gene expression. Gene expression changes mapped to 219 molecular interaction pathways and 1620 gene ontology groups. Bayesian network modeling identified effects of infection on several interrelated signaling pathways including MAPK, Phosphatidylinositol, mTOR, Calcium, Toll-like Receptor, CCR3, Wnt, TGF-β, and Regulation of Actin Cytoskeleton and Apoptosis that were used to model of host-pathogen interactions. Comparison of WT and MUT demonstrated significantly different patterns of host response at early time points of infection (15 minutes, 30 minutes and one hour) within phosphatidylinositol, CCR3, Wnt, and TGF-β signaling pathways and the regulation of actin cytoskeleton pathway.鼠伤寒沙门氏菌（Salmonella enterica Serovar Typhimurium, S. Typhimurium）可引发人类及犊牛出现伴有腹泻的小肠结肠炎，并导致多形核白细胞（polymorphonuclear cell, PMN）向肠黏膜内浸润。由致病岛Ⅰ（Pathogenicity Island I）编码的沙门氏菌Ⅲ型分泌系统（Salmonella Type III Secretion System, T3SS）可将沙门氏菌效应蛋白SipA、SopA、SopB、SopD及SopE2转运至上皮细胞内，且对于腹泻的诱发必不可少。上述效应蛋白协同作用，可诱导肠液分泌以及C-X-C趋化因子的转录，从而将PMN招募至感染部位。尽管目前已明确了各效应蛋白与体外培养宿主细胞间的单个分子相互作用，但它们在肠液分泌与炎症反应中的协同作用仍有待进一步阐明。本研究推测，相较于未感染的牛回肠组织，对比牛肠黏膜对野生型沙门氏菌以及SipA、SopABDE2效应蛋白突变株的应答反应，将有望揭示此前未被发现的与腹泻相关的宿主细胞通路。为明确这些毒力因子的协同效应，本研究采用牛结扎回肠环模型，借助牛基因芯片（bovine microarray）检测了感染12小时内宿主对野生型鼠伤寒沙门氏菌（WT）以及ΔsipA、sopABDE2突变株（MUT）的应答反应。研究采用标准基因芯片分析方法与动态贝叶斯网络建模方法（dynamic Bayesian network modeling approach, DBN）对数据进行分析。两种分析方法均证实，沙门氏菌感染可诱导免疫应答相关基因以及全新基因的表达上调。基因表达变化共映射至219个分子相互作用通路以及1620个基因本体（gene ontology）组。贝叶斯网络建模明确了感染对多条相互关联的信号通路的调控作用，包括丝裂原活化蛋白激酶（MAPK）、磷脂酰肌醇（Phosphatidylinositol）、雷帕霉素靶蛋白（mTOR）、钙离子信号通路、Toll样受体（Toll-like Receptor）、CCR3、Wnt、转化生长因子β（TGF-β）、肌动蛋白细胞骨架调控及细胞凋亡通路，上述通路被用于构建宿主-病原体互作模型。野生型菌株与突变株的对比分析显示，在感染早期（15分钟、30分钟及1小时），宿主在磷脂酰肌醇、CCR3、Wnt、TGF-β信号通路以及肌动蛋白细胞骨架调控通路中的应答模式存在显著差异。