肠上皮细胞感知新的PAMP激活NF-κB信号通路的机制研究

The intestinal epithelium separates the vast microbiota from tissue of intestinal lumen and can differentiate commensal bacteria from pathogens. It can initiate an inflammatory response upon detection of pathogens, but the mechanism remains poorly understood. Many pathogens use type III secretion system (TTSS) to deliver virulence factors into infected cells to subvert host cell functions. Our previously study showed that enteropathogenic E. coli (EPEC) and related pathogens secrete so many effectors that inhibit the NF-κB activation. Then we speculate that EPEC employ these effectors to antagonize NF-κB activation by EPEC infection. Large scale genetic screen identified a gene that catalyzes a small compound formation which is involved in LPS synthesis pathway in all gram negative bacteria which is necessary for NF-κB activation. The small compound synthetized in vitro is sufficient for the activation of NF-κB signaling pathway when secreted by pathogen TTSS or transfected into mammalian epithelium cells which suggests that it is new PAMP for immunity signal transduction. These preliminary results promote further study that involves two important questions. The first one is the detection of the small compound by epithelial cells which is secreted by infecting bacteria or through other ways, leading to NF-κB activation. The second one involves the signal transduction pathways that activate NF-κB by the new PAMP. We propose to identify the receptor of the small compound and elucidate the ways epithelial cells respond to pathogen infection through NF-κB activation. This research will expose new basic knowledge about how to discriminate pathogen-commensal bacteria which are essential for the development of new strategies for prevention and treatment of inflammatory bowel diseases. In addition, the proposed project will also shed light on the principles guiding the infection strategy of EPEC and related pathogens, thus contributing to the development of new approaches to treat infection by these pathogens.

肠道表皮细胞将肠道内微生物菌群和肠腔外组织分隔开，检测致病菌的存在并激活免疫反应，然而其区分肠道共生菌和致病菌的具体的机制还不是很清楚。肠致病大肠杆菌利用三型分泌系统分泌许多抑制NF-κB激活的效应蛋白，因此推测EPEC感染可能需要效应蛋白来抑制细菌感染引起的NF-κB激活。通过大规模的遗传筛选，我们发现一个LPS合成通路上的基因是激活NF-κB信号通路必需的，这个基因存在于所有革兰氏阴性细菌中，催化一个小分子的合成。体外合成的小分子通过三型分泌系统或者转染进入细胞后足以激活NF-κB通路表明这个小分子可能是一个激活免疫通路的新的PAMP。接下来的研究一是寻找新的PAMP在宿主细胞内的受体，二是探明小分子激活NF-κB信号通路的机制。这项研究将将帮助人们更好的理解肠道表皮细胞如何区分共生菌和致病菌，对炎性肠病的治疗提供新的理论基础，也有助于了解EPEC及相关致病菌的感染策略以及开发新疗法。

肠致病性大肠杆菌和耶尔森菌等诸多拥有三型分泌系统（T3SS）的革兰氏阴性致病菌可借助三型分泌系统分泌致病因子激活宿主的NF-κB信号通路，但具体的分子机制还没有搞清楚。研究人员通过对大肠杆菌和耶尔森菌进行大规模转座子介导的细菌遗传筛选，以及生物化学分析等方法，发现细菌中的七碳糖代谢中间产物HBP和ADP-heptose（二磷酸腺苷庚糖）都能导致细菌三型分泌系统依赖的NF-κB通路的激活及炎症细胞因子的产生。ADP-heptose广泛存在于几乎所有的革兰氏阴性细菌及部分革兰氏阳性菌中。在非感染的情况下，将ADP-heptose直接添加至细胞培养基中，即可明显激活宿主细胞内NF-κB通路。为进一步研究细胞感知ADP-heptose的分子机制，研究人员在人胚肾细胞系中进行了基于流式细胞分选的CRISPR-Cas9全基因组筛选，最终发现了ALPK1-TIFA信号通路对于ADP-heptose激活NF-κB是必需的。ALPK1是细胞质内的一个激酶，包含N-端结构域和C-端激酶结构域，中间由一个长柔性序列相连。经过一系列的生物化学实验分析，研究人员发现ALPK1的N-端和C-端结构域之间具有组成性的相互作用，并且ALPK1的N-端结构域可以作为受体直接结合ADP-heptose。这项研究揭示，宿主细胞内的全新激酶受体蛋白ALPK1可以识别细菌来源的ADP-heptose，代表了一种全新的、保守存在于脊椎动物中的抗细菌天然免疫模式识别通路，这一发现也丰富和改变了人们对LPS相关分子诱导炎症反应的机制；鉴于ADP-heptose具有自主进入哺乳动物细胞的能力，该研究也为开发新的免疫调节剂和疫苗佐剂提供了新的概念和方法。