Impact of dysbiotic and symbiotic catabolism of luminal amino acids on intestinal epithelial barrier function and inflammation

美国有300多万成年人患有炎症性肠病(IBD)，包括克罗恩病 疾病(CD)和溃疡性结肠炎(UC)。IBD的特点是肠道微生物区系失调，上皮细胞受损 屏障功能、慢性肠道炎症和粘膜细胞因子增加。上皮屏障功能是 由一系列围绕紧密连接(TJ)、粘连连接和 桥粒。关键上皮屏障的破坏允许腔内容物获得免疫 特权隔室，从而促进了IBD的发病。微生物区系诱导的肠道微生物区系 粘膜炎症扰乱了细胞间的连接和上皮的动态平衡特性，从而导致 一种受损的上皮屏障。然而，我们对共生刺激的分子基础的了解 细胞间连接蛋白的功能、上皮细胞动态平衡与受损上皮细胞的修复 肠道炎症时的屏障是非常有限的。因此，这项提案的总体目标是确定 特定共生细菌和细菌代谢产物调节细胞功能的机制 细胞间连接蛋白，并保护免受上皮屏障损害和损伤。我们的初步数据 确定了共生的均匀类杆菌主要分解精氨酸产生多胺 亚精胺，促进健康屏障。相比之下，非生态型变形杆菌主要是大肠杆菌。 分解赖氨酸合成多胺身体碱，这会损害肠道通透性。细菌多胺是 调节多种细胞过程的脂肪胺。根据我们的初步数据，我们假设 微生物多胺通过激活TRPV和调节TJ糖基化来调节上皮屏障功能，a 肠上皮屏障中Claudin和ZO蛋白的翻译后修饰。多胺可以激活 瞬时受体潜力受体(TRPV)调节细胞功能。在目标1中，我们将剖析 共生多胺激活的TRPV1和非生物身体碱诱导的TRPV3介导的TJ调节 流程。在目标2中，我们将阐明微生物多胺驱动的TJ蛋白的SUMO化最终在 扰动屏障函数。最后，在目标3中，我们将确定小分子和流明的影响。 代谢物，它抑制身体合成肠道细菌的酶，从而抑制 肠道发炎。这个项目在概念和技术上都是创新的。它将雇佣有创造力的 使用TRPV1和TRPV3基因敲除小鼠和一种新的病原体特异性抗菌剂。对…的调查 肠道细菌产生的多胺对上皮细胞TRPV和TJ的影响是一种新的SUMO化方法。 这些研究的完成将对肠道上皮屏障的分子基础提供清晰的见解 共生体氨基酸分解代谢调控与开发新思路、新原则的产生 共生细菌代谢产物开发治疗IBD和其他胃肠道疾病，具有很高的 NIDDK研究优先。

Over 3 million adults in the U.S. suffer from inflammatory bowel diseases (IBD), which encompasses Crohn’s disease (CD) and ulcerative colitis (UC). IBD is characterized by dysbiotic gut microbiota, compromised epithelial barrier function, chronic intestinal inflammation, and increased mucosal cytokines. Epithelial barrier function is regulated by a series of intercellular junctions that encompass the tight junction (TJ), adherens junction, and desmosomes. Disruption of the critical epithelial barrier allows access of luminal contents to immunologically privileged compartments, thereby contributing to IBD pathogenesis. The dysbiotic gut microbiota-induced mucosal inflammation perturbs intercellular junctions and epithelial homeostatic properties, thereby resulting in a compromised epithelial barrier. However, our knowledge of the molecular basis of commensal-stimulated intercellular junction proteins’ function, epithelial homeostasis, and restoration of the compromised epithelial barrier during intestinal inflammation is very limited. Thus, the overall goals of this proposal are to identify mechanisms by which specific commensal bacteria and bacterial metabolic products regulate functions of intercellular junction proteins, and protect from epithelial barrier compromise and injury. Our preliminary data determined that the symbiotic Bacteroides uniformis predominantly catabolizes arginine to produce polyamine spermidine, which promotes a healthy barrier. In contrast, dysbiotic Proteobacterial species E. coli predominantly catabolizes lysine to synthesize polyamine cadaverine, which impairs gut permeability. Bacterial polyamines are aliphatic amines that regulate multiple cellular processes. Based on our preliminary data, we hypothesize that microbial polyamines regulate epithelial barrier functions by activating TRPV and regulating TJ SUMOylation, a post-translational modification of claudin and ZO proteins in the gut epithelial barrier. Polyamines can activate Transient Receptor Potential Receptors (TRPVs) to regulate cellular functions. In Aim 1, we will dissect the symbiotic polyamine-activated TRPV1 and dysbiotic cadaverine-elicited TRPV3-mediated TJ regulatory processes. In Aim 2, we will elucidate microbial polyamine-driven SUMOylation of TJ proteins eventuating in the perturbed barrier function. Finally, in Aim 3, we will determine the impact of small molecules and luminal metabolites, which inhibit cadaverine synthesizing enzymes of the dysbiotic gut bacteria and thereby dampen intestinal inflammation. This project is both conceptually and technically innovative. It will employ the creative use of Trpv1 and Trpv3 knockout mice and a novel pathogen-specific antibacterial agent. The investigation of the effects of gut bacterially produced polyamines on epithelial TRPV and TJ SUMOylation approach is novel. Completion of these studies will provide clear insights into the molecular basis of intestinal epithelial barrier regulation by commensals’ amino acid catabolism and engender new ideas and proof-of-principle of exploiting commensal bacterial metabolites to develop therapeutics for IBD and other gastrointestinal diseases, a high NIDDK research priority.