Microbiota based mechanisms of post-infection irritable bowel syndrome

摘要 在美国，六分之一的成年人患有慢性且经常致残的肠易激综合征症状。 （IBS）。肠道感染是感染后IBS（PI-IBS）发展的既定风险因素。的 肠道含有多种蛋白酶，我们发现PI-IBS患者具有显著的 粪便蛋白水解活性（PA）高于对照组。更重要的是，PA与失去 肠屏障功能和患者症状加重。我们发现患有PI-IBS的患者， 具有高PA的患者在感染后不久就开始显著丧失微生物多样性。关键微生物 分类群丢失了，尤其是来自Alistipes属的。使用元蛋白质组学，我们发现驱动PA进入的蛋白酶 这些病人都是人类。为了了解微生物群的丧失是否会影响宿主 蛋白酶，我们使用无菌小鼠。健康人体微生物群在无菌小鼠中的定植 （人源化）导致PA的显著下降，表明微生物抑制宿主蛋白酶 因此具有维持肠道健康的作用。然而，来自高PA的生态失调的微生物群 缺少特定微生物的患者不能抑制PA。我们假设阿里斯蒂普斯和 其它缺失的细菌在抑制PA中起关键作用。我们计划对鉴定出的候选细菌进行测试 目的1中PA调节的初步实验和种间相互作用。接下来，我们确定 微生物的损失如何导致蛋白酶的抑制不良。未结合胆红素是丝氨酸的抑制剂 蛋白酶和微生物β-葡萄糖醛酸苷酶使胆红素去结合。我们发现，PI-IBS患者高 PA具有较低的粪便微生物β-葡萄糖醛酸酶活性。此外，他们有较低的水平， 胆红素去结合的产物。β-葡萄糖醛酸苷酶是一个大家族的微生物酶，具有不同的 不同底物的来源、结构和催化效率。我们假设特定的 微生物β-葡萄糖醛酸苷酶将导致胆红素的去缀合受损。在第二章中，我们将分析 我们的PI-IBS患者的宏基因组学数据，高PA和低PA存在微生物β-葡萄糖醛酸苷酶 以及确定这些粪便样品用于胆红素去结合的功效。此外，我们将 从Alistipes和其他细菌分类群产生纯化的β-葡萄糖醛酸苷酶，用于评估胆红素去缀合 体外药效接下来，我们已经表明使用富含低PA的Alistipes的粪便微生物群转移 在高PA人源化小鼠中，微生物群可以抑制PA，这为使用微生物群进行蛋白酶 抑制和纠正肠屏障功能。在目标3中，我们将使用共同住房策略， 人源化高PA小鼠和低PA小鼠之间的微生物群转移，并确定屏障功能障碍是否与 高PA状态可以反转。此外，我们将确定屏障途径的变化，离子 新微生物群植入后紧密连接蛋白的选择性和表达。总之，这些目标 将研究微生物通过调节肠道蛋白酶对PI-IBS病理生理学的影响。识别 可以导致蛋白酶抑制和屏障功能恢复的基于微生物群的策略， 有益于肠易激综合征和其他与微生物生态失调有关的疾病。

ABSTRACT One in six adults in the U.S. suffers from chronic and often disabling symptoms of irritable bowel syndrome (IBS). Intestinal infections are an established risk-factor for development of post-infection IBS (PI-IBS). The intestinal tract contains a variety of proteases, and we have discovered that PI-IBS patients have significantly higher fecal proteolytic activity (PA) than controls. More importantly, PA associates strongly with loss of intestinal barrier function and worse symptoms for the patients. We found that patients who develop PI-IBS and have high PA have a significant loss of microbial diversity that starts soon after the infection. Key microbial taxa are lost, especially from the Alistipes genus. Using metaproteomics, we found that proteases driving PA in these patients are of human origin. In order to understand if loss of microbiota could be affecting host proteases, we used germ-free mice. Colonization of germ-free mice with healthy human microbiota (humanization) results in a significant decline of PA suggesting commensal microbes inhibit host proteases and thus have a role in maintaining intestinal health. However, the dysbiotic microbiota from the high PA patients that are missing specific microbes were unable to suppress PA. We hypothesize that Alistipes and other missing bacteria play a critical role in suppression of PA. We plan to test the candidate bacteria identified in the preliminary experiments and inter-species interactions in PA regulation in Aim 1. Next, we determined how loss of microbes result in poor inhibition of proteases. Unconjugated bilirubin is an inhibitor of serine proteases and microbial β-glucuronidases deconjugate bilirubin. We found that the PI-IBS patients with high PA have lower fecal microbial β-glucuronidase enzymatic activity. Additionally, they have lower levels of end products of bilirubin deconjugation. β-glucuronidases are a large family of microbial enzymes with varying sources, structures and catalytic efficacies for different substrates. We hypothesize that loss of specific microbial β-glucuronidases will result in impaired deconjugation of bilirubin. In Aim 2, we will analyze metagenomics data from our PI-IBS patients with high and low PA for presence of microbial β-glucuronidases as well as determine the efficacy of these fecal samples for bilirubin deconjugation. Additionally, we will generate purified β-glucuronidases from Alistipes and other bacterial taxa for assessing bilirubin deconjugation efficacy in vitro. Next, we have shown that fecal microbiota transfer using an Alistipes enriched low PA community can suppress PA in high PA humanized mice providing a rationale for using microbiota for protease suppression and correcting intestinal barrier function. In Aim 3, we will use cohousing strategies to allow microbiota transfer between humanized high and low PA mice and determine if barrier dysfunction associated with a high PA state can be reversed. Furthermore, we will determine changes in barrier pathways, ionic selectivity and expression of tight junction proteins upon engraftment of new microbiota. Together, these aims will examine microbial influence on PI-IBS pathophysiology via regulation of intestinal proteases. Identification of microbiota-based strategies that can result in protease inhibition and restoration of barrier function can be beneficial for IBS and other conditions associated with microbial dysbiosis.