Microbiota based mechanisms of post-infection irritable bowel syndrome

摘要 在美国，每六个成年人中就有一个患有肠易激综合征的慢性且经常致残的症状 (IBS)。肠道感染是感染后肠易激综合征(PI-IBS)的一个公认的危险因素。这个 肠道中含有多种蛋白水解酶，我们发现PI-IBS患者有明显的 粪便蛋白水解酶活性(PA)高于对照。更重要的是，PA与损失 肠屏障功能改善，症状加重。我们发现，发生PI-IBS和 PA高的人在感染后不久就会显著丧失微生物多样性。关键微生物 分类群丢失了，特别是泽泻属。利用元蛋白质组学，我们发现驱动PA的蛋白酶 这些患者来自人类。为了了解微生物区系的丧失是否会影响宿主 我们使用的是无菌小鼠。无菌小鼠在健康人体微生物区系中的定植 (人源化)导致PA显著下降，表明共生微生物抑制宿主蛋白酶 从而在维持肠道健康方面发挥作用。然而，来自高PA的非生物微生物区系 缺少特定微生物的患者无法抑制PA。我们假设泽泻和 其他缺失的细菌在抑制PA的过程中发挥着关键作用。我们计划测试已确定的候选细菌 在目标1中的PA调节的初步实验和种间交互作用中。接下来，我们确定 微生物的流失如何导致对蛋白水解酶的抑制不佳。未结合胆红素是丝氨酸的抑制剂 蛋白水解酶和微生物β-葡萄糖苷酸酶去结合胆红素。我们发现PI-IBS患者具有较高的 PA具有较低的粪便微生物β-葡萄糖苷酸酶活性。此外，它们的终端级别较低 胆红素脱除产品。β-葡萄糖醛酸苷酶是一大类微生物酶，具有不同的 不同底物的来源、结构和催化效率。我们假设损失了特定的 微生物的β-葡萄糖醛酸苷酶将导致胆红素的去结合能力受损。在目标2中，我们将分析 我们的高PA和低PA的PI-IBS患者的微生物β-葡萄糖醛酸苷酶的宏基因组学数据 以及确定这些粪便样本对胆红素去卷曲的效果。此外，我们还将 泽泻和其他细菌产生纯化的β-葡萄糖醛酸苷酶用于评估胆红素的脱除 体外药效。接下来，我们已经证明了使用泽泻属富含低PA的粪便微生物区系转移 群落可以抑制高PA人源化小鼠的PA，为利用微生物区系进行蛋白酶提供了理论基础 抑制和纠正肠道屏障功能。在目标3中，我们将使用合住策略来允许 人源化高PA和低PA小鼠之间的微生物区系转移和确定屏障功能障碍是否相关 具有高PA的状态可以反转。此外，我们将确定屏障途径的变化，离子 紧密连接蛋白在植入新微生物区系时的选择性和表达。总而言之，这些目标 将通过调节肠道蛋白水解酶来研究微生物对PI-IBS病理生理的影响。鉴定 可以导致蛋白酶抑制和屏障功能恢复的基于微生物区系的策略 有益于肠易激综合征和其他与微生物失调相关的疾病。

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.