Identification of sialic acid-degrading microbiome proteins during colitis

摘要 我们的目标是开发和应用化学生物学方法，这将有助于识别和 本发明的目的在于表征负责消化道粘液的消化道微生物组酶。肠 肠道内有大量多样的肠道细菌，称为肠道微生物组， 代谢、免疫发育和体内平衡以及上皮细胞血管生成所必需的。健康 对于个体来说，大多数微生物都被粘液的稳定分泌所控制， 主要由交联和高度O-糖基化的粘蛋白组成。炎症性肠道疾病 (IBD)包括溃疡性结肠炎和克罗恩病，肠道微生物设法渗入宿主肠道， 粘蛋白核心、上皮和固有层。由此产生的微生物成分的识别， 宿主的先天性和适应性免疫反应驱动与所有形式相关的肠道炎症 IBD。我们认为从健康状态到结肠炎的进展是通过粘液的微生物侵蚀开始的 层和结肠炎相关肠道微生物，包括致病性和感染性细菌和病毒（即，C. 艰难梭菌、巨细胞病毒），具有异常的蛋白质功能， 比健康个体中发现的微生物降解。我们的假设得到了最近发现的支持， 在结肠炎的鼠模型中，发现唾液酸降解酶活性水平升高。在该提议中， 我们将集中于阐明微生物蛋白质，识别和降解唾液酸，一个丰富的和最 粘液聚糖上末端表达的碳水化合物。我们认为，虽然粘液聚糖的许多层 在健康个体中，唾液酸有助于微生物防御的下面，唾液酸的去除是 肠道的微生物渗透为了验证这一假设，我们开发了基于UV-光活化唾液酸的 化学探针，不可逆地标记来自微生物组样品的所有宿主和细菌蛋白， 结合唾液酸（即，唾液酸酶、凝集素、转运蛋白、转移酶）。所有探针结合的蛋白质都经历 基于亲和性的富集和随后的蛋白质鉴定以及用质谱法（MS）定量。 在这里，我们将采用我们的唾液酸探针和创新的方法，以一个完善的过继性T细胞转移 慢性结肠炎小鼠模型，并直接比较我们的元蛋白质组学结果与对照组的结果 在相同条件下饲养的小鼠。在健康小鼠和结肠炎小鼠之间观察到的差异可能涉及 哪些酶对人类IBD至关重要，并有助于实现我们识别微生物的长期目标 促进结肠炎的蛋白质。重要的是，我们完全有资格实现我们提出的目标，因为Wolan 实验室率先开发和应用化学探针和基于MS的元蛋白质组学， 找到大肠菌群中大量存在的细菌半胱氨酸蛋白酶。所述探针、方法和 这里产生的结果将为研究宿主-微生物相互作用带来令人兴奋的新机会。

ABSTRACT Our goal is to develop and apply chemical biological methodologies that will assist in the identification and characterization of gut microbiome enzymes responsible for the degradation of the gut mucus. The intestinal tract harbors an enormous and diverse collection of commensal bacteria, termed the gut microbiome, that are essential for metabolism, immune development and homeostasis, and epithelial cell angiogenesis. In healthy individuals, the majority of microbes are held at bay in the gut lumen by the steady secretion of mucus, which primarily consists of crosslinked and heavily O-glycosylated mucin proteins. In inflammatory bowel diseases (IBD), including ulcerative colitis and Crohn’s disease, the gut microbes manage to infiltrate the host intestinal mucin protein core, epithelium, and lamina propria. The resulting recognition of microbial constituents by the host’s innate and adaptive immune responses drives the intestinal inflammation that is associated with all forms of IBD. We posit that progression from a healthy state to colitis commences via microbial erosion of the mucus layer and that colitis-associated gut microbes, including pathogenic and infectious bacteria and viruses (i.e., C. difficile, cytomegalovirus), harbor aberrant protein functionalities that are far more adept at host mucus layer degradation than microbes found in healthy individuals. Our hypothesis is supported by recent discoveries that, in murine models of colitis, elevated levels of sialic acid-degrading enzymatic activity are found. In this proposal, we will focus on elucidating microbial proteins that recognize and degrade sialic acid, an abundant and the most terminally expressed carbohydrate on mucus glycans. We believe that, while the many layers of mucus glycans beneath sialic acid aid in microbial defense in healthy individuals, removal of sialic acid is the first overall step in microbial infiltration of the gut. To test this hypothesis, we have developed UV-photoactivatable sialic acid-based chemical probes that irreversibly label all host and bacterial proteins from microbiome samples capable of binding sialic acid (i.e., sialidases, lectins, transporters, transferases). All probe-bound proteins are subjected to affinity-based enrichment and subsequent protein identification and quantitation with mass spectrometry (MS). Here, we will employ our sialic acid probes and innovative methods to a well-established adoptive T cell transfer murine model of chronic colitis and directly compare our metaproteomics results to those obtained from control mice raised under identical conditions. Differences observed between healthy and colitic mice may implicate which enzymes are critical for human IBD and assist in achieving our long-term goal of identifying microbial proteins that promote colitis. Importantly, we are well qualified to accomplish our proposed aim, as the Wolan laboratory spearheaded the development and application of chemical probes and MS-based metaproteomics to find the bacterial cysteine proteases that are overly abundant in colitic microbiomes. The probes, methods, and results generated here will lead to exciting new opportunities for studying host-microbe interactions.