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，以帮助识别和 负责降解肠道粘液的肠道微生物组酶的特征。肠道 肠道中含有大量和多样化的共生细菌，被称为肠道微生物组，它们是 对新陈代谢、免疫发育和动态平衡以及上皮细胞血管生成至关重要。在健康中 对于个体来说，大多数微生物通过粘液的稳定分泌在肠腔内被挡在门外，这种粘液 主要由交联型和高度O-糖基化的粘蛋白组成。在炎症性肠病中 (IBD)，包括溃疡性结肠炎和克罗恩病，肠道微生物设法渗透到宿主肠道 粘蛋白核心、上皮和固有层。由此产生的对微生物成分的识别 宿主的先天和获得性免疫反应驱动与各种形式相关的肠道炎症 IBD的症状。我们假设，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。 结肠炎相关肠道微生物，包括致病和感染性细菌和病毒(即C. 艰难梭菌、巨细胞病毒)具有异常的蛋白功能，这些功能在宿主粘液层上更为熟练 比在健康个体中发现的微生物更容易降解。我们的假设得到了最近发现的支持， 在小鼠结肠炎模型中，发现唾液酸降解酶活性水平升高。在这份提案中， 我们将专注于阐明识别和降解唾液酸的微生物蛋白质，唾液酸是一种丰富和最 在粘液多聚糖上末端表达的碳水化合物。我们认为，虽然多层粘液多聚糖 在唾液酸有助于健康个体的微生物防御的情况下，唾液酸的去除是 肠道微生物渗入。为了验证这一假设，我们开发了基于紫外光激活的唾液酸 化学探针，不可逆地标记微生物样本中的所有宿主和细菌蛋白，能够 结合唾液酸(即唾液酸酶、凝集素、转运体、转移酶)。所有与探针结合的蛋白质都受到 基于亲和力的富集法和随后的蛋白质鉴定和质谱仪(MS)定量。 在这里，我们将使用我们的唾液酸探针和创新方法来进行公认的采用T细胞转移 建立小鼠慢性结肠炎模型，并直接将我们的代谢蛋白质组学结果与对照组的结果进行比较 在相同条件下饲养的小鼠。在健康和结肠炎小鼠之间观察到的差异可能与 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，作为狼人，我们完全有资格实现我们提出的目标 实验室率先开发和应用化学探针和基于MS的代谢蛋白质组学 找出在大肠菌群中过度丰富的细菌半胱氨酸蛋白酶。探测器、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.