Identification of microbiome proteins associated with IBD

描述（由申请人提供）：我们的提案旨在通过鉴定和表征与炎症性肠病（IBD）相关的细菌蛋白质和酶活性，将远端肠道微生物组研究领域从基因组学基础推进到定量蛋白质组学。人体肠道内有大量多样的肠道细菌，称为肠道微生物组，它们对人体代谢、免疫发育和体内平衡以及上皮细胞血管生成至关重要。最近的研究表明，IBD微生物群中细菌产生的代谢物水平异常，表明促炎倾向。我们假设代谢物的变化是由于功能失调的IBD相关远端肠道微生物组引起的细菌分泌的酶活性水平改变。为了验证这一假设，我们建议设计和使用反应性化学探针，不可逆地标记酶家族，并允许这些标记的蛋白质从复杂的蛋白质组中分离。这种富集技术与质谱法（MS）结合使用，用于蛋白质鉴定和定量。由于这是初步的概念验证元蛋白质组学研究，我们将这些化学标记技术用于IL 10-/-敲除IBD小鼠模型并分析鼠远端肠道微生物组。将探测IL 10-/-小鼠微生物群，以与从相同遗传谱系的正常小鼠收获的细菌进行直接比较。为此，我们将利用SILAM（哺乳动物的稳定同位素标记）来确保定量测量这些小鼠模型的健康和IBD相关肠道细菌之间的鉴定的蛋白质。重要的是，在健康和IBD相关小鼠群体之间观察到的蛋白质水平的任何差异都可以预测人类IBD中蛋白质和酶功能的偏差，因为宏基因组学分析已经显示出人类和小鼠微生物组中细菌种类和分布的高度相关性。我们创新的化学生物学和元蛋白质组学方法是建立基础的先决条件：1）可以询问IBD患者的微生物组改变，2）可以提高我们对微生物组稳态和肠道炎症改变的理解，3）可以增强我们操纵肠道细菌对抗各种人类疾病的能力。我们的努力的成功结果将导致第一次定量蛋白质组学分析的微生物组相关疾病，可以适应人类，以及导致范式转变的方法，以化学，生物学和生物医学调查的微生物组。

DESCRIPTION (provided by applicant): Our proposal seeks to advance the field of distal gut microbiome research from its basis in genomics into quantitative proteomics through identification and characterization of bacterial proteins and enzymatic activities that are associated with inflammatory bowel diseases (IBD). The human intestinal tract harbors an enormous and diverse collection of commensal bacteria, termed the gut microbiome, that are essential for human metabolism, immune development and homeostasis, and epithelial cell angiogenesis. Recent studies demonstrate that aberrant levels of bacterially produced metabolites occur in IBD microbiota and are indicative of a pro-inflammatory predisposition. We hypothesize that the changes in metabolites are due to altered levels of bacterially secreted enzymatic activities arising from a dysfunctional IBD-associated distal gut microbiome. To test this hypothesis, we propose to design and use reactive chemical probes that irreversibly label enzymatic families and permit the isolation of these tagged proteins from complex proteomes. This enrichment technique is used in combination with mass spectrometry (MS) for protein identification and quantitation. As this is an initial proof-of-concept meta-proteomic study, we wil employ these chemical labeling techniques to an IL10-/- knockout IBD mouse model and analyze the murine distal gut microbiome. The IL10-/- mice microbiota will be probed for direct comparison to bacteria harvested from normal mice of an identical genetic lineage. To this end, we will exploit the use of SILAM (stable isotope labeling of mammals) to ensure quantitative measurement of identified proteins between healthy and IBD- associated commensal bacteria of these mouse models. Importantly, any differences in protein levels observed between healthy and IBD-associated mice populations may forecast deviations in proteins and enzyme functions in human IBD as metagenomic analyses have shown a high correlation of bacterial species and distribution within the microbiomes of human and mouse. Our innovative chemical biology and meta-proteomic methodologies are prerequisites to establish a foundation on which: 1) altered microbiomes of humans with IBD can be interrogated, 2) our understanding of microbiome homeostasis and alteration in intestinal inflammation can be improved, and 3) our ability to manipulate gut bacteria to combat a variety of human ailments can be enhanced. A successful outcome of our efforts will result in the first quantitative proteomic analysis of a microbiome-associated disease that can be adapted to humans, as well as result in a paradigm shift in approaches to chemical, biological, and biomedical investigations of the microbiome.