Identification of microbiome proteins associated with IBD

描述(由申请人提供)：我们的建议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。人体肠道中含有大量多样的共生细菌，称为肠道微生物组，对人体新陈代谢、免疫发育和动态平衡以及上皮细胞血管生成至关重要。最近的研究表明，细菌产生的代谢物的异常水平发生在IBD微生物区系中，并表明有促炎倾向。我们假设代谢物的变化是由于与IBD相关的功能失调的肠道微生物群引起的细菌分泌酶活性水平的改变。为了验证这一假设，我们建议设计和使用反应性化学探针，这些探针不可逆地标记酶家族，并允许从复杂的蛋白质组中分离这些标记的蛋白质。这种浓缩技术与质谱仪(MS)相结合用于蛋白质的鉴定和定量。由于这是一项初步的概念验证元蛋白质组学研究，我们将使用这些化学标记技术来建立IL10/基因敲除的IBD小鼠模型，并分析小鼠远端肠道微生物组。将对IL10-/-小鼠的微生物群进行探测，以便与从具有相同基因谱系的正常小鼠身上获得的细菌进行直接比较。为此，我们将利用SILAM(哺乳动物稳定同位素标记)来确保对这些小鼠模型的健康和IBD相关共生细菌之间识别的蛋白质进行定量测量。重要的是，在健康和IBD相关小鼠群体之间观察到的任何蛋白质水平的差异都可能预测人类IBD中蛋白质和酶功能的偏差，因为元基因组分析表明细菌种类和在人类和小鼠微生物群中的分布高度相关。我们创新的化学生物学和偏蛋白组学方法是建立基础的先决条件，在此基础上：1)可以询问患有IBD的人类微生物群的变化；2)可以提高我们对微生物组动态平衡和肠道炎症变化的了解；3)我们可以增强操纵肠道细菌对抗各种人类疾病的能力。我们努力的成功结果将导致对一种可以适应人类的微生物组相关疾病进行第一次定量蛋白质组分析，并导致微生物组化学、生物学和生物医学研究方法的范式转变。

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.