Microbiome Driven Proteolysis as a Contributing Factor to Severity of Ulcerative Colitis Disease Activity

溃疡性结肠炎（UC）是一种以结肠粘膜炎症为特征的慢性疾病。UC具有显著的全球负担，其特征在于针对肠道微生物群的异常免疫应答。目前的治疗选择仅针对宿主炎症通路，并且通常在管理疾病方面无效。目前，北美有近100万人患有UC，其在人类中的患病率在全球范围内继续增长。大量病例的增加，加上缺乏有效的治疗方法，强调了研究新的UC治疗干预措施的必要性。我们的研究小组最近通过来自40名UC患者的6个基于粪便或血清的组学数据集表征了控制UC的宿主-微生物组相互作用，这些患者显示了广泛的临床、内窥镜和组织学疾病活动。经过广泛的分析，六个数据集为一个中心假设提供了有力的证据：普通拟杆菌蛋白酶可以驱动UC疾病的严重程度。元蛋白质组学确定了B。vulgatus蛋白酶作为严重性的区别特征。霰弹枪宏基因组学指导分类推断，并揭示了B。vulgatus关联主要由蛋白质调节驱动，而不是微生物丰度。在患者血清中发现的丝氨酸蛋白酶抑制剂的丰度表明蛋白酶的重要性。后肽组显示与UC疾病严重程度相关的肽片段增加。一个独立的210人队列验证了B之间的强联系。普通蛋白酶和UC疾病严重程度。验证我们的假设，我们证明了B。在体外实验中，普通肠杆菌可破坏肠上皮通透性，而蛋白酶抑制剂足以恢复上皮屏障。B的单定殖。将无菌IL-10缺陷型小鼠接种到无菌IL-10缺陷型小鼠中证明了定殖在这些动物中诱导结肠炎，并且蛋白酶抑制足以防止结肠炎发展。此外，移植来自具有过量B的UC患者的粪便。将普通酵母蛋白酶导入无菌小鼠中诱导依赖于蛋白酶活性的结肠炎（米尔斯等人，Nature Microbiology 2021）。基于我们已发表的工作，我们制定了两个可验证的假设，以进一步建立在这些新的发现：1）蛋白水解是一个中心机制利用的B。在体外和体内诱导与UC相关的结肠炎和2）B。普通蛋白酶降解肠屏障的关键组分。本申请的具体目的是定义基因组背景、作用，并开发针对B的特异性抑制剂。UC中的普通蛋白酶（目的1），表征B的靶标和动力学。vulgatus介导的UC蛋白水解（目的2）。为了完成这项提案，我们将使用最先进的生化，细胞培养，细菌遗传学和体内模型的多方面方法。最终，这项工作有可能在UC基础和转化研究中开辟新天地。

Ulcerative colitis (UC) is a chronic disease characterized by inflammation of the mucosa of the colon. UC has a significant global burden, and is characterized by an aberrant immune response directed towards the gut microbiota. Current treatment options exclusively target host inflammatory pathways and are often ineffective in managing disease. Nearly 1 million individuals in North America are currently living with UC and its prevalence in humans continues to grow worldwide. The considerable number of growing cases, coupled with the lack of an effective therapy, stresses the need for investigation into new therapeutic interventions against UC. Our group recently characterized host-microbiome interactions governing UC through six fecal or serum based –omic datasets from 40 UC patients displaying a wide range of clinical, endoscopic, and histologic disease activity. After broad-scale analyses, the six datasets provided powerful evidence towards a central hypothesis: Bacteroides vulgatus proteases can drive UC disease severity. Metaproteomics pinpointed B. vulgatus proteases as a distinguishing feature of severity. Shotgun metagenomics guided taxonomic inferences and revealed that the B. vulgatus association was driven primarily by protein regulation as opposed to microbial abundances. An abundance of serine protease inhibitors found in the patient serum suggested the importance of proteases. The metapeptidome showed increased peptide fragments correlated with UC disease severity. An independent 210-person cohort validated the strong connection between B. vulgatus proteases and UC disease severity. Testing our hypothesis, we demonstrate B. vulgatus can disrupt intestinal epithelial permeability in vitro, and protease inhibition was sufficient to restore epithelial barrier. Monocolonization of B. vulgatus into germfree IL-10 deficient mice demonstrated colonization induces colitis in these animals and protease inhibition is sufficient to prevent colitis development. Furthermore, transplantation of feces from UC patients with over-abundant B. vulgatus proteases into germ-free mice induced colitis dependent on protease activity (Mills et al., Nature Microbiology 2021). Based on our published work, we have formulated two testable hypotheses to further build on these new findings: 1) proteolysis is a central mechanism utilized by B. vulgatus to induce colitis in vitro and in vivo relevant to UC and 2) B. vulgatus proteases degrade key components of the intestinal barrier. The specific aims of this application are to define the genomic context, roles, and develop specific inhibitors against B. vulgatus proteases in UC (Aim 1), characterize the targets and dynamics of B. vulgatus mediated proteolysis in UC (Aim 2). To complete this proposal, we will use a multifaceted approach of state-of-the-art biochemical, cell culture, bacterial genetics, and in vivo models. Ultimately, this work has the potential to break new ground in UC basic and translational research.