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，其特点是针对肠道微生物群的异常免疫反应。目前的治疗方案专门针对宿主炎症途径，在控制疾病方面往往无效。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。越来越多的病例，加上缺乏有效的治疗方法，强调需要研究针对UC的新治疗措施。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描述了UC的宿主-微生物组相互作用，这些数据集显示了广泛的临床、内镜和组织学疾病活动。经过大规模的分析，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。宏蛋白质组学指出普通芽孢杆菌蛋白酶是其严重程度的显著特征。霰弹枪宏基因组学指导了分类推断，并揭示了B. vulgatus的关联主要是由蛋白质调控而不是微生物丰度驱动的。在患者血清中发现大量丝氨酸蛋白酶抑制剂，表明蛋白酶的重要性。后肽丘显示肽片段增加与UC疾病严重程度相关。一个独立的210人队列验证了普通双球菌蛋白酶与UC疾病严重程度之间的密切联系。为了验证我们的假设，我们证明了普通双球菌可以在体外破坏肠上皮的通透性，蛋白酶抑制足以恢复上皮屏障。将普通双球菌单定殖到无菌IL-10缺陷小鼠体内，表明定殖可诱导结肠炎，而蛋白酶抑制足以防止结肠炎的发生。此外，将含有过量普通芽孢杆菌蛋白酶的UC患者的粪便移植到无菌小鼠体内，可诱导依赖蛋白酶活性的结肠炎（Mills等人，Nature Microbiology, 2021）。基于我们已发表的工作，我们提出了两个可测试的假设，以进一步建立这些新发现：1)蛋白质水解是普通双球菌在体外和体内诱导与UC相关的结肠炎的中心机制；2)普通双球菌蛋白酶降解肠道屏障的关键成分。本申请的具体目的是定义基因组背景，作用，并开发针对UC中普通双球菌蛋白酶的特异性抑制剂（目的1），表征UC中普通双球菌介导的蛋白水解的靶点和动力学（目的2）。为了完成这一建议，我们将采用多方面的方法，包括最先进的生化、细胞培养、细菌遗传学和体内模型。最终，这项工作有可能在加州大学的基础研究和转化研究中开辟新的领域。

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.