Interrogating the Cellular Mechanisms of Host-Pathogen Interactions during IBD

描述(申请人提供)：炎症性肠病(IBD)被定义为严重的、慢性的肠道粘膜炎症，导致呕吐、腹泻、出血、腹痛和体重减轻。目前的估计表明，每年有300万到400万人患有IBD，而且被诊断出的人更多。炎症性肠病的病因(S)尚不清楚，可能是多因素造成的。许多证据表明，IBD的启动涉及肠道上皮异常的宿主-微生物相互作用。一种通常与IBD患者的肠道粘膜有关而不是来自健康人的微生物被称为粘附性侵袭性大肠杆菌(AIEC)。AIEC是一种新的致病类型，其定义是在缺乏与其他致病大肠杆菌相关的已知毒力因子的情况下，它们黏附和侵袭上皮细胞的能力。对AIEC感染的细胞反应还没有进行全面的分析，也不清楚IBD肠道环境是否选择了人类感染AIEC。这项资助的总体目标是：A)定义和比较感染AIEC(目标1)和感染IBD(目标2)的人上皮细胞中蛋白质组如何改变，以及B)确定观察到的任一种情况(目标1和2)的蛋白质丰度差异是否会导致上皮细胞更容易受到AIEC感染。在体外，感染了AIEC的细胞，而不是其他致病类型或共生大肠杆菌，增加了自噬途径的蛋白质标记物的水平。自噬是一种细胞固有的反应，它降解细胞内的内容物，这种反应可以在细胞内感染细菌时被激活。人类自噬基因的多态也与明显更高的IBD风险相关。因此，我认为在感染IBD和感染AIEC的人上皮细胞中，自噬途径中的蛋白质水平发生了变化，导致细胞内感染的异常控制。为了研究这一假设，我将结合使用定量质谱学的无偏蛋白质组学方法和互补的功能获得和功能丧失方法来评估特定宿主蛋白丰度的变化是否必要和/或足以影响aIEC的附着、侵袭或细胞内。 坚持不懈。这些研究将确定AIEC和IBD诱导的宿主蛋白质变化的规模，这是前所未有的，并将阐明参与AIEC反应和IBD诱导的途径。我们对限制AIEC感染的宿主细胞内在机制(S)的研究也可能揭示新的细胞药物靶点，这些靶点可能有助于开发能够抑制细胞内感染和/或减少炎症性肠病发病机制中的微生物成分的“基于宿主的”抗生素。通过确定患者IBD发病过程中涉及的细胞变化，我们的建议还可能导致识别IBD生物标记物，以便于早期诊断，从而中断IBD发病的慢性循环。

DESCRIPTION (provided by applicant): Inflammatory bowel disease (IBD) is defined as severe, chronic inflammation of the intestinal mucosa, resulting in vomiting, diarrhea, bleeding, abdominal pain and weight loss. Current estimates suggest that 3-to-4 million people suffer from IBD and more are diagnosed each year. The cause(s) of IBD is not known and likely to be multi-factorial. Many lines of evidence suggest that the initiation of IBD involves abnormal host-microbial interactions at the intestinal epithelium. One microorganism that is commonly associated with the intestinal mucosa of IBD patients but not from healthy subjects is called adherent-invasive E. coli (AIEC). AIEC are a new pathotype defined by their ability to adhere to and invade epithelial cells in the absence of known virulence factors associated with other pathogenic E. coli. A comprehensive analysis of the cellular response to AIEC infection has not been performed, and it is not clear whether the IBD gut environment selects for AIEC infection in humans. The overall goals of this grant are to A) define and compare how the proteome is altered in human epithelial cells infected with AIEC (Aim 1) and affected with IBD (Aim 2), and B) determine whether any of the observed differences in protein abundance from either condition (Aim 1&2) causes epithelial cells to become more permissive to AIEC infection. In vitro, cells infected with AIEC, but not other pathotypes or commensal E. coli, have increased levels of a protein marker of the autophagy pathway. Autophagy is a cell-intrinsic response that degrades cellular contents, which can be activated in response to intracellular infection with bacteria. Polymorphisms in autophagy genes in humans are also associated with a significantly higher risk of IBD. Therefore, I propose that altered levels of proteins in the autophagy pathway occur in human epithelial cells affected with IBD and infected with AIEC, leading to aberrant control of intracellular infection. To investigate this hypothesis, I will combine an unbiased proteomic approach using quantitative mass spectrometry with a complementary gain-of-function and loss-of-function approach to assess whether changes in the abundance of specific host proteins are necessary and/or sufficient to affect AIEC attachment, invasion, or intracellular persistence. These studies will identify AIEC- and IBD-induced changes to host proteins on a scale never previously possible and will elucidate pathways that are involved in the response to AIEC and in the induction of IBD. Our studies to characterize the host cell-intrinsic mechanism(s) that restrict AIEC infection may also reveal novel cellular drug targets that may be useful in developing "host- based" antibiotics capable of inhibiting intracellular infection, and/or diminishing the microbial component of IBD pathogenesis. By identifying cellular changes involved in the onset of IBD in patients, our proposal may also lead to the identification of IBD biomarkers to facilitate early diagnosis, a goal for interrupting the chronic cycle of IBD pathogenesis.