Interrogating the Cellular Mechanisms of Host-Pathogen Interactions during IBD

描述（由申请方提供）：炎症性肠病（IBD）定义为肠粘膜的严重慢性炎症，导致呕吐、腹泻、出血、腹痛和体重减轻。目前的估计表明，有300万至400万人患有IBD，每年有更多的人被诊断出来。IBD的原因尚不清楚，可能是多因素的。许多证据表明，IBD的起始涉及肠上皮细胞处的异常宿主-微生物相互作用。一种通常与IBD患者的肠粘膜相关但不来自健康受试者的微生物被称为粘附侵袭性E。coli（AIEC）。AIEC是一种新的致病型，在缺乏与其他致病性大肠杆菌相关的已知毒力因子的情况下，它们能够粘附并侵入上皮细胞。杆菌尚未对AIEC感染的细胞反应进行全面分析，目前尚不清楚IBD肠道环境是否选择人类AIEC感染。本基金的总体目标是：A）定义和比较感染AIEC（目标1）和IBD（目标2）的人上皮细胞中蛋白质组的改变，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导致上皮细胞对AIEC感染更加宽容。在体外，AIEC感染细胞，而不是其他致病型或大肠杆菌。自噬途径的蛋白质标记物水平增加。自噬是降解细胞内容物的细胞内在反应，其可以响应于细胞内细菌感染而被激活。人类自噬基因的多态性也与IBD的风险显著升高相关。因此，我认为自噬途径中蛋白质水平的改变发生在患有IBD和感染AIEC的人类上皮细胞中，导致细胞内感染的异常控制。为了研究这一假设，我将结合联合收割机一个公正的蛋白质组学方法，使用定量质谱与互补的功能获得和功能丧失的方法，以评估是否有必要和/或足够的变化，在特定的宿主蛋白质的丰度影响AIEC的附着，入侵，或细胞内 坚持不懈这些研究将确定AIEC和IBD诱导的宿主蛋白质的变化，其规模前所未有，并将阐明参与AIEC反应和IBD诱导的途径。我们表征限制AIEC感染的宿主细胞内在机制的研究还可以揭示新的细胞药物靶标，其可以用于开发能够抑制细胞内感染和/或减少IBD发病机制的微生物组分的“基于宿主的”抗生素。通过鉴定参与IBD患者发病的细胞变化，我们的建议也可能导致IBD生物标志物的鉴定，以促进早期诊断，这是中断IBD发病机制慢性周期的目标。

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.