Novel cytoskeletal mechanisms of pathogenic bacteria interactions with intestinal epithelium

摘要 肠上皮细胞（IEC）是抵抗肠道致病菌的第一道防线。然而 细菌病原体发展了不同的策略来定植肠上皮， 肠胃疾病一种策略，用于'附着和抹去'细菌（AEB），如 肠致病性E.大肠埃希菌（EPEC）和肠出血性大肠杆菌（E.大肠埃希菌（EHEC），涉及细菌粘附到IEC 而不进入宿主细胞。另一种策略是粘附性侵袭性E.大肠杆菌（AIEC）或沙门氏菌 导致细菌内化到IEC中。这些不同的殖民路线承载着一个关键的机制， 相似性：它们需要上皮肌动蛋白细胞骨架的重排以使细菌附着， 入侵病原体诱导的细胞骨架重塑的一个重要但未充分研究的机制涉及一种 主要肌动蛋白细胞骨架马达，非肌肉肌球蛋白II（NM-II）。我们的初步数据显示 细菌-IEC相互作用的NM-II依赖性调节的双重性。药理学和遗传学抑制 在体外和体内，NM-II增加AEB对IEC的附着，但抑制AIEC的侵袭。这些截然不同的角色 NM-II在IEC-细菌相互作用中的作用可能反映了利用两种不同的NM-II依赖性功能： 丝收缩性和肌动蛋白丝交联活性，由不同的病原体。这些令人兴奋的数据提供了 以下创新假设的科学前提：NM-II在调节肠粘膜中起双重作用， 病原体通过抑制AEB附着于上皮细胞与肠上皮相互作用，但 促进入侵的病原菌进入上皮。 本研究的主要目的是：（1）确定NM-II在调节肠上皮细胞凋亡中的作用; 上皮细胞与附着和消退细菌的相互作用;（2）剖析NM-II驱动的 入侵细菌进入肠上皮细胞的内在化。我们将研究EPEC、EHEC、AIEC和 沙门氏菌在模型IEC细胞系、类器官衍生的IEC单层的体外和肠内定植 啮齿类柠檬酸杆菌、AIEC和沙门氏菌在体内定植。NM-II活性将被调节， 药理学和遗传学方法。遗传方法将靶向两种主要的上皮NM-II亚型， NM-IIA和NM-IIC，通过使用CRISPR/Cas9基因选择性下调其在人IEC中的表达 编辑和RNA干扰。体内NM-II功能将通过使用具有肠或肠上皮细胞的小鼠来检查。 上皮特异性敲除NM-IIA或完全敲除NM-IIC。为了确定哪种NM-II活性是 对于细菌-IEC相互作用至关重要，我们将利用IEC细胞和表达NM-IIA突变体的转基因小鼠 缺乏肌动蛋白丝交联或丝收缩活动。意义：提案将 揭示了了解肠上皮细胞如何控制致病性细菌感染的新见解 并将确定治疗干预的新靶点，以治疗由肠道病原体引起的疾病。

ABSTRACT Intestinal Epithelial Cells (IEC) provide the first line of defense against enteric pathogenic bacteria. However bacterial pathogens developed different strategies to colonize intestinal epithelium causing severe gastrointestinal disorder. One strategy, used by ‘attaching and effacing’ bacteria (AEB), such as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), involves bacterial adhesion to IEC without entering the host cells. The other strategy used by adherent Invasive E. coli (AIEC) or Salmonella results in bacterial internalization into IEC. These different colonization routes bear a key mechanistic similarity: they require rearrangements of the epithelial actin cytoskeleton to enable bacterial attachment and invasion. One important but understudied mechanism of pathogen-induced cytoskeletal remodeling involves a major actin cytoskeleton motor, non-muscle myosin II (NM-II). Our preliminary data demonstrate a striking dualism of NM-II-dependent regulation of bacterial-IEC interactions. Pharmacologic and genetic inhibition of NM-II increases AEB attachment to IEC, but inhibits AIEC invasion in vitro and in vivo. These contrasting roles of NM-II in IEC-bacterial interactions likely reflect exploiting two different NM II-dependent functions: its actin filament contractility and actin filament cross-linking activity, by different pathogens. This exciting data provides a scientific premise for the following innovative hypothesis: NM-II plays a dual role in regulating enteric pathogen interactions with intestinal epithelium by inhibiting AEB attachment to epithelial cells, but promoting epithelial entry of invading pathogenic bacteria. This hypothesis will be tested in the following Aims: (1) to determine the roles of NM-II in regulating intestinal epithelial cell interactions with attaching and effacing bacteria; (2) to dissect the mechanisms of NM-II-driven internalization of invading bacteria into intestinal epithelial cells. We will study EPEC, EHEC, AIEC and Salmonella colonization of model IEC cell lines, organoid-derived IEC monolayers in vitro and intestinal colonization by Citrobacter rodentium, AIEC and Salmonella in vivo. NM-II activity will be modulated by pharmacologic and genetic approaches. The genetic approach will target two major epithelial NM-II isoforms, NM-IIA and NM-IIC, by selectively downregulating their expression in human IEC using CRISPR/Cas9 gene editing and RNA interference. In vivo NM-II functions will be examined by using mice with either intestinal epithelial specific knockout of NM-IIA, or total knockout of NM-IIC. To determine which NM-II activity is essential for bacterial-IEC interactions, we will utilize IEC cells and transgenic mice expressing NM-IIA mutants deficient in either actin filament cross-linking, or filament contraction activities. Significance: the proposal will reveal novel insights into understanding how the intestinal epithelium controls pathogenic bacterial infections and will identify new targets for therapeutic interventions to treat diseases caused by enteric pathogens.