Novel cytoskeletal mechanisms of pathogenic bacteria interactions with intestinal epithelium

摘要 肠上皮细胞(IEC)是抵御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，导致严重的 胃肠道疾病。其中一种策略是“附着和清除”细菌(AEB)，例如 肠致病性大肠杆菌(EPEC)和肠出血性大肠杆菌(EHEC)涉及细菌对IEC的黏附 而不进入宿主细胞。粘附性侵袭性大肠杆菌(AIEC)或沙门氏菌使用的另一种策略 导致细菌内化到IEC。这些不同的殖民路线承载着一个关键的机制 相似性：它们需要重新排列上皮肌动蛋白细胞骨架，以实现细菌附着和 入侵。病原体诱导的细胞骨架重塑的一个重要但尚未被研究的机制涉及 主要肌动蛋白细胞骨架运动，非肌肉肌球蛋白II(NM-II)。我们的初步数据显示， NM-II依赖的细菌-IEC相互作用调节的二元性。药理和遗传抑制作用 NM-II促进AEB与IEC的黏附，但在体内外抑制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对NM-II两种不同依赖功能的利用：其肌动蛋白 细丝的收缩能力和肌动蛋白细丝的交联活性，受不同病原体的影响。这一令人兴奋的数据提供了 以下创新假说的科学前提：NM-II在肠道调节中具有双重作用 病原体通过抑制AEB与肠上皮细胞的附着而与肠上皮细胞相互作用，但 促进入侵病原菌的上皮细胞进入。 这一假说将在以下目的中得到检验：(1)确定NM-II在调节肠道中的作用 上皮细胞与附着和清除细菌的相互作用；(2)剖析NM-II驱动的机制 侵入的细菌内化到肠上皮细胞。我们将研究EPEC、EHEC、AIEC和 沙门氏菌在模型IEC细胞系、有机类IEC单层细胞和肠道中的定植 轮状柠檬酸杆菌、AIEC和沙门氏菌在体内的定植NM-II的活动将由 药理学和遗传学方法。遗传方法将针对两种主要的上皮NM-II亚型， 利用CRISPR/Cas9基因选择性下调NM-IIA和NM-IIC在人IEC中的表达 编辑和RNA干扰。在体内，NM-II的功能将通过使用小鼠的肠道或 NM-IIA的上皮特异性敲除，或NM-IIC的完全敲除。要确定哪个NM-II活动是 对于细菌-IEC的相互作用，我们将利用IEC细胞和表达NM-IIA突变体的转基因小鼠 缺乏肌动蛋白微丝交联或微丝收缩活性。意义：该提案将 揭示了解肠道上皮如何控制病原性细菌感染的新见解 并将确定治疗干预措施的新目标，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.