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Novel cytoskeletal mechanisms of pathogenic bacteria interactions with intestinal epithelium

病原菌与肠上皮相互作用的新细胞骨架机制

基本信息

批准号：
10516636
负责人：
Andrei Ivanovich Ivanov
金额：
$ 50.68万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-15 至 2027-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10516636
关键词：
Actins Antibiotic Therapy Attenuated Automobile Driving Bacteria Bacterial Adhesion Bacterial Antibiotic Resistance Bacterial Attachment Site Bacterial Infections CRISPR/Cas technology Cell Communication Cell Line Cell surface Cells Chemicals Chronic Citrobacter rodentium Cytoskeleton Data Disease Economic Burden Elderly Enteral Epithelial Epithelial Cells Escherichia coli Escherichia coli EHEC Event Filament Gastroenteritis Gastrointestinal Diseases Genes Genetic Health Hemolytic-Uremic Syndrome Human Immunocompromised Host In Vitro Infant Infection Inflammatory Inflammatory Bowel Diseases Intestinal Diseases Intestinal Mucosa Invaded Knock-out Knockout Mice Mediating Microfilaments Modeling Molecular Molecular Chaperones Morbidity - disease rate Motor Motor Activity Mus Myosin ATPase Myosin Type II Organoids Outcome Pathogenesis Pathogenicity Pathway interactions Patients Persons Pharmacology Play Prevention Process Protein Isoforms Proteins Public Health RNA Interference Regulation Role Route Salmonella Salmonella enterica Severities Shigella Symptoms Systemic disease Testing Therapeutic Intervention Transgenic Mice Ursidae Family base blebbistatin cohort crosslink enteric infection enteric pathogen enteropathogenic Escherichia coli gastrointestinal genetic approach genetic manipulation gut colonization improved in vivo inhibitor innovation insight intestinal epithelium monolayer mortality mutant new therapeutic target non-muscle myosin novel novel therapeutic intervention novel therapeutics pathogen pathogenic bacteria polymerization receptor

项目摘要

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.

抽象的肠上皮细胞 (IEC) 是抵御肠道致病菌的第一道防线。然而细菌病原体发展出不同的策略来定植肠上皮，导致严重的胃肠道疾病。 “附着和消除”细菌 (AEB) 使用的一种策略，例如肠致病性大肠杆菌 (EPEC) 和肠出血性大肠杆菌 (EHEC)，涉及细菌粘附到 IEC 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为 IEC。这些不同的殖民路线具有关键的机制相似之处：它们需要上皮肌动蛋白细胞骨架的重新排列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出惊人的细菌与 IEC 相互作用的 NM-II 依赖性调节的二元性。药理学和遗传抑制 NM-II 增加 AEB 对 IEC 的附着，但在体外和体内抑制 AIEC 入侵。这些对比鲜明的角色 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：其肌动蛋白不同病原体的肌丝收缩性和肌动蛋白丝交联活性。这个令人兴奋的数据提供了以下创新假设的科学前提：NM-II 在调节肠道中发挥双重作用通过抑制 AEB 对上皮细胞的附着来抑制病原体与肠上皮的相互作用，但是促进入侵病原菌进入上皮细胞。该假设将通过以下目的进行检验：（1）确定NM-II在调节肠道中的作用上皮细胞与附着和消除细菌的相互作用； (2)剖析NM-II驱动机制将入侵的细菌内化到肠上皮细胞中。我们将学习 EPEC、EHEC、AIEC 和 IEC 细胞系模型、体外和肠道类器官衍生的 IEC 单层的沙门氏菌定植啮齿类柠檬酸杆菌、AIEC 和沙门氏菌在体内定植。 NM-II 活性将受到调节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， NM-IIA 和 NM-IIC，通过使用 CRISPR/Cas9 基因选择性下调其在人类 IEC 中的表达编辑和RNA干扰。体内 NM-II 功能将通过使用具有任一肠道的小鼠进行检查 NM-IIA 的上皮特异性敲除，或 NM-IIC 的完全敲除。确定 NM-II 的活性对于细菌-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肌动蛋白丝交联或丝收缩活性缺陷。意义：该提案将揭示了解肠上皮如何控制致病细菌感染的新见解并将确定治疗肠道病原体引起的疾病的治疗干预的新目标。