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：
10663379
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/10663379
关键词：
Acceleration Actins Adherent Invasive Escherichia coli 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 Cells Epithelium 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 Play Polymers 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 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 pharmacologic 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）提供了针对肠道致病细菌的第一道防线。然而细菌病原体开发了不同的策略来定植肠上皮，导致严重胃肠道疾病。一种策略，是通过“附着和效率”细菌（AEB）所使用的，例如肠病毒大肠杆菌（EPEC）和肠ho骨大肠杆菌（EHEC）涉及对IEC的细菌粘附不输入宿主单元。辅助侵入性大肠杆菌（AIEC）或沙门氏菌使用的另一种策略导致细菌内部化到IEC。这些不同的殖民路线具有关键机制相似性：它们需要上皮肌动蛋白细胞骨架的重排才使细菌附着和入侵。病原体诱导的细胞骨架重塑的一种重要但了解的机制涉及主要的肌动蛋白细胞骨架运动，非肌肉肌球蛋白II（NM-II）。我们的初步数据证明了罢工 NM-II依赖性细菌相互作用的二元论。药理学和遗传抑制 NM-II增加了AEB对IEC的附着，但抑制了AIEC在体外和体内的侵袭。这些对比的角色 IEC - 细菌相互作用中的NM-II可能反映了利用两个不同的NM II依赖性功能：其肌动蛋白不同病原体的细丝收缩和肌动蛋白交联活动。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Enter中起双重作用病原体与肠上皮相互作用，通过抑制AEB附着在上皮细胞上，但促进入侵致病细菌的上皮进入。该假设将在以下目的中进行检验：（1）确定NM-II在调节肠道中的作用上皮细胞与附着和效率细菌的相互作用；（2）剖析NM-II驱动的机制入侵细菌将细菌的内在化到肠上皮细胞中。我们将研究EPEC，EHEC，AIEC和 Model IEC细胞系的沙门氏菌定植，体外和肠道中衍生的IEC单层IEC单层在体内通过柠檬酸杆菌，AIEC和沙门氏菌定植。 NM-II活动将由药理学和遗传方法。遗传方法将针对两个主要的上皮NM-II同工型，即 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。在体内NM-II功能将通过使用两种肠的小鼠检查 NM-IIA的上皮特异性敲除或NM-IIC的总敲除。确定哪种NM-II活动是对于细菌相互作用所必需的，我们将利用表达NM-IIA突变体的IEC细胞和转基因小鼠缺乏肌动蛋白丝交联或细丝合同活动。意义：提案将揭示新的见解，以了解肠上皮如何控制致病细菌感染并将确定治疗由肠道病原体引起的治疗疾病的新目标。