EHEC-induced actin rearrangement and Stx2 translocation across epithelium

EHEC 诱导的肌动蛋白重排和 Stx2 跨上皮易位

基本信息

批准号：
8207883
负责人：
JOHN M LEONG
金额：
$ 24.75万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-01-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8207883
关键词：
Actins Address Alleles Anemia Animal Model Apical Bacteria Bacterial Toxins Bacterial Translocation Bacteriophages Binding Blood Circulation Body Weight decreased Cells Cessation of life Citrobacter rodentium Cytoskeleton DNA Sequence Rearrangement Development Disease Employee Strikes Epithelial Epithelial Cells Epithelium Escherichia coli EHEC Escherichia coli Infections Escherichia coli O157 Europe F-Actin Hemolytic-Uremic Syndrome In Vitro Infection Integration Host Factors Intestines Japan Kidney Kidney Failure Lesion Life Measures Mediating Modeling Mus North America Outcome Study Pathogenesis Permeability Process Production Proteins Regulation Structure Surface System Systemic disease Testing Toxin Virulence Virulence Factors absorption cellular microvillus enteropathogenic Escherichia coli foodborne pathogen in vivo Model intestinal epithelium monolayer mouse model mutant novel pathogen public health relevance research study

项目摘要

DESCRIPTION (provided by applicant): Enterohemorrhagic E. coli (EHEC) O157:H7 produces the phage-encoded toxin Stx, which can be absorbed from the intestine and cause life-threatening hemolytic uremic syndrome (HUS), featuring anemia, thromobcytopenia and renal failure. The absorption of Stx across the intestinal barrier is poorly understood. Given that the epithelial cytoskeleton is integral to the regulation of transcytotic and paracellular transport, Stx translocation may be facilitated by the ability of EHEC to dramatically alter host filamentous (F-) actin. EHEC, along with enteropathogenic E. coli (EPEC) and the natural mouse pathogen Citrobacter rodentium, trigger the formation of attaching and effacing (AE) lesions on epithelial cells. These lesions are characterized by intimate attachment and rearrangement of F-actin to form "pedestals" beneath bound bacteria. The ability to generate AE lesions requires the type III translocation of bacterial effector proteins, and the effectors Tir and EspFU have been implicated in the formation of actin pedestals. In addition, EspFU can promote the disruption of epithelial barriers in vitro. We identified the activities of Tir and EspFU required for pedestal formation and have isolated numerous mutants that are incapable of promoting localized actin assembly. Recently, to examine the pathogenesis of Stx-mediated disease, we generated a murine model for EHEC infection utilizing C. rodentium lysogenized with a phage encoding Stx2, an allele of Stx particularly associated with HUS. Mice infected with C. rodentium (?Stx2) suffer Stx-dependent renal damage, weight loss and eventual death. We have also constructed derivatives of C. rodentium (?Stx2) that express EHEC virulence factors, including Tir and/or EspFU, and a pilot experiment was suggestive that the ability to generate cytoskeletal changes may enhance virulence in this model. To test the hypothesis that EHEC mediated actin rearrangement contributes to the disruption of intestinal epithelial barrier function and enhancement of Stx-mediated disease, we will pursue the following aims: 1.Assess the ability of EHEC-mediated actin rearrangement to enhance Stx2 translocation across polarized epithelial cells in vitro. Polarized monolayers will be infected with (a non-Stx- producing) EHEC expressing wild type Tir and EspFU, or with derivatives that are incapable of triggering actin rearrangement. Purified Stx2d will be added to the apical surface and its translocation across infected monolayers of T84 intestinal epithelial cells will be measured. 2. Assess the ability of EspFU to enhance Stx2-mediated disease in a mouse model for EHEC. Mice will be orally infected with C. rodentium (?Stx2) expressing wild type TirEHEC and EspFU, or with derivatives of these effectors that specifically lack activities required for triggering actin rearrangement. Bacterial colonization, intestinal permeability, and systemic disease will be assessed. PUBLIC HEALTH RELEVANCE: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen in North America, Europe and Japan (79). The life-threatening systemic manifestations of infection are due to the absorption of bacterial toxin from the intestine (51, 52). EHEC binds to cells of the gut wall and triggers the host cell to generate striking pedestal-like structures beneath bound bacteria (33), a process that may promote the absorption of toxin into the mammalian bloodstream. These studies investigate whether the ability of EHEC to induce pedestals promotes systemic disease.

描述（由申请人提供）：肠出血性大肠杆菌 (EHEC) O157:H7 产生噬菌体编码的毒素 Stx，该毒素可从肠道吸收并导致危及生命的溶血性尿毒症综合征 (HUS)，其特征为贫血、血小板减少症和肾衰竭失败。人们对 Stx 穿过肠道屏障的吸收知之甚少。鉴于上皮细胞骨架对于转细胞和细胞旁运输的调节是不可或缺的，EHEC 显着改变宿主丝状 (F-) 肌动蛋白的能力可能会促进 Stx 易位。 EHEC 与肠致病性大肠杆菌 (EPEC) 和天然小鼠病原体啮齿类柠檬酸杆菌一起引发上皮细胞上附着和擦除 (AE) 病变的形成。这些病变的特点是 F-肌动蛋白紧密附着和重新排列，在结合的细菌下方形成“基座”。产生 AE 损伤的能力需要细菌效应蛋白的 III 型易位，效应蛋白 Tir 和 EspFU 与肌动蛋白基座的形成有关。此外，EspFU可以在体外促进上皮屏障的破坏。我们鉴定了基座形成所需的 Tir 和 EspFU 的活性，并分离出许多无法促进局部肌动蛋白组装的突变体。最近，为了研究 Stx 介导的疾病的发病机制，我们利用编码 Stx2（一种与 HUS 特别相关的 Stx 等位基因）的噬菌体溶原化的啮齿类梭菌，建立了 EHEC 感染的小鼠模型。感染啮齿类念珠菌 (?Stx2) 的小鼠会遭受 Stx 依赖性肾损伤、体重减轻并最终死亡。我们还构建了表达 EHEC 毒力因子（包括 Tir 和/或 EspFU）的 C. rodentium (?Stx2) 衍生物，初步实验表明，产生细胞骨架变化的能力可能会增强该模型的毒力。为了检验 EHEC 介导的肌动蛋白重排会破坏肠上皮屏障功能并增强 Stx 介导的疾病这一假设，我们将追求以下目标： 1.评估 EHEC 介导的肌动蛋白重排在体外增强 Stx2 跨极化上皮细胞易位的能力。极化单层将被表达野生型 Tir 和 EspFU 的（不产生 Stx 的）EHEC 或无法触发肌动蛋白重排的衍生物感染。将纯化的 Stx2d 添加到顶端表面，并测量其在受感染单层 T84 肠上皮细胞中的易位。 2. 评估 EspFU 在 EHEC 小鼠模型中增强 Stx2 介导的疾病的能力。小鼠将被口服感染表达野生型 TirEHEC 和 EspFU 的啮齿类 C. rodentium (?Stx2)，或这些效应器的衍生物，这些效应器特别缺乏触发肌动蛋白重排所需的活性。将评估细菌定植、肠道通透性和全身性疾病。公共卫生相关性：肠出血性大肠杆菌 (EHEC) O157:H7 是北美、欧洲和日本的一种重要食源性病原体 (79)。感染的危及生命的全身表现是由于肠道吸收细菌毒素所致(51, 52)。 EHEC 与肠壁细胞结合，并触发宿主细胞在结合的细菌下方产生引人注目的基座状结构 (33)，这一过程可能会促进哺乳动物血液吸收毒素。这些研究调查了肠出血性大肠杆菌诱导基座的能力是否会促进全身性疾病。