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

• 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
• 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

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(申请人提供)：肠出血性大肠杆菌(EHEC)O157：H7产生噬菌体编码毒素STX，可从肠道吸收，导致危及生命的溶血性尿毒症综合征(HUS)，特征是贫血、血小板减少和肾功能衰竭。对STX通过肠道屏障的吸收知之甚少。鉴于上皮细胞骨架对跨细胞和细胞旁运输的调节是不可或缺的，EHEC显著改变宿主丝状(F-)肌动蛋白的能力可能会促进STX的易位。EHEC与肠道致病大肠杆菌(EPEC)和自然的小鼠病原体轮状柠檬酸杆菌一起，触发上皮细胞附着和消失(AE)病变的形成。这些损伤的特点是F-肌动蛋白紧密附着和重排，在结合的细菌下形成“基座”。发生AE损伤的能力需要细菌效应器蛋白的III型易位，而效应器TIR和EspFu与肌动蛋白底座的形成有关。此外，EspFu在体外可促进上皮屏障的破坏。我们鉴定了底座形成所需的TIR和EspFu的活性，并分离出了许多不能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，我们建立了一种EHEC感染的小鼠模型，该模型利用编码STX的Stx2的噬菌体溶原化的轮状芽孢杆菌，Stx2是STX的一个与HUS特别相关的等位基因。感染轮状芽胞杆菌(？Stx2)的小鼠会出现STX依赖性的肾脏损伤、体重减轻和最终死亡。我们还构建了表达EHEC毒力因子(包括TIR和/或EspFu)的轮状芽胞杆菌(？Stx2)的衍生物，初步实验表明，在这个模型中，产生细胞骨架变化的能力可能会增强毒力。为了验证EHEC介导的肌动蛋白重排有助于破坏肠上皮屏障功能和增强STX介导的疾病的假说，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促进Stx2跨极化上皮细胞易位的能力。极化的单层将感染(不产生STX的)表达野生型TIR和EspFu的EHEC，或者感染不能触发肌动蛋白重排的衍生物。将纯化的Stx2d加入到根尖表面，并测量其跨感染的T84肠上皮细胞单层的移位。 2.在EHEC小鼠模型中评估EspFu增强Stx2介导疾病的能力。小鼠将被口服感染表达野生型TirEHEC和EspFu的轮状芽孢杆菌(？Stx2)，或这些效应物的衍生物，这些效应物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的食源性病原体(79)。威胁生命的全身感染表现是由于肠道吸收了细菌毒素(51，52)。EHEC与肠壁细胞结合，并触发宿主细胞在结合的细菌(33)下产生引人注目的基座状结构，这一过程可能会促进毒素吸收到哺乳动物的血液中。这些研究调查了EHEC诱导基座的能力是否促进了全身性疾病。