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.

性状（由申请方提供）：肠出血性大肠杆菌。大肠杆菌（EHEC）O 157：H7产生噬菌体编码的毒素Stx，该毒素可从肠道吸收并引起危及生命的溶血性尿毒综合征（HUS），其特征为贫血、血小板减少和肾衰竭。Stx通过肠屏障的吸收知之甚少。鉴于上皮细胞骨架是不可或缺的跨细胞和细胞旁运输的调节，肠出血性大肠杆菌的能力，以显着改变宿主丝状（F-）肌动蛋白的Stx易位可能是促进。肠出血性大肠杆菌沿着肠致病性大肠杆菌。大肠杆菌（EPEC）和天然小鼠病原体啮齿类柠檬酸杆菌（Citrobacter rodentium），触发上皮细胞上的附着和消失（AE）损伤的形成。这些病变的特征是F-肌动蛋白的紧密附着和重排，在结合的细菌下面形成“纤维”。产生AE病变的能力需要细菌效应蛋白的III型易位，并且效应子Tir和EspFU已经涉及肌动蛋白酶的形成。此外，EspFU可以促进体外上皮屏障的破坏。我们确定了所需的基座形成的Tir和EspFU的活动，并已分离出许多突变体，不能促进本地化的肌动蛋白组装。最近，为了研究Stx介导的疾病的发病机制，我们利用C.用编码Stx 2的噬菌体溶原化的啮齿目动物，Stx 2是与HUS特别相关的Stx的等位基因。感染C.啮齿动物（？Stx 2）遭受Stx依赖性肾损伤、体重减轻和最终死亡。我们还构造了C的导数。啮齿动物（？Stx 2）表达EHEC毒力因子，包括Tir和/或EspFU，并且初步实验表明产生细胞骨架变化的能力可能增强该模型中的毒力。为了检验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介导的疾病的假设，我们将追求以下目标： 1.评估体外EHEC介导的肌动蛋白重排增强Stx 2跨极化上皮细胞易位的能力。极化的单层细胞将被表达野生型Tir和EspFU的（非Stx产生的）EHEC或不能触发肌动蛋白重排的衍生物感染。将纯化的Stx 2d添加到顶端表面，并测量其穿过T84肠上皮细胞的感染单层的易位。 2.评估EspFU在EHEC小鼠模型中增强Stx 2介导的疾病的能力。 小鼠经口感染C.啮齿动物（？Stx 2）表达野生型TirEHEC和EspFU，或与这些效应物的衍生物，其特异性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）O 157：H7是北美、欧洲和日本的一种重要食源性病原体（79）。危及生命的感染的全身表现是由于从肠道吸收细菌毒素（51，52）。肠出血性大肠杆菌与肠壁细胞结合，并触发宿主细胞在结合的细菌下方产生引人注目的管状结构（33），这一过程可能促进毒素吸收到哺乳动物血流中。这些研究调查了肠出血性大肠杆菌诱导肠道菌群的能力是否促进了系统性疾病。