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（由申请人提供）：肠ha骨大肠杆菌（EHEC）O157：H7产生噬菌体编码的毒素STX，可以从肠道中吸收并引起威胁生命的溶血性​​尿毒症综合征（HUS），具有贫血，血管症，血栓症和肾衰竭和肾脏失败。众所周知，STX在整个肠壁上的吸收知之甚少。鉴于上皮细胞骨架是跨环细胞和细胞细胞转运的调节不可或缺的一部分，因此EHEC显着改变宿主丝状（F-）肌动蛋白的能力可以促进STX易位。 EHEC，以及肠病大肠杆菌（EPEC）和天然小鼠病原体柠檬酸杆菌啮齿动物，触发在上皮细胞上的附着和胚胎（AE）病变的形成。这些病变的特征是F-肌动蛋白的紧密依恋和重排以在结合细菌下形成“基座”。产生AE病变的能力需要细菌效应子蛋白的III型易位，并且TIR和ESPFU的效应子与肌动蛋白基座的形成有关。此外，ESPFU可以促进体外上皮屏障的破坏。我们确定了基座形成所需的TIR和ESPFU的活性，并隔离了许多无法促进局部肌动蛋白组装的突变体。最近，为了检查STX介导的疾病的发病机理，我们利用用噬菌体编码STX2（phage odeogen）裂解了EHEC感染的鼠模型，这是STX的噬菌体，这是STX的等位基因，特别是与HUS相关。感染了啮齿动物梭菌（？stx2）的小鼠遭受STX依赖​​性肾脏损伤，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）的啮齿动物（？STX2）的衍生物，而试验实验暗示着产生细胞骨架变化的能力可能会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肠道上皮屏障功能的中断和STX介导疾病的增强的假设，我们将追求以下目的： 1.填写EHEC介导的肌动蛋白重排的能力，可以在体外增强跨偏光上皮细胞的STX2易位。偏振单层将被表达野生型TIR和ESPFU的EHEC感染（非STX产生），或无法触发肌动蛋白重排的衍生物。纯化的STX2D将被添加到顶部表面，并将测量其在T84肠上皮细胞的受感染单层中的易位。 2。评估ESPFU在EHEC小鼠模型中增强STX2介导的疾病的能力。 小鼠将口服啮齿动物念珠菌（？stx2），表达野生型Tirehec和ESPFU，或以这些效应子的衍生物的衍生物，这些效应子特别缺乏触发肌动蛋白重排所需的活动。将评估细菌定殖，肠道渗透性和全身性疾病。 公共卫生相关性：肠ha骨大肠杆菌（EHEC）O157：H7是北美，欧洲和日本的重要食物传播病原体（79）。威胁生命的系统感染是由于肠道吸收细菌毒素的原因（51，52）。 EHEC与肠壁的细胞结合，并触发宿主细胞以在结合细菌下产生醒目的基座状结构（33），这一过程可能促进毒素吸收到哺乳动物的血液中。这些研究研究了EHEC诱导基座的能力是否促进了全身性疾病。