Shiga toxin activity in human intestinal organoids

 描述（由申请方提供）：滋贺毒素（Stx）是产Stx大肠埃希菌（STEC）的主要毒力因子，STEC是食源性疾病的主要原因。每年，STEC，包括E.大肠杆菌O157：H7在美国引起超过100，000例感染性水样和血性腹泻，约10%的感染者发展为危及生命的肾脏疾病，即溶血性尿毒综合征（HUS）。HUS的症状可以在用纯化的Stx治疗的灵长类动物中复制。在HUS中，微血管系统中的血小板血栓形成损害了流向肾脏的血流，并且当红细胞挤压通过闭塞的血管时被机械剪切时，发生溶血性贫血。由于尚不清楚的原因，幼儿比成人更容易患上HUS，这是儿童急性肾衰竭的最常见原因。目前，还没有治疗HUS的全身表现。此外，抗生素诱导STEC产生更大量的Stx，抗生素治疗与更严重的疾病结果相关。 该领域的一个主要障碍是缺乏复制人类疾病的易处理的哺乳动物模型，因此关于Stx蛋白毒素或STEC病原体对健康、分化的人类肠组织的作用知之甚少。我们开发了一种令人兴奋的新实验模型来研究Stx和STEC对人类肠道的影响。干细胞诱导的人类肠道类器官（iHIO）忠实地代表了分化的人类肠道组织。我们的初步研究表明，人类肠道类器官对注射到管腔中的Stx敏感。此外，虽然无害的E.大肠杆菌可以在管腔内复制而不造成损害，致病性E. coli O157：H7破坏肠上皮屏障。 该提案将使用人类肠道类器官作为一种新的模型系统来研究与Stx和STEC相关的致病过程。我们将用无害的E.大肠杆菌和致病性O157：H7菌株，具有或不具有产生Stx的能力。目标1将侧重于疾病的宿主参数，目标2将侧重于细菌毒力因子的相对贡献：目标1。宿主对细菌感染和Stx表达的反应是什么？目标2. Stx与致病性O157：H7表型在介导肠道损伤方面的相对贡献是什么？

 DESCRIPTION (provided by applicant): Shiga toxin (Stx) is the primary virulence factor of Stx producing Eschrichia coli (STEC), a leading cause of food-borne illnesses. Annually, STEC, including E. coli O157:H7, cause over 100,000 cases of infectious watery and bloody diarrhea in the U.S. About 10% of the infected progress to the life-threatening kidney disorder, hemolytic uremic syndrome (HUS). The symptoms of HUS can be replicated in primates treated with purified Stx. In HUS, platelet thrombus formation in the microvasculature compromises blood flow to the kidney, and hemolytic anemia develops when red blood cells are mechanically sheared as they squeeze through the occluded vessels. For reasons that are not clear, young children are more likely to develop HUS than adults, and it is the most common cause of acute kidney failure in children. Currently, there is no treatment for the systemic manifestations of HUS. In addition, antibiotics induce STEC to produce higher quantities of Stx, and antibiotic treatment is correlated with more severe disease outcome. A major barrier in the field is the lack of tractable mammalian models that replicate human disease, and as a consequence little is known about the actions of Stx protein toxin or the STEC pathogens on healthy, differentiated human intestinal tissues. We have developed an exciting new experimental model to study the effects of Stx and STEC on the human intestinal tract. Stem cell 'induced human intestinal organoids' (iHIOs) faithfully represent differentiated human intestinal tissue. Our preliminary studies show human intestinal organoids are sensitive to Stx injected into the lumen. Furthermore, while harmless E. coli can replicate in the lumen without causing damage, pathogenic E. coli O157:H7 destroy the intestinal epithelial barrier. This proposal will use human intestinal organoids as a novel model system to study the pathogenic processes associated with Stx and STEC. We will compare infection by harmless E. coli and pathogenic O157:H7 strains, with or without the ability to produce Stx. Aim 1 will focus on the host parameters of disease and Aim 2 will focus on the relative contribution of the bacterial virulence factors: Aim 1. What is the host response to bacterial infection and Stx expression? Aim 2. What is the relative contribution of Stx versus the pathogenic O157:H7 phenotype in mediating intestinal damage?