Environmentally-induced virulence changes in enterhemorrhagic escherichia coli

本申请旨在资助研究肠出血性大肠杆菌O157：H7的应激诱导毒力变化。这种肠道病原体是出血性腹泻、出血性结肠炎和溶血性尿毒综合征的主要原因。在摄入过程中，肠出血性大肠杆菌暴露于几种应激，包括胃酸以及肠内的胆汁盐和短链脂肪酸。它也可能在酸洗肉尸体时受到压力。我们已经发现，酸和胆盐应激显著增强EHEC和一种密切相关的病原体肠致病性大肠杆菌（EPEC）与人上皮细胞的粘附。我们还确定了一种细菌蛋白和宿主细胞脂质，在酸应激EPEC的增强粘附中发挥作用。这项建议的目标是确定基因和途径，包括压力诱导的肠出血性大肠杆菌毒力的变化。使用DNA微阵列，我们将检查暴露于酸，胆汁盐，和短链脂肪酸之前和之后的肠出血性大肠杆菌的mRNA表达谱，以确定细菌蛋白负责的应力诱导的毒力变化。将通过定量实时PCR确认差异基因表达，并通过2D凝胶电泳和电喷雾串联质谱验证蛋白质变化。为了证实应激诱导基因在肠出血性大肠杆菌毒力中的作用，我们将构建单个基因缺陷的突变体，并测试它们在应激暴露前后毒力表型的变化。我们还将使用转座子诱变鉴定毒力基因的调节因子。应激诱导的肠出血性大肠杆菌O157：H7毒力变化可对疾病的发展和进展产生深远影响，因此为调节该病原体的毒力潜力提供了令人兴奋的靶点。这种靶向方法通过允许选择性减毒EHEC O157：H7，同时保持在预防感染中发挥作用的非致病性肠道细菌，从而认识到肠道细菌的价值。该研究还将确定和描述与亚致死环境压力（包括食品加工实践）相关的感染风险。

This application is for funding to support the investigation of stress-induced virulence changes in enterohemorrhagic (EHEC) E.coli O157:H7. This enteric pathogen is a leading cause of bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. During ingestion, EHEC is exposed to several stresses including gastric acid as well as bile salts and short chain fatty acids in the intestine. It can also be exposed to stress during acid-washing of meat carcasses. We have found that acid and bile salt stress significantly enhance adhesion of EHEC and a closely related pathogen, enteropathogenic E.coli(EPEC),to human epithelial cells. We have also identified a bacterial protein and a host cell lipid that play a role in the enhanced adhesion of acid-stressed EPEC. The goal of this proposal is to identify genes and pathways encompassing stress-induced EHEC virulence changes. Using DNA microarrays, we will examine the mRNA expression profiles of EHEC before and after exposure to acid, bile salts, and short chain fatty acids to identify bacterial proteins responsible for the stress-induced virulence changes. Differential gene expression will be confirmed by quantitative real-time PCR and protein changes will be validated by 2D gel electrophoresis and electrospray tandem mass spectrometry. To confirm the role of stress-induced genes in EHEC virulence, we will construct mutants defective in the individual genes and test them for changes in virulence phenotype before and after stress exposure. We will also identify regulators of the virulence genes using transposon mutagenesis. Stress-induced changes in virulence of EHEC O157:H7 can have a profound impact on the development and progression of the disease and therefore provide exciting targets to modulate the virulence potential of this pathogen. This targeted approach recognizes the value of commensal bacteria by permitting selective attenuation of EHEC O157:H7 while maintaining the nonpathogenic, commensal bacteria that play a role in preventing infection. The research will also identify and characterize the infection risk associated with sublethal environmental stress including food processing practices.