DEVELOPMENT OF AN ENTEROPATHOGENIC ESCHERIA COLI VACCINE

本项目的目的是利用生物发光成像技术来确定肠道致病性大肠杆菌在人体肠道中的定植部位。coli（EPEC）。EPEC菌株B171-8的两个变体将被修饰以使这些细菌产生光。我们对特定的突变体进行了基因工程改造，从而产生了EPEC菌株的同基因变体，并在斯坦福大学的GCRC中测试了它们对人类志愿者的致病性。这些研究的结果证实，功能性BFP是EPEC毒力所必需的，并且产生非功能性但形态正常的BFP的变体（B171- 8 Fma）的毒力约低200倍。我们现在试图确定致病菌在感染过程中与人体肠道相互作用的确切部位，并确定致病性较低的变体是否在肠道的同一区域定植。我们计划转化B171-8和B171- 8 Fma与pUC 19为基础的质粒含有一个10 kb的插入编码来自土壤细菌发光杆菌的lux操纵子。这些发光生物体将被人类志愿者摄入，志愿者将使用增强型变化耦合器件（ICCD）相机对他们的眼睛进行成像。该系统可以检测单个光子-允许通过哺乳动物组织监测发光细胞的灵敏度。使用鼠伤寒沙门氏菌的类似研究已在小鼠肠道中成功进行。我们预计，ICCD摄像机的最新技术进步将使我们能够识别在感染过程中的不同时间EPEC最严重的人类肠道区域。

The purpose of this project is to use biolumiescent imaging to identify the site in the human intestine colonized by the enteropathogenic E. coli (EPEC). Two variants of the EPEC strain B171-8 will be modified to render these bacteria light-producing. We have genetically engineered specific mutants, thereby producing isogenic variants of this EPEC bacterial strain, and tested their pathogenicity on human volunteers in the GCRC at Stanford. The results of those studies establish that functional BFP are required for EPEC virulence and that a variant (B171-8Fma) elaborating a non-functional, but morphologically normal, BFP was approximately 200-fold less virulent. We now seek to determine the exact site at which pathogenic bacteria interact with the human intestinal tract during infection and to determine if the less-pathogenic variant colonizes the same region of the intestine. We plan to transform both B171-8 and B171-8Fma with a pUC19-based plasmid containing a 10 kb insert encoding the lux operon derived from the soil bacterium Photorhabdus luminescens. These luminescent organisms will be ingested by human volunteers who will have their abdomens imaged using an intensified change coupled device (ICCD) camera. This system can detect single photons - a sensitivity that allows for monitoring light emitting cells through mammalian tissues. Similar studies using Salmonella typhimurium have been successfully performed in the mouse intestine. We anticipate that the recent technological advances in the ICCD camera will allow us to identify the regions of the human intestine most heavily colonized by EPEC at various times during the course of the infection.