DEVELOPMENT OF AN ENTEROPATHOGENIC ESCHERIA COLI VACCINE

本项目的目的是利用生物荧光成像技术鉴定肠致病性大肠杆菌（EPEC）在人肠中的定植位点。EPEC菌株B171-8的两个变体将被修改以使这些细菌产生光。我们已经对特定的突变体进行了基因工程改造，从而产生了这种EPEC菌株的等基因变体，并在斯坦福大学GCRC的人类志愿者身上测试了它们的致病性。这些研究的结果表明，功能性BFP是EPEC毒力所必需的，而B171-8Fma变体产生了一种无功能但形态正常的BFP，其毒力大约降低了200倍。我们现在试图确定致病细菌在感染期间与人类肠道相互作用的确切位置，并确定致病性较低的变异是否在肠道的同一区域定植。我们计划用puc19为基础的质粒转化B171-8和B171-8Fma，该质粒含有一个10 kb的插入片段，编码来自土壤细菌光habdus luminescens的lux操纵子。这些发光生物将被人类志愿者摄入，他们将使用强化变化耦合装置（ICCD）相机对腹部进行成像。该系统可以探测到单个光子，这种灵敏度可以通过哺乳动物组织监测发光细胞。利用鼠伤寒沙门氏菌进行的类似研究已在小鼠肠道中成功进行。我们预计，ICCD相机的最新技术进步将使我们能够识别在感染过程中不同时间被EPEC最严重定植的人类肠道区域。

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.