DEVELOPMENT OF AN ENTEROPATHOGENIC ESCHERIA COLI VACCINE

本项目的目的是使用生物发光成像来确定肠道致病性大肠杆菌(EPEC)在人体肠道中的定植部位。EPEC菌株B171-8的两个变种将被改造，使这些细菌发光。我们已经对特定的突变体进行了基因工程，从而产生了这种EPEC细菌菌株的同基因变体，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，并且一个突变体(B171-8Fma)阐述了一个无功能但形态正常的BFP，其毒力大约低200倍。我们现在试图确定感染期间病原菌与人类肠道相互作用的确切位置，并确定致病性较低的变体是否在肠道的同一区域定居。我们计划用一个基于pUC19的质粒转化B171-8和B171-8Fma，该质粒含有一个10kb的插入片段，编码来自土壤细菌Photorhabdus Lighescens的lux操纵子。这些发光生物将被人类志愿者摄取，他们的腹部将使用增强型变化耦合设备(ICCD)相机进行成像。该系统可以检测单个光子--一种允许通过哺乳动物组织监测发光细胞的敏感度。使用鼠伤寒沙门氏菌的类似研究已经在小鼠的肠道中成功地进行了。我们预计，ICCD相机的最新技术进步将使我们能够识别感染过程中不同时间内EPEC最严重的肠道定植区域。

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.