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.
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