CARBOHYDRATE METABOLISM OF E COLI

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The specific aim of this project is to: 1) develop capillary electrophoresis-based assays capable of resolving complex mixtures of carbohydrates and 2) apply the assays to mucus extracts supplied from Conway's laboratory to determine their carbohydrate content. More specifically, the preference for metabolism of carbohydrates from mucus extracts on which Escherichia coli (E. coli) has been culture will be determined. This project will be a collaborative effort between Dr. Tyrrell Conway of the University of Oklahoma and Dr. Tim Smith at Southeastern Oklahoma State University. Conway's whole-genome expression profiling of E. coli revealed genes which were induced by conditions designed to mimic the nutrient availability in the mammalian intestine; it was found that primarily metabolic pathways were turned on. Mutational analysis in mouse colonization assays revealed several sugars that appeared to be important for E. coli to colonize. An important extension of these findings is to identify the sugars that are available in mucus and to determine the order of E. coli preference for the individual mucus-derived sugars. The proposed metabolomics study will utilize capillary electrophoresis to monitor the carbohydrate metabolism of various E. coli strains and specific mutants cultured on synthetic and authentic mucus. Dr. Conway's laboratory will generate time-course samples of wild type and mutant cultures. Dr. Smith's laboratory will develop the bioanalytical assays based around capillary electrophoresis necessary to determine the carbohydrate content in the extracts. The goal is to determine the exact metabolic preference of colonized E. coli. This collaboration will provide a more in depth understanding of how gastrointestinal pathogens acquire the nutrients necessary to infect their host and begin the disease process and should build on the knowledge of this model system.