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Enterohemorrhagic Escherichia coli (EHEC) is the most devastating among the various pathogenic E. coli strains, causing watery/bloody diarrhea, hemorrhagic colitis and life-threatening hemolytic uremic syndromein human patients. Here we first used differential 2-DE gel display analysis to compare proteomic profiles from bacteria cultured in vitro and bacteria isolated from the large bowel of infected gnotobiotic piglets (in vivo). Approximately 760 unique proteins were identified from 2-DE gel spots. Among them, about 160 proteins were differentially expressed (1.5-fold change) at 95% statistical confidence in the in vitro versus in vivo comparative analysis of the whole cell lysates. Several outer membrane proteins (OmpA, OmpX and OmpW) and Lac Z were increased significantly or not present under the in vivo or in vitro conditions respectively. Two in vivo up-regulated enzymes (FumB and GlpB) contributed to energy generation pathways under anaerobic conditions. In addition, four enzymes (GalM, GalE, GalT, GalK) that were involved in the biosynthesis of the lipopolysaccharide components of the outer membrane were increased under the in vivo conditions. The significantly increased proteins in vivo may be associated with specific functions, such as attaching and effacing of the host intestinal epithelial villi, escaping from/ subverting the host immune responses, coping with the ambientenvironments (acid, temperature, oxygen, nutrients, and other stresses) of the piglets' gut etc. Characterizing these proteins functionally and immunologically could result in the discovery of novel targets for anti-infective and new antigens for vaccine development. An alternative approach-MudPIT (2D-LC-MS/MS) was also used to increase the coverage of low abundance EHEC proteins, but not yet applied to differential quantitation with APEX.