Construction and evaluation of chimeric heat-labile toxin B subunit and N-terminal(1–75) fragment of colonization factor antigen I gene of enterotoxigenic Escherichia coli

Abstract

A significant percentage of enterotoxigenic Escherichia coli (ETEC) diarrheal disease worldwide is caused by colonization factor antigen I-producing isolates of ETEC either as infant diarrhea with high mortality or traveler’s diarrhea. Bacterial adhesion to intestinal epithelial cells and elaboration of enterotoxins are the main steps in pathogenesis of ETEC, and antibodies to these antigens have been associated with protection. Colonization factor antigen I (CFA/I) is the most frequently encountered adhesin in epidemiological studies. In this study, a DNA fragment encoding the first 25 amino acids of the N-terminal end of CFA/I B subunit was genetically fused to the C-terminal end of the B subunit of heat-labile enterotoxin gene of Escherichia coli with five glycine residues as a linker and expressed in E. coli. The N-terminal fragment was selected because it is relatively conserved among several ETEC fimbriae and strongly reacts with monoclonal antibodies against CFA/I, and several B-cell epitopes have been mapped to this region. The B subunit of heat labile enterotoxin (LTB) of E. coli is a potent immunogen and also elicits antitoxin response. The recombinant protein was recognized by anti-LTB MAb and anti-rCfaB polyclonal antibody and bound GM1 gangliosides receptors in enzyme-linked immunosorbant assay (ELISA). Polyclonal antibody raised in rabbit reacted with the dissociated as well as intact fimbriae expressed on the surface of bacteria in western blot and ELISA, respectively, and blocked fluid accumulation in the rabbit ileal loop assay. This demonstrates that the rLTB-CfaB(1–25) protein could be used for production of anti-LTB and anti-CfaB and that combining the two fragments has not impaired the function of either part. Moreover, complete lack of fluid accumulation in rabbit ileal loops might have resulted from both anti-adhesive and anti-toxin activity of the antibodies raised against the fusion protein.