Expression of a pilin subunit BfpA of the bundle-forming pilus of enteropathogenic Escherichia coli in an aroA live salmonella vaccine strain

Enteropathogenic Escherichia coli (EPEC) is a major cause of childhood diar rhea in developing countries and is a leading cause of severe diarrheal ill ness among Brazilian infants. As one approach to constructing a vaccine can didate against diarrhea caused by EPEC, we evaluated whether the pilin subu nit (BfpA) of the bundle-forming pilus (BFP could be expressed by a live Sa lmonella vaccine strain. Several copies of the coding region of BfpA (bfpA) were amplified by PCR from a preparation of the EAF plasmid of EPEC strain B171 and cloned into plasmid vectors. An intact copy of bfpA was subcloned into the heat inducible prokaryotic expression vector pCYTEXP1, and the re sulting pBfpA was used to transform the aroA S. typhimurium strain SL3261, generating SL3261(pBfpA). The recombinant vaccine strain was able to expres s, but not to process, rBfpA as evidenced by a prominent 21 kDa protein tha t crossreacted with anti-BFP antiserum found only in extracts of heat-treat ed SL3261(pBfpA), but not in strains of untreated SL3261(pRfpA) or SL3261 n ot carrying the plasmid. Furthermore, rBfpA accumulation was not toxic to t he Salmonella host, as evidenced by similar plating efficiencies between in duced and uninduced strains of SL3261(pBfpA). Finally, SL3161(pBfpA) orally administered to BALB/c mice was capable of eliciting a sustained and vigor ous humoral immune response to BfpA, achievable even with a single oral dos e of approximately 10(9) organisms. Therefore, this pilin product may serve as a potential immunogen as part of a live combined vaccine strategy to pr event two of the major public health problems in Brazil - salmonellosis and EPEC childhood diahrrea. (C) 1999 Elsevier Science Ltd. All rights reserve d.