An Escherichia coli-Enterococcus faecalis shuttle vector as a tool for theconstruction of a group B Streptococcus heterologous mutant expressing thebeta antigen (Bac) of the C protein complex

Group B streptococci (GBS) represent a very important group of human pathog ens. So far little is known about the mechanisms by which these bacteria ca n cause disease and the bacterial factors involved. One putative virulence factor is the beta antigen of the C protein complex (Bac), which can bind t o the Fc region of human IgA. Its binding function might represent an impor tant virulence mechanism. However, the genetic manipulation df this group o f bacteria, necessary to prove involvement of bacterial factors in pathogen esis, is still in its infancy. We therefore tested the pAM401 vector system for its suitability in the construction of a heterologous expression mutan t using the Bac protein as a model antigen. The bac gene, including its own promoter, was cloned into the Escherichia coli-Enterococcus faecalis shutt le vector pAM401 and was stably maintained extrachromosomally in the bac-de ficient GBS strain 335. Expression of Bac was assessed by extracting the pr otein from transformed 335(pPJTU1) cells, negative controls (335 wild-type, 335(pAM401)) and other Bac-expressing GBS strains (A909, LA239). Blots of the extracted proteins probed with IgA, polyclonal sera and a monoclonal an tibody raised against Bac clearly revealed expression of the 130-kDa protei n in the transformed GBS 335(pPJTU1) cells. The correct processing and surf ace anchoring of the expressed Bac was demonstrated by binding of I-125-lab elled IgA to whole cells. Strain 335(pPJTU1) bound 12 times as much IgA com pared to the parental strain LA239 and the GBS 335 negative controls, and a total of 25% compared to the high-level-expressing strain A909. Our studie s show that the pAM401 shuttle vector can be used for stable heterologous e xpression of surface proteins in GBS. Our strategy is also of major importa nce for the complementation of deletion mutants in GBS and other Gram-posit ive human pathogens to fulfill Koch's postulates. The Bac mutant constructe d in this study, 335(pPJTU1), can be used in animal models to assess the im portance of Bac in GBS pathogenesis. (C) 1999 Federation of European Microb iological Societies. Published by Elsevier Science B.V. All rights reserved .