An Escherichia coli-Enterococcus faecalis shuttle vector as a tool for the construction of a group B Streptococcus heterologous mutant expressing the beta antigen (Bac) of the C protein complex.
Group B streptococci (GBS) represent a very important group of human pathogens. So far little is known about the mechanisms by which these bacteria can cause disease and the bacterial factors involved. One putative virulence factor is the beta antigen of the C protein complex (Bac), which can bind to the Fc region of human IgA. Its binding function might represent an important virulence mechanism. However, the genetic manipulation of this group of bacteria, necessary to prove involvement of bacterial factors in pathogenesis, is still in its infancy. We therefore tested the pAM401 vector system for its suitability in the construction of a heterologous expression mutant using the Bac protein as a model antigen. The bac gene, including its own promoter, was cloned into the Escherichia coli-Enterococcus faecalis shuttle vector pAM401 and was stably maintained extrachromosomally in the bac-deficient GBS strain 335. Expression of Bac was assessed by extracting the protein 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 antibody raised against Bac clearly revealed expression of the 130-kDa protein in the transformed GBS 335(pPJTU1) cells. The correct processing and surface anchoring of the expressed Bac was demonstrated by binding of (125)I-labelled IgA to whole cells. Strain 335(pPJTU1) bound 12 times as much IgA compared 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 studies show that the pAM401 shuttle vector can be used for stable heterologous expression of surface proteins in GBS. Our strategy is also of major importance for the complementation of deletion mutants in GBS and other Gram-positive human pathogens to fulfill Koch's postulates. The Bac mutant constructed in this study, 335(pPJTU1), can be used in animal models to assess the importance of Bac in GBS pathogenesis.
Kreikemeyer
B
B
Department of Microbial Pathogenesis and Vaccine Research, GBF-National Research Centre for Biotechnology, Braunschweig, Germany. bernd.kreikemeyer@medizin.uni-ulm.de
Jerlström
P G
PG
D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D018274:Electroporation / Q000379:methods; D013293:Enterococcus faecalis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D007070:Immunoglobulin A / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D013292:Streptococcus agalactiae / Q000235:genetics* / Q000276:immunology / Q000378:metabolism / Q000472:pathogenicity; D014169:Transformation, Bacterial
D000942:Antigens, Bacterial; D007070:Immunoglobulin A; D007141:Immunoglobulin Fc Fragments