Characterization of Fusobacterium necrophorum subsp. necrophorum outer membrane proteins

Abstract

Liver abscesses are of major economic importance to the cattle industry. These are mainly associated with the presence of Fusobacterium necrophorum, a non-spore forming and Gram-negative anaerobe. There are two main subspecies, F. necrophorum subspecies necrophorum and subsp. funduliforme, and they differ molecularly, morphologically, biochemically and in virulence. Previous studies have shown that the outer membrane proteins (OMP) of F. necrophorum subsp. necrophorum are important for its successful binding to immobilized bovine adrenal gland capillary endothelial (EJG) cells. In this study, a 42.4 kDa OMP of F. necrophorum subsp. necrophorum with the highest binding capacity to EJG cells was characterized. The gene was cloned into pFLAG-CTS vector and the proteins were subsequently expressed on the surface of E. coli BL21 DE3 cells. When E. coli carrying the recombinant plasmid (SM 2013) was induced using IPTG, there was significant enhancement in the binding to immobilized EJG cells compared to both uninduced SM 2013 and the E. coli carrying control vector only. When fixed EJG cells were incubated with purified native OMP, SM 2013 showed lowered levels of binding, compared to the uninduced SM 2013 and the E. coli carrying control vector only. Pre-incubation of induced SM 2013 with polyclonal antibodies made against the OMP reduced the binding to immobilized EJG cells to uninduced SM 2013 levels. This gain of function by recombinant E. coli confirms the ability of this protein to act as an adhesion to help binding of F. necrophorum subsp. necrophorum to host cells.