Characterization of Fusobacterium necrophorum subsp. necrophorum outer membrane proteins

Area of Science:

• Veterinary Microbiology
• Bacterial Pathogenesis
• Molecular Biology

Background:

• Liver abscesses in cattle pose significant economic challenges.
• Fusobacterium necrophorum, a Gram-negative anaerobe, is a primary causative agent.
• Outer membrane proteins (OMP) of F. necrophorum subsp. necrophorum are implicated in host cell adhesion.

Purpose of the Study:

• To characterize a specific 42.4 kDa OMP from F. necrophorum subsp. necrophorum with high binding capacity to bovine endothelial cells.
• To confirm the adhesive properties of this OMP through recombinant expression and functional assays.

Main Methods:

• Cloning the OMP gene into a pFLAG-CTS vector for expression in E. coli BL21 DE3.
• Inducing protein expression with IPTG and assessing binding to immobilized bovine adrenal gland capillary endothelial (EJG) cells.
• Utilizing purified native OMP and polyclonal antibodies for further binding validation.

Main Results:

• Recombinant E. coli expressing the OMP (SM 2013) showed significantly enhanced binding to EJG cells after induction.
• Purified native OMP demonstrated reduced binding, while antibody pre-incubation inhibited binding of induced SM 2013.
• The gain of function in recombinant E. coli confirmed the OMP's role as an adhesion molecule.

Conclusions:

• The characterized 42.4 kDa OMP is a crucial adhesin for F. necrophorum subsp. necrophorum.
• This OMP facilitates the binding of F. necrophorum subsp. necrophorum to host cells, contributing to pathogenesis.
• Targeting this adhesin could offer novel strategies for controlling F. necrophorum infections in cattle.