Identification and immunogenicity of Mannheimia haemolytica S1 outer membrane lipoprotein PlpF

Abstract

Immunity against Mannheimia haemolytica requires antibodies against leukotoxin (LKT) and bacterial cell surface antigens, most likely immunogenic outer membrane proteins (OMPs). Five immunogenic outer membrane lipoproteins identified and characterized in M. haemolytica were designated Pasteurella lipoproteins (Plp) A, -B, -C, -D and -E. Using immunoproteomics, we identified a heretofore-uncharacterized M. haemolytica immunogenic outer membrane lipoprotein that we designated PlpF, which was previously designated in the published sequence as a conserved hypothetical protein. We cloned and expressed rPlpF from two M. haemolytica serotype 1 strains (SAC159 and SAC160) and demonstrated a variable number of perfect (KKTEED) or imperfect (KKaEEa) repeats between residues 41 and 76 on the N-terminus. Antigenicity plots predicted the N-terminus repeat region to be highly antigenic. The plpF gene in multiple M. haemolytica S1, S2, and S6 isolates varied in the number of repeats from three to seven. C-terminal region was highly conserved. Immunization of mice with SAC159 or SAC160 demonstrated immunogenicity in a dose-response manner. Immunization of calves demonstrated an increase in antibodies to PlpF, and rPlpF antibodies stimulated complement-mediated killing of M. haemolytica. Because calves had pre-existing anti-M. haemolytica antibodies due to prior natural exposure, functionality of the anti-PlpF antibody responses were demonstrated by marked reduction of complement-mediated killing by blocking of anti-PlpF antibodies with rPlpF In conclusion, PlpF might have vaccination potential against M. haemolytica infection in cattle.