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Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins
Published on: July 1, 2011
Identification and immunogenicity of Mannheimia haemolytica S1 outer membrane lipoprotein PlpF
Sahlu Ayalew1, Binu Shrestha, Marie Montelongo
1Department of Veterinary Pathobiology, Oklahoma State University, Stillwater, OK 74078, USA. sahlu.ayalew@okstate.edu
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.
Insights
A novel outer membrane lipoprotein, PlpF, was identified in Mannheimia haemolytica. Antibodies against PlpF demonstrated immunogenicity and complement-mediated killing, suggesting its potential as a vaccine candidate against bovine respiratory disease.
Area of Science:
- Veterinary Immunology
- Bacteriology
- Molecular Biology
Background:
- Mannheimia haemolytica causes significant economic losses in cattle due to respiratory disease.
- Immunity relies on antibodies targeting leukotoxin (LKT) and outer membrane proteins (OMPs).
- Previous studies identified five Pasteurella lipoproteins (Plp A-E) but left other immunogenic OMPs uncharacterized.
Purpose of the Study:
- To identify and characterize novel immunogenic outer membrane lipoproteins in Mannheimia haemolytica.
- To investigate the immunogenicity and functional activity of a newly identified lipoprotein, PlpF.
- To assess the potential of PlpF as a vaccine candidate against M. haemolytica infections in cattle.
Main Methods:
- Immunoproteomics was used to identify PlpF.
- Recombinant PlpF (rPlpF) was cloned and expressed from M. haemolytica serotype 1 strains.
- Antigenicity plots and sequence analysis were performed on the plpF gene.
- Mice and calves were immunized with rPlpF to assess immunogenicity and antibody functionality.
- Complement-mediated killing assays were conducted to evaluate antibody efficacy.
Main Results:
- A previously uncharacterized immunogenic outer membrane lipoprotein, PlpF, was identified.
- The N-terminus of PlpF exhibited a variable number of repeats (3-7) and was predicted to be highly antigenic.
- Immunization of mice and calves with rPlpF elicited a dose-dependent immune response, increasing anti-PlpF antibodies.
- Anti-PlpF antibodies from immunized calves stimulated complement-mediated killing of M. haemolytica.
- Blocking experiments confirmed the functional activity of anti-PlpF antibodies in complement-mediated killing.
Conclusions:
- PlpF is a novel, immunogenic outer membrane lipoprotein of Mannheimia haemolytica.
- Antibodies against PlpF are capable of mediating complement-dependent bacterial killing.
- PlpF shows significant potential as a vaccine candidate to protect cattle against M. haemolytica infections.
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