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Characterization of a heat-modifiable outer membrane protein of Haemophilus somnus
Y Tagawa1, M Haritani, H Ishikawa
1National Institute of Animal Health, Ibaraki, Japan.
Abstract:
In immunoblot analysis, a murine monoclonal antibody (MAb), 27-1, which was produced to an outer membrane protein (OMP) of Haemophilus somnus, showed that a major OMP is heat modifiable, having a molecular mass of 28 kDa when the N-lauroylsarcosine-insoluble OMP preparation was solubilized at 60 degrees C and a mass of 37 kDa when the OMP preparation was solubilized at 100 degrees C. The heat-modifiable OMP reacted intensely with convalescent sera obtained from calves with experimental H. somnus pneumonia in immunoblot analysis. Immunoelectron microscopic and antibody absorption studies revealed that the MAb 27-1 epitope was not surface exposed on the intact bacterium. However, a decrease in antibody reactivity to the heat-modifiable OMP in immunoblot analysis after absorption of convalescent serum with intact bacterial cells of H. somnus suggests that a surface-exposed portion of the heat-modifiable OMP is expressed on the intact bacterium. MAb 27-1 reacted with 45 of 45 strains of H. somnus tested in immunoblot analysis. The apparent molecular mass of the antigen varied among strains, and five reactivity patterns demonstrated by MAb 27-1 were observed. MAb 27-1 also reacted with six species in the family Pasteurellaceae, Escherichia coli, and Salmonella dublin, but not with the other eight species of gram-negative bacteria. The heat-modifiable OMP of H. somnus showed immunological cross-reactivity with the OmpA protein of E. coli K-12 and significant N-terminal amino acid sequence homology with the OmpA proteins of gram-negative bacteria. We conclude that a major, 37-kDa heat-modifiable OMP of H. somnus, which elicits an antibody response in H. somnus-infected animals, is a common antigen among H. somnus strains tested and is structurally related to the OmpA protein of E. coli.
Insights
A major outer membrane protein (OMP) of Haemophilus somnus is heat-modifiable and common among strains. This OMP elicits an antibody response in infected animals and is structurally related to E. coli OmpA.
Area of Science:
- Microbiology
- Immunology
- Bacterial Pathogenesis
Background:
- Haemophilus somnus causes significant disease in cattle, including pneumonia.
- Outer membrane proteins (OMPs) are crucial for bacterial structure and host-pathogen interactions.
- Understanding H. somnus OMPs can lead to improved diagnostics and vaccines.
Purpose of the Study:
- To characterize a major heat-modifiable outer membrane protein (OMP) of Haemophilus somnus.
- To investigate the antigenicity and distribution of this OMP among H. somnus strains.
- To determine the structural relationship of this OMP to known bacterial proteins.
Main Methods:
- Immunoblot analysis using a murine monoclonal antibody (MAb 27-1) against H. somnus OMP.
- Heat-modifiable properties assessed by solubilizing OMP preparations at different temperatures (60°C vs. 100°C).
- Immunoelectron microscopy, antibody absorption studies, and N-terminal amino acid sequencing.
Main Results:
- A major heat-modifiable OMP of H. somnus (37 kDa at 100°C, 28 kDa at 60°C) was identified.
- This OMP reacted strongly with convalescent sera from H. somnus-infected calves.
- MAb 27-1 recognized all 45 tested H. somnus strains, indicating it's a common antigen.
- The OMP showed cross-reactivity and sequence homology with the OmpA protein of Escherichia coli.
Conclusions:
- A major 37-kDa heat-modifiable OMP of H. somnus is a common antigen across strains and elicits an immune response in infected animals.
- This OMP is structurally related to the OmpA protein family found in other Gram-negative bacteria.
- Further characterization of this OMP could aid in developing targeted diagnostics and therapeutics for H. somnus infections.