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Purification and partial characterization of the major outer membrane protein of Haemophilus somnus
Y Tagawa1, H Ishikawa, N Yuasa
1Division of Systematic Diagnosis Research, National Institute of Animal Health, Ibaraki, Japan.
Abstract:
We purified the major outer membrane protein (MOMP), which is the most abundant OMP (with an apparent molecular mass of 40 kDa), from Haemophilus somnus strain 8025. The method involves solubilization of the MOMP with Zwittergent 3-14 and further purification accomplished by ion-exchange and molecular-sieve chromatographies. The amino-terminal sequence of the MOMP showed considerable similarity to those of porin proteins from other gram-negative bacteria. The MOMP of H. somnus is immunogenic to rabbits and calves. Hyperimmune sera from rabbits and calves reacted with both the MOMP and lipopolysaccharides in enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. The rabbit antiserum to the MOMP was cross-reactive with whole-cell preparations from strains 8025, D1238, NT2301, and 540 at a band with a molecular mass of 40 kDa in immunoblot analysis, although the reactivity of the rabbit antiserum with strain 540 was lower than those with the other strains tested. Two murine monoclonal antibodies (MAbs) to the MOMP were developed. ELISA with the OMP fractions as the antigens showed that one MAb was cross-reactive with the four strains but that the other MAb was reactive with the three strains other than strain 540. These results indicate that the MOMP of H. somnus possesses at least two antigenic determinants and that the MOMP of strain 540 is antigenically different from those of the other strains. The antigenic heterogeneity of the H. somnus MOMP has implications regarding the development of a serotyping system with MAbs that is based on the MOMP epitopes.
Insights
We purified the major outer membrane protein (MOMP) from Haemophilus somnus, revealing antigenic differences between strains. This discovery impacts the development of diagnostic tools for this important bacterium.
Area of Science:
- Microbiology
- Immunology
- Protein Chemistry
Background:
- Haemophilus somnus is a significant pathogen in livestock.
- Outer membrane proteins (OMPs) are crucial for bacterial structure and interaction with the host.
- The major outer membrane protein (MOMP) is the most abundant OMP in H. somnus.
Purpose of the Study:
- To purify and characterize the MOMP of Haemophilus somnus strain 8025.
- To investigate the immunogenicity and antigenic properties of H. somnus MOMP.
- To explore the potential of MOMP for developing diagnostic and serotyping tools.
Main Methods:
- Solubilization of MOMP using Zwittergent 3-14.
- Purification via ion-exchange and molecular-sieve chromatography.
- Amino-terminal sequencing, enzyme-linked immunosorbent assay (ELISA), immunoblot analysis, and monoclonal antibody (MAb) development.
Main Results:
- Purified MOMP showed similarity to porins from other gram-negative bacteria.
- H. somnus MOMP is immunogenic in rabbits and calves, with hyperimmune sera reacting to MOMP and lipopolysaccharides.
- Rabbit antiserum cross-reacted with multiple H. somnus strains, but reactivity varied, particularly with strain 540.
- Two murine MAbs identified distinct antigenic determinants on MOMP, with one MAb showing strain-specific reactivity.
Conclusions:
- The MOMP of H. somnus possesses at least two antigenic determinants.
- Significant antigenic heterogeneity exists among H. somnus MOMP isolates, with strain 540 being antigenically distinct.
- MOMP epitopes offer potential for developing a serotyping system for H. somnus.