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Immunogenic outer-membrane proteins of Haemophilus influenzae type b in infection
P R Langford1, A E Williams, E R Moxon
1Molecular Infectious Diseases Group, Institute of Molecular Medicine, Oxford, UK.
Abstract:
Outer-membrane proteins (OMPs) from Haemophilus influenzae type b (strain Eagan), grown both in vitro (broth) and in vivo (rat intra-peritoneal), were separated by SDS-PAGE. The major OMPs were present in both growth conditions although the amounts of OMP a and OMP d were reduced in rat-grown organisms. There were strong additional bands in in-vivo-grown organisms at 51 and 92 kDa. Antiserum was raised in rabbits against in-vivo-grown bacteria, and absorbed with lysates of in-vitro-grown bacteria. This serum was used in Western blot analysis of OMPs from in-vitro- and in-vivo-grown cells to identify immunogenic proteins present in infection. These infection-associated OMPs had apparent molecular masses of 43 kDa, 48 kDa, 81 kDa and greater than 200 kDa. Bands of reactivity, of the same molecular mass as some of these, were found on immunoblots when rat and human convalescent sera were used as the source of primary antibody. In particular, a band of 81 kDa was recognized by pooled rat and three human convalescent sera.
Insights
Outer-membrane proteins (OMPs) from Haemophilus influenzae type b were analyzed under different growth conditions. Specific infection-associated OMPs were identified, offering potential targets for diagnostics or therapeutics.
Area of Science:
- Microbiology
- Immunology
- Bacterial Pathogenesis
Background:
- Haemophilus influenzae type b (Hib) is a significant pathogen.
- Outer-membrane proteins (OMPs) are crucial for bacterial structure and interaction with the host.
- Understanding Hib OMPs during infection is vital for developing effective interventions.
Purpose of the Study:
- To compare the OMP profiles of Hib grown in vitro and in vivo.
- To identify immunogenic OMPs associated with Hib infection using specific antisera.
- To investigate the reactivity of convalescent sera against Hib OMPs.
Main Methods:
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for OMP separation.
- Western blot analysis using rabbit antiserum raised against in vivo-grown bacteria.
- Absorption of antiserum with in vitro-grown bacterial lysates.
- Analysis with rat and human convalescent sera.
Main Results:
- Major OMPs were conserved between in vitro and in vivo growth, with reduced levels of OMP a and OMP d in vivo.
- Distinct additional protein bands were observed in bacteria grown in vivo (51 and 92 kDa).
- Infection-associated OMPs identified by Western blot were 43, 48, 81 kDa, and >200 kDa.
- An 81 kDa OMP was recognized by pooled rat and human convalescent sera.
Conclusions:
- Hib exhibits distinct OMP profiles depending on growth conditions, with specific proteins upregulated during infection.
- The identified infection-associated OMPs, particularly the 81 kDa protein, are potential targets for diagnostic and immunotherapeutic strategies against Hib.
- Convalescent sera recognize specific Hib OMPs, indicating their immunogenicity during natural infection.