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Purification and characterization of an outer membrane protein adhesin from Haemophilus parainfluenzae HP-28
C H Lai1, C Bloomquist, W F Liljemark
1Department of Diagnostic School of Dentistry, University of Minnesota, Minneapolis 55455.
Abstract:
Outer membranes were isolated from Haemophilus parainfluenzae HP-28 by a mild extraction method followed by Sephadex G-150 gel filtration chromatography. The first peak (pool 1) recovered contained an activity which inhibited adherence of HP-28 cells to saliva-coated spheroidal hydroxyapatite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of pool 1 revealed a dominant protein band of 34 kDa. The SDS-PAGE-purified 34-kDa protein was excised from the gel and used for antibody preparation in rabbits. The antiserum produced was analyzed by immunoblot and was shown to be monospecific for the 34-kDa protein. Anti-34-kDa protein antibody was purified from the rabbit antiserum by protein A-Sepharose 6MB affinity chromatography. This antibody was then cross-linked to protein A-Sepharose 6MB to construct a second affinity column. The 34-kDa proteins were purified from outer membranes by this affinity chromatography. The 34-kDa protein was homogeneous, as confirmed by SDS-PAGE, isoelectric focusing, and reverse-phase chromatography analyses. Fab and Fc fragments of the purified anti-34-kDa protein antibodies were prepared by papain digestion, followed by carboxymethyl cellulose chromatography. Fab fragments from the anti-34-kDa protein antibody and the affinity-purified 34-kDa protein both showed significant inhibition of parent H. parainfluenzae HP-28 cell adherence to experimental salivary pellicle and to Streptococcus sanguis SA-1.
Insights
A 34 kDa protein from Haemophilus parainfluenzae outer membranes inhibits bacterial adherence. Antibodies targeting this protein also blocked adherence, suggesting its role in adhesion mechanisms.
Area of Science:
- Microbiology
- Immunology
- Biochemistry
Background:
- Haemophilus parainfluenzae adheres to host tissues, contributing to infections.
- Understanding the molecular mechanisms of bacterial adherence is crucial for developing therapeutic strategies.
Purpose of the Study:
- To isolate and characterize a protein from Haemophilus parainfluenzae HP-28 outer membranes involved in bacterial adherence.
- To generate antibodies against the identified protein and evaluate their efficacy in inhibiting adherence.
Main Methods:
- Outer membranes were extracted and fractionated using Sephadex G-150 gel filtration.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) identified a 34 kDa protein.
- Antibodies were generated against the 34 kDa protein and purified using affinity chromatography.
- Fab fragments of antibodies and the purified protein were tested for adherence inhibition.
Main Results:
- A 34 kDa protein was isolated and found to inhibit the adherence of Haemophilus parainfluenzae HP-28 to saliva-coated hydroxyapatite.
- Monospecific antibodies were successfully raised against the 34 kDa protein.
- Both the purified 34 kDa protein and its specific Fab fragments significantly inhibited bacterial adherence.
Conclusions:
- The 34 kDa outer membrane protein of Haemophilus parainfluenzae HP-28 plays a significant role in bacterial adherence.
- Targeting this protein with antibodies or its fragments represents a potential therapeutic approach to prevent H. parainfluenzae colonization.