Related Experiment Videos
Purification and characterization of fimbriae isolated from Bordetella pertussis
Abstract:
Fimbriae were detached from Bordetella pertussis by mechanical shearing and purified by successive precipitations with ammonium sulfate, phosphate buffer (pH 6.0), and magnesium chloride. In each of these purification steps, the fimbriae aggregated into bundles as seen by electron microscopy. These aggregates could be disaggregated at pH 9.5. By electron microscopy, the purified fimbriae appeared as long filaments with a diameter of 5 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fimbriae showed a single protein subunit with a molecular weight of 22,000. The purified fimbriae did not have hemagglutinating activity when assayed with several types of erythrocytes, and they were antigenically, chemically, and structurally distinct from the filamentous hemagglutinin of B. pertussis. The purified fimbriae were also identified as serotype 2 agglutinogens, since antibody to the purified fimbriae agglutinated B. pertussis strains serotyped as 1.2.4, 1.2.3, or 1.2.3.6 but did not agglutinate those serotyped as 1.3.6.
Insights
Researchers purified fimbriae from Bordetella pertussis, finding they are distinct from filamentous hemagglutinin and act as serotype 2 agglutinogens. These purified fimbriae are key for understanding Bordetella pertussis serotyping.
Area of Science:
- Microbiology
- Immunology
- Protein Biochemistry
Background:
- Bordetella pertussis causes whooping cough.
- Fimbriae are surface appendages involved in bacterial adhesion and virulence.
- Understanding B. pertussis surface structures is crucial for vaccine development and diagnostics.
Purpose of the Study:
- To isolate and characterize fimbriae from Bordetella pertussis.
- To determine the antigenic and structural properties of purified fimbriae.
- To investigate the role of fimbriae in B. pertussis serotyping.
Main Methods:
- Mechanical shearing for fimbriae detachment.
- Ammonium sulfate, phosphate buffer, and magnesium chloride precipitation for purification.
- Electron microscopy for structural analysis.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for subunit analysis.
- Hemagglutination assays and antibody agglutination tests for functional and antigenic characterization.
Main Results:
- Purified fimbriae appeared as 5 nm diameter filaments, aggregating into bundles disaggregable at pH 9.5.
- SDS-PAGE revealed a single protein subunit of 22,000 molecular weight.
- Purified fimbriae lacked hemagglutinating activity and were distinct from filamentous hemagglutinin.
- Fimbriae were identified as serotype 2 agglutinogens, cross-reacting with specific B. pertussis serotypes.
Conclusions:
- The study successfully purified and characterized Bordetella pertussis fimbriae.
- These fimbriae are structurally and antigenically distinct from filamentous hemagglutinin.
- The purified fimbriae serve as serotype 2 agglutinogens, important for B. pertussis strain identification.