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Tn5-induced lipopolysaccharide mutations in Bordetella pertussis that affect outer membrane function
M L Turcotte1, Denis Martin2, Bernard R Brodeur2
1Department of Medical Microbiology and Immunology, University of Alberta, 1-69 Medical Sciences Bldg, Edmonton, Alberta, Canada T6G 2H7.
Microbiology (Reading, England)
|July 1, 1997
Summary
Researchers identified new Bordetella pertussis mutants with altered lipopolysaccharide (LPS) structures. These changes affect antibiotic sensitivity and may impact bacterial virulence and vaccine development.
Area of Science:
- Microbiology
- Immunology
- Genetics
Background:
- Lipopolysaccharide (LPS) structure is critical for Bordetella pertussis virulence and immune evasion.
- Understanding LPS biosynthesis and regulation is key to developing effective pertussis vaccines and treatments.
Purpose of the Study:
- To identify and characterize Tn5 insertion mutants of Bordetella pertussis with altered lipopolysaccharide (LPS) phenotypes.
- To investigate the genetic basis and functional consequences of LPS structural changes.
Main Methods:
- Screening of Tn5 insertion mutants using an LPSB-specific monoclonal antibody (mAb).
- Analysis of LPS phenotypes using silver-stained SDS-PAGE.
- Epitope exposure assessment via binding assays with LPSA- and LPSB-specific mAbs.
- Genetic mapping of Tn5 insertions using PFGE and Southern blotting.
Main Results:
- Nine distinct LPS phenotypes were identified in ten mutants, with six exhibiting novel LPS bands (IntA and IntB).
- Mutants showed altered epitope exposure on LPS, both cell-free and on whole cells.
- Seven Tn5 insertions mapped to the bpl gene region involved in LPS biosynthesis; three mapped elsewhere.
- Mutants MLT2 and MLT3 showed accelerated avirulence, despite Tn5 insert locations distant from the bvglASR locus.
- LPS alterations correlated with changes in reactivity to strain-specific mAbs and antibiotic sensitivity.
Conclusions:
- Tn5 mutagenesis effectively generated Bordetella pertussis mutants with diverse and undocumented LPS structures.
- LPS alterations impact bacterial surface epitope exposure, antibiotic sensitivity, and potentially virulence.
- The study implicates both structural and regulatory genes in LPS production and highlights novel genetic loci involved in LPS modification.