Lipopolysaccharides (LPS) of Bordetella pertussis are important modulators of the immune system. Interaction of the lipid A region of LPS with the Toll-like receptor 4 (TLR4) complex causes dimerization of TLR4 and activation of downstream nuclear factor kappaB (NFkappaB), which can lead to inflammation. We have previously shown that two strains of B. pertussis, BP338 (a Tohama I-derivative) and 18-323, display two differences in lipid A structure. 1) BP338 can modify the 1- and 4'-phosphates by the addition of glucosamine (GlcN), whereas 18-323 cannot, and 2) the C3' acyl chain in BP338 is 14 carbons long, but only 10 or 12 carbons long in 18-323. In addition, BP338 lipid A can activate TLR4 to a greater extent than 18-323 lipid A. Here we set out to determine the genetic reasons for the differences in these lipid A structures and the contribution of each structural difference to the ability of lipid A to activate TLR4. We show that three genes of the lipid A GlcN modification (Lgm) locus, lgmA, lgmB, and lgmC (previously locus tags BP0399-BP0397), are required for GlcN modification and a single amino acid difference in LpxA is responsible for the difference in C3' acyl chain length. Furthermore, by introducing lipid A-modifying genes into 18-323 to generate isogenic strains with varying penta-acyl lipid A structures, we determined that both modifications increase TLR4 activation, although the GlcN modification plays a dominant role. These results shed light on how TLR4 may interact with penta-acyl lipid A species.
Lipopolysaccharide, structure, genetics, lipid A, Bordetella pertussis, lpxA, Toll-like receptor 4
NCBI PubMed ID: 23467413Publication DOI: 10.1074/jbc.M112.434365Journal NLM ID: 2985121RPublisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Correspondence: rachelf@mail.ubc.ca
Institutions: Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada, Equipe Structure et Activités des Endotoxines, UMR 8621 du Centre National de la Recherche Scientifique, Institut de Génétique et Microbiologie (IGM), Université de Paris-Sud, 91405 Orsay cedex, France
Methods: PCR, DNA techniques, MALDI-MS, genetic methods, statistical analysis, bioinformatic analysis, SDS-Tricine-PAGE, HEK-blue TLR4 activation assay
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