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Genetic and phenotypic characterization of novel Bordetella pertussis lipid A modifications

University of British Columbia

Lipopolysaccharide (LPS) is a component of the outer membrane in most Gram-negative bacteria. The lipid A region of LPS anchors the molecule to the outer membrane and forms the first barrier between Gram-negative bacteria and the extracellular environment. Lipid A is also important for bacterial interaction and activation of host immune cells through binding to Toll-like receptor 4 (TLR4), activation of which results in a downstream inflammation response. The work presented in this thesis explores the genetic basis for different lipid A structures and the effects of this structural variability on activation of TLR4 and resistance to cationic antimicrobial peptides (CAMPs). Penta-acyl lipid A from B. pertussis strain BP338 is modified with glucosamine (GlcN), and mutational analyses revealed that LgmA, LgmB, and LgmC are required for this modification. Bioinformatic analysis suggests the following hypothetical model: LgmA transfers N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to the carrier lipid C55P, LgmC removes the acetyl group, and LgmB transfers GlcN from C55P to the lipid A phosphate. Glycosyltransferase assays with LgmA-expressing E. coli membranes show that LgmA transfers GlcNAc onto a lipid, therefore supporting the first step in this model. Site-directed mutagenesis has also identified a putative active site in LgmA and LgmC. Killing assays show the GlcN modification in B. pertussis increases resistance to numerous CAMPs and to outer membrane perturbation by EDTA. Lipid A from B. pertussis strain 18-323 exhibits low levels of TLR4 activation. Lipid A of strain 18-323 has no GlcN modification (due to an incomplete lgm locus) and a shorter C3’ acyl chain compared to strain BP338 (due to a difference in LpxA). Complementation of 18-323 with BP338 lipid A-modifying genes lpxA and/or the lgm locus increase TLR4 activation, though the GlcN modification had a dominant effect. In hexa-acyl E. coli lipid A, shortening the C3 and C3’ acyl chains had been shown to decrease TLR4 activation and resistance to polymyxin B, but increase activation of the limulus amebocyte lysate assay. Thus, varying the structure of lipid A, in both B. pertussis and E. coli, can affect TLR4 activation and resistance of the bacteria to CAMPs.

Text

2015-08-31

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/50063

Microbiology and Immunology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/