Biosynthesis and Structure of the Burkholderia cenocepacia K56-2 Lipopolysaccharide Core Oligosaccharide: TRUNCATION OF THE CORE OLIGOSACCHARIDE LEADS TO INCREASED BINDING AND SENSITIVITY TO POLYMYXIN B

Burkholderia cenocepacia is an opportunistic pathogen that displays a remarkably high resistance to antimicrobial peptides. We hypothesize that high resistance to antimicrobial peptides in these bacteria is because of the barrier properties of the outer membrane. Here we report the identification of...

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Bibliographic Details
Published in:The Journal of biological chemistry 2009-08, Vol.284 (32), p.21738-21751
Main Authors: Ortega, Ximena, Silipo, Alba, Saldías, M. Soledad, Bates, Christa C, Molinaro, Antonio, Valvano, Miguel A
Format: Article
Language:English
Subjects:
Animals
Burkholderia - metabolism
Carbohydrate Sequence
Cell Line
Cloning, Molecular
Drug Resistance, Bacterial
Glycobiology and Extracellular Matrices
Humans
Lipopolysaccharides - metabolism
Mice
Molecular Sequence Data
Mutation
Oligosaccharides - chemistry
Plasmids - metabolism
Polymyxin B - pharmacology
Protein Binding
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Summary:Burkholderia cenocepacia is an opportunistic pathogen that displays a remarkably high resistance to antimicrobial peptides. We hypothesize that high resistance to antimicrobial peptides in these bacteria is because of the barrier properties of the outer membrane. Here we report the identification of genes for the biosynthesis of the core oligosaccharide (OS) moiety of the B. cenocepacia lipopolysaccharide. We constructed a panel of isogenic mutants with truncated core OS that facilitated functional gene assignments and the elucidation of the core OS structure in the prototypic strain K56-2. The core OS structure consists of three heptoses in the inner core region, 3-deoxy-D-manno-octulosonic acid, D-glycero-D-talo-octulosonic acid, and 4-amino-4-deoxy-L-arabinose linked to D-glycero-D-talo-octulosonic acid. Also, glucose is linked to heptose I, whereas heptose II carries a second glucose and a terminal heptose, which is the site of attachment of the O antigen. We established that the level of core truncation in the mutants was proportional to their increased in vitro sensitivity to polymyxin B (PmB). Binding assays using fluorescent 5-dimethylaminonaphthalene-1-sulfonyl-labeled PmB demonstrated a correlation between sensitivity and increased binding of PmB to intact cells. Also, the mutant producing a heptoseless core OS did not survive in macrophages as compared with the parental K56-2 strain. Together, our results demonstrate that a complete core OS is required for full PmB resistance in B. cenocepacia and that resistance is due, at least in part, to the ability of B. cenocepacia to prevent binding of the peptide to the bacterial cell envelope.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M109.008532