Burkholderia cenocepacia is an opportunistic pathogen that displays a remarkably high resistance to antimicrobial peptides (APs). We hypothesize that high resistance to APs in these bacteria is due to 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 3 heptoses in the inner core region, 3-deoxy-D-manno-octulosonic acid (Kdo), D-glycero-D-talo-octulosonic acid (Ko), and 4-amino-4-deoxy-L-arabinose (L-Ara4N) linked to Ko. Also, glucose is linked to heptose I while 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 dansyl-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 to 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.
Lipopolysaccharide, structure, O-antigen, core oligosaccharide, outer membrane, macrophages, Polymyxin B, Burkholderia cenocepacia
NCBI PubMed ID: 19525227Publication DOI: 10.1074/jbc.M109.008532Journal NLM ID: 2985121RPublisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Correspondence: mvalvano@uwo.ca
Institutions: Infectious Diseases Research Group, Siebens-Drake Research Institute, Department of Microbiology and Immunology, Department of Medicine, University of Western Ontario, London, Ontario N6A 5C1, Canada, Dipartimento di Chimica Organica e Biochimica, Universitá di Napoli, Complesso Universitario Monte Sant'angelo, Via Cintia 4, 80126 Napoli, Italy
Methods: 13C NMR, 1H NMR, NMR-2D, mild acid hydrolysis, MALDI-TOF MS, composition analysis, NMR-1D, genetic methods, statistical analysis
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