We reported previously that the core oligosaccharide region of the lipopolysaccharide (LPS) is essential for optimal adhesion of Actinobacillus pleuropneumoniae, an important swine pathogen, to respiratory tract cells. Rough LPS and core LPS mutants of A. pleuropneumoniae serotype 1 were generated by using a mini-Tn10 transposon mutagenesis system. Here we performed a structural analysis of the oligosaccharide region of three core LPS mutants that still produce the same O-antigen by using methylation analyses and mass spectrometry. We also performed a kinetic study of proinflammatory cytokines production such as interleukin (IL)-6, tumor necrosis factor-alpha, IL1-β, MCP-1, and IL8 by LPS-stimulated porcine alveolar macrophages, which showed that purified LPS of the parent strain, the rough LPS and core LPS mutants, had the same ability to stimulate the production of cytokines. Most interestingly, an in vitro susceptibility test of these LPS mutants to antimicrobial peptides showed that the three core LPS mutants were more susceptible to cationic peptides than both the rough LPS mutant and the wild type parent strain. Furthermore, experimental pig infections with these mutants revealed that the galactose (Gal I) and d,d-heptose (Hep IV) residues present in the outer core of A. pleuropneumoniae serotype 1 LPS are important for adhesion and overall virulence in the natural host, whereas deletion of the terminal GalNAc-Gal II disaccharide had no effect. Our data suggest that an intact core-lipid A region is required for optimal protection of A. pleuropneumoniae against cationic peptides and that deletion of specific residues in the outer LPS core results in the attenuation of the virulence of A. pleuropneumoniae serotype 1
Lipopolysaccharide, LPS, oligosaccharide, core, host, strain, structural, terminal, tract, virulence, serotype, O-antigen, analysis, O antigen, cell, Research, type, core oligosaccharide, infection, wild type, specific, spectrometry, mutant, mutants, biological, structural analysis, region, mass spectrometry, methylation, production, transposon, rough, interleukin, pathogen, purified, natural, core-lipid A region, disaccharide, galactose, cells, experimental, mutagenesis, effect, peptides, ability, susceptibility, Actinobacillus, adhesion, Actinobacillus pleuropneumoniae, PDF, protection, cytokines, rough LPS, macrophages, in vitro, macrophage, LPS core, peptide, test, cytokine, antimicrobial, alveolar, alveolar macrophage, attenuation, cationic, necrosis, pig, porcine, respiratory, swine, tumor, tumor necrosis factor-alpha
NCBI PubMed ID: 16188878Journal NLM ID: 2985121RPublisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
Institutions: Groupe de Recherche sur les Maladies Infectieuses du Porc and the Departement de Pathologie et Microbiologie, Faculte de Medecine Veterinaire, Universite de Montreal, St-Hyacinthe, Quebec J2S 7C6, Canada, Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A OR6, Canada
Methods: serological methods
Found