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-beta, 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.