Isolation and characterization of mini-Tn10lipopolysaccharide mutants ofActinobacillus pleuropneumoniaeserotype 1

Abstract

Lipopolysaccharide (LPS) has previously been identified as the major adhesin of Actinobacillus pleuropneumoniae involved in adherence to porcine respiratory tract cells. The purpose of the present study was to isolate and characterize mutants in LPS biosynthesis by using a mini-Tn10 transposon mutagenesis system. Seven mutants appeared to possess a rough LPS (among which two had similar Southern blot profiles) while one mutant (#5.1) expressed the high-molecular-mass LPS, but as visualized by Tricine SDS-PAGE, showed an additional band in the core-lipid A region. The LPS mutants showed sensitivity to pig serum to various degrees, while the parent strain was serum-resistant. Use of piglet frozen tracheal sections indicated that, surprisingly, the rough LPS mutants adhered similarly or in greater numbers than the parent strain. However, the LPS mutant #5.1 adhered significantly less than the parent strain and was also less virulent in pigs. The gene affected by mini-Tn10 in LPS mutant #5.1 is galU, the structural gene for UTP-alpha-D-glucose-1-phosphate uridylyltransferase, involved in LPS core biosynthesis. Complementation analysis confirmed that the phenotypic characteristics of LPS mutant #5.1 are the result of the inactivation of the galU gene. Our data suggest that although the presence of O-antigen does not seem to be essential, an intact core-lipid A region might be required for adherence of A. pleuropneumoniae to porcine respiratory tract cells. To the best of our knowledge, these mutants represent the first isogenic mutants of A. pleuropneumoniae defective in LPS biosynthetic genes.

Truncation of the Lipopolysaccharide Outer Core Affects Susceptibility to Antimicrobial Peptides and Virulence of Actinobacillus pleuropneumoniae Serotype 1

“…For the kinetic study the PAMs were classified into the following three groups: unstimulated cells as a negative control, cells stimulated with 1 g/ml purified LPS, or 10 9 CFU/ml of heat-killed bacteria and incubated at 37°C in 5% CO 2 . Purification of LPS was performed according to the Darveau-Hancock procedure (30,31), and the products were separated and analyzed by SDS-PAGE and immunoblotting as described previously (13). Purified LPS of the wild type parent strain and all the LPS mutants (TABLE ONE) were used in this study.…”

“…We have previously generated four core LPS and seven rough LPS mutants of A. pleuropneumoniae serotype 1 by using a mini-Tn10 mutagenesis system, and the gene affected by the transposon was identified in each of the mutants (13)(14)(15). Characterization of the rough LPS mutants allowed us to identify a cluster of 13 genes involved in O-polysaccharide biosynthesis in A. pleuropneumoniae serotype 1 (15).…”

“…Characterization of the rough LPS mutants allowed us to identify a cluster of 13 genes involved in O-polysaccharide biosynthesis in A. pleuropneumoniae serotype 1 (15). The genes affected in the core LPS mutants were galU in mutant 5.1 (13), which encodes a UTP-␣-D-glucose-1-phosphate uridylyltransferase, a gene involved in outer core elongation with galactose in mutant CG1 (14) and a gene coding for a D-glycero-D-manno-heptosyltransferase in mutants CG3 and CG5 (14). All these core LPS mutants still express an O-chain, although their core oligosaccharide is apparently truncated, * This work was supported in part by the Natural Sciences and Engineering Research based on gel mobility (14).…”

“…All these core LPS mutants still express an O-chain, although their core oligosaccharide is apparently truncated, * This work was supported in part by the Natural Sciences and Engineering Research based on gel mobility (14). Characterization of the surface properties of these LPS mutants demonstrated that O-antigen deletions in rough mutants have no effect on the adhesion of the bacteria to frozen tracheal sections of pigs, whereas an intact core oligosaccharide region seems to be required for optimal adherence (7,13). Most interestingly, in vivo experiments in pigs showed that the core LPS mutant 5.1 that still produces an O-antigen was less virulent, whereas no difference was observed between the rough mutant 27.1 lacking O-antigen and the parent strain (13)(14)(15).…”

“…Characterization of the surface properties of these LPS mutants demonstrated that O-antigen deletions in rough mutants have no effect on the adhesion of the bacteria to frozen tracheal sections of pigs, whereas an intact core oligosaccharide region seems to be required for optimal adherence (7,13). Most interestingly, in vivo experiments in pigs showed that the core LPS mutant 5.1 that still produces an O-antigen was less virulent, whereas no difference was observed between the rough mutant 27.1 lacking O-antigen and the parent strain (13)(14)(15). These observations suggest that the LPS core oligosaccharide could play a major role in colonization and pathogenesis of A. pleuropneumoniae in pigs.…”