Isolation and characterization of mini-Tn10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1

Lipopolysaccharide (LPS) has previously been identified as the major adhesi n of Actinobacillus pleuropneumoniae involved in adherence to porcine respi ratory tract cells. The purpose of the present study was to isolate and cha racterize mutants in LPS biosynthesis by using a mini-Tn10 transposon mutag enesis system. Seven mutants appeared to possess a rough LPS (among which t wo had similar Southern blot profiles) while one mutant (#5.1) expressed th e 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 sensiti vity to pig serum to various degrees, while the parent strain was serum-res istant. Use of piglet frozen tracheal sections indicated that, surprisingly , the rough LPS mutants adhered similarly or in greater numbers than the pa rent strain. However, the LPS mutant #5.1 adhered significantly less than t he parent strain and was also less virulent in pigs. The gene affected by m ini-Tn10 in LPS mutant #5.1 is galU, the structural gene for UTP-alpha-D-gl ucose-1-phosphate uridylyltransferase, involved in LPS core biosynthesis. C omplementation analysis confirmed that the phenotypic characteristics of LP S 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 essent ial, an intact core-lipid A region might be required for adherence of A. pl europneumoniae to porcine respiratory tract cells. To the best of our knowl edge, these mutants represent the first isogenic mutants of A. pleuropneumo niae defective in LPS biosynthetic genes.