Isolation and characterization of mini-Tn10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1.
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.
Rioux
S
S
Département de pathologie et microbiologie, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, QC, Canada.
Galarneau
C
C
Harel
J
J
Frey
J
J
Nicolet
J
J
Kobisch
M
M
Dubreuil
J D
JD
Jacques
M
M
D000189:Actinobacillus Infections / Q000662:veterinary*; D016977:Actinobacillus pleuropneumoniae / Q000145:classification / Q000235:genetics* / Q000472:pathogenicity; D000818:Animals; D001422:Bacterial Adhesion / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D012703:Serotyping; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology*; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000235:genetics
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; C121039:galU protein, E coli