Molecular and chemical characterization of the lipopolysaccharide O-antigen and its role in the virulence of Yersinia enterocolitica serotype O:8.
The Y. enterocolitica O:8(YeO8) O-antigen repeat units consist of five sugar residues: N-acetyl-D-galactosamine (GalNAc), D-galactose (Gal), D-mannose (Man), L-fucose (Fuc), and 6-deoxy-D-gulose (6d-Gul). The nucleotide sequence of the O-antigen gene cluster of the YeO8 strain 8081-c was determined. Altogether, 18 open reading frames (ORFs) were identified and shown to be essential for O-antigen biosynthesis. We previously characterized the 3'-end of the O-antigen gene cluster and identified four genes: two for GDP-Man biosynthesis, one for UDP-Gal biosynthesis, and one for O-antigen polymerase. Based on sequence similarity, Tn5-insertion phenotypes and chemical analysis, the 14 new genes were assigned the following functions: four genes are involved in the biosynthesis of CDP-6d-Gul and two in GDP-Fuc biosynthesis. Five gene products were assigned sugar transferase functions and one gene product was similar to Wzx, the O-antigen flippase. Two genes remained unassigned. By genetic complementation we also showed that YeO8 O-antigen biosynthesis was dependent on N-acetyl-glucosaminyl:undecaprenylphosphate transferase (GlcNAc transferase), the WecA (formerly known as Rfe) protein. Data obtained from chemical-composition analysis suggest that in addition to being GlcNAc transferase, WecA may also function as a GalNAc transferase. Using a restriction-deficient derivative of Y. enterocolitica O:8 strain 8081, a rough mutant, designated 8081-R2, was isolated. 8081-R2 was complemented in trans with a cloned O-antigen gene cluster restoring surface O-antigen expression. The virulence of the wild-type strain and that of the complemented strain were significantly higher (approx. 100-fold) than that of the rough mutant in an orally infected mouse model, showing that YeO8 O-antigen is a virulence factor.
Zhang
L
L
Turku Immunology Centre, Department of Medical Microbiology, University of Turku, Finland. lijuan@hancock.microbiology.ubc.ca
Radziejewska-Lebrecht
J
J
Krajewska-Pietrasik
D
D
Toivanen
P
P
Skurnik
M
M
D000818:Animals; D001435:Bacteriophages; D001483:Base Sequence; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D008070:Lipopolysaccharides / Q000737:chemistry; D051379:Mice; D008811:Mice, Inbred DBA; D008969:Molecular Sequence Data; D005810:Multigene Family; D016254:Mutagenesis, Insertional; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D019081:O Antigens; D010641:Phenotype; D011991:Receptors, Virus; D012703:Serotyping; D014166:Transferases / Q000235:genetics; D014774:Virulence; D015008:Yersinia enterocolitica / Q000472:pathogenicity*
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D008070:Lipopolysaccharides; D019081:O Antigens; D011991:Receptors, Virus; D014166:Transferases; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase