Role of the Escherichia coli O157:H7 O side chain in adherence and analysis of an rfb locus.
Shiga-toxigenic Escherichia coli strains belonging to serotype O157 are important human pathogens, but the genetic basis of expression of the O157 antigen and the role played by the lipopolysaccharide O side chain in the adherence of this organism to epithelial cells are not understood. We performed TnphoA mutagenesis on E. coli O157:H7 strain 86-24 to identify a mutant (strain F12) deficient in O-antigen expression. Nucleotide sequence analysis demonstrated that the transposon inserted within an open reading frame with significant homology to rfbE of Vibrio cholerae O1 (U. H. Stroeher, L. E. Karageorgos, R. Morona, and P. A. Manning, Proc. Natl. Acad. Sci. USA 89:2566-2570, 1992), which is postulated to encode perosamine synthetase. This open reading frame was designated rfbE(EcO157:H7). The guanine-plus-cytosine fraction (0.35) suggests that rfbE(EcO157:H7) may have originated in a species other than E. coli. rfbE(EcO157:H7) is conserved in nontoxigenic E. coli O157 strains expressing a variety of other flagellar antigens but is not found in E. coli O55:H7 strains, which are more closely related to E. coli O157:H7. Strain F12 was significantly more adherent to HeLa cells in a quantitative adherence assay than was its E. coli O157:H7 parent, but they did not differ in other phenotypes. Restoration of the expression of the O side chain by complementation of the TnphoA mutation in strain F12 by a plasmid expressing intact rfbE(EcO157:H7) reduced the adherence of the hyperadherent strain F12. We conclude that rfbE(EcO157:H7) is necessary for the expression of the O157 antigen, that acquisition of E. coli rfb genes occurred independently in E. coli O157:H7 and unrelated O157 strains, and that the O side chain of E. coli O157:H7 lipopolysaccharide interferes with the adherence of E. coli O157:H7 to epithelial cells.
Bilge
S S
SS
Department of Pediatrics, University of Washington School of Medicine, Seattle, USA.
Vary
J C
JC
Dowell
S F
SF
Tarr
P I
PI
D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D019453:Escherichia coli O157 / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D019081:O Antigens / Q000032:analysis; D000637:Transaminases / Q000737:chemistry / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D004251:DNA Transposable Elements; D019081:O Antigens; D000637:Transaminases; D002238:Carbohydrate Epimerases; C061272:perosamine synthetase