Purification and characterization of two outer membrane antigens from Escherichia coli O157:H7

Escherichia coli O157:H7 has emerged as an important foodborne pathogen, causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) with increasing frequency since 1982. Two low molecular weight outer membrane antigens expressed by E. coli O157:H7, which bind monoclonal antibody 4E8C12 were purified to homogeneity by ammonium sulfate precipitation, Sephacryl S-100 HR gel filtration, and DEAE-Sephadex A-25 ion-exchange column chromatography. The first 20 N-terminal amino acid sequences of the two antigens were very similar and were determined to be M-N-Q-D-E-A-G-X-N-F-Q-F-K-G-X-V-K-E-Q, which has 90% homology with a deduced amino acid sequence from E. coli K-12 Yjbj gene. PCR sequencing of the corresponding gene revealed that the nucleotide sequence is 100% homologous to the E. coli K-12 Yjbj gene. Studies using anti-lipid A antibody and an endotoxin-removing gel revealed that the two antigens are lipid A-associated or lipopolysaccharide (LPS) core components. Another monoclonal antibody (MAb 5F9C8) specific for many serotypes of enterohemorrhagic E. coli (EHEC) was produced. Western blot revealed that MAb 5F9C8 reacted with five outer membrane antigens with apparent molecular weights of 6, 8, 10, 11, and 12 kDa, respectively. Transposon phoA mutagenesis studies revealed that MAb 5F9C8 recognizes different epitopes other than MAb 4E8C12, which is more specific for more virulent EHEC serotypes, including O157:H7, O26:H11, and O111. The data indicate that the antigenic presentation of the two antigens that bind MAb 4E8C12 is very complex and the expression of the antigens may be multiple gene-regulated. It is suggested that either a special epitope located at the LPS core region or a special three dimensional structure contributes to the antigenic reactivity recognized by MAb 4E8C12.