Isolation and characterization of the localized adherence factor of enteropathogenic Escherichia coli

Abstract

The binding factor of enteropathogenic Escherichia coli O111:H- responsible for localized adherence (LA) on HeLa cells was investigated. Inhibition of LA by carbohydrates and lectins showed that the reactive epitope on HeLa cells contains N-acetylgalactosamine units. Treatment of bacteria with EDTA for extraction of lipopolysaccharides eliminated these polymers as binding factors. Such treatment also caused a marked increase in adhesion suggesting steric hindrance by lipopolysaccharides of the LA factor binding capacity. Immunoblotting with rabbit antibodies showed a strong reaction with two components with approximate molecular sizes of 29 and 32 kilodaltons (kDa) present in the outer membrane preparations of bacteria. Both the absorbed rabbit immune serum and the outer membrane preparation of the bacteria inhibited bacterial adhesion by 100%. Outer membrane components were isolated from an N-acetylgalactosamine-agarose column by elution with KSCN, labeled with 125I, and immunoprecipitated with absorbed rabbit hyperimmune antiserum. The only component precipitated was the protein doublet at 29 to 32 kDa corresponding to the components detected by immunoblotting. The predominant component was always the 32-kDa polypeptide. We conclude that this component of the outer membrane is the best candidate for the LA factor in enteropathogenic E. coli.

The outer membrane protein of enteropathogenic Escherichia coli, described as the 'localised adherence factor', is OmpF and probably not involved in adhesion to HEp-2 cells

“…This would be particularly likely to be the case for the transmembrane porin proteins. The above reactions closely resembled those of Scaletsky et al [10] where an antiserum, absorbed with a non-adhesive strain of E. coli; also reacted predominantly with a 32 kDa protein and to a lesser extent with a 29 kDa OMP. Unfortunately, Scaletsky et al [10] did not provide evidence as to the reaction of their absorbed antiserum with the non-adherent strain used for absorbing the antiserum.…”

“…Strains of E. coli, previously used for studying localised adhesion of EPEC to HEp-2 cells [8], were examined for the presence of the 32 kDa OMP described by Scaletsky et al [10] as the 'localised adherence factor'. The SDS-PAGE OMP profile of strain E20517 (Fig.…”

“…Furthermore, OmpsF, C and A are known to be chromosomally determined. Scaletsky et al [10] suggested that an OMP of 32 kDa was a candidate EPEC adhesin based on the reaction of an antiserum raised to an adhesive strain of EPEC (serogroup 0111) and absorbed with a homologous non-adhesive strain. For our studies we used an antiserum raised to strain E20513, which carries EAF genes and adheres to HEp-2 cells [8], and absorbed this antiserum with the EAF plasmid-cured strain 60R556.…”

“…Recently, Scaletsky et al [10] reported that the EPEC localized adherence factor was an OMP of 32 kDa, the possession of which enabled bacteria to adhere to HeLa cells via HeLa cell membrane components containing N-acetylgalactosamine. In the present study we examined strains of EPEC for this factor.…”

Modulation of lectinophagocytosis of Escherichia coli by variation of pH and temperature

“…The addition of this sugar during phagocytic assays at 37 or 41³C caused inhibition of phagocytosis of EPEC (Table 3). Since the outer membrane of enter-opathogenic E. coli bears a polypeptide which binds to N-acetylgalactosamine residues on HeLa cells [16] we suggest that this polypeptide can participate in phagocytosis of EPEC. Microorganisms produce multiple adhesins in order to colonize diverse surfaces and these proteins are also involved in non-opsonic phagocytosis of many microbial pathogens.…”