Molecular analysis of Shiga toxigenic Escherichia coli O111:H- proteins which react with sera from patients with hemolytic-uremic syndrome

Abstract

Western blot analysis was used to assess the reactivity of convalescent-phase sera from patients who were associated with an outbreak of hemolytic-uremic syndrome (HUS) caused by fermented sausage contaminated with Shiga toxin-producing Escherichia coli (STEC). The predominant STEC isolated from HUS patients belonged to serotype O111:H-, and reactivity to O111:H- whole-cell lysates, treated or untreated with proteinase K, was examined. As expected, all five serum samples demonstrated a marked anti-lipopolysaccharide response, but several protein bands were also immunoreactive, particularly one with an apparent size of 94 kDa. One convalescent-phase serum sample was subsequently used to screen an O111:H- cosmid bank and 2 of 900 cosmid clones were found to be positive, both of which contained a similar DNA insert. Western blot analysis of one of these clones identified three major immunoreactive protein bands of approximately 94, 70, and 50 kDa. An immune response to the three proteins was detectable with all five convalescent-phase serum samples but not with healthy human serum. Immunoreactive 94- and 50-kDa species were produced by a deletion derivative of the cosmid containing a 7-kb STEC DNA insert. Sequence analysis of this region indicated that it is part of the locus for enterocyte effacement, including the eaeA gene which encodes intimin. The deduced amino acid sequence of the O111:H- intimin was 88.6% identical to intimin from O157:H7 STEC, and the most divergent region was the 200 residues at the carboxyl terminus, which were only 75% identical. Such variation may be antigenically significant as serum from a HUS patient infected only with the O111:H- STEC reacted with intimin from an enteropathogenic E. coli O111 strain, as well as several other eaeA-positive STEC isolates, but not with an eaeA-positive STEC belonging to serotype O157:H-. Sera from two of the other HUS patients also failed to react with intimin from this latter strain. However, intimin from O157:H- STEC did react with serum from a patient infected with both O111:H- and O157:H- STEC.

FIG. 1

Western immunoblot analysis of convalescent-phase sera from HUS patients. Convalescent-phase sera from five HUS patients were reacted with electrophoresed undigested extracts of O111:H⁻ STEC 95NR1 (UD) or with extracts which had been digested with proteinase K (D) as described in Materials and Methods. The positions of the protein size markers are indicated at right.

FIG. 2

Reactivity of HUS patient serum with various STEC and reference E. coli strains. Serum from HUS patient 1 was used to probe Western blots of undigested E. coli lysates as described in Materials and Methods. Lanes: 1, 95NR1 (O111:H⁻, eaeA positive); 2, EPEC 87A (O111, eaeA positive); 3, EDL933 (O157:H7, eaeA positive); 4, EDL933-Cu (O157:H7, eaeA positive, 60-MDa plasmid negative); 5, 95ZG1 (O26, eaeA positive); 6, 95SF2 (O157:H⁻, eaeA positive); 7, 94CR (O48:H21); 8, MW13 (O98); 9, MW10 (O113); 10, 95AS1 (O128); 11, 95PM2 (O123); 12, 95HE4 (O91); 13, E. coli DH5α. The positions of the protein size markers are indicated at left.

FIG. 3

Reactivity of HUS patient serum with E. coli DH5α clones. Serum from HUS patient 1 was used to probe Western blots of 95NR1 and of E. coli DH5α carrying the cosmid vector (pPM2101), the immunoreactive cosmid pEV267, its deletion derivative pEV283, and two pBC SK subclones of pEV283 (pEV284 and pEV285; see Fig. 4 for map). The positions of the protein size markers are indicated at left.

FIG. 4

Locations of ORFs within the region of 95NR1 DNA cloned in pEV283 and in subclones pEV284 and pEV285. The cosmid deletion derivative pEV283 was constructed by digestion of pEV267 with HindIII followed by religation. This deleted approximately 30 kb of 95NR1 insert DNA 5′ to the HindIII site shown. Subclones pEV284 and pEV285 are in pBC SK.

FIG. 5

Alignment of the deduced amino acid sequence of intimin from O111:H⁻ STEC 95NR1 with that previously published for intimin from an O157:H7 STEC isolate (33) and that of a partial sequence for intimin from an O111:H8 STEC isolate (18). Identical residues are represented by dots, while the dash indicates the absence of an amino acid.

FIG. 6

Additional Western blot analysis. Serum from HUS patient 2, who was infected with both O111:H⁻ and O157:H⁻ STEC, was used to probe Western blots of undigested E. coli lysates as described in Materials and Methods. Lanes: 1, 95NR1 (O111:H⁻, eaeA positive); 2, EPEC 87A (O111, eaeA positive); 3, EDL933 (O157:H7, eaeA positive); 4, EDL933-Cu (O157:H7, eaeA positive, 60-MDa plasmid negative); 5, 95SF2 (O157:H⁻, eaeA positive); 6, 95ZG1 (O26, eaeA positive); 7, MW13 (O98); 8, 94CR (O48:H21); 9, MW10 (O113); 10, 95PM2(O123); 11, 95AS1 (O128). The positions of the protein size markers are indicated at left.