Production of the subtilase AB5 cytotoxin by Shiga toxin-negative Escherichia coli

Abstract

The subtilase cytotoxin (SubAB) is an AB(5) toxin described in certain Shiga toxin (Stx)-producing Escherichia coli (STEC) strains that usually lack the locus for enterocyte effacement (LEE). We report for the first time the production of SubAB by two Stx-negative E. coli strains, isolated from unrelated cases of childhood diarrhea. The characterization of the SubAB-coding genes showed a 90% nucleotide sequence similarity with that of the prototype subAB, located on the virulence plasmid of the STEC O113 strain 98NK2 (pO113). In both strains, subAB was physically associated with tia, an invasion genetic determinant of enterotoxigenic E. coli. The strains were negative for the saa gene, encoding an adhesin located on pO113 and present in many of the SubAB-positive strains described so far. PCR screening of 61 STEC and 100 Stx-negative E. coli strains in our collection revealed the presence of subAB in five LEE-negative STEC strains but not in the Stx-negative strains. subAB was contiguous to tia in three of the positive strains, which were all negative for saa. These results indicate that SubAB production is not restricted to STEC and suggest that a subAB-tia putative pathogenicity island is involved in the dissemination of subAB genes, as an alternative to plasmid pO113.

FIG. 1.

ClustalW comparison of the amino acidic sequences of the A and B subunits of SubAB from strain ED 591 (this study) and the reference strain 98NK2. The amino acidic substitutions are indicated by the corresponding letters. The residues involved in the catalytic activity in the A subunit and those forming the receptor-binding pocket in the B subunit are indicated (shaded gray).

FIG. 2.

Molecular architecture of the subAB locus in the subAB-tia-positive strains. The primers' positions and orientations are indicated by arrows.

FIG. 3.

PCR amplification of tia in strains ED 32, ED 591, ED 81, ED 97, ED 99, ED 186, and ED 424 and in the negative control (lanes 3 to 10, respectively) using the primer pair tia_sense/tia_lo. Molecular weight markers are indicated in lanes 1 and 2 (1 kb and 100 bp, respectively). Refer to Fig. 1 and Table 1 for primer location.