Cloning and Sequencing of Two New Verotoxin 2 Variant Genes of Escherichia coli Isolated from Cases of Human and Bovine Diarrhea

Abstract

We cloned and sequenced two new Verotoxin 2 (VT2) variant genes: one from an Escherichia coli strain from a case of bovine diarrhea and the other from an E. coli strain from a patient with diarrhea. The nucleotide and amino acid sequences of these two genes were highly homologous with, but distinct from those of the VT2, VT2vha, VT2vhb, SLT-IIv (VT2vp1) and SLT-Hva (VT2vp2) genes. Their nucleotide sequences were much more closely homologous to that of VT2vh than to that of VT2vp. Search for these two new genes in other Verocytotoxin-producing E. coli strains resulted in the isolation of 2 strains carrying one of the new VT2 variant genes, one strain from Tokyo and the other from Canada. Strains of Verocytotoxin-producing Escherichia coli (VTEC) are associated with human diseases such as diarrhea, hemorrhagic colitis and hemolytic uremic syndrome, dysentery in calves and edema disease of pigs (11). Verotoxins which have been well characterized include VT1 (or Shiga-like toxin I, SLT-I) (3,4, 10,18,19,27), VT2 (or Shiga-like toxin II, SLT-II) (17,24,27,30) and several variants of VT2, such as 13,14,29), VT2vha (8,21), VT2vhb (8,21) and SLT-IIva (6). VT1 is identical (28), or almost identical (3, 4, 10) with Shiga toxin of Shigella dysenteriae type 1 in the nucleotide sequence of its gene and amino acid sequence. VT2 is distinct from VT1 and Shiga toxin and is not neutralized by polyclonal antisera against VT1 and Shiga toxin (24, 27, 30). The overall nucleotide sequence homologies of the VT2 gene with those of the VT1 and Shiga toxin genes are approximately 55-60% (9, 16, 17). The VT2 and SLT-IIv genes have 91% homology in nucleotide sequences (7,29). The nucleotide sequences of the VT2vha and VT2vhb genes are nearly identical, showing 99% overall homology (8). The sequences of the genes of the A and B subunits of VT2vha show 98.6 and 95.5% homology, respectively, with those of the VT2 gene (8). The nucleotide sequence of the SLT-IIva gene is most closely homologous with that of the SLTIIv gene: the nucleotide sequences of the A and B subunits of the SLT-IIva gene have 70.6 and 98% homology, respectively, with those of the SLT-IIv gene (6). To simplify the names of VT2 variants, in this paper, we tentatively named SLTIIv and SLT-IIva as VT2vp1 and VT2vp2, respectively.During studies to develop methods for identification of VTEC strains such as the DNA colony hybridization test with poly-and oligonucleotide probes, bead-enzyme-linked immunosorbent assay (bead-ELISA) and the polymerase chain reaction (PCR) with common primers for various VTs and specific primers for each VT (12) (Yamasaki et al, submitted), we found several VTEC strains that possibly carried new VT2 variants genes. In this paper, we report cloning and sequencing of the genes of two new VT2 variants.

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