A novel pathogenicity island integrated adjacent to the thrW tRNA gene of avian pathogenic Escherichia coli encodes a vacuolating autotransporter toxin

Abstract

We report the complete nucleotide sequence and genetic organization of the Vat-encoding pathogenicity island (PAI) of avian pathogenic Escherichia coli strain Ec222. The 22,139-bp PAI is situated adjacent to the 3' terminus of the thrW tRNA gene, has a G+C content of 41.2%, and includes a bacteriophage SfII integrase gene, mobile genetic elements, two open reading frames with products exhibiting sequence similarity to known proteins, and several other open reading frames of unknown function. The PAI encodes an autotransporter protein, Vat (vacuolating autotransporter toxin), which induces the formation of intracellular vacuoles resulting in cytotoxic effects similar to those caused by the VacA toxin from Helicobacter pylori. The predicted 148.3-kDa protein product possesses the three domains that are typical of serine protease autotransporters of Enterobacteriaceae: an N-terminal signal sequence of 55 amino acids, a 111.8-kDa passenger domain containing a modified serine protease site (ATSGSG), and a C-terminal outer membrane translocator of 30.5 kDa. Vat has 75% protein homology with the hemagglutinin Tsh, an autotransporter of avian pathogenic E. coli. A vat deletion mutant of Ec222 showed no virulence in respiratory and cellulitis infection models of disease in broiler chickens. We conclude that the newly described PAI and Vat may be involved in the pathogenicity of avian septicemic E. coli strain Ec222 and other avian pathogenic E. coli strains.

FIG. 1.

Genetic map of the VAT-PAI with 22,139 bp of unique DNA downstream of the tRNA gene thrW and upstream of the yagU gene of E. coli K-12 MG1655. Numbers indicate ORFs described in Table 2. ORFs have not been drawn to scale.

FIG. 2.

Structure of the Vat precursor protein (not drawn to scale). The protein consists of 1,377 amino acids, with a signal sequence (SS) of 5.7 kDa, the Vat cytotoxin of 111.8 kDa, and a C-terminal outer membrane translocator of 30.5 kDa. A, signal sequence cleavage site; B, two cysteine residues; C, atypical serine protease motif; D, region that is deleted in vat mutant strain Ec222VPΔ2; E, outer membrane translocator cleavage site.

FIG. 3.

Alignment of the sequence of the predicted Vat protein (accesion number AY151282) with those of its closest homologues, Tsh (AJ223631), SepA (Z48219), EspP (X97542), Pet (AF056581), and Sat (AF289092). Residues identical in all six sequences are shaded in dark grey and those residues conserved in at least three sequences are shaded in light grey. The asterisk at residue 56 indicates the first amino acid of the mature Vat protein. The serine protease motif is underlined.

FIG. 3.

Alignment of the sequence of the predicted Vat protein (accesion number AY151282) with those of its closest homologues, Tsh (AJ223631), SepA (Z48219), EspP (X97542), Pet (AF056581), and Sat (AF289092). Residues identical in all six sequences are shaded in dark grey and those residues conserved in at least three sequences are shaded in light grey. The asterisk at residue 56 indicates the first amino acid of the mature Vat protein. The serine protease motif is underlined.

FIG. 3.

Alignment of the sequence of the predicted Vat protein (accesion number AY151282) with those of its closest homologues, Tsh (AJ223631), SepA (Z48219), EspP (X97542), Pet (AF056581), and Sat (AF289092). Residues identical in all six sequences are shaded in dark grey and those residues conserved in at least three sequences are shaded in light grey. The asterisk at residue 56 indicates the first amino acid of the mature Vat protein. The serine protease motif is underlined.

FIG. 4.

Vacuolation of CEF cells caused by culture supernatant of E. coli Ec222. (A) Vacuoles (arrows) were evident after 6 h of exposure of CEF cells to a 1/16 dilution of the filter-sterilized supernatant of an overnight culture of wild-type E. coli Ec222. The cells were stained with neutral red, which accumulated in the vacuoles. (B) Normal appearance of CEF cells 6 h after treatment as described for panel A except that the E. coli strain was Ec222ΔVP, a vat deletion mutant derivative of Ec222. Bars, 10 μm.