Identification and pathogenomic analysis of an Escherichia coli strain producing a novel Shiga toxin 2 subtype

Abstract

Shiga toxin (Stx) is the key virulent factor in Shiga toxin-producing Escherichia coli (STEC). To date, three Stx1 subtypes and seven Stx2 subtypes have been described in E. coli, which differed in receptor preference and toxin potency. Here, we identified a novel Stx2 subtype designated Stx2h in E. coli strains isolated from wild marmots in the Qinghai-Tibetan plateau, China. Stx2h shares 91.9% nucleic acid sequence identity and 92.9% amino acid identity to the nearest Stx2 subtype. The expression of Stx2h in type strain STEC299 was inducible by mitomycin C, and culture supernatant from STEC299 was cytotoxic to Vero cells. The Stx2h converting prophage was unique in terms of insertion site and genetic composition. Whole genome-based phylo- and patho-genomic analysis revealed STEC299 was closer to other pathotypes of E. coli than STEC, and possesses virulence factors from other pathotypes. Our finding enlarges the pool of Stx2 subtypes and highlights the extraordinary genomic plasticity of E. coli strains. As the emergence of new Shiga toxin genotypes and new Stx-producing pathotypes pose a great threat to the public health, Stx2h should be further included in E. coli molecular typing, and in epidemiological surveillance of E. coli infections.

Figure 1

Phylogenetic tree of Stx2 subtypes by the neighbor-joining method. The neighbor-joining tree was inferred from comparison of combined (A and B) holotoxin amino acid sequences of all Stx2 subtypes. Numbers on the tree indicate bootstrap values calculated for 1000 subsets for branch points >50%. Bar, 0.05 substitutions per site. Stx2 subtypes are indicated by different colors. An extended version of this tree is available as Fig. S2.

Figure 2

Amino acid alignment of Stx2h (A and B) subunits against other Stx2 subtypes. Amino acid conserved with all Stx2 sequences are indicated with an asterisk. Differences between sequences are indicated in black letter.

Figure 3

Induction of Stx2h production in STEC299. (A) mRNA expression by qRT-PCR. The relative levels of expression under non-induction and induction conditions were relative to the expression level in Xuzhou21 before induction by mitomycin C which was arbitrary set at 1.0. The relative value was averaged from three independent experiments. Error bars represent the standard errors. (B) Vero toxicity assay. The cytotoxicity was detected after 24 h incubation exposure to the induced supernatants overnight. Xuzhou21 was used as positive control; MG1655 was used as negative control. (C) Western blot assays with an anti-Stx2 rabbit polyclonal antibody on whole cells and supernatants from STEC299 induced with mitomycin C with a final concentration of 0.5 μg/ml. Xuzhou21 was used as positive control.

Figure 4

Architecture of Stx2h-converting phages and genomic comparison with other Stx2 phages. The figure shows comprehensive analyses of all Stx2 subtypes converting prophages. Corresponding CDSs are colored as indicated. Integration sites of each phage are presented in parentheses.

Figure 5

The phylogenetic relationship of the strain STEC299 with the other 32 reference strains. (A) ClonalFrame tree of the stains inferred from the concatenated ribosomal protein gene sequences that are single-copy and shared (n = 53) by the 33 strains. Three independent and converged runs were merged and a 95% consensus tree was presented in the final graph. (B) Neighbor-net phylogeny generated from wgMLST allele profiles of 2,321 loci that shared by all the strains. The uncertainty and incompatibilities in the dataset were shown as networks. (C) Gubbins tree generated with the concatenated sequences of all the shared loci found in wgMLST analysis. STEC strains are highlighted in red on the three different trees.