Evolutionary genetics of a new pathogenic Escherichia species: Escherichia albertii and related Shigella boydii strains

Abstract

A bacterium originally described as Hafnia alvei induces diarrhea in rabbits and causes epithelial damage similar to the attachment and effacement associated with enteropathogenic Escherichia coli. Subsequent studies identified similar H. alvei-like strains that are positive for an intimin gene (eae) probe and, based on DNA relatedness, are classified as a distinct Escherichia species, Escherichia albertii. We determined sequences for multiple housekeeping genes in five E. albertii strains and compared these sequences to those of strains representing the major groups of pathogenic E. coli and Shigella. A comparison of 2,484 codon positions in 14 genes revealed that E. albertii strains differ, on average, at approximately 7.4% of the nucleotide sites from pathogenic E. coli strains and at 15.7% from Salmonella enterica serotype Typhimurium. Interestingly, E. albertii strains were found to be closely related to strains of Shigella boydii serotype 13 (Shigella B13), a distant relative of E. coli representing a divergent lineage in the genus Escherichia. Analysis of homologues of intimin (eae) revealed that the central conserved domains are similar in E. albertii and Shigella B13 and distinct from those of eae variants found in pathogenic E. coli. Sequence analysis of the cytolethal distending toxin gene cluster (cdt) also disclosed three allelic groups corresponding to E. albertii, Shigella B13, and a nontypeable isolate serologically related to S. boydii serotype 7. Based on the synonymous substitution rate, the E. albertii-Shigella B13 lineage is estimated to have split from an E. coli-like ancestor approximately 28 million years ago and formed a distinct evolutionary branch of enteric pathogens that has radiated into groups with distinct virulence properties.

FIG. 1.

Phylogenetic relationships of Shigella B13 strains and the E. albertii lineage. A neighbor-joining tree (MEGA2) was constructed by using the Tamura-Nei model with gamma correction for the distribution of rates based on 14 housekeeping genes. The E. albertii lineage (A) includes E. albertii, Shigella B13, and Shigella B7. On the basis of the rate of synonymous substitutions for E. coli and S. enterica (4.7 × 10⁻⁹ per site per year), the E. albertii lineage shared a common ancestor with E. coli and Shigella pathogenic groups ∼28 million years ago (mya). Atypical S. boydii strains express the Shigella B13 antigen and do not cluster with the E. albertii lineage but are more similar in their MLST profile to E. coli and Shigella groups. Shigella B13 strain 5216-70 belongs to EIEC group 2 (B), and four atypical Shigella B13 strains belong to their own cluster within the diversity of E. coli and Shigella (C). The number of strains examined is in parentheses. NT, nontypeable; EHEC, enterohemorrhagic E. coli.

FIG. 2.

Intimin variants of the E. albertii-Shigella B13 lineage. (A) A neighbor-joining tree (MEGA2) based on nucleotide sequences of the eae central conserved region of intimins of E. coli shows that the sequences of the intimin gene homologues of E.albertii-Shigella B13 strains are most similar to the sequence of the E. coli iota2 allele. (B) Haplotype plots of the nucleotide differences in the 3′ part of the eae gene encoding the Int280 region. The sequences of E. albertii and Shigella B7 strains are similar to the sequence of the iota2 allele of E. coli in the central conserved region but are most similar to the sequences of the alpha and zeta alleles of E. coli in the Int280 region. (C) Percent amino acid similarities among representative intimin variants. The lower triangular matrix shows pairwise similarity values for the central conserved region, and the upper triangular matrix shows values for the Int280 region. The shading highlights close similarities to the intimin domains of the E. albertii-Shigella B13 lineage (tan). C.rod, C. rodentium.

FIG. 3.

Multiple sequence alignment of CdtBs from representatives of the five described E. coli alleles, CDT I (44), CDT II (37), CDT III (36), CDT IV (49), and CDT V (4, 22), and representatives of three distinct alleles of the E. albertii lineage (B13, E.alb., and B7). At amino acid position 41, shown here in a box, Shigella B13 strains are predicted to translate a stop codon rather than a tryptophan codon due to a single G-to-A transition at the second codon position. Conserved residues thought to be important for enzymatic activity (11, 35) are highlighted in gray. Arrowheads indicate residues that were mutated and shown to completely or partially abolish the cell cycle arrest activity of CDT (11, 35).

FIG. 4.

Cytopathic effects of bacterial cell extracts on HeLa cell monolayers. Cytoplasmic distension and nuclear abnormalities were apparent 72 h after HeLa cells were exposed to E. albertii and Shigella B7-related strains (A), whereas Shigella B13 strains did not appear to produce active toxin (B). Included for comparison are CDT-negative E. coli K-12 (C) and CDT-positive E. coli 493/89 (D). Magnification, ×40.