Short-tailed stx phages exploit the conserved YaeT protein to disseminate Shiga toxin genes among enterobacteria

Abstract

Infection of Escherichia coli by Shiga toxin-encoding bacteriophages (Stx phages) was the pivotal event in the evolution of the deadly Shiga toxin-encoding E. coli (STEC), of which serotype O157:H7 is the most notorious. The number of different bacterial species and strains reported to produce Shiga toxin is now more than 500, since the first reported STEC infection outbreak in 1982. Clearly, Stx phages are spreading rapidly, but the underlying mechanism for this dissemination has not been explained. Here we show that an essential and highly conserved gene product, YaeT, which has an essential role in the insertion of proteins in the gram-negative bacterial outer membrane, is the surface molecule recognized by the majority (ca. 70%) of Stx phages via conserved tail spike proteins associated with a short-tailed morphology. The yaeT gene was initially identified through complementation, and its role was confirmed in phage binding assays with and without anti-YaeT antiserum. Heterologous cloning of E. coli yaeT to enable Stx phage adsorption to Erwinia carotovora and the phage adsorption patterns of bacterial species possessing natural yaeT variants further supported this conclusion. The use of an essential and highly conserved protein by the majority of Stx phages is a strategy that has enabled and promoted the rapid spread of shigatoxigenic potential throughout multiple E. coli serogroups and related bacterial species. Infection of commensal bacteria in the mammalian gut has been shown to amplify Shiga toxin production in vivo, and the data from this study provide a platform for the development of a therapeutic strategy to limit this YaeT-mediated infection of the commensal flora.

FIG. 1.

Subcloning of the open reading frame associated with adsorption of φ24_B to MRL1. Construct pUCR1 was one of the four original clones that complemented the φ24_B phage adsorption defect. All four clones shared the two open reading frames present in pUCR1; the solid box represents yaeT, and the striped box represents skp. When fragments from pUCR1 were subcloned into pUC19 or pUC18 (indicated by the vector promoter direction: leftward, pUC19; rightward, pUC18), only the complete yaeT gene found in pUCR1 and pUCR1D complemented the MRL1 adsorption defect. Restriction sites are as follows: X, XhoI; H, HincII; K, KpnI; P, PstI.

FIG. 2.

Phylogenetic tree of YaeT orthologues for most of the gram-negative bacteria for which there are complete genome sequences. Homologues of YaeT were identified by BLASTP analyses and used to identify all homologues of YaeT possessing at least 30% amino acid identity. The sequences were aligned and then subjected to a maximum parsimony analysis using ARB (29). Branches were validated through 100 bootstrap analyses. Scale bar = 1% amino acid changes. The Enterobacteriaceae is indicated by larger type, and a few unusual members of this family are indicated by arrows. The asterisk indicates a bacterial species that clusters with an unrelated bacterial family.

FIG. 3.

Multiple alignment of YaeT from members of the Enterobacteriaceae. Clustal X (46) was used to align YaeT sequences from eight members of the Enterobacteriaceae against the orthologue from Neisseria meningitidis, for which a structure has been proposed (51). The alignments were used to identify regions possessing the greatest similarities and disparities; the latter are mostly associated with the carboxyl terminus and correspond to predicted extracellular loops. The conservation of residues is indicated above the alignments as follows: asterisk, complete identity; colon, conservation of a strong group (46); period, conservation of a weak group (46).

FIG. 4.

SDS-PAGE and Western blot identification of purified histidine-tagged recombinant YaeT. (A) Coomassie blue-stained SDS-PAGE gel. (B) Western blot analysis using rabbit anti-H-YaeT (1:50,000). Lane 1, nickel affinity-purified H-YaeT; lane 2, whole-cell lysates of E. coli strain MC1061.

FIG. 5.

Inhibition of φ24_B adsorption using rabbit anti-H-YaeT antiserum. A total of 5 × 10⁷ MC1061 cells were incubated with no serum (bar 1), rabbit preimmune serum (1:5 dilution) (bar 2), or rabbit anti-H-YaeT serum (1:5 dilution) (bar 3) prior to infection with φ24_B (5 × 10⁸ PFU). The error bars indicate the standard errors of the means (n = 15). In a separate experiment, using the same numbers of cells and phage, various dilutions of the anti-H-YaeT serum were added to the cells prior to infection with φ24_B (bar 4, none; bar 5, 1:600 dilution; bar 6, 1:60 dilution; bar 7, 1:6 dilution) The error bars indicate the standard errors of the means (n = 5).

FIG. 6.

Complementation of a natural yaeT mutant confers the ability to support φ24_B adsorption. (A) SDS-PAGE analysis of total cell protein from E. carotovora subsp. atroseptica (lane 2), E. coli strain MC1061 (lane 2), and E. carotovora subsp. atroseptica carrying the construct pKT230yaeT (lane 3). The region corresponding to 90 kDa is indicated on the left. (B) Western blot analysis of total cell proteins from panel A. (C) Phage binding to YaeT on the surface of E. carotovora carrying pKT230yaeT. Data were calculated for plasmid-free and pKT230-containing strains challenged with 3.4 × 10⁷ phage particles. The x axis indicates different total numbers of E. carotovora cells, as follows: bar 1, 1.5 × 10⁸ cells bearing pKT230; and bars 2, 3, and 4, 1.5 × 10⁸, 3 × 10⁸, and 5 × 10⁸ cells carrying pKT230yaeT, respectively. The error bars indicate the standard errors of the means (n = 5).

FIG. 7.

Relationship between φ24_B phage adsorption and yaeT sequence variation with the predicted protein conformation in Enterobacteriaceae strains. (A) Proposed configuration of YaeT in the E. coli LPS membrane. The black loops are the predicted extracellular loops; the gray segments are predicted outer membrane (OM)-spanning regions; and the open segments are predicted periplasmic (Peri.) regions of YaeT. The extracellular loops are numbered from the amino terminus. (B) Identification of amino acid substitutions that correlate with the loss of Stx phage adsorption ability. The highlighted sequences are the changes predicted to be responsible for the loss of the ability to support phage adsorption. The sequences are sequences of, from top to bottom, S. flexneri, S. sonnei, E. coli strain MC1061, S. enterica serovar Choleraesuis strain SC-B67, C. rodentium strain ICC168, E. carotovora subsp. atroseptica strain SCRI1043, and P. luminescens subsp. laumondii strain TTO1.

FIG. 8.

Variations in tail spike proteins that recognize YaeT as an adsorption target. Amplification primers were designed against the gene sequence encoding the tail spike protein (the J gene orthologue) of φ24_B and 933W (VTTF1-Fwd, GTTGTTGTTTCGGGGACG; VTUTF-Rev, TCATTCTCCTGTTCTGCC; VTTF3-Fwd, TGCAGAGGAAAGCTCGAC; VTTF3-Rev, GCAGCCTCTTCTGCCTTT). The primers were used in combinations of VTTF1-Fwd with VTUTF-Rev or VTTF3-Rev and of VTTF3-Fwd with VTUTF-Rev or VTTF3-Rev (combinations 1, 3, 2, and 4, respectively). All variations of tail spike proteins identified by this means were capable of adsorption to E. coli MC1061, and this adsorption could be blocked with the anti-YaeT sera. Three different amplification profiles were found for the short-tailed phages listed in the table in panel B. The majority (70%) of phages possessed amplification profile A, which results from the production of all four amplification products of the anticipated size. Profile C was found only rarely and is characterized by the failure of the VTUTF-Rev primer to produce any products in combination with either 5′ primer, while the products of VTTF3-Rev were ∼800 bp larger than expected.