Enterohemorrhagic Escherichia coli O157:H7 produces Tir, which is translocated to the host cell membrane but is not tyrosine phosphorylated

Abstract

Intimate attachment to the host cell leading to the formation of attaching and effacing (A/E) lesions is an essential feature of enterohemorrhagic Escherichia coli (EHEC) O157:H7 pathogenesis. In a related pathogen, enteropathogenic E. coli (EPEC), this activity is dependent upon translocation of the intimin receptor, Tir, which becomes tyrosine phosphorylated within the host cell membrane. In contrast, the accumulation of tyrosine-phosphorylated proteins beneath adherent EHEC bacteria does not occur, leading to questions about whether EHEC uses a Tir-based mechanism for adherence and A/E lesion formation. In this report, we demonstrate that EHEC produces a functional Tir that is inserted into host cell membranes, where it serves as an intimin receptor. However, unlike in EPEC, in EHEC Tir is not tyrosine phosphorylated yet plays a key role in both bacterial adherence to epithelial cells and pedestal formation. EHEC, but not EPEC, was unable to synthesize Tir in Luria-Bertani medium but was able to secrete Tir into M9 medium, suggesting that Tir synthesis and secretion may be regulated differently in these two pathogens. EHEC Tir and EPEC Tir both bind intimin and focus cytoskeletal rearrangements, indicating that tyrosine phosphorylation is not needed for pedestal formation. EHEC and EPEC intimins are functionally interchangeable, but EHEC Tir shows a much greater affinity for EHEC intimin than for EPEC intimin. These findings highlight some of the differences and similarities between EHEC and EPEC virulence mechanisms, which can be exploited to further define the molecular basis of pedestal formation.

FIG. 1

Protein secretion profiles of EHEC O157:H7 strains. (A) Secreted proteins derived from equal amounts of EHEC 86-24 (wild type), UMD619 (intimin mutant), CVD451 (SepB mutant), and EHECΔtir standing cultures grown to an OD₆₀₀ of 0.8 were analyzed by SDS–12% PAGE and stained with Coomassie blue. Arrows mark the positions of known EHEC-secreted proteins. (B) EHEC O157:H7 strain 86-24 was cultured in LB medium, Dulbecco MEM (DMEM), or M9 minimal medium and grown to an OD₆₀₀ of 0.7 to 0.8. TCA-precipitated culture supernatants or bacterial cell lysates derived from equivalent amounts of bacteria were resolved by SDS–12% PAGE, transferred to nitrocellulose, and probed with polyclonal rat anti-EHEC Tir antisera. Molecular mass markers are in kilodaltons.

FIG. 2

Clustal W alignment of the EHEC O157:H7 strain 86-24 Tir protein sequence (AF125993) with those of EPEC O127:H6 strain E2348/69 Tir (AF013122) and EHEC O26:− Tir (AJ223063). Amino acid residues identical in all three proteins are indicated by shading, the two predicted transmembrane domains are underlined, and the tyrosine residues are indicated by asterisks.

FIG. 3

Immunofluorescence microscopy of HeLa cells following a 6-h infection with wild-type EHEC O157:H7 strain 86-24 or EHECΔtir or a 9 h infection with EHECΔtir/T7-Tir. Fixed and permeabilized cells were colabelled with either anti-PY antisera and Texas red-phalloidin (B, C, E, F, H, and I) or anti-T7 antisera and Texas red-phalloidin (K and K) or singly labelled with anti-O157 antisera (M and N) to illustrate bacterial adherence. Arrowheads in J, K, and L indicate the localization of anti-T7 antisera with the actin pedestals. (A, D, G, and J) Phase-contrast images of the sections used for immunofluorescence.

FIG. 4

EHEC Tir is translocated to the host cell but not tyrosine phosphorylated. HeLa cells were infected with either EHEC UMD619 or EPEC CVD206, and Triton X-100-soluble membrane fractions were prepared, treated with alkaline phosphatase (Alk Phos), and resolved by SDS–8% PAGE. After being blotted to nitrocellulose, the samples were probed with anti-EHEC Tir (a), anti-EPEC Tir (b), or anti-PY (c) antisera. Molecular mass markers are in kilodaltons.

FIG. 5

Intimin binds to secreted EHEC Tir. Gel overlay of diluted EHEC culture supernatants treated with equal amounts of HeLa cell membrane extracts. (Left) Samples were resolved by SDS–12% PAGE, transferred to nitrocellulose, overlaid with 100 μg of EHEC T7-intimin fusion protein, and probed with anti-T7 antisera. (Right) The same blot was reproved with anti-EHEC Tir antisera. Dilutions of EHEC culture supernatants are indicated above the panels, and molecular mass markers are in kilodaltons.

FIG. 6

Intimin binds interchangeably to EHEC Tir and EPEC Tir. ELISA wells were coated with EPEC- or EHEC-secreted proteins, incubated with increasing concentrations of intimin, and visualized with o-phenylenediamine. (A) EPEC-T7 intimin (triangles) or EHEC-T7 intimin (diamonds) binding to culture supernatants from EPEC/pCVD450 (open symbols) or EPECΔtir (filled symbols). (B) EHEC-T7 intimin (squares) or EPEC-T7 intimin (circles) binding to EHEC 86-24 (open symbols) or EHECΔtir (filled symbols). Data are presented as means ± standard deviations for triplicate points from one representative experiment.

FIG. 7

Immunofluorescence microscopy of epitope-tagged intimin binding to EHEC- or EPEC-infected cells. HeLa cells infected with either EPEC CVD206 or EHEC UMD619 were incubated with equal amounts of both T7-EHEC intimin and MBP-EPEC intimin. Bound fusion proteins were visualized with anti-T7 and anti-MBP antisera labelled with either fluorescein isothiocyanate (EHEC intimin) or Texas red (EPEC intimin).

FIG. 8

Heterologous Tir-intimin binding results in A/E lesion formation. HeLa cells were infected with either the EHEC or the EPEC intimin mutant UMD619 or UMD207 alone (E and F) or coinfected for 6 h with EPEC UMD207 plus EHECΔtir (A), EHEC UMD619 plus EHECΔtir (B), EHEC UMD619 plus EPECΔtir (C), or EPEC UMD207 plus EPECΔtir (D). Cells were fixed for fluorescence microscopy and labelled with either Texas red-phalloidin to visualize actin rearrangements (middle panels) or anti-PY antisera (right panels). (Left panels) Phase-contrast images of the same sections as those used for immunofluorescence.

FIG. 9

Hairpin model for EHEC Tir structure. EHEC Tir is predicted to be an integral membrane protein with two transmembrane domains (TMD’s). Both the amino and carboxy termini are predicted to reside within the host cell, and intimin is thought to bind to a putative extracellular loop.