PMC

Expression of tccP2_O26 in EDL933 ΔtccP restores actin polymerization in infected HeLa cells; TccP2-HA is recruited to the site of bacterial attachment. Scale bar, 2 μm.

Deletion of tccP2 in O111 EPEC results in a loss in the ability to trigger actin pedestal formation in Nck-deficient cells. The wild-type (WT) phenotype is restored by expression of tccP2_O26. All strains tested were capable of initiating the formation of actin pedestals on Nck-proficient cells. Bar, 2 μm.

(A) Sequence alignment of the C-terminal region of Tir from EPEC with the Y474 equivalents from EHEC O26, O111, and O103. The Nck binding site is underlined. (B) Immunofluorescent staining showing Tir tyrosine phosphorylation (αTyr-P) at sites of induced actin polymerization following infection of HeLa cells with E2348/69, an O26 strain expressing E2348/69-like Tir, but not with O157:H7 EDL933. Bar, 5 μm.

(A) DNA and protein sequence alignment of the 5′ region/N terminus of tccP2/TccP2 from typical O157 (strain Sakai) and atypical O157 (strain 980938). tccP2 in Sakai has a single-base-pair deletion at position 28 (indicated by a hyphen), resulting in frameshift mutation downstream of codon 10. All of the typical O157 (H7 and H−) strains we examined contain the same deletion in tccP2. The Shine-Dalgarno (SD) sequences are indicated by underlining. (B) Multiple-sequence alignment of TccP and TccP2 of representative O157, O26, O111, and O103 EHEC strains. Strain names are indicated in parentheses. The unique N terminus involved in protein translocation is shaded gray, and the proline-rich repeats are shaded black. The proline-rich repeats and the partial repeat in the TccP proteins are indicated by arrows and a dashed arrow, respectively. The position of the frameshift in TccP2 of the O157 Sakai strain, which corresponds to codon 10 of the intact TccP2, is indicated by an asterisk. In silico translation of Sakai tccP2 from codon 11 revealed that it potentially encodes a TccP2 protein containing five proline-rich repeats.

Expression and secretion of TccP and TccP2 proteins in EHEC strains overexpressing PchA. Whole-cell lysates (P) and culture supernatants (S) were prepared from EHEC strains and analyzed by Western blotting using rabbit TccP antiserum. Positions of TccP and TccP2 proteins are indicated by asterisks and arrowheads, respectively. The amount of sample loaded onto the gel was standardized by bacterial cell concentrations that were estimated from OD₆₀₀ values; each whole-cell lysate corresponds to 1 × 10⁸ cells, and each supernatant is derived from the culture containing 2 ×10⁹ cells. (A) TccP is not detected in culture supernatants of wild-type (WT) Sakai; secretion and detection of TccP is enhanced in Sakai Δsep but abolished in Sakai ΔsepL/ΔescR (left panel). Overexpression of intact TccP2_O157 from pTB101-tccP2 leads to protein secretion from Sakai ΔsepL (the endogenous tccP2 of Sakai is a pseudogene) but not from Sakai ΔsepL/ΔescR (right panel). (B) Detection of TccP and TccP2 in clinical EHEC isolates: O157:H7 strains 990281, 990570, and 981795; O26:H11 strains 11368 and ED411; O111:H− strains 11109 and 11128; and O103:H2 strains 12009 and PMK5. The presence or absence of tccP genes in each strain and the predicted molecular masses of each TccP protein are indicated; strain 990570 appears to be a nonexpressor. The immunoreactive bands at about 42 kDa are likely to be bacterial immunoglobulin-binding proteins, as they reacted in the absence of primary antibody (data not shown).