TccP2-mediated subversion of actin dynamics by EPEC 2 - a distinct evolutionary lineage of enteropathogenic Escherichia coli

Abstract

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in developing countries. While colonizing the gut mucosa, EPEC triggers extensive actin-polymerization activity at the site of intimate bacterial attachment, which is mediated by avid interaction between the outer-membrane adhesin intimin and the type III secretion system (T3SS) effector Tir. The prevailing dogma is that actin polymerization by EPEC is achieved following tyrosine phosphorylation of Tir, recruitment of Nck and activation of neuronal Wiskott-Aldrich syndrome protein (N-WASP). In closely related enterohaemorrhagic E. coli (EHEC) O157 : H7, actin polymerization is triggered following recruitment of the T3SS effector TccP/EspF(U) (instead of Nck) and local activation of N-WASP. In addition to tccP, typical EHEC O157 : H7 harbour a pseudogene (tccP2). However, it has recently been found that atypical, sorbitol-fermenting EHEC O157 carries functional tccP and tccP2 alleles. Interestingly, intact tccP2 has been identified in the incomplete genome sequence of the prototype EPEC strain B171 (serotype O111 : H-), but it is missing from another prototype EPEC strain E2348/69 (O127 : H7). E2348/69 and B171 belong to two distinct evolutionary lineages of EPEC, termed EPEC 1 and EPEC 2, respectively. Here, it is reported that while both EPEC 1 and EPEC 2 triggered actin polymerization via the Nck pathway, tccP2 was found in 26 of 27 (96.2 %) strains belonging to EPEC 2, and in none of the 34 strains belonging to EPEC 1. It was shown that TccP2 was: (i) translocated by the locus of enterocyte effacement-encoded T3SS; (ii) localized at the tip of the EPEC 2-induced actin-rich pedestals in infected HeLa cells and human intestinal in vitro organ cultures ex vivo; and (iii) essential for actin polymerization in infected Nck-/- cells. Therefore, unlike strains belonging to EPEC 1, strains belonging to EPEC 2 can trigger actin polymerization using both Nck and TccP2 actin-polymerization signalling cascades.

Fig. 1.

(a) Schematic representation of the genomic localization of tccP and ECs1126 (renamed tccP2) in EHEC O157 : H7 (strain Sakai). tccP is located on prophage Sp14 (top) and tccP2 on Sp4 (bottom). (b) Multiple sequence alignment of TccP of EHEC O157 : H7 (strain Sakai) and EPEC O119 : H6 (strain ICC199), and TccP2 of EPEC O111 : H− (strain B171) and EPEC O111 : H2 (strains CB03454, CB03447 and CB07077). The unique N terminus is shaded grey and the PRRs are shaded black. The complete unit of a PRR and a partial C-terminal repeat are indicated by arrows and a dashed arrow, respectively. The TccP sequence of ICC199 was taken from Whale et al. (2006) (accession no. DQ206456), and that for TccP2 of B171 from its unfinished genome sequence (AAJX01000044.1). Note that the tccP2 gene is not annotated in the B171 genome sequence.

Fig. 2.

(a) TccP was detected with TccP antiserum in bacterial whole-cell lysates of EHEC EDL933 and EPEC ICC199, but not EDL933ΔtccP or EPEC E2348/69. TccP2 was also detected using TccP antiserum in lysates of EPEC O111 : H2 strain ICC215 and EPEC O111 : NM strain B171. (b) TccP2 is a T3SS-translocated effector. Translocation of the EPEC B171 effector protein TccP2 into live HeLa cells using TEM-1 fusion and fluorescence microscopy is shown. HeLa cells were infected with wild-type EPEC E2348/69 carrying pCX340 (negative control) (i), and E2384/69 (ii) and E2348/69ΔescN (iii) strains expressing TccP2__B171–TEM fusion protein. β-Lactamase activity in HeLa cells was revealed by the blue fluorescence emitted by the cleaved CCF2 product (cells infected with E2348/69 expressing TccP2–TEM), whereas CCF2 emitted a green fluorescence (cells infected with ΔescN mutant expressing TccP2–TEM).

Fig. 3.

(a) Widespread Tir_(Y-P), labelled in green, was observed at the site of bacterial adhesion after infection of HeLa cells with strains E2348/69, B171 and ICC215, but not with EDL933. Bar, 2 μm. (b) FAS. EDL933, ICC199 and B171, but not E2348/69, triggered efficient actin polymerization following infection of Nck1–/Nck2– fibroblasts. All strains tested, including E2348/69, efficiently induced actin polymerization in infected Nck1+/Nck2+ fibroblasts. The asterisk indicates a statistically significant difference in the percentage of pedestals triggered on Nck1−/Nck2− fibroblasts, compared to that triggered on Nck1+/Nck2+ fibroblasts by EPEC E2348/69 (P<0.05). Bacteria, labelled in red, were detected with anti-intimin or anti-EHEC antibodies for EPEC E2348/69, ICC199 and B171 strains, and EHEC EDL933, respectively. Actin was labelled in green with Oregon Green-conjugated phalloidin. Bar, 2 μm.

Fig. 4.

(a) Nck1−/Nck2− fibroblasts were infected with tccP⁻/tccP2⁻ EPEC 1 strain E2348/69 and E2348/69(pICC365-tccP2__B171). Expression of TccP2__B171 compensated for the absence of Nck, enabling efficient and significantly different (P<0.05) production of actin-rich pedestals at the site of bacterial attachment. Bar, 2 μm. (b) B171 and its isogenic B171ΔtccP2 mutant strain induced actin polymerization at a similar efficiency in infected HeLa cells. Bar, 2 μm. (c) Efficient actin polymerization was detected following infection of Nck1−/Nck2− fibroblasts with B171, but not with B171ΔtccP2. Wild-type phenotype was restored by complementation of B171ΔtccP2 with pICC281 (tccP) or pICC365 (tccP2) (data not shown). Bacteria, labelled in red, were detected with anti-intimin β or anti-intimin α antibodies for B171 and E2348/69, respectively. Actin was labelled in green with phalloidin–Oregon Green. Bar, 2 μm.

Fig. 5.

(a) Nck and TccP2 were simultaneously recruited and co-localized at the site of strain B171(pICC365-tccP2__B171)-induced actin assembly beneath adherent bacteria during infection of HeLa cells. Nck was labelled in green using an anti-Nck antibody, TccP2–HA was labelled in far red with an anti-HA mAb, and actin was labelled in red, using rhodamine-conjugated phalloidin. Bacteria and cell nuclei were visualized with Hoechst stain (blue). Separate monochrome images of the UV, far-red, red and green fluorescence channels are shown, as well as merged images of all channels (right column). Bar, 10 μm. (b) Tir and TccP2 co-localized at the site of B171ΔtccP2(pICC365-tccP2__B171) attachment during infection of N-WASP^+/+ and N-WASP^−/− fibroblasts. However, induced actin assembly was only detected beneath adherent bacteria during infection of N-WASP^+/+ fibroblasts. Tir was labelled in red, TccP2–HA was labelled in green, and actin was labelled in far red (shown in blue). Bacteria were visualized with Hoechst stain (shown in monochrome).

Fig. 6.

Both wild-type and ΔtccP2 B171 strains induced A/E lesions in intestinal IVOC. Scanning electron micrographs of duodenal mucosa infected with B171 and B171ΔtccP2 are shown. A non-infected sample was included as a negative control. Bars, 5 μm.

Fig. 7.

Nck was recruited to the site of IVOC adhesion of strains B171 and B171ΔtccP2, while TccP2 was found only under adherent B171. Terminal ileal mucosa was infected with B171 and B171ΔtccP2 for 8 h, and cryosections were processed for immunofluorescence. Staining was performed for TccP2 (green in merged image, upper two panels) or Nck (green, lower two panels). Bacteria and cell nuclei were visualized by propidium iodide (PI) stain (red). Epithelial cells were counterstained with anti-cytokeratin (labelled blue). Separate monochrome images of the red and green fluorescence channels (left and middle column respectively) are shown, as well as merged images of all channels (right column).

Fig. 8.

FAS and recruitment of TccP2 to the site of bacterial adhesion. HeLa cells were infected with strain EDL933ΔtccP for 5 h. Following fixation and permeabilization, bacteria were labelled in blue. Actin was detected by rhodamine–phalloidin, and TccP2–HA was labelled in green. Strain ICC185, unable to form actin-rich pedestals during infection, was complemented by both TccP2__B171 (pICC365) and TccP2__ICC215 (pICC366). TccP2 was detected beneath adherent ICC185(pICC365) and ICC185(pICC366), but not ICC185, and co-localized with polymerized actin at the tip of the triggered actin pedestal. Bar, 2 μm.

Fig. 9.

Summary of actin-polymerization cascades induced by EPEC 1, EPEC 2 and EPEC O119 : H6. All EPEC lineages translocate Tir that once inserted into the host-cell membrane serves as a receptor for the bacterial adhesin intimin. Intimin-mediated clustering of Tir triggers phosphorylation of tyrosine residue Y474 and concurrent recruitment of Nck. Nck recruits and activates N-WASP, leading to Arp2/3-dependent actin polymerization and pedestal formation. EPEC 2 and ‘non-1 non-2 lineage’ O119 : H6 EPEC, but not EPEC 1 bacteria, also translocate TccP2 and TccP, respectively, and are capable of recruiting N-WASP directly and triggering Nck-independent actin-pedestal formation. Note that TccP does not bind Tir directly, but via an unidentified host-encoded adaptor (indicated by a question mark).