EspO1-2 regulates EspM2-mediated RhoA activity to stabilize formation of focal adhesions in enterohemorrhagic Escherichia coli-infected host cells

Abstract

Enterohemorrhagic Escherichia coli (EHEC) Sakai strain encodes two homologous type III effectors, EspO1-1 and EspO1-2. These EspO1s have amino acid sequence homology with Shigella OspE, which targets integrin-linked kinase to stabilize formation of focal adhesions (FAs). Like OspE, EspO1-1 was localized to FAs in EHEC-infected cells, but EspO1-2 was localized in the cytoplasm. An EHEC ΔespO1-1ΔespO1-2 double mutant induced cell rounding and FA loss in most of infected cells, but neither the ΔespO1-1 nor ΔespO1-2 single mutant did. These results suggested that EspO1-2 functioned in the cytoplasm by a different mechanism from EspO1-1 and OspE. Since several type III effectors modulate Rho GTPase, which contributes to FA formation, we investigated whether EspO1-2 modulates the function of these type III effectors. We identified a direct interaction between EspO1-2 and EspM2, which acts as a RhoA guanine nucleotide exchange factor. Upon ectopic co-expression, EspO1-2 co-localized with EspM2 in the cytoplasm and suppressed EspM2-mediated stress fiber formation. Consistent with these findings, an ΔespO1-1ΔespO1-2ΔespM2 triple mutant did not induce cell rounding in epithelial cells. These results indicated that EspO1-2 interacted with EspM2 to regulate EspM2-mediated RhoA activity and stabilize FA formation during EHEC infection.

Figure 1. EspO1-1 and EspO1-2 stabilize FAs and the actin cytoskeleton in EHEC-infected cells.

(A) Alignment of OspE2 homologs EspO1-1 and EspO1-2 in EHEC strain Sakai. EspO1-1 and EspO1-2 have 59% amino-acid sequence identity, and also have extensive homology with OspE2 (29% and 25% amino acid sequence identity to OspE2, respectively). Asterisks indicate amino acid residues that are conserved in either OspE1 and OspE2 or in EspO1-1 and EspO1-2. Black highlight indicates an amino-acid residue conserved in both OspE1 and OspE2 and either EspO1-1, EspO1-2, or both EspO1-1 and EspO1-2. Arrowhead indicates the tryptophan, which is involved in ILK binding, at amino acid residue 77 of EspO1s and residue 68 of OspEs. (B) Morphological change in HeLa cells uninfected or infected with EHEC. HeLa cells were infected with EHEC wild-type (WT), an ΔespO1-1 mutant (ΔespO1-1), an ΔespO1-2 mutant (ΔespO1-2), an ΔespO1-1ΔespO1-2 double mutant (ΔespO1-1ΔespO1-2), an ΔespO1-1ΔespO1-2/pEspO1-1 or an ΔespO1-1ΔespO1-2/pEspO1-2. At 4 h post-infection, the cells were fixed and Giemsa-stained. (C) Percent cell rounding in cells infected by WT, ΔespO1-1ΔespO1-2, ΔespO1-1 or ΔespO1-2 shown in (B) and quantified (>50 cells, n≥3). Data are the mean and S.D. *P<0.001. The percent of cell rounding of uninfected cells was <3%. (D) Formation of actin filaments and focal adhesions (FAs) in HeLa cells infected with EHEC. HeLa cells were infected with WT, ΔespO1-1ΔespO1-2, ΔespO1-1 or ΔespO1-2. At 4 h post-infection, the cells were fixed and processed for confocal laser scanning microscopy using rhodamine-phalloidin to visualize actin filaments (red), anti-vinculin antibody to visualize FAs (green), and DAPI to visualize DNA/chromosomes (blue).

Figure 2. Localization of EspO1-1 and EspO1-2 in EHEC-infected cells.

(A) HeLa cells were infected with ΔespO1-1ΔespO1-2 carrying pK19-HA or ΔespO1-1ΔespO1-2 carrying a plasmid with the gene for either HA-tagged EspO1-1 (pEspO1-1-HA) or EspO1-2 (pEspO1-2-HA) and, at 4 h post-infection, were analyzed by immunofluorescence staining. Cells infected with ΔespO1-1ΔespO1-2 carrying pEspO1-1-HA or ΔespO1-1ΔespO1-2 carrying pEspO1-2-HA were processed for confocal laser scanning microscopy using Alexa 488-conjugated phalloidin to visualize actin filaments (green), and anti-HA antibody to visualize HA-tagged proteins (red). (B) HeLa cells were infected with WT carrying pK19-HA, or ΔespO1-1ΔespO1-2 carrying pK19-HA, pEspO1-1-HA or pEspO1-2-HA. Epithelial cells were infected with these strains for 4 h and immunofluorescence-stained with anti-FAK antibody to visualize focal adhesions (green) and anti-HA antibody (red). (C) HeLa cells were infected with WT carrying pK19-HA, pEspO1-1-HA or pEspO1-2-HA. (D) HeLa cells were infected with ΔespO1-1 carrying pK19-HA or pEspO1-1-HA. (E) HeLa cells were infected with ΔespO1-2 carrying pK19-HA or pEspO1-2-HA. Epithelial cells were infected with these strains for 4 h and immunofluorescence-stained with Alexa 488-conjugated phalloidin (green) and anti-HA antibody (red).

Figure 3. Interaction of EspO1-2 with EspM2.

(A) GST-EspO1-2 binding assays were performed using cell lysates expressing Flag-tagged Map, EspM2, EspM2 or EspG. Proteins that bound to GST-EspO1-2 and GST-alone were analyzed by SDS-PAGE followed by immunoblotting with anti-Flag antibody. (B) To map the EspM2-binding site in EspO1-2, a series of truncated GST-EspO1-2 peptides was used in binding assays with cell lysate expressing Flag-tagged EspM2. (C) Co-localization of RFP-EspO1-2 and GFP-EspM2 in HeLa cells. HeLa cells were transfected with pRFP-EspO1-2, pRFP-EspO1-2 1–60aa or pRFP (red) and pEGFP-EspM2 (green).

Figure 4. Interaction between EspO1-2 and EspM2 inhibits formation of stress fibers induced by EspM2.

(A) HeLa cells transfected with pREP, pRFP-EspO1-2, pRFP-EspO1-2 W77A or pRFP-EspO1-2 W77H and pEGFP or pEGFP-EspM2 were immunostained with Alexa 350-conjugated phalloidin to visualize actin filaments and stress fibers (upper panels). Percent of transfected cells in which formation of dense stress fibers, as shown in the upper right panel, was induced (lower graph). (B) To investigate the effect of the EspO1-2 Trp77 residue on EspM2 binding, GST-EspO1-2 W77A and GST-EspO1-2 W77H, each with an amino acid substitution for EspO1-2 Trp77, were used in binding assays with cell lysate expressing Flag-tagged EspM2.

Figure 5. Enhancement of RhoA signaling activity induces cell rounding.

(A) Involvement of EspO1-1 and EspO1-2 in the RhoA-ROCK signaling pathway in EHEC-infected cells. HeLa cells were infected with the EHEC WT, an ΔespO1-1ΔespO1-2 double mutant, an ΔespO1-1 mutant or an ΔespO1-2 mutant. At 1 and 3 h post-infection, a sample of cells was lysed and GTP-RhoA, the active form of RhoA, was co-precipitated with the GST-Rho binding domain (RBD) of rhotekin. The amount of RhoA bound to RBD (GTP-RhoA) and total RhoA in each cell lysate was analyzed by immunoblotting with anti-RhoA antibodies. The intensity of bands was quantified using the Image J program, and the results are expressed as fold increases in the ratio for GTP-RhoA/Total RhoA in uninfected cells (n = 3). (B) Effect of the ROCK inhibitor Y27632 on the morphological change of EHEC-infected cells. Untreated HeLa cells and HeLa cells treated with Y27632 for 1 h were infected with EHEC WT or the ΔespO1-1ΔespO1-2 double mutant. At 3 h post-infection, the cells were fixed and stained with Giemsa. The percent of cell rounding in cells infected by WT or ΔespO1-1ΔespO1-2 was quantified (>50 cells, n≥3). (C) Effect by expression of dominant-negative RhoA (T19N) in transfected cells on the morphological change of EHEC-infected cells. HeLa cells transfected with GFP or GFP-tagged RhoA T19N were infected with EHEC WT or the ΔespO1-1ΔespO1-2 double mutant at 48 h post-transfection. At 3 h post-infection, the cells were fixed and stained with Giemsa. The percent of cell rounding in cells infected by WT or ΔespO1-1ΔespO1-2 was quantified (>50 cells, n≥3).

Figure 6. Contribution of EspM2 to cell rounding and actin cytoskeleton structure disruption in the infected cells.

(A) HeLa cells were infected with an ΔespO1-1ΔespO1-2ΔespM2 triple mutant and, at 4 h post-infection, stained with rhodamine-phalloidin to visualize actin filaments. (B) The percent of cells shown in (A) in which cell rounding was induced and the actin cytoskeleton was disrupted was quantified (>50 cells, n≥3). Data are the mean and S.D. *P<0.001.

Figure 7. A proposed model for the role of EspO1-2 in an EHEC-infected cell.

EspO1-2 interacts with EspM2 in EHEC-infected cells to suppress EspM2-enhanced host RhoA activity.