EspH Suppresses Erk by Spatial Segregation from CD81 Tetraspanin Microdomains

CD81 is not required for F-actin recruitment at infection sites. CRISPR-control (Control) and CRISPR CD81KO (CD81KO) HeLa cells were infected with the EPEC wt for the indicated times. The cells were then fixed, and the level of clustering of F-actin at infection sites was determined as indicated in Materials and Methods and in the legend to Fig. 1A. Representative images (left) and the results of quantitative analysis of protein clustering at infection sites (right) are presented. P values refer to the comparison with infected CRISPR control cells. ns, nonsignificant statistical difference (P > 0.05). DIC, differential interference contrast. Bars = 10 μm.

Screening for bacterial effectors that mediate CD81 clustering at infection sites. (A) Screening for LEE and non-LEE effectors involved in CD81 clustering. HeLa cells were infected with the indicated EPEC strains (see Table S1 in the supplemental material), and the levels of CD81 and F-actin clustering at infection sites were determined as described in the legend to Fig. 1A. (B) EspH is involved in CD81 clustering. HeLa or Caco-2 cells were infected with EPEC ΔespH or EPEC ΔespH+EspH strain (Table S1), and EspH expression was induced using IPTG. Cells were processed for confocal imaging as described in the legend to Fig. 1A. Representative images (left) and the results of clustering analysis of CD81 and F-actin (right) are presented. Results are the mean ± SE from 3 experiments. P values refer to the comparison with EPEC wt-infected cells. ***, P < 0.0005; **, P < 0.005; *, P < 0.05; ns, nonsignificant statistical difference (P > 0.05). Bars = 15 μm.

EspH reduces pErk levels, and CD81 counteracts this effect. (A) pErk levels are suppressed in EPEC wt-infected but not EPEC escV-infected HeLa and Caco-2 cells. Cells were infected with EPEC for 90 min at 37°C or left untreated. Cells were lysed and processed for pErk and tErk detection by Western blotting, as described in Materials and Methods. Representative Western blots are shown. (B) EspH mediates pErk suppression. HeLa cells were infected with the indicated EPEC strains for 90 min at 37°C or left uninfected. The cells were then lysed and processed for quantitative Western blotting of pErk levels, as described in the legend to panel A and Materials and Methods. Results are the mean ± SE from 3 experiments. P values refer to the comparison with uninfected cells. (C) CD81 counteracts EspH-mediated pErk suppression. CD81KO or CD81KO–mEmerald-CD81 HeLa cells were infected with the indicated EPEC strains and subjected to quantitative Western blotting of pErk levels, as described in Materials and Methods. Results are the mean ± SE from 3 experiments. P values refer to the comparison with infected CRISPR control cells. ***, P < 0.0005; **, P < 0.005; *, P < 0.05.

The C-terminal 38-amino-acid segment of EspH is important for pErk increase. (A) Secondary structure prediction of EspH. Prediction of the secondary structure of EspH in EPEC (GenBank accession number SLM08785), EHEC (GenBank accession number ACG59626), and Citrobacter rodentium (GenBank accession number CBG89721) was performed using the JPred4 protein secondary structure prediction server (68). The results were viewed using the Jalview (version 2.10.3b1) program (69). The C-terminal segment (amino acids 114 to 168) is shown. jnetpred predicts secondary structures in EspH. Red tubes and green arrows represent helices and sheets, respectively; JnetCONF shows the confidence of prediction (from 0 to 9; a higher value means a higher-confidence prediction); JnetPSSM refers to Jnet PSIBLAST pssm-based profile prediction; red tubes and green arrows indicate helices and sheets, respectively. The C-terminal part of EspH containing the C-terminal 38-amino-acid segment and a highly conserved predicted α-helix, which was deleted to generate the EspH_Δ130–168 mutant, is indicated with a green box. (B) pErk levels are significantly decreased in host cells infected with EPEC ΔespH+EspH compared to cells infected with EPEC ΔespH or EPEC EspH_Δ130–168. HeLa cells were infected with the indicated EPEC strain, and pErk levels were determined as described in the legend to Fig. 4. Results are the mean ± SE from 3 independent experiments. P values refer to the comparison with EPEC ΔespH-infected cells. (C) pErk levels are decreased in EspH-eGFP-expressing cells compared to eGFP- or EspH EspH_Δ130–168-eGFP-expressing cells. HeLa cells were transfected with plasmids expressing the indicated eGFP. Cells were lysed and analyzed for pErk levels by Western blotting as described in the legend to Fig. 4. eGFP expression levels in the lysates were detected using anti-eGFP antibodies. The results are the mean ± SE from 3 experiments. P values refer to the comparison with eGFP-transfected cells. (D) Phospho-p38 levels were not significantly altered in cells infected with EPEC ΔespH, EPEC ΔespH+EspH, or EPEC EspH_Δ130–168. HeLa cells were infected with the indicated EPEC strains, and phospho-p38 levels normalized to total p38 levels were determined by Western blotting. Results are the mean ± SE from 3 independent experiments. P values refer to the comparison with EPEC ΔespH-infected cells. (E) Analysis of Rho GTPase activity. HeLa cells were transfected with plasmids expressing eGFP, EspH-eGFP, or EspH_Δ130–168-eGFP. The cells were then analyzed for Rho activity by the luciferase-based assay, as described in Materials and Methods. Data were normalized to those for eGFP-expressing cells. P values refer to the comparison with eGFP-transfected cells. In control experiments, Rho GTPases were inhibited by the Rho inhibitor I, and the results were compared to those for dimethyl sulfoxide-treated cells (inset). The results are the mean ± SE for 12 measurements. ***, P < 0.0005; **, P < 0.005; ns, nonsignificant statistical difference (P > 0.05).

EspH moves out of the CD81 microdomain. (A and B) Localization of translocated EspH with respect to mEmerald-CD81. mEmerald-CD81-expressing cells were infected for 30 (A) or 90 (B) min at 37°C. Cells were then fixed and stained with anti-SBP antibodies to label EspH. Cells were imaged by confocal microscopy, and an intensity profile was created along a drawn line over CD81-EspH clusters, as exemplified in the boxed areas. The plot represents the average of intensity profile values measured for ∼20 different such clusters. a.u., arbitrary units. Bars = 15 μm. (C) Distribution of ectopically expressed eGFP tagged EspH with respect to immunostained CD81. HeLa cells expressing EspH-eGFP or EspH_Δ130–168-eGFP were immunostained with the anti-CD81 5A6 antibody and imaged by confocal microscopy. The representative confocal image shows EspH and CD81 located at the plasma membrane (arrows) or at a central aggregate (arrowheads). The fluorescence level of eGFP and immunostained CD81 positioned in the central CD81 aggregate of EspH-eGFP- and EspH_Δ130–168-eGFP-expressing cells was quantified by a method similar to the quantification method applied for estimating the clustering of CD81 and F-actin at infection sites (Fig. 1). The results are the mean ± SE for 25 measurements. P values refer to the comparison with EspH-eGFP transfected cells. ***, P < 0.0005; ns, nonsignificant statistical difference (P > 0.05). Bar = 10 μm.

EspH suppresses TNF-α-induced pErk levels, and CD81 counteracts this effect. (A) Ectopic expression of EspH-eGFP specifically suppresses the TNF-α-induced pErk. HeLa cells were either transfected with the indicated eGFP-encoding constructs or remained untransfected. Cells were then exposed (+) or not exposed (No Ligand) to EGF, TNF-α, or IL-1β ligands, as described in Materials and Methods. Cells were lysed, and pErk levels were detected by quantitative Western blotting, as described in the legend to Fig. 4 and Materials and Methods. The expressed eGFPs were detected using anti-GFP antibodies. The results are the mean ± SE from three experiments. P values refer to the comparison with untransfected cells. (B) Ectopically expressed mutant EspH with a 38-amino-acid C-terminal truncation fails to suppress the TNF-α-evoked pErk. HeLa cells were transfected with plasmids expressing eGFP, EspH-eGFP, or EspH-eGFP_Δ130–168. The cells were then challenged with TNF-α, lysed, and subjected to determination of changes in pErk levels by quantitative Western blotting. The results are the mean ± SE from three experiments. P values refer to the comparison with eGFP-transfected cells. (C) Translocated EspH with a 38-amino-acid C-terminal truncation fails to suppress TNF-α-evoked pErk in infected cells. HeLa cells were infected with EPEC ΔespH, EPEC ΔespH+EspH, or EPEC ΔespH+EspH_Δ130–168, which were not preactivated, for 3 h at 37°C. Thereafter, bacterium-containing medium was replaced with fresh TNF-α (25 ng/ml)-containing DMEM (+TNF-α) for one set of cells, while for other cells, the bacterium-containing medium was replaced with just plain DMEM (−TNF-α). The cells were then incubated for a further 30 min at 37°C and processed for Western blotting analyses. The results are the mean ± SE from 3 experiments. P values refer to the comparison with untreated (−TNF-α) EPEC-ΔespH-infected cells. (D) CD81 counteracts the ability of EspH to suppress TNF-α-stimulated pErk. CRISPR control, CD81KO, and CD81KO–mEmerald-CD81 cells were transfected with the indicated eGFP-encoding constructs, and the relative changes in pErk levels were determined as described in the legend to panel B. The results are the mean ± SE from 3 experiments. P values refer to the comparison with eGFP-transfected cells. ***, P < 0.0005; **, P < 0.005; *, P < 0.05; ns, nonsignificant statistical difference (P > 0.05).