Nck adaptors, besides promoting N-WASP mediated actin-nucleation activity at pedestals, influence the cellular levels of enteropathogenic Escherichia coli Tir effector

Abstract

Enteropathogenic Escherichia coli (EPEC) binding to human intestinal cells triggers the formation of disease-associated actin rich structures called pedestals. The latter process requires the delivery, via a Type 3 secretion system, of the translocated Intimin receptor (Tir) protein into the host plasma membrane where binding of a host kinase-modified form to the bacterial surface protein Intimin triggers pedestal formation. Tir-Intimin interaction recruits the Nck adaptor to a Tir tyrosine phosphorylated residue where it activates neural Wiskott-Aldrich syndrome protein (N-WASP); initiating the major pathway to actin polymerization mediated by the actin-related protein (Arp) 2/3 complex. Previous studies with Nck-deficient mouse embryonic fibroblasts (MEFs) identified a key role for Nck in pedestal formation, presumed to reflect a lack of N-WASP activation. Here, we show the defect relates to reduced amounts of Tir within Nck-deficient cells. Indeed, Tir delivery and, thus, pedestal formation defects were much greater for MEFs than HeLa (human epithelial) cells. Crucially, the levels of two other effectors (EspB/EspF) within Nck-deficient MEFs were not reduced unlike that of Map (Mitochondrial associated protein) which, like Tir, requires CesT chaperone function for efficient delivery. Interestingly, drugs blocking various host protein degradation pathways failed to increase Tir cellular levels unlike an inhibitor of deacetylase activity (Trichostatin A; TSA). Treatments with TSA resulted in significant recovery of Tir levels, potentiation of actin polymerization and improvement in bacterial attachment to cells. Our findings have important implications for the current model of Tir-mediated actin polymerization and opens new lines of research in this area.

Keywords: A/E, Attaching and effacing; Ab, Polyclonal antibody; Actin; BFP, Bundle-forming pili; Cmp, Chloramphenicol; CrkII, CT10 regulator of kinase; CrkL, Crk-like; Ctr., Control; EHEC, Enterohaemorrhagic Escherichia coli; EPEC; EPEC, Enteropathogenic Escherichia coli; Esp, EPEC-secreted proteins; FBS, Fetal bovine serum; GBD, GTPase-binding domain; HDAC, Histone deacetylases; HeLa, Human cervical epithelial cancer cell line; IRSp53, Insulin receptor tyrosine kinase substrate p53; IRTKS, Insulin receptor tyrosine kinase substrate; LEE, Locus of enterocyte effacement; MEFs, Mouse embryonic fibroblasts; MOI, Multiplicity of infection; Map, Mitochondrial associated protein; MoAb, Monoclonal antibody; N-WASP; N-WASP, Neural Wiskott–Aldrich syndrome protein; NF-kB, Nuclear factor kB; NPF, Nucleation promoting factor; Nck; Nck, Non-catalytic tyrosine kinase; Nle, Non-LEE effectors; PRD, Proline-rich domain; SH3, Src homology 3; T3SS, Type 3 secretion system; TNF- α, Tumor necrosis factor-α; TSA; TSA, Trichostatin A; Tir; Tir, Translocated Intimin receptor; WB, Western Blot; WT, Wild type.; bacterial adhesion; pedestals.

Figure 1.

Analysis of Tir homeostasis within cells with reduced expression of Nck adaptor. (A) WT, Nck-deficient cells (Nck -/-) and Nck1 reconstituted cells (R) were infected with EPEC for 3 h or left uninfected as a negative control. Cell monolayers were lysed in imidazole buffer and fractionated by centrifugation. The membrane fraction (pellet) was resuspended in Laemmli sample buffer. One third of the membrane fraction and one fiftieth of the cytoplasmic fraction were analyzed by WB with anti-Tir MoAb, anti-EspF Ab and, as a loading control, with anti-actin MoAb. The genotype of the cells used was corroborated by blotting with anti-Nck MoAb. (B) HeLa cells were treated using siRNA with oligonucleotides against Nck or with a control oligonucleotide (Ctr.) and infected with EPEC for 3 h. Cell monolayers were lysed in 1% Triton X-100 lysis buffer and the soluble supernatants (contain cytoplasmic and membrane fractions) were blotted with anti-Tir Ab. The arrow indicates the fully-modified Tir form. Actin and tubulin were used as loading controls and the levels of Nck were analyzed by blotting with anti-Nck MoAb. The graph represents the quantification of the 3 Tir bands normalized to actin. (C) Quantification of the number of pedestals of a total of 100 HeLa cells treated with siRNAs for Nck (black bar) compared to control oligonucleotide treated cells (Ctr., white bar). Graphs represent mean ± SD. Statistical analysis was performed using the Student´s t-test from 3 independent experiments; **, P˂0.01.

Figure 2.

EPEC adhesion to Nck-deficient cells. WT, Nck-deficient cells (Nck −/−) and Nck1 reconstituted cells (R) were infected for 3 h with EPEC strains Δeae (deleted gene encoding Intimin), Δtir (deleted gene encoding Tir) and, as a control, Δtir + ptir (Δtir complemented with Tir). As a control HeLa cell infections were performed in parallel. Cell monolayers were lysed in 1% Triton X-100 lysis buffer. The soluble supernatants that contain the cytoplasmic and membrane fractions (A) and the insoluble pellets that contain the attached bacteria (B) were subjected to SDS-PAGE and blotted with the indicated antibodies. Actin was used as a loading control. DnaK is an EPEC chaperone used as a control for the bacterial protein content.

Figure 3.

Stability of Tir present within Nck-deficient MEFs. (A) WT and Nck-deficient cells (Nck−/−) were infected with EPEC for 3 h or left uninfected as a negative control. Then the cells were treated with gentamicin for 6 hour time-course. Cell monolayers from a single well of a 6-well-plate were collected at indicated times by adding Laemmli sample buffer and lysates were blotted with anti-Tir MoAb. (B) WT and Nck−/− cells were infected with EPEC for 15 minutes at an MOI of 300. Cells were then treated with chloramphenicol (+ Cmp.) or vehicle as a control (− Cmp.) for 1, 2 or 3 h. Monolayers were lysed in 1% Triton X-100 lysis buffer. The soluble supernatants that contain the cytoplasmic and membrane fractions were blotted with anti-Tir Ab and anti-EspF Ab. Actin was used as a loading control. For better visualization of Tir the intensity of the image was decreased in the − Cmp WB. Arrows indicate the fully-modified Tir form.

Figure 4.

Phosphorylation status and levels of Tir phosphorylation-deficient mutants within Nck-deficient MEFs. (A) WT, Nck-deficient cells (Nck −/−) and Nck1 reconstituted cells (R) were infected with EPEC for 3 h. Cell monolayers were lysed in modified Ripa buffer and the cell lysates (Lys) were used to perform immunoprecipitation (IP) experiments using an anti-Tir MoAb coupled to magnetic beads. Sequential WB was performed with a generic anti-phosphotyrosine MoAb (in red) and with anti-Tir Ab (in green). The merge of both images is shown. The isotype control IP (IgG) and the cell lysates are shown. (B and C) WT, Nck −/− cells and Nck1 reconstituted cells (R) were infected with EPEC strains for 3 h. EPEC Δtir strain complemented with WT Tir (Δtir + ptir), tyrosine phosphorylation deficient Tir mutant (Δtir + ptir Y454F,Y474F), serine phosphorylation deficient Tir mutant (Δtir + ptir S434A,S463A) and tyrosine and serine phosphorylation deficient Tir mutant (Δtir + ptir Y454F,Y474F/S434,S463A). Cell monolayers were lysed in 1% Triton X-100 lysis buffer. The soluble supernatants that contain the cytoplasmic and membrane fractions (B) and the insoluble pellets that contain the attached bacteria (C) were subjected to SDS-PAGE and blotted with the indicated antibodies. Actin was used as a loading control.

Figure 5.

The levels of Map effector within infected-Nck-deficient cells are decreased compared to WT cells. WT, Nck-deficient cells (Nck -/-) and Nck1 reconstituted cells (R) were infected for 3 h with WT EPEC or with WT EPEC that expresses HA tagged-Map (WT+pSKT7-map-HA). Cell monolayers were lysed in 1% Triton X-100 lysis buffer. The soluble supernatants that contain the cytoplasmic and membrane fractions were analyze for WB with anti-Tir Ab, anti-HA MoAb and with anti-EspF Ab. Actin was used as a loading control.

Figure 6.

Trichostatin A treatments increase bacterial attachment and the levels of Tir in EPEC-infected MEFs. (A) WT, Nck-deficient cells (Nck−/−) and Nck1 reconstituted cells (R) were treated with Trichostatin A (TSA, 5 μM) for 16 hours prior to and during infection with EPEC for 3h. Cell monolayers were lysed in 1% Triton X-100 lysis buffer. The soluble supernatants that contain the cytoplasmic and membrane fractions and the insoluble pellets that contain the attached bacteria were subjected to SDS-PAGE and blotted with the indicated antibodies. Actin was used as a loading control, blotting with anti-Nck MoAb was performed to confirm the genotype of the MEFs used and with anti-DnaK MoAb as an indicator of bacterial attachment. (B) Confocal immunofluorescence images of WT and Nck −/− cells treated with TSA for 16 h and infected with EPEC for 3 h. Actin staining with TRITC-phalloidin is shown in red. DAPI was used to stain EPEC. The merged images shown in the last column were generated with Leica software. Pictures were taken at 600X magnification and scale bar represents 20 μm. Insets are 4X digital zoom images. (C) Quantification of the number of attached bacteria and pedestals of 100 cells from the immunofluorescence images. Graph represents mean ± SD. Statistical analysis using Student´s t-test for the number of attached bacteria and Mann Whitney test for pedestal number from at least 3 independent experiments is shown. *, P < 0.05; **, P < 0.01.

Figure 7.

Immunofluorescence staining of Tir within MEFs treated with Trichostatin. (A) Confocal immunofluorescence images of WT, Nck-deficient cells (Nck −/−) and Nck1 reconstituted cells (R) treated with Trichostatin A (TSA, 5 μM) for 16 h (or left untreated as a control) and infected with EPEC for 3 h. Immunofluorescence staining was done using anti-Tir MoAb (in green) and DAPI to stain EPEC (in blue). The merged images shown in the last column were generated with Leica software. Pictures were taken at 600× magnification and scale bar represents 20 μm.