Journal: Cell Communication and Signaling : CCS

Article Title: The signaling pathway of Campylobacter jejuni -induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

doi: 10.1186/1478-811X-9-32

Figure Lengend Snippet: C. jejuni -triggered Cdc42 activation is time-dependent and requires intact lipid rafts . (A) Quantification of Cdc42 activity during the course of infection. INT-407 cells were infected with wt C. jejuni 81-176 for indicated periods of time. The presence of active Cdc42-GTP was quantified by G-Lisa and GST-CRIB pulldown. One hundred % of GTPase activity corresponds to the highest amount of detected Cdc42-GTP level (right lane). Similar quantities of total Cdc42 and GAPDH were confirmed by Western blotting. (B) Effect of Cdc42 expression knockdown on C. jejuni invasion. INT-407 cells were transfected with Cdc42-siRNA as well as a scrambled siRNA as control. After 48 hours, cells were infected with C. jejuni for 6 hours. Intracellular bacteria were quantified by gentamicin protection assays. Immunoblotting with α-Cdc42 antibody confirmed down-regulation of the protein. GAPDH expression levels were determined as control. (C) Effects of MβCD targeting lipid rafts on host cell internalization of C. jejuni . INT-407 monolayers were pre-incubated with the indicated concentrations of MβCD for 30 min, followed by 6 hours infection with wt C. jejuni 84-25. Intracellular C. jejuni were quantified by gentamicin protection assays. The presence of active Cdc42-GTP was analyzed by CRIB-GST pulldown and quantified. One hundred % of activity corresponds to the highest amount of detected Cdc42-GTP level (lane 2). Similar quantities of total Cdc42 and GAPDH were confirmed by Western blotting. (*) P ≤ 0.05 and (**) P ≤ 0.005 were considered as statistically significant as compared to the control.

Article Snippet: siRNA directed against human DOCK180, Vav2 and siRNA containing a scrambled control sequence were purchased from Santa Cruz. siRNA against human Cdc42 was synthesised as 5'-TTCAGCAATGCAGACAATTAA-3'.

Techniques: Activation Assay, Activity Assay, Infection, Western Blot, Expressing, Knockdown, Transfection, Control, Bacteria, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: The signaling pathway of Campylobacter jejuni -induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

doi: 10.1186/1478-811X-9-32

Figure Lengend Snippet: Importance of fibronectin, integrin-β1, FAK and Src kinases expression on C. jejuni invasion . The following cells lines were infected with wt C. jejuni 81-176 for 6 hours. (A) Fibronectin-deficient cells (Fn -/- ) and corresponding floxed wt cells (Fn +/+ ), (B) integrin-β1-deficient cells (GD25) and GD25 stably re-expressing wt integrin-β1A (GD25-β1A) cells, (C) FAK-deficient cells (FAK -/- ) and FAK -/- cells stably re-expressing wt FAK and (D) Src kinase-deficient cells (SYF -/- ) and SYF -/- cells stably re-expressing wt c -src . Intracellular C. jejuni were quantified by gentamicin protection assays, and Cdc42 activation by CRIB-GST pulldowns. (**) P ≤ 0.005 was considered as statistically significant. Fibronectin, integrin-β1, FAK and Src expression was analyzed by immunoblotting. GAPDH expression levels were determined as loading control.

Article Snippet: siRNA directed against human DOCK180, Vav2 and siRNA containing a scrambled control sequence were purchased from Santa Cruz. siRNA against human Cdc42 was synthesised as 5'-TTCAGCAATGCAGACAATTAA-3'.

Techniques: Expressing, Infection, Stable Transfection, Activation Assay, Western Blot, Control

Journal: Cell Communication and Signaling : CCS

Article Title: The signaling pathway of Campylobacter jejuni -induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

doi: 10.1186/1478-811X-9-32

Figure Lengend Snippet: C. jejuni invasion is impaired in cells expressing integrin-β1 point mutations exhibiting defects in fibronectin fibril organisation or FAK phosphorylation . (A) Integrin-β1-deficient cells (GD25) and GD25 stably re-expressing mutated integrin subunit β1A (GD25-β1 ATT788-9AA or GD25-β1 AY783/795F or wild-type β1A (GD25-β1A) cells were infected with wt C. jejuni 81-176 for 6 hours. Intracellular C. jejuni were quantified by gentamicin protection assays. (B) The presence of active Cdc42-GTP was quantified by CRIB-GST pulldowns. One hundred % of activity corresponds to the highest amount of detected Cdc42-GTP level. (**) P ≤ 0.005 was considered as statistically significant. Similar quantities of total Cdc42 and GAPDH were confirmed by Western blotting.

Article Snippet: siRNA directed against human DOCK180, Vav2 and siRNA containing a scrambled control sequence were purchased from Santa Cruz. siRNA against human Cdc42 was synthesised as 5'-TTCAGCAATGCAGACAATTAA-3'.

Techniques: Expressing, Phospho-proteomics, Stable Transfection, Infection, Activity Assay, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: The signaling pathway of Campylobacter jejuni -induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

doi: 10.1186/1478-811X-9-32

Figure Lengend Snippet: Importance of the CadF and flagellar apparatus for C. jejuni -induced activation of Cdc42 and bacterial invasion . (A) FAK +/+ and FAK -/- cells were infected with wt C. jejuni F38011 or isogenic F38011Δ cadF for the indicated periods of time. Quantification of Cdc42-GTP levels by CRIB-GST pulldown during the course of infection. One hundred % of activity corresponds to the highest amount of detected Cdc42-GTP level (lane 4). ( B) FAK-positive cells were infected with the indicated strains in a time-course. The presence of bound, active Cdc42-GTP was analyzed in CRIB-GST pulldown assays followed by Western blotting using α-Cdc42 antibody. Similar quantities of individual GTPases at every time point were confirmed by Western blotting using equivalent volumes of cell lysates. (C) Quantification of Cdc42-GTP levels during the course of infection. One hundred % of activity corresponds to the highest amount of detected Cdc42-GTP level (lane 3). The amount of intracellular bacteria was quantified by gentamicin protection assays under the same experimental conditions. (**) P ≤ 0.005 were considered as statistically significant as compared to the control.

Article Snippet: siRNA directed against human DOCK180, Vav2 and siRNA containing a scrambled control sequence were purchased from Santa Cruz. siRNA against human Cdc42 was synthesised as 5'-TTCAGCAATGCAGACAATTAA-3'.

Techniques: Activation Assay, Infection, Activity Assay, Western Blot, Bacteria, Control

Journal: Cell Communication and Signaling : CCS

Article Title: The signaling pathway of Campylobacter jejuni -induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

doi: 10.1186/1478-811X-9-32

Figure Lengend Snippet: Importance of guanine exchange factor Vav2 for C. jejuni -induced Cdc42 activation . INT-407 cells were transfected for 48 hours with siRNA for Vav2 (A), Tiam-1 (B) or DOCK180 (C) as well as a scrambled siRNA as control. Immunoblotting with the indicated antibodies confirmed knockdown of the respective proteins. GAPDH expression levels were determined as control. Quantification of Cdc42 GTPase activity after infection with wt C. jejuni 81-176 for 6 hours. The presence of bound, active Cdc42-GTP was analyzed in CRIB-GST pulldown assays followed by Western blotting using α-Cdc42 antibody. One hundred % of activity corresponds to the highest amount of detected Cdc42-GTP level (lane 1).

Article Snippet: siRNA directed against human DOCK180, Vav2 and siRNA containing a scrambled control sequence were purchased from Santa Cruz. siRNA against human Cdc42 was synthesised as 5'-TTCAGCAATGCAGACAATTAA-3'.

Techniques: Activation Assay, Transfection, Control, Western Blot, Knockdown, Expressing, Activity Assay, Infection

Journal: Cell Communication and Signaling : CCS

Article Title: The signaling pathway of Campylobacter jejuni -induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

doi: 10.1186/1478-811X-9-32

Figure Lengend Snippet: Downregulation, elimination or i nterference with important Vav2 functions reduces the uptake of C. jejuni in host cells . (A) INT-407 cells were transfected with siRNA against Vav2 or a scrambled siRNA as control. After 48 hours, cells were infected with wt C. jejuni 81-176 for 6 hours. Intracellular bacteria were quantified by gentamicin protection assays. (B) The presence of active Rac1-GTP and Cdc42-GTP was quantified by CRIB-GST pulldowns. One hundred % of activity corresponds to the highest amount of detected GTPase-GTP level. (C) INT-407 cells were transfected with indicated Myc-tagged or (D) GFP-tagged Vav2 constructs. After 48 hours, cells were infected with wt C. jejuni 81-176 for 6 hours. Intracellular bacteria were quantified by gentamicin protection assays. Expression of the individual Vav2 constructs was verified by Western blot analysis. GAPDH expression levels were determined as control. (E) Vav2-deficient cells (Vav1/2 -/- ) or Vav2-expressing control fibroblasts (Vav1/2 +/+ ) were infected for 6 hours with C. jejuni . Intracellular and cell-associated bacteria were quantified by gentamicin protection assays. (*) P ≤ 0.05 and (**) P ≤ 0.005 were considered as statistically significant.

Article Snippet: siRNA directed against human DOCK180, Vav2 and siRNA containing a scrambled control sequence were purchased from Santa Cruz. siRNA against human Cdc42 was synthesised as 5'-TTCAGCAATGCAGACAATTAA-3'.

Techniques: Transfection, Control, Infection, Bacteria, Activity Assay, Construct, Expressing, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: The signaling pathway of Campylobacter jejuni -induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

doi: 10.1186/1478-811X-9-32

Figure Lengend Snippet: Importance of FAK, EGFR, PDGFR and PI3-kinase activities for C. jejuni -induced activation of Cdc42 and bacterial invasion . (A) INT-407 monolayers were pre-incubated for 30 min with the indicated pharmacological inhibitors and infected with C. jejuni for 6 hours. Intracellular C. jejuni were quantified by gentamicin protection assays. The presence of active Cdc42-GTP was quantified by CRIB-GST pulldowns. One hundred % of activity corresponds to the highest amount of detected Cdc42-GTP level (lane 2). (B) Effect of overexpression of dominant-negative forms of PDGFR and EGFR on C. jejuni uptake. 48 hours post transfection INT-407 cells were infected with C. jejuni for 6 hours. Intracellular bacteria were quantified by gentamicin protection assays. Expression of the individual constructs was verified by Western blotting. GAPDH expression levels were determined as control. (*) P ≤ 0.05 and (**) P ≤ 0.005 were considered as statistically significant.

Article Snippet: siRNA directed against human DOCK180, Vav2 and siRNA containing a scrambled control sequence were purchased from Santa Cruz. siRNA against human Cdc42 was synthesised as 5'-TTCAGCAATGCAGACAATTAA-3'.

Techniques: Activation Assay, Incubation, Infection, Activity Assay, Over Expression, Dominant Negative Mutation, Transfection, Bacteria, Expressing, Construct, Western Blot, Control

Journal: Cell Communication and Signaling : CCS

Article Title: The signaling pathway of Campylobacter jejuni -induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

doi: 10.1186/1478-811X-9-32

Figure Lengend Snippet: Model for C. jejuni -induced signaling leading to Cdc42 activation and bacterial invasion . C. jejuni adheres to host cells via the fibronectin-binding protein CadF, which acts as a bridge engaging the integrin-β1 receptor. Integrin occupancy and clustering in lipid rafts leads to recruitment and activation of the non-receptor tyrosine kinase FAK. Phosphorylation of FAK and Src triggers a cascade of signals resulting in the formation of protein complexes leading to activation of other signaling factors as indicated. Assembly of integrin-dependent signal complexes leads to phosphorylation and transactivation of PDGFR and EGFR, followed by stimulation of PI3-K and Vav2. Activated Vav2 then induces the activation of Cdc42. This signaling potentially causes localized actin and/or microtubule rearrangements at the site of C. jejuni entry, resulting in bacterial uptake. In addition to CadF, the C. jejuni flagellum also appears to play a role in the described signal cascades. If the flagellum participates by sole bacterial motility, by translocating bacterial effector proteins or targeting a host receptor directly is not yet clear and needs to be investigated in future studies.

Article Snippet: siRNA directed against human DOCK180, Vav2 and siRNA containing a scrambled control sequence were purchased from Santa Cruz. siRNA against human Cdc42 was synthesised as 5'-TTCAGCAATGCAGACAATTAA-3'.

Techniques: Activation Assay, Binding Assay, Phospho-proteomics