RNAi screen reveals an Abl kinase-dependent host cell pathway involved in Pseudomonas aeruginosa internalization - PubMed (original) (raw)
RNAi screen reveals an Abl kinase-dependent host cell pathway involved in Pseudomonas aeruginosa internalization
Julia F Pielage et al. PLoS Pathog. 2008.
Abstract
Internalization of the pathogenic bacterium Pseudomonas aeruginosa by non-phagocytic cells is promoted by rearrangements of the actin cytoskeleton, but the host pathways usurped by this bacterium are not clearly understood. We used RNAi-mediated gene inactivation of approximately 80 genes known to regulate the actin cytoskeleton in Drosophila S2 cells to identify host molecules essential for entry of P. aeruginosa. This work revealed Abl tyrosine kinase, the adaptor protein Crk, the small GTPases Rac1 and Cdc42, and p21-activated kinase as components of a host signaling pathway that leads to internalization of P. aeruginosa. Using a variety of complementary approaches, we validated the role of this pathway in mammalian cells. Remarkably, ExoS and ExoT, type III secreted toxins of P. aeruginosa, target this pathway by interfering with GTPase function and, in the case of ExoT, by abrogating P. aeruginosa-induced Abl-dependent Crk phosphorylation. Altogether, this work reveals that P. aeruginosa utilizes the Abl pathway for entering host cells and reveals unexpected complexity by which the P. aeruginosa type III secretion system modulates this internalization pathway. Our results furthermore demonstrate the applicability of using RNAi screens to identify host signaling cascades usurped by microbial pathogens that may be potential targets for novel therapies directed against treatment of antibiotic-resistant infections.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Figure 1. P. aeruginosa entry into Drosophila S2 cells mimics entry into mammalian cells.
(A) Invasion of PAK into Drosophila S2 cells is actin-dependent. Drosophila S2 cells were treated with 10 µM Cytochalasin D and infected with PAK for 2 h at 28°C. The number of internalized bacteria was determined by performing an invasion assay. Results are normalized to untreated cells. ***p<0.001 compared to untreated cells. (B) The T3SS effector proteins ExoS and ExoT inhibit P. aeruginosa entry into S2 cells. Invasion of PAK and PAKΔSΔT into S2 cells after 2 h at 28°C were compared. The results were normalized with respect to invasion of PAK. ***p<0.001 compared to invasion of PAK into S2 cells.
Figure 2. P. aeruginosa invasion into mammalian cells is dependent on Abl tyrosine kinases.
(A) HeLa cells were infected with PAKΔSΔT for 1 h in the presence of the Abl inhibitor Gleevec (0–50 µM), and bacterial invasion was quantified. The results are normalized with respect to invasion in untreated cells. **p<0.01, ***p<0.001 compared to untreated cells. (B) Abl−/−Arg−/− fibroblasts and parental 3T3 fibroblasts were infected with PAK or PAKΔSΔT for 1 h and bacterial uptake was measured by invasion assays. *p<0.05, ***p<0.001 compared to 3T3 fibroblasts. (C) HeLa cells were treated with Abl or control (c) siRNA. Cell lysates immunoblotted with an anti-Abl-antibody show decreased Abl protein levels compared to control siRNA-treated or untreated cells. GAPDH was used as loading control. (D) Standard invasion and adhesion assays were used to quantify the effect of Abl or control siRNA treatment on invasion and adhesion of PAKΔSΔT at 1 h into HeLa cells. The results are normalized with respect to invasion in untreated cells. *p<0.05 compared to control siRNA-treated cells.
Figure 3. Abl-dependent Crk phosphorylation is required for P. aeruginosa invasion.
(A) HeLa cells were treated with Crk or control (c) siRNA and cell lysates were immunoblotted with an anti-CrkI/II-antibody. GAPDH was used as loading control. (B) Standard invasion assays were used to quantify the effect of Crk or control siRNA treatment of invasion of PAK or PAKΔSΔT at 1 h into HeLa cells. *p<0.05 compared to control RNA-treated cells. (C–E) HeLa cells were infected with the indicated strains for the indicated times, lysed and immunoblotted with anti-phospho-CrkII-Y221 and anti-CrkI/II. (F, G) HeLa cells were infected with PAKΔSΔT for indicated times in the presence of 30 µM Gleevec (F) or after treatment with Abl or control siRNA (G) and cell lysates were immunoblotted with anti-phospho-CrkII-Y221 and anti-CrkI/II. (H) HeLa cells were transfected with pCAGGS-CrkII and pCAGGS–CrkII-Y221F for 16 h and cell lysates were immunoblotted with an anti-CrkI/II antibody. GAPDH was used as loading control. (I) Standard invasion assays were used to quantify the effect of over-expression of CrkII or CrkII-Y221F on invasion of PAKΔSΔT at 1 h into HeLa cells. **p<0.01 compared to cells over-expressing wild type CrkII.
Figure 4. Rac1 and Cdc42 are required for invasion of P. aeruginosa into mammalian cells.
(A) HeLa cells were treated with Rac1, Cdc42 or control (c) siRNA. Cell lysates were immunoblotted with anti-Rac1 and anti-Cdc42, respectively. GAPDH was used as loading control. (B) Internalization of PAK, PAKΔS, PAKΔT and PAKΔSΔT at 1 h was quantified in HeLa cells treated with control siRNA or siRNA against Rac1 or Cdc42. *p<0.05, **p<0.01 compared to control RNA-treated cells for each bacterial strain. (C–F) GTPase activation assays. HeLa cells were infected with PAK, PAKΔSΔT, PAKΔS and PAKΔT for the indicated times. Lysates were incubated with Pak1-PBD to precipitate GTP-bound Cdc42 and GTP-bound Rac1. The bound proteins and cell lysates were examined by immunoblotting with anti-Cdc42 (C) and anti-Rac1 (E), respectively. The experiments were performed 3 times and a typical gel is shown. (D, F) The GTPases activation assays to assess Cdc42 (D) or Rac1 (F) activation upon infection with PAK, PAKΔSΔT, PAKΔS and PAKΔT were quantified by densitometry. Shown are the mean values +/- SD from three independent experiments.
Figure 5. Kinetics of internalization of PAK into mammalian cells.
Invasion of PAK, PAKΔSΔT, PAKΔS and PAKΔT in HeLa cells at the indicated times was quantified. Shown are the mean +/− SD from three independent experiments.
Figure 6. Invasion of P. aeruginosa is Pak1 dependent.
(A) HeLa cells were treated with Pak1 or control (c) siRNA. Cell lysates were immunoblotted with anti-Pak1. GAPDH was used as loading control. (B) Internalization of PAKΔSΔT at 1 h was quantified in HeLa cells treated with control siRNA or siRNA against Pak1. *p<0.05 compared to control siRNA-treated cells. (C) HeLa cells were treated with Pak1 and/or Abl siRNA or control siRNA. Cell lysates immunoblotted with anti-Pak1 and anti-Abl. GAPDH was used as loading control. (D) Internalization of PAKΔSΔT at 1 h was quantified in HeLa cells treated with control siRNA or siRNA against Pak1, Abl, and Pak1+Abl. Results are normalized to control siRNA-treated HeLa cells. *p<0.05, ***p<0.001 compared to control siRNA-treated cells. (E) MDCK cells were induced for 18 h to express human Pak1 (Pak1wt), a dominant-negative (Pak1DN) or a constitutively active (Pak1CA) mutant of Pak1 by removal of doxycycline. Invasion of PAKΔSΔT at 1 h was quantified and the results were normalized with respect to uninduced cells. *p<0.05, ***p<0.001 compared to uninduced cells.
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