The phosphoinositol-3-kinase-protein kinase B/Akt pathway is critical for Pseudomonas aeruginosa strain PAK internalization - PubMed (original) (raw)
The phosphoinositol-3-kinase-protein kinase B/Akt pathway is critical for Pseudomonas aeruginosa strain PAK internalization
A Kierbel et al. Mol Biol Cell. 2005 May.
Abstract
Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P3 accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells.
Figures
Figure 1.
Pharmacological inhibitors of PI3K block PAK internalization. Day 1 MDCK cells were pretreated with the indicated concentration of LY for 1 h. Standard bacterial invasion (A) or adhesion assays (B) were carried out in the presence of the indicated concentration of the drug. Shown are the results of least three independent experiments, each performed in triplicate. Error bars indicate SEM. *p < 0.01, **p < 0.001 compared with untreated cells, Student's two-tailed t test.
Figure 2.
Exogenous addition of PIP(3,4,5)P3 is sufficient to promote bacterial invasion through the apical surface. PIP(3,4,5)P3 complexed with histone (30 μM) was added to the apical surface of filter-grown day 3 MDCK cells for 5 min. The lipid was removed by washing and standard invasion (A) and adhesion assays (B) were performed. Shown are the results of least three independent experiments, each performed in triplicate. Error bars indicate SEM. *p < 0.001 compared with the control cells, Student's two-tailed t test.
Figure 3.
PH-Akt-GFP, a probe for PtdIns(3,4,5)P3, accumulates at the site of bacterial entry. Day 1 filter-grown MDCK cells stably transfected with PH-Akt-GFP (green) were infected for 30 min with Syto59-labeled PAK (red). The samples were fixed and analyzed by epifluorescence (A and B) and confocal microscopy (C–F). A and B, conventional fluorescence microscopy of bound bacteria in the absence (A) or presence (B) 50 μM LY. Bar, 1 μm. C–F, confocal (X-Z sections) of infected (C and D) or uninfected cells (E and F). Bar, 10 μm. In uninfected cells (E), PtdIns(3,4,5)P3 is localized to the basolateral membrane. In the presence of apically applied PAK, PtdIns(3,4,5)P3 accumulates at the apical site of bacteria binding (A, apical view; C, X-Z section). Treatment with 50 μM LY decreases the frequency of apical accumulation of PH-Akt-GFP at the site of bacterial binding to 30% of the control (B, D, and G). The graph in G shows the combined data for three different experiments. Error bars indicate SEM. *p < 0.01 compared with the control cells, Student's two-tailed t test.
Figure 4.
Akt is phosphorylated upon infection with PAK. Day 1 MDCK cells were infected with PAK; at the indicated times postinfection, host cell lysates prepared and immunoprecipitated with anti-Akt followed by immunoblotting with anti-Akt or an antibody specific for Akt phosphorylated on serine 473. (A) Levels of phosphorylated and total Akt (Takt) of untreated cells (U), cells infected with PAK (MOI of 200) for 1 h, or cells treated with 10 ng/ml EGF for 10 min. (B) Phosphorylation of AKT increases at increasing MOI and can be blocked by the PI3K inhibitor LY. (C) Relative to total AKT, an increase in phosphorylation of AKT is detected within 30 min of exposure to bacteria and peaks at 1 h. Gels are representative of at least three independent experiments.
Figure 5.
SH-5, a selective inhibitor of AKT activation, blocks PAK uptake. Day 1 MDCK cells were treated for 2 h with SH-5 (10 μM), an inhibitor that specifically blocks AKT phosphorylation, and standard adhesion (open bars) and internalization assays (closed bars) were performed. Results are representative of at least three independent experiments, each performed in triplicate. Error bars indicate SEM. *p < 0.001 compared with untreated cells, Student's two-tailed t test.
Figure 6.
siRNA-mediated AKT depletion abrogates PAK uptake. Akt protein was depleted in HeLa cells transfection with Akt siRNA. (A) Western blot of lysates transfected with a control siRNA or Akt siRNA to two of the three Akt isoforms for 48 h. Both total (TAkt) and phosphorylated Akt were effectively depleted. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as loading control. (B) Standard adhesion (open bars) and internalization assays (closed bars) were performed. Results correspond to at least three independent experiments, each performed in triplicate. Error bars indicate SEM. *p < 0.05 compared with control siRNA, Student's two-tailed t test.
Figure 7.
Akt is sufficient to restore invasion in the absence of PI3K signaling. Invasion and adhesion assays were performed in MDCK cells stably transfected with CA Akt or with vector alone (control). Cells were exposed to carrier or to 50 and 100 μM LY. Shown are the combined results from five independent experiments, each performed in triplicate. Error bars indicate SEM. *p < 0.05 comparing the CA Akt cell line treated with LY to control cells treated with LY, Student's two-tailed t test. **p < 0.01 comparing the control cells treated with LY to control cells treated with carrier alone, Student's t test.
References
- Bette-Bobillo, P., Giro, P., Sainte-Marie, J., and Vidal, M. (1998). Exoenzyme S from P. aeruginosa ADP ribosylates rab4 and inhibits transferrin recycling in SLO-permeabilized reticulocytes. Biochem. Biophys. Res. Commun. 244, 336-341. -PubMed
- Burgering, B. M., and Coffer, P. J. (1995). Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376, 599-602. -PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Miscellaneous