Pseudomonas aeruginosa exploits a PIP3-dependent pathway to transform apical into basolateral membrane - PubMed (original) (raw)

Pseudomonas aeruginosa exploits a PIP3-dependent pathway to transform apical into basolateral membrane

Arlinet Kierbel et al. J Cell Biol. 2007.

Abstract

Pseudomonas aeruginosa, an important human pathogen, preferentially binds and enters injured cells from the basolateral (BL) surface. We previously demonstrated that activation of phosphatidylinositol 3-kinase (PI3K) and Akt are necessary and sufficient for P. aeruginosa entry from the apical (AP) surface and that AP addition of phosphatidylinositol 3,4,5-trisphosphate (PIP3) is sufficient to convert AP into BL membrane (Kierbel, A., A. Gassama-Diagne, K. Mostov, and J.N. Engel. 2005. Mol. Biol. Cell. 16:2577-2585; Gassama-Diagne, A., W. Yu, M. ter Beest, F. Martin-Belmonte, A. Kierbel, J. Engel, and K. Mostov. 2006. Nat. Cell Biol. 8:963-970). We now show that P. aeruginosa subverts this pathway to gain entry from the AP surface. In polarized monolayers, P. aeruginosa binds near cell-cell junctions without compromising them where it activates and recruits PI3K to the AP surface. Membrane protrusions enriched for PIP3 and actin accumulate at the AP surface at the site of bacterial binding. These protrusions lack AP membrane markers and are comprised of BL membrane constituents, which are trafficked there by transcytosis. The end result is that this bacterium transforms AP into BL membrane, creating a local microenvironment that facilitates its colonization and entry into the mucosal barrier.

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Figures

Figure 1.

Figure 1.

Attachment of P. aeruginosa to the AP membrane of MDCK cells triggers the formation of membrane protrusions. (a) Scanning electron microscopy of MDCK cells infected with P. aeruginosa for 30 min. (left and middle) Top-down views of membrane protrusions. The host membrane is indicated by asterisks, whereas bacteria are indicated by the arrowheads. (right) Lower magnification of the picture in the middle panel showing that bacteria were typically observed to adhere to the cell–cell junctions. (b and c) Confocal micrographs of MDCK cells expressing GFP-PH-Akt (green; a marker for PIP3) infected with PKH26 red–labeled P. aeruginosa. (b) XZ sections and 3D reconstruction of samples fixed 30 min after the addition of bacteria. The host cell nuclei are stained with DAPI (blue). PIP3-containing protrusions are observed at the site of bacterial binding to the AP surface. The MDCK cells form a single monolayer of cells. (c) XZ sections of samples fixed 2 h after the addition of bacteria. The bacteria were allowed to adhere for 1 h, nonadherent bacteria were removed by washing, and extracellular bacteria were killed by the addition of amikacin for 1 h. The bacteria are fully internalized; no AP protrusions or AP PIP3 are observed. Bars, 10 μm.

Figure 2.

Figure 2.

PI3K and filamentous actin are recruited to the protrusions at the site of bacterial attachment. Confocal micrographs of wild-type MDCK (a) or MDCK cells expressing GFP-PH-Akt infected with PKH26 red–labeled P. aeruginosa for 30 min (b). XZ sections revealed the recruitment of PI3K (green; a) and filamentous actin (blue; b) to the bacteria-induced protrusions at the site of AP binding. Bars, 10 μm.

Figure 3.

Figure 3.

_P. aeruginosa_–triggered AP protrusions have BL membrane characteristics. XZ and 3D reconstructions of confocal micrographs of MDCK cells expressing GFP-PH-Akt infected with PKH26 red–labeled P. aeruginosa for 30 min showing both vertical (XZ) sections as well as 3D reconstructions of a portion of the monolayer. (a) PIP3 (GFP-PH-Akt; green) is recruited to the AP bacteria-induced protrusion, whereas the AP protein gp135 (purple) is absent. (b and c) The BL marker proteins p58 (purple; b) and β-catenin (purple; c) are recruited to the AP bacteria-induced protrusions. The 3D reconstructions show that BL components appear to surround the bacterial aggregate. Bars, 10 μm.

Figure 4.

Figure 4.

P. aeruginosa induces the relocalization of proteins from the BL to the AP surface by transcytosis. (a, left) The BL surface of confluent MDCK cells was biotinylated for 30 min at 4°C. Cells were infected with P. aeruginosa expressing GFP for 30 min at 37°C, fixed, and the AP and BL surfaces were probed with fluorescent streptavidin (red). Biotinylated BL proteins are only detectable on the BL surface and at the sites of bacterial attachment on the AP surface. (a, right) The relocalization of BL proteins is reduced by LY. Cells were preincubated with 100 μM LY for 1 h. The drug was also present during biotinylation and infection. (b) The diffusion from the AP to the BL surface of the small molecule FITC-inulin was measured in uninfected cells, cells infected with P. aeruginosa for 30 min, or after exposure to 10 mM EDTA for 10 min. AP addition of P. aeruginosa decreased the monolayer permeability. Error bars represent SD. (c) XZ section of MDCK cells infected with GFP–P. aeruginosa and stained with an antibody against an extracellular domain of the BL protein β1-integrin (red). The cells were then fixed and stained with DAPI (blue) to visualize the nuclei. The addition of bacteria did not permit diffusion of the antibody to the BL surface, suggesting that the tight junctions were not disrupted upon bacterial binding. (d) BL surface biotinylation of MDCK T23 cells previously infected with recombinant adenovirus encoding HA-tagged DN dynamin II was performed as described in panel a. DN dynamin II (blue) is expressed both in AP and BL membranes. The left panel shows a typical example of the relocalization of biotinylated BL proteins to the site of AP bacterial attachment to a nontransfected cell. The right panel shows that in cells expressing DN dynamin II, the AP accumulation of biotinylated proteins was inhibited. Bars, 10 μm.

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