Molecular Biology of the Cell
Vol. 16, 2577–2585, May 2005
The Phosphoinositol-3-Kinase–Protein Kinase B/Akt
Pathway Is Critical for Pseudomonas aeruginosa Strain
A. Kierbel,*†A. Gassama-Diagne,‡§?K. Mostov,‡§?and J. N. Engel*†?
Departments of *Medicine,†Microbiology and Immunology,‡Anatomy, and§Biochemistry; and
?Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143
Submitted August 19, 2004; Revised February 23, 2005; Accepted February 28, 2005
Monitoring Editor: Jennifer Lippincott-Schwartz
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)P3accumulated 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.
Pseudomonas aeruginosa is one of the leading causes of nos-
ocomial infections in humans (reviewed in Engel, 2003). This
Gram negative opportunistic pathogen causes acute infec-
tions of the respiratory and urinary tract, skin, and eye in the
setting of preexisting epithelial tissue damage and/or host
immunocompromise. P. aeruginosa is also a cause of chronic
lung infections and ultimately death in patients with cystic
Although usually considered an extracellular pathogen,
?50% of clinical, laboratory, and environmental P. aerugi-
nosa isolates demonstrate measurable internalization in vivo
as well as in vitro (Chi et al., 1991; Fleiszig et al., 1994, 1995,
1997b, 1998; Hirakata et al., 1998; Grassme ´ et al., 2000). These
two different phenotypes correlate with the differences in
type III secreted effectors (reviewed in Engel, 2003). Both
classes of strains are virulent in animal models of P. aerugi-
nosa infection. The noninvasive, cytotoxic strains secrete
ExoU (Hauser et al., 1998), a potent phospholipase (Sato et
al., 2003), and ExoT, a bifunctional enzyme with N-terminal
GAP activity toward Rho family GTPases (Krall et al., 2000;
Kazmierczak and Engel, 2002) and C-terminal ADP ribosyl-
transferase (ADPRT) activity toward Crk (Sun and Barbieri,
2003). Both domains of ExoT contribute to its anti-internal-
ization activity (Garrity-Ryan et al., 2000; Garrity-Ryan et al.,
2004). The invasive strains are much less cytotoxic due to the
loss of the ExoU gene (Allewelt et al., 2000). Interestingly,
these strains secrete ExoT and a closely related protein ExoS
that also possesses an N-terminal GAP domain whose sub-
strates include Rho family GTPases (Goehring et al., 1999;
Pederson et al., 1999) and a C-terminal ADPRT domain
whose targets include Ras, Ral, Rabs, and Rho family
GTPases (Bette-Bobillo et al., 1998; Ganesan et al., 1999; Riese
et al., 2001; Fraylick et al., 2002). Why this class of strains is
able to enter into nonphagocytic cells in the presence of two
potential anti-internalization factors is still enigmatic. This
contradictory phenotype can be partly explained by rela-
tively inefficient translocation of ExoS and ExoT into host
cells and the increased ability of these strains to survive
intracellularly (Ha and Jin, 2001).
Much remains to be learned about the mechanism and
role of P. aeruginosa invasion into epithelial cells. It has been
suggested that invasion may permit the bacteria to penetrate
the epithelial cell layer to reach the bloodstream and dis-
seminate to distant organs or to escape recognition by the
host immune system. Bacterial invasion also may benefit the
host, as seen in respiratory cell shedding of infected cells
(Pier et al., 1997). Likewise, the involvement of host signal
transduction pathways in P. aeruginosa internalization is
poorly understood. Several host cell receptors for P. aerugi-
nosa internalization have been suggested, including aGM1
(de Bentzmann et al., 1996), fibronectin and the integrin ?5?1
(Roger et al., 1999), and the cystic fibrosis transmembrane
regulator (Pier et al., 1997). It has been shown that invasion
results in tyrosine phosphorylation of several host proteins,
including caveolin (Zaas et al., 2005) and may involve src-
family tyrosine kinases (Evans et al., 1998; Esen et al., 2001).
One candidate host phosphoprotein is phosphoinositide
3-kinase (PI3K) (Esen et al., 2001)
PI3Ks are a highly conserved subfamily of lipid kinases that
catalyze the addition of a phosphate molecule specifically to
This article was published online ahead of print in MBC in Press
on March 16, 2005.
Address correspondence to: J. N. Engel (Jengel@medicine.ucsf.edu).
© 2005 by The American Society for Cell Biology 2577
the 3-position of the inositol ring of phosphoinositides to gen-
phatidylinositol-3,4-bisphosphate [PtdIns(3,4)P2], and phos-
phatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] (reviewed
in Vanhaesebroeck and Alessi, 2000). These short-lived phos-
pholipids modulate the actin cytoskeleton and function as scaf-
folds to which specific effectors that regulate membranes are
recruited. Modification of phosphoinositides by kinases and
phosphatases permits their precise temporal and spatial con-
For class IA PI3Ks, activation and subsequent phosphor-
ylation of membrane tyrosine kinase receptors recruit the
p85 regulatory subunit of PI3K to the membrane by binding
to its Src homology (SH)2 domain. On translocation to the
membrane, the p110 catalytic subunit of PI3K is activated,
leading to increased levels of 3-phosphoinositides, which
recruit effector proteins to the plasma membrane by binding
to a pleckstrin homology (PH) domain. PI3Ks modulate
many cytoskeleton-based cellular processes, including adhe-
sion, spreading, macropinocytosis, and phagocytosis. PI3K
has been shown to be necessary for the invasion of epithelial
cells by several bacteria, including Listeria monocytogenes
(Ireton et al., 1996), Helicobacter pylori (Kwok et al., 2002), and
Escherichia coli K1 (Reddy et al., 2000).
Both PtdIns(3,4)P2and PtdIns(3,4,5)P3have been shown
to activate one of the main downstream targets of PI3K, the
serine threonine protein kinase B (PKB), also known as Akt
(Burgering and Coffer, 1995; Vanhaesebroeck and Alessi,
2000). On binding to phosphoinositides by its PH domain,
Akt is recruited to the membrane where it is phosphorylated
by PDK1 at threonine 473 and at serine 308, leading to
activation of its kinase activity. The PI3K/Akt signaling
pathway is involved in diverse processes such as vesicular
trafficking, mitogenesis, and cell survival (Coffer et al., 1998).
Although Akt has shown to be activated during the entry of
several bacterial pathogens, in no case has it been shown to
be required (Ireton et al., 1996; Steele-Mortimer et al., 2000;
Coombes and Mahony, 2002; Martinez and Hultgren, 2002).
In the present work, we have addressed the involvement
of PI3K and PKB/Akt in the entry of the invasive ExoS and
ExoT producing P. aeruginosa strain PAK. Using comprehen-
sive strategies, we demonstrate that PI3K and Akt are critical
for PAK entry into nonphagocytic cells. To the best of our
knowledge, this is the first example of a bacterial pathogen
that requires Akt for entry.
MATERIALS AND METHODS
Binding and Internalization Assays
Madin-Darby canine kidney (MDCK) cells (1 ? 106cells/well; clone II ob-
tained from Dr. Keith Mostov, University of California, San Francisco, San
Francisco, CA) were cultured in minimal essential medium (MEM) containing
5% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) in six-well culture
plates or on 12-mm Transwell filters (0.4-?m pore size; Corning Glassworks,
Corning, NY) and incubated for 24 h (day 1 MDCK cell monolayers) (unless
otherwise indicated) at 37°C with 5% CO2. P. aeruginosa strain PAK (obtained
from J. Mattick, University of Queensland, Brisbane, Australia) was routinely
grown shaking overnight in Luria-Bertani broth at 37°C. These stationary
phase bacteria were diluted in MEM-lite (Hauser et al., 1998) and added to the
MDCK cells at a multiplicity of infection (MOI) of 30 unless otherwise
indicated. Adhesion and internalization assays were performed as described
previously (Kazmierczak et al., 2001).
MDCK cells (4 ? 106cells) were seeded onto 10-cm plates for 24 h. The cells
were washed and placed in serum-free MEM for ?17 h. Stationary phase
grown PAK were added for 1 h unless otherwise indicated. The infected
monolayers were washed with cold phosphate-buffered saline (PBS) contain-
ing 1 mM sodium orthovanadate (Sigma-Aldrich, St. Louis, MO). Cells were
lysed in modified radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris,
pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 0.5%
deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 50 mM NaF, 0.1 mM
okadaic acid (Sigma-Aldrich), 1 mM phenylmethylsulfonyl fluoride (Sigma-
Aldrich), and proteinase inhibitor tablets (Complete; Roche Diagnostics, In-
dianapolis, IN) for 20 min. The cell lysates were centrifuged at 16,000 ? g for
20 min. To preclear the cell lysate the supernatant was mixed with 20 ?l of
protein G-Sepharose (4 Fast Flow; Amersham Biosciences, Piscataway, NJ),
and the protein content was determined using protein assay reagent (bicin-
choninic acid; Pierce Chemical, Rockford, IL). The cleared lysate (300–400 ?g
of protein) was incubated with Akt antibody (Cell Signaling Technology,
Beverly, MA) overnight at 4°C and incubated for 1 h with protein G-Sepha-
rose. The immune complexes were washed three times with modified RIPA
buffer without deoxycholate, eluted in SDS sample buffer, electrophoresed on
10% SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride
membranes. The membranes were blocked with PBS containing 0.05% Tween
20 (PBST) and 5% nonfat milk for 1 h at room temperature and then incubated
overnight at 4°C with an antibody that recognizes Akt phosphorylated on
serine 473 (Cell Signaling Technology). The membranes were washed with
PBST and incubated with horseradish peroxidase-conjugated secondary an-
tibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at
room temperature and developed using a enhanced chemiluminescence kit
(Amersham Biosciences). Membranes were then stripped and reprobed with
an antibody that recognizes all forms of Akt (Cell Signaling Technology).
Primary antibodies were diluted 1/1000 and secondary antibodies 1/3000.
MDCK (1 ? 106cells/well) and HeLa cells (3 ? 105cells/well) were grown in
six-well plates in MEM supplemented with 5 or 10% FBS, respectively, for
24 h. Drug treatments were carried out in serum-free medium. Unless other-
wise indicated, cells were preincubated for 1 h with MEM containing
LY294002 (LY) (Sigma-Aldrich) or wortmannin (Sigma-Aldrich) or for 2 h
with MEM containing the Akt inhibitor SH-5 (Calbiochem, San Diego, CA).
Adhesion and invasion assays were performed as detailed above.
Small Interfering RNA (siRNA)-mediated Akt Depletion
Akt and control siRNA were purchased from Cell Signaling Technology.
HeLa cells grown in 10-cm dishes to 50% confluence were transfected with
100 nM siRNA according to the manufacturer’s instructions. Forty-eight
hours after transfection, the standard adhesion and invasion assays were
performed. In parallel, lysates were immunoblotted with Akt antibody to
evaluate the efficiency of siRNA-mediated protein depletion.
PtdIns(3,4,5)P3was added to the cells via a shuttle system (Echelon, San Jose,
CA). Long-chain (Di-C16) synthetic phosphoinositides were freshly prepared
as a complex with histone (Weiner et al., 2002) and added to the apical domain
of the monolayer of cells for 5 min. The lipid was removed by washing, and
the cells were immediately infected with PAK. Standard adhesion and inva-
sion assays were performed.
MDCK cells (1 ? 106cells/transwell) stably transfected with the pleckstrin
homology domain of Akt fused to green fluorescent protein (PH-Akt-GFP), a
probe for PtdIns(3,4,5)P3(Yu et al., 2003), were cultured on Transwell filters
for 24 h. PAK was incubated for 5 min at 37°C with 20 ?M Syto 59 (Molecular
Probes, Eugene, OR), a red fluorescent stain that stains nucleic acids, and then
washed and resuspended in MEM. The presence of this dye minimally
affected invasion (our unpublished data). Syto 59-labeled bacteria were incu-
bated with the MDCK cells (MOI of 500) for 30 min, washed three times with
PBS, and fixed with 4% paraformaldehyde for 30 min at room temperature.
Samples were examined with a Zeiss 510 LSM confocal microscope or with a
Nikon TE2000 inverted microscope. Images were collected as TIFF files and
analyzed with Adobe PhotoShop.
The PI3K Pathway Is Necessary for PAK Internalization
into Epithelial Cells
To address the possibility that the PI3K signaling pathway is
involved in the internalization of P. aeruginosa strain PAK,
we assayed the effect of two structurally unrelated cell-
permeable, low-molecular-weight inhibitors, LY and wort-
mannin, on bacterial entry into epithelial cells. These drugs
inhibit PI3K activity by different mechanisms (Vlahos et al.,
1994; Ui et al., 1995).
Confluent MDCK cells grown for 24 h (day 1 MDCK cell
monolayers) were pretreated either with MEM containing
A. Kierbel et al.
Molecular Biology of the Cell2578
Weiner, O. D., Neilsen, P. O., Prestwich, G. D., Kirschner, M. W., Cantley, L. C.,
and Bourne, H. R. (2002). A PtdInsP(3)- and Rho GTPase-mediated positive
feedback loop regulates neutrophil polarity. Nat. Cell Biol. 4, 509–513.
Wilkowsky, S. E., Barbieri, M. A., Stahl, P., and Isola, E. L. (2001). Trypano-
soma cruzi: phosphatidylinositol 3-kinase and protein kinase B activation is
associated with parasite invasion. Exp. Cell Res. 264, 211–218.
Yamaguchi, T., and Yamada, H. (1991). Role of mechanical injury on airway
surface in the pathogenesis of Pseudomonas aeruginosa. Am. Rev. Respir. Dis.
Yu, W., O’Brien, L. E., Wang, F., Bourne, H., Mostov, K. E., and Zegers, M. M.
(2003). Hepatocyte growth factor switches orientation of polarity and mode of
movement during morphogenesis of multicellular epithelial structures. Mol.
Biol. Cell 14, 748–763.
Zaas, D., Duncan, M., Li, G., Wright, J. R., and Abraham, S. N. (2005).
Pseudomonas invasion of type I pneumocytes is dependent on the expression
and phosphorylation of caveolin-2. J. Biol. Chem. 280, 4864–4872.
Zahm, J. M., Chevillard, M., and Puchelle, E. (1991). Wound repair of human
surface respiratory epithelium. Am. J. Respir. Cell. Mol. Biol. 5, 242–248.
Zenni, M. K., Giardina, P. C., Harvey, H. A., Shao, J., Ketterer, M. R., Lubaroff,
D. M., Williams, R. D., and Apicella, M. A. (2000). Macropinocytosis as a
mechanism of entry into primary human urethral epithelial cells by Neisseria
gonorrhoeae. Infect. Immun. 68, 1696–1699.
PI3K-Protein Kinase B/Akt Pathway
Vol. 16, May 2005 2585