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
dimethyl sulfoxide (DMSO) (control), MEM containing LY
(2–100 ?M), or MEM containing wortmannin (100 nM) for
1 h. The cells were then cocultivated for 1 h in the presence
of drugs with stationary phase grown PAK, and standard
bacterial adhesion and invasion assays were performed. Fig-
ure 1A shows that LY blocked PAK internalization in a
dose-dependent manner. LY (50 ?M; Figure 1) or 100 nM
wortmannin (our unpublished data) reduced entry to ?30%
compared with control-treated cells, whereas 100 ?M LY
virtually abolished bacterial internalization. The inhibitory
effect on invasion was not due to a decrease in adhesion.
Rather, exposure for 1 h to 50 ?M LY slightly increased
adhesion (Figure 1B), although adhesion was not affected by
shorter treatment times with LY (our unpublished data).
Treatment with these inhibitors also blocked PAK uptake in
HeLa cells (our unpublished data). Incubation of the bacteria
with the drugs alone did not affect bacterial viability (our
unpublished data). Likewise, exposure of the MDCK cells
monolayers to the PI3K inhibitors did not results in cell
death, as assayed by LDH release (our unpublished data).
Together, these results suggest that PI3K activity is required
for PAK internalization into epithelial cells
Apical Addition of a PI3K Product PtdIns(3,4,5)P3
Enhances the Internalization of PAK
PI3K phosphorylates the 3?-OH position of the inositol ring
of phosphatidyl inositol to generate PtdIns3P, PtdIns(3,4)P2,
and PtdIns(3,4,5)P3. PtdIns(4,5)P2is the preferred substrate
of class I PI3Ks. PtdIns(3,4)P2 is then generated from
PtdIns(3,4,5)P3via the action of 5?-inositol phosphatases.
Both PtdIns(3,4)P2and PtdIns(3,4,5)P3have been shown to
activate the downstream effector Akt (Vanhaesebroeck and
Exogenous phosphoinositides complexed to a cationic car-
rier molecule such as histone can be delivered to mamma-
lian cells and have been shown to cause the recruitment of
endogenous PI3K to the host cell membrane. This results in
the activation of a positive feedback loop that is sufficient to
synthesize sufficient new endogenous PtdIns(3,4,5)P3to
elicit physiological responses, including phosphorylation of
Akt (Ozaki et al., 2000; Weiner et al., 2002; our unpublished
data). The apical surface of highly polarized MDCK cells
(such as those plated as confluent monolayers and subse-
quently incubated for 3 d) is known to be devoid of detect-
able PtdIns(3,4,5)P3(Watton and Downward, 1999). We
used this technique (Ozaki et al., 2000) to determine whether
the apical delivery of phosphoinositides was sufficient to
enhance PAK invasion into polarized MDCK cells.
Three-day-old confluent MDCK cells grown as polarized
monolayers were exposed apically to 30 ?M PtdIns(3,4,5)P3
complexed with histone for 5 min. Under these conditions
the PI3K pathway is activated (our unpublished data). The
lipid was removed by washing, the MDCK cells were in-
fected with PAK for 1 h, and standard adhesion and inva-
sion assays were performed. Figure 2 shows that apical
addition of PtdIns(3,4,5)P3stimulated invasion of PAK ap-
proximately twofold above addition of histone alone (A)
without affecting bacterial adhesion (B). Together, these re-
sults suggest that PtdIns(3,4,5)P3is necessary and sufficient
to promote PAK entry into MDCK cells.
Products of PI3K Accumulate at the Site of Bacterial
Binding and Entry
It is now recognized that specific protein domains bind to
phosphoinositides. Fusions of these domains to fluorescent
proteins such as GFP provide a useful tool to visualize the
subcellular localization of specific phosphoinositides (Lem-
mon, 2003). We used the PH domain of Akt fused to GFP
(PH-Akt-GFP) as a spatial probe to examine whether
PtdIns(3,4,5)P3accumulates at the site of bacterial entry
(Servant et al., 2000). Day 1 MDCK cell monolayers express-
ing PH-Akt-GFP, a probe for PtdIns(3,4,5)P3, were coculti-
vated with Syto59-labeled PAK for 30 min. A relatively high
MOI (500) was used to maximize the number of adherent
bacteria. The cells were then fixed, and the samples were
analyzed by confocal and conventional fluorescence micros-
copy. In uninfected cells, the fusion protein localized to the
basolateral surface of MDCK cells (Figure 3E). On apical
infection with PAK, PH-Akt-GFP accumulated at the apical
surface of the cell, at the site where the bacteria bound
(Figure 3, A and C). Interestingly, the bacteria were often
observed to attach as aggregates.
To determine whether the localized accumulation of
PtdIns(3,4,5)P3required PI3K activity, we pretreated the
MDCK cells expressing PH-Akt-GFP with 50 ?M LY for 1 h.
As shown in Figure 3, B and D, LY treatment inhibits this
process. The effect of LY on the accumulation of PH-Akt-
GFP in the vicinity of adherent bacteria was quantified by
conventional fluorescence microscopy. As shown in the
graph in Figure 3G, pretreatment with 50 ?M LY decreased
the frequency of PH-Akt-GFP colocalization with bacteria to
30% of control (p ? 0.01). Exposure to LY did not alter the
ization. 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.
Pharmacological inhibitors of PI3K block PAK internal-
PI3K-Protein Kinase B/Akt Pathway
Vol. 16, May 20052579
localization of the fusion protein in uninfected cells (Figure
3F). We conclude that PAK is able to activate PI3K and
recruit PtdIns(3,4,5)P3to the site of entry.
The PI3K/Akt Pathway Is Activated upon Infection with
An important target of activated PI3K is the serine and
threonine kinase Akt. Activation of PI3K by growth factors
results in stimulation of Akt kinase activity, which correlates
with phosphorylation of serine 473 and threonine 308 (Van-
haesebroeck and Alessi, 2000). Because activation of PI3K
and subsequent phosphorylation of Akt has been implicated
in the entry of a subset of intracellular pathogens (Reddy et
al., 2000; Coombes and Mahony, 2002), the phosphorylation
status of Akt upon PAK infection was addressed. Day 1
MDCK cell monolayers were infected with PAK (MOI of
200) for 60 min in serum-free media. As a positive control,
MDCK cells were exposed to epidermal growth factor (EGF)
(10 ng/ml) for 10 min. Total Akt was immunoprecipitated
from soluble whole cell lysates and sequentially immuno-
blotted with a monoclonal anti-phospho AktSer473antibody
followed by a monoclonal antibody recognizing total Akt.
As expected, EGF treatment robustly increased the amount
of phosphorylated Akt (Figure 4A). Phosphorylation of Akt
also was strongly increased upon infection with PAK in both
a dose-dependent (Figure 4B) and time-dependent (Figure
4C) manner. Maximal phosphorylation was observed at 1 h
mote bacterial invasion through the apical surface. PIP(3,4,5)P3com-
plexed with histone (30 ?M) 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 exper-
iments, each performed in triplicate. Error bars indicate SEM. *p ?
0.001 compared with the control cells, Student’s two-tailed t test.
Exogenous addition of PIP(3,4,5)P3is sufficient to pro-
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 ab-
sence (A) or presence (B) 50 ?M LY. Bar, 1 ?m. C–F, confocal (X-Z
sections) of infected (C and D) or uninfected cells (E and F). Bar, 10
?m. In uninfected cells (E), PtdIns(3,4,5)P3is localized to the baso-
lateral membrane. In the presence of apically applied PAK,
PtdIns(3,4,5)P3accumulates at the apical site of bacteria binding (A,
apical view; C, X-Z section). Treatment with 50 ?M 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.
PH-Akt-GFP, a probe for PtdIns(3,4,5)P3, accumulates at
A. Kierbel et al.
Molecular Biology of the Cell 2580
postinfection (Figure 4C), which correlates well with the
kinetics of PAK internalization. Activation of Akt by PAK
was completely inhibited by pretreatment with 100 ?M LY
(Figure 4B). Lower concentrations of LY (50 ?M) incom-
pletely blocked invasion (Figure 1A) and Akt phosphoryla-
tion (our unpublished data). These results demonstrate that
PAK induction of Akt phosphorylation was dependent upon
Akt Activation Mediates PAK Entry
We used pharmacological inhibitors and siRNA-mediated
protein depletion to determine whether Akt activation is
necessary for PAK entry. Day 1 MDCK monolayers were
pretreated for 2 h with 10 ?M SH-5, a specific inhibitor of the
serine/threonine kinase activity of Akt (Kozikowski et al.,
2003), and then incubated with PAK in the presence of the
drug. Standard adhesion and internalization assays were
performed. Figure 5 demonstrates that SH-5 decreased bac-
terial internalization almost fivefold without affecting adher-
ence. The inhibitor did not affect bacterial or host cell via-
bility (our unpublished data).
Because pharmacological inhibitors can have nonspecific
effects, we confirmed these results by determining the effect
of Akt depletion on PAK invasion. These experiments were
performed in HeLa cells because siRNA can be efficiently
introduced into these cells by transfection. Because Akt has
known antiapoptotic effects, we first established that trans-
fection of HeLa cells with a control or Akt siRNA did not
affect cell viability, as determined by LDH release, and cell
number, as measured by direct cell counting (our unpub-
lished data). Immunoblot analysis of lysates from HeLa cells
transfected with a control siRNA or the Akt siRNA for 48 h
demonstrated that phosphorylated Akt and total Akt pro-
tein levels were diminished approximately fourfold (Figure
6A). Parallel samples transfected with siRNA (Akt or con-
trol) were cocultivated with PAK for 1 h and standard
bacterial adhesion and invasion assays were performed (Fig-
ure 6B). Partial depletion of Akt diminished PAK entry into
HeLa cells by 40% without having an effect on bacterial
adherence. Although there have been numerous examples of
microorganism entry that requires PI3K (Steele-Mortimer et
al., 2000; Coombes and Mahony, 2002), to the best of our
knowledge, this is the first time that Akt has been shown to
mediate internalization of bacteria.
Akt Is Sufficient to Restore Invasion in the Absence PI3K
To determine whether Akt is sufficient to promote invasion
of PAK, we used an MDCK cell line that expresses a consti-
tutively active (CA) allele of Akt that lacks the pleckstrin
homology domain and whose phosphorylation sites (serine
473 and threonine 308) have been changed to aspartic acid
(Stokoe et al., 1997). Figure 7 shows the PAK adhesion and
invasion assays performed with MDCK cells stably trans-
fected with CA Akt. In the absence of LY, invasion was not
augmented by expression of CA Akt. However, expression
of CA Akt could overcome the LY-mediated inhibition of
invasion. Together, these experiments suggest that CA Akt
is sufficient to allow efficient PAK internalization in the
presence of a PI3K inhibitor.
Microbial pathogens have developed diverse strategies to
adapt and survive within their specific hosts. Intracellular
pathogens commonly hijack host signaling pathways for
invasion, intracellular motility, and intracellular replication
and survival. In all cases, the infection process requires a
MDCK cells were infected with PAK; at the indicated times postin-
fection, 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 phos-
phorylated 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.
Akt is phosphorylated upon infection with PAK. Day 1
uptake. Day 1 MDCK cells were treated for 2 h with SH-5 (10 ?M),
an inhibitor that specifically blocks AKT phosphorylation, and stan-
dard adhesion (open bars) and internalization assays (closed bars)
were performed. Results are representative of at least three inde-
pendent experiments, each performed in triplicate. Error bars indi-
cate SEM. *p ? 0.001 compared with untreated cells, Student’s
two-tailed t test.
SH-5, a selective inhibitor of AKT activation, blocks PAK
PI3K-Protein Kinase B/Akt Pathway
Vol. 16, May 20052581
precise subversion of the cellular machinery to establish a
replicative or survival niche. Although microbial exploi-
tation of the host cytoskeleton and associated proteins has
been well described, we are just beginning to appreciate
that lipids and lipid metabolism also are targeted by
pathogenic organisms (reviewed in Pizarro-Cerda and
Cossart, 2004). In particular, phosphoinositides, short-
lived lipids whose production at specific membrane loca-
tions in the cell enables the tightly controlled recruitment
or activation of diverse cellular effectors involved in cell
motility or phagocytosis, are modulated by microorgan-
isms. Modification of phosphoinositides by kinases and
phosphatases allows the precise control of their temporal
and spatial distribution.
The carefully orchestrated sequential localized synthesis
of phosphoinositides is particularly well established for
phagocytosis. PtdIns(4,5)P2and the enzyme required for its
synthesis accumulates in pseudopods during extension of
the phagocytic cup and coincides with actin enrichment
(Botelho et al., 2000), suggesting that localized production of
PtdIns(4,5)P2is required for the phagocytic process. As the
phagosome seals, PtdIns(4,5)P2and actin disappear, and
PtdIns(3,4,5)P5accumulates locally, only to disappear con-
comitantly with the sealing of the phagosomal vacuole (Cox
et al., 1999; Marshall et al., 2001).
Modulation of host phosphoinositides and activation of
Akt are increasingly appreciated in microbial entry. For
example, InlA- and InlB-mediated entry of Listeria mono-
cytogenes (Ireton et al., 1996), FimH-mediated entry of
uropathogenic E. coli (Martinez et al., 2000), and the entry
of C. pneumoniae (Coombes and Mahony, 2002) require
PI3K and result in the activation of Akt. Enteropathogenic
E. coli are able to avoid uptake into cultured macrophages
by inhibiting the activation of PI3K and Akt (Celli et al.,
2001). Through the action of SigD, an inositol phospha-
tase, Salmonella typhimurium entry activates PI3K and Akt,
although engagement of this pathway is not required for
its entry (Steele-Mortimer et al., 2000). However, in none
of these cases has activation of Akt been shown to be
necessary for entry. Interestingly, both PI3K and Akt are
necessary for the entry of the protozoan parasite Trypano-
soma cruzi into nonphagocytic and phagocytic cells
(Wilkowsky et al., 2001).
In this work, we have investigated the entry pathway of
P. aeruginosa strain PAK and now demonstrate that for
postexponential phase grown bacteria, activation of PI3K
and Akt is both necessary and sufficient for bacterial entry
into cultured epithelial cells. Pharmacological inhibitors
of PI3K and Akt, as well as depletion of Akt, blocked
invasion. The effect of Akt depletion on bacterial entry
was only partial; this finding could be explained by the
persistence of residual Akt activity or by the requirement
for other downstream targets of PI3K in PAK uptake.
Addition of PtdIns(3,4,5)P3complexed to a cationic carrier
to polarized MDCK cells was sufficient to augment PAK
internalization. Accumulation of PtdIns(3,4,5)P3, nor-
mally found at the basolateral surface, at the site of api-
cally entering bacterial was observed, suggesting that P.
aerguinosa entry involves recruitment, either by transloca-
tion from the basolateral surface and/or by new synthe-
sis, of PtdIns(3,4,5)P3. Because LY blocked both the accu-
mulation of PtdIns(3,4,5)P3and bacterial entry, these
results suggest that PAK-mediated activation of PI3K and
subsequent localized accumulation of PtdIns(3,4,5)P3is
necessary for bacterial invasion. Expression of CA Akt in
the presence of a PI3K inhibitor was sufficient to restore
bacterial entry. To the best of our knowledge, this is the
first time that Akt has been shown to mediate internaliza-
tion of a bacterium.
It is interesting that addition of PtdIns(3,4,5)P3only aug-
mented PAK entry into highly polarized MDCK cells but not
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. Glycer-
aldehyde-3-phosphate dehydrogenase (GAPDH) is used as loading
control. (B) Standard adhesion (open bars) and internalization as-
says (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, Stu-
dent’s two-tailed t test.
siRNA-mediated AKT depletion abrogates PAK uptake.
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 ?M LY. Shown are the
combined results from five independent experiments, each per-
formed 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
Akt is sufficient to restore invasion in the absence of PI3K
A. Kierbel et al.
Molecular Biology of the Cell2582
into incompletely polarized confluent monolayers (our un-
published data). Although the increase in PAK internaliza-
tion was modest, it is remarkable that any effect was seen,
given that the exogenous phosphoinositides were present
only for a brief period. There are numerous suggestions in
the literature that P. aeruginosa entry into cultured epithelial
cells occurs preferentially at the basolateral surface in polar-
ized monolayers or is enhanced in less well polarized or
wounded epithelium (Yamaguchi and Yamada, 1991; Zahm
et al., 1991; Tsang et al., 1994; de Bentzmann et al., 1996;
Fleiszig et al., 1997a, 1998; Kazmierczak et al., 2004). It is
possible that apical localization PtdIns(3,4,5)P3in incom-
pletely polarized cells accounts for their increased suscepti-
bility to P. aeruginosa invasion; we are currently investigat-
ing the molecular basis for
PtdIns(3,4,5)P3on highly polarized versus incompletely po-
We note that the most of the bacteria were bound as
aggregates; these clusters of bacteria were commonly asso-
ciated with PtdIns(3,4,5)P3. These bacterial aggregates may
represent pack swarming, a bacterial chemotaxic response to
host cell factors released upon damage by the bacterial type
III secretion apparatus (Dacheux et al., 2001).
How might activation of the PI3K and Akt pathway
lead to PAK internalization? Both PI3K and Akt have been
shown to modify the cytoskeleton dynamics, to be in-
volved in the regulation of membrane traffic, and to have
direct roles in macropinocytosis, a pathway used by some
pathogenic bacteria for cellular entry (Ojcius et al., 1998;
Zenni et al., 2000). The regulatory subunit P85 of PI3K
contains an amino-terminal SH3 domain and two SH2
domains that allow the p85 subunit to recruit multiple
intracellular signaling molecules at the same time, possi-
bly to the site of bacterial entry. Several proteins involved
in modulating the actin cytoskeleton have PH domains,
such as guanine exchange factors for Rho family GTPases.
The downstream targets of Akt are numerous and affect
diverse host cell processes including cell survival, tran-
scription, metabolic, and host defense pathways (re-
viewed in Vanhaesebroeck and Alessi, 2000). Of interest,
Akt also has been shown to stimulate Rab5-dependent
endocytosis. The identification of other PI3K effectors and
the specific targets of Akt involved in PAK internalization
are under study.
One possible category of PI3K effectors is the Rho fam-
ily GTPases, although the relationship of Rho family GT-
Pases to PI3K and Akt activation is complex. Ras and Rho
family GTPases seem to act both upstream and down-
stream of PI3K and Akt (Nobes and Hall, 1995; Ren and
Schwartz, 1998; Weiner et al., 2002). For entry of strain
PA103, we have previously shown a requirement for Rho
family GTPases that is antagonized by the type III trans-
located effector ExoT (Kazmierczak et al., 2001; Kazmierc-
zak et al., 2004); we are currently investigating the role of
Rho family GTPases in PAK entry. Interestingly, Exo S, a
type III secretion-dependent effector translocated into
host cells by invasive P. aeruginosa strains, is able to
inactivate Ras via ADP ribosylation (Ganesan et al., 1998).
ExoS also has been shown to abrogate EGF-mediated
activation of Akt- (Henriksson et al., 2000) and Rab5-
mediated endocytosis (Barbieri et al., 2001). It will be
interesting to determine whether PAK stimulates Rab5-
In summary, we provide the first example of a bacterial
pathogen that exploits and requires the PI3K and Akt path-
way for internalization. Future work will be directed to
determining how the bacteria activate this pathway and
what downstream targets of Akt are involved.
We thank Drs. David Stokoe and Emma Shtivelman and members of the
Engel and Mostov laboratories for advice and encouragement. We thank Dr.
Emma Shtivelman for the cell lines expressing constitutively active Akt and
Dr. Orion Weiner for help with the PtdIns(3,4,5)P3lipid transfer experiments.
This work was supported by National Institutes of Health grants HL-55980 (to
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