Cellular Microbiology (2007) 9(1), 84–96
doi:10.1111/j.1462-5822.2006.00769.x
First published online 2 August 2006
Leishmania promastigotes activate PI3K/Akt signalling
to confer host cell resistance to apoptosis
Aaron Ruhland, Nicole Leal and Peter E. Kima*
Department of Microbiology and Cell Science, University
of Florida, Gainesville, FL 326111, USA.
Summary
Previous reports have shown that cells infected with
promastigotes of some Leishmania species are
resistant to the induction of apoptosis. This would
suggest that either parasites elaborate factors that
block signalling from apoptosis inducers or that
parasites engage endogenous host signalling pathways that block apoptosis. To investigate the latter
scenario, we determined whether Leishmania infection results in the activation of signalling pathways
that have been shown to mediate resistance to apoptosis in other infection models. First, we showed
that infection with the promastigote form of Leishmania major, Leishmania pifanoi and Leishmania
amazonensis activates signalling through p38
mitogen-activated protein kinase (MAPK), NFkB and
PI3K/Akt. Then we found that inhibition of signalling
through the PI3K/Akt pathway with LY294002 and
Akt IV inhibitor reversed resistance of infected bone
marrow-derived macrophages and RAW 264.7 macrophages to potent inducers of apoptosis. Moreover,
reduction of Akt levels with small interfering RNAs
to Akt resulted in the inability of infected macrophages to resist apoptosis. Further evidence of the
role of PI3K/Akt signalling in the promotion of cell
survival by infected cells was obtained with the
finding that Bad, which is a substrate of Akt,
becomes phosphorylated during the course of infection. In contrast to the observations with PI3K/Akt
signalling, inhibition of p38 MAPK signalling with
SB202190 or NFkB signalling with wedelolactone
had limited effect on parasite-induced resistance to
apoptosis. We conclude that Leishmania promastigotes engage PI3K/Akt signalling, which confers to
the infected cell, the capacity to resist death from
activators of apoptosis.
Received 1 March, 2006; revised 29 May, 2006; accepted 8 June,
2006. *For correspondence. E-mail [email protected]; Tel.
(+1) 352 392 0384; Fax (+1) 352 392 5922.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
Introduction
One strategy that intracellular pathogens employ to
ensure survival of their host cell is the prevention of apoptosis or programmed cell death, which can be induced by
a wide range of stimuli (Gao and Abu Kwaik, 2000; Heussler et al., 2001a). A few reports have shown that infection
of macrophages with Leishmania parasites confers on
macrophages the capacity to resist apoptosis induced by
diverse stimuli (Moore and Matlashewski, 1994; Aga
et al., 2002; Akarid et al., 2004; Lisi et al., 2005). Infection
with Leishmania major promastigotes blocks the release
of apoptosis effectors from the mitochondria, when
bone marrow-derived macrophages (BMDMs) are either
treated with staurosporine or when they are deprived of
macrophage colony stimulating factor (Akarid et al.,
2004). Macrophages from both susceptible (BALB/c) and
resistant (C57BL/6) mice were shown to be equally resistant to apoptosis after infection with these parasites,
which suggested that this phenomenon is not dependent
on the genetic background of the host. Infection with
another Leishmania species, Leishmania donovani, also
prevents apoptosis of macrophages that are deprived of
macrophage colony stimulating factor (Moore and Matlashewski, 1994). This raises the likelihood that Leishmania employ a common mechanism to block apoptosis of
their infected cell. However, the mechanism(s) by which
Leishmania parasites achieve this inhibition of apoptosis
is not known. One report suggested that because infection with L. donovani promastigotes induces macrophages to release cytokines, one of these cytokines, most
likely tumour necrosis factor (TNFa), might act in an autocrine manner to confer the antiapoptotic effect observed
(Moore and Matlashewski, 1994). But a separate study
with L. major did not confirm such a role for TNF (Akarid
et al., 2004). Further, TNF has most often been found to
have a proapoptotic effect (Ashkenazi and Dixit, 1998).
In recent years, studies on cell signalling have shown
that engaging some signalling pathways can result in the
prolongation of cell survival. Some of these pathways
include signalling through phosphatidyl inositol-3-kinase
(PI3K) and the downstream kinase, protein kinase B
(PKB) also known as Akt (Fresno Vara et al., 2004); signalling that activates the transcription factor NFkB
(Shishodia and Aggarwal, 2002); and signalling through
the mitogen-activated protein kinases (MAPK) (Park
Leishmania activate the PI3K/Akt pathway 85
et al., 2002; Zhang et al., 2005). Some of these signalling
pathways have been shown to be engaged during Leishmania infections. However, the primary focus of most of
those studies was the analysis of the overall host
response in experimental infections. For example, NFkB
signalling was shown to play a role in the generation of a
protective response against Leishmania (Mason et al.,
2004). In those studies, the role of NFkB was found to
affect T cell maturation and effector cell generation. Similarly, using p85–/– mice, a role for PI3K signalling during an
L. major infection was shown (Fukao et al., 2002). That
study focused on defective production of IL-12 in the
absence of PI3K signalling in dendritic cells. Could the
engagement of any of these signalling pathways by Leishmania parasites account for the observation that infected
macrophages resist apoptosis?
Leishmania are dimorphic organisms that preferentially
infect macrophages in their mammalian hosts. Natural
infections are initiated by the promastigote form, which
transform into amastigotes within macrophages over a
period of 24 to 72 h. Depending on the Leishmania
species and the characteristics of the host, infection with
Leishmania results in lesions either at cutaneous sites or
in visceral organs. Inflammatory cells are recruited to
these lesions where chemokines and cytokines have
been detected (Soong et al., 1996, Moll, 1997). The fact
that parasitized cells establish themselves and persist in
this rich milieu of immunologic activity suggests that there
are parasite-controlled mechanisms that prevent both
parasites and infected cells from being killed.
In the studies described here, we evaluate which of
three signalling pathways, p38 MAPK, NFkB or PI3K is
engaged by Leishmania promastigotes to block apoptosis
of their host cells. First we determined whether infection of
macrophages with promastigotes of L. major or promastigotes of the Leishmania mexicana complex engage signalling through these pathways during infection. As all
three signalling pathways were found to be engaged, we
then determined using chemical inhibitors, which of these
pathways is critical for parasite-induced protection from
apoptosis. The observation that blocking of signalling
through the PI3K/Akt pathway reversed the antiapoptotic
effect of these parasites was confirmed in studies using
small inhibitory RNAs (siRNAs) to reduce Akt levels in the
infected cells.
Results
Leishmania major, L. mexicana pifanoi and Leishmania
amazonensis promastigotes block apoptosis of
macrophages induced with campothecin
Two major pathways of apoptosis have been described
(Danial and Korsmeyer, 2004). These are: (i) the
mitochondrial-dependent pathway, also called the intrinsic
pathway, which is triggered by DNA damage or stress and
results in the release of cytochrome c from the mitochondria, and (ii) the extrinsic pathway, which is dependent on
the engagement of members of the TNF family of receptors called death receptors. These pathways converge at
the level of caspases, which are cytosolic proenzymes that
are activated in a cascade to eventually carry out DNA
fragmentation. Recent evidence has shown that there is
cross-talk between these pathways and that mitochondria
are most likely involved in both pathways (Li et al., 1998).
Since it had previously been shown that Leishmania promastigotes prevent apoptosis by inducers of the mitochondrial pathway (Akarid et al., 2004), we elected to induce
apoptosis in these studies with campothecin or actinomycin D, which are known inducers of the mitochondrial
pathway. Experiments with these compounds produced
comparable results and so results from both inducers are
shown. Apoptosis was assessed by changes in caspase 3
activation and by assessment of DNA fragmentation after
labelling for terminal deoxynucleotidase (TDT). As Fig. 1A
shows, treatment of RAW 264.7 macrophages with 2 mM
campothecin resulted in significant activation of caspase 3
as compared to the level of caspase 3 that was found in
resting cells and in cells treated with vehicle (DMSO). In
contrast, there was limited activation of caspase 3 in
macrophages that were first infected for 4 h with promastigotes of L. major, L. pifanoi or L. amazonensis before
incubation with campothecin. Experiments using actinomycin D yielded identical results (not shown). Both longer
and shorter infection times prior to apoptosis induction
were also assessed. As Fig. 1A shows that cells infected
for 12 and 24 h were also significantly resistant to apoptosis induction. However, infected cells became progressively less resistant to apoptosis with the duration of the
infection. This was interpreted to imply that the signalling
events that mediate this phenomenon are more pronounced at earlier times after infection. Similar observations were made with the B10R macrophage line (data not
shown).
For another measure of apoptosis, DNA fragmentation
as a result of apoptosis induction was determined by
TUNEL using bromolated deoxyuridine triphosphate
nucleotide (Br-dUTP) labelling of DNA breaks in the presence of terminal deoxynucleotide transferase (TdT). In
this method, cells with DNA breaks incorporate Br-dUTP,
which is detected by fluorescence-activated cell (FACS)
analysis following reactivity with an anti-BrdU monoclonal
antibody (see Experimental procedures). As Fig. 1B
shows, 71% of cells that were induced to undergo apoptosis by incubation with campothecin incorporated
Br-UTP and fluoresced well above background, which is
an indication that they were indeed undergoing apoptosis.
However, when macrophages were infected for 4 h with
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 9, 84–96
86
A
B
A. Ruhland, N. Leal and P. E. Kima
Fig. 1. Infection with Leishmania
promastigotes confers resistance to apoptosis
induced by campothecin or actinomycin D.
RAW 264.7 macrophages were infected with
Leishmania promastigotes at a multiplicity of
infection (moi) of 10:1 as described in
Experimental procedures. After 4 h free
parasites were washed off. At that time, some
cultures were treated with 2 mM campothecin
while other cultures were treated with
campothecin after 12 or 24 post infection.
After an additional 7 h from the time of
campothecin treatment, either cell lysates
were prepared and assayed for caspase 3
activity (A) or cells were harvested and DNA
fragmentation was assayed by TUNEL
analysis (B). For TUNEL analysis, the marker
for histogram analysis was set to exclude
background FITC fluorescence measured
from resting macrophages. Cells that
fluoresced above this threshold channel
number were considered apoptotic. Vehicle
controls received DMSO alone. The results of
the caspase assays are the compilation of at
least three experiments and significance was
determined by Students t-test (P < 0.05). The
histograms from TUNEL assays are typical of
three similar experiments.
C. RAW 264.7 macrophages were incubated
with L. major promastigotes that had been
treated with 1% paraformaldehyde for 30 min
prior to infection. After 4 h, free parasites
were removed and cells were treated with
actinomycin D for 7 h prior to TUNEL staining.
Results are representative of two similar
experiments.
C
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 9, 84–96
Leishmania activate the PI3K/Akt pathway 87
promastigotes of L. major, L. pifanoi or L. amazonensis
prior to apoptosis induction, there was limited incorporation of Br-UTP in response to treatment with campothecin.
The histograms from infected cells were comparable to
those of control cells that were not induced. It is noteworthy that unlike live parasites, dead parasites did not block
the induction of apoptosis. The histograms in Fig. 1C
show that after induction of apoptosis with actinomycin D,
the proportion of cells with fragmented DNA in cultures
that were incubated with paraformaldehyde (PFA) fixed
parasites (Fig. 1C, panel D), was almost identical to that
found in uninfected cells cultured with actinomycin
(Fig. 1C, panel B). In contrast, in cultures incubated with
live parasites before actinomycin treatment, there was
minimal evidence of DNA fragmentation (Fig. 1C, panel
C). This would indicate that resistance to apoptosis is an
active process that requires the presence of live
parasites. Once again, similar observations were made
with B10R cells (not shown). Taken together, using two
methods to evaluate apoptosis in two macrophage lines,
we found that promastigotes of L. major as well as promastigotes of members of the L. mexicana complex
blocked apoptosis of macrophages in response to potent
inducers of apoptosis.
Infection with Leishmania promastigotes engages the
PI3-kinase/Akt pathway, MAPK p38 and the NFkB
pathways
To initiate an analysis of the mechanism that underlies
apoptosis resistance, we first determined whether infection with Leishmania promastigotes activates signalling
pathways that have been shown to prolong cell survival in
other systems. Signalling through the PI3K/Akt, p38
MAPK or NFkB pathways were thus evaluated. Evidence
that parasite infection activates the PI3K/Akt pathway was
determined by assessing changes in phosphorylation of
Akt, a downstream serine-threonine kinase of PI3K. We
assessed phosphorylation of Akt at position Ser473, which
has been shown to be necessary for PI3K/Akt signal
transduction (Yang et al., 2002). Macrophages were
infected with Leishmania promastigotes and Akt phosphorylation was assessed at discrete times by Western
blotting. The blots were first probed for phospho-Akt and
then stripped and re-probed for Akt protein. Figure 2A
shows that within 15 min of exposure to parasites, there
was a significant rise in Akt phosphorylation as compared
to the level of this molecule in resting macrophages.
Phospho-Akt was induced in response to infection with
L. major, L. amazonensis and L. pifanoi (not shown). The
figure shows that phosphorylated Akt persists well above
background for 24 h. Infection had no effect on native Akt
levels. Interestingly, as the representative blot and densitometric scan compiled from at least three experiments
show, there is a reproducible increase in phosphorylated
Akt levels at 24 h post infection. To confirm that the phosphorylated Akt observed in these experiments was indeed
the result of signalling through the PI3K/Akt pathway, representative infections with L. major were initiated in the
presence of LY294002, an inhibitor of PI3K signalling. As
Fig. 2B shows, at 30 min post infection, there was appreciable reduction of Akt phosphorylation when infections
were performed in the presence of the PI3K inhibitor, as
compared to Akt levels in infections without inhibition. This
inhibitor also reduces the basal level of phosphorylated
Akt in resting cells. These results show that PI3K/Akt
signalling is initiated after promastigotes contact macrophages and that it is sustained over a period of several
hours. We should note that when included at the time of
infection LY294002 inhibits parasite internalization (Morehead et al., 2002). But in the experiments described here,
this inhibitor as well as the other inhibitors used in this
study, are added well after infections are established and
were not found to affect parasite viability (Kima et al.,
unpubl. obs.).
Several studies of intracellular pathogenesis have
implicated NFkB signalling in the inhibition of apoptosis
of infected cells (Heussler et al., 2002; Payne et al.,
2003). A critical event in signalling through the NFkB
pathway is the phosphorylation of IkB by IkB kinase,
which results in the release of NFkB from suppression by
IkB (Ghosh and Karin, 2002). We determined that NFkB
signalling is activated during Leishmania infections of
macrophages by assessing phosphorylation of IkB in
Western blots. Blots were first probed with antibodies to
phospho- IkB, followed by stripping and probing with an
antibody to native IkB. Figure 2C shows that as early as
15 min after infection with L. major, IkB phosphorylation
is induced. Infection with L. pifanoi (not shown) and
L. amazonensis induced IkB phosphorylation as well,
albeit with differences in the time course of induction.
There was no change in the levels of native IkB. The
observed changes in IkB phosphorylation following infection were verified by performing infections in the presence of wedelolactone, an inhibitor NFkB signalling
(Kobori et al., 2004). As Fig. 2D shows, phosphorylation
of IkB was blocked when L. major infection was performed in the presence of this inhibitor. These studies
show that infection with promastigotes of L. major, L. pifanoi and L. amazonensis results in the activation of the
NFkB signalling pathway.
Signalling through mitogen associated protein MAP
kinases (MAPK) occurs in response to several agents
(Han et al., 1994). Three major cascades of MAPK are
known. The c-Jun NH(2)-terminal kinase (JNK) cascade,
the extracellular signal-regulated kinase (ERK) cascade,
and the p38 MAPK cascade. In these studies we focused
on one of the MAPK family members, p38 MAPK. We
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 9, 84–96
88
A. Ruhland, N. Leal and P. E. Kima
A
B
C
D
E
Fig. 2. Multiple signalling cascades are induced upon infection with Leishmania promastigotes. RAW 264.7 macrophages were infected with
Leishmania promastigotes at an moi of 10:1 as described in Experimental procedures. At the indicated time points, cell lysates were collected
and equal amounts (50 mg) were resolved by SDS-PAGE and analysed by Western blotting.
A. Blots were probed with antibodies to phospho Akt, then stripped and re-probed with antibody to native Akt. The relative density of the
phospho-Akt bands from several blots was obtained and plotted.
B. The dependence of Akt phosphorylation on PI3K signalling during infection was determined when infections were performed in the
presence of LY294002. Lysates were prepared after a 30 min infection. The blots were stripped and reprobed with b-actin.
C. Blots were probed with antibodies to phospho IkB-a. These blots were stripped and probed with antibodies to native IkB-a.
D. Leishmania major infection was performed in the presence of increasing concentrations of wedelolactone (IkK inhibitor). Lysates were
prepared after 30 min infection. Blots were probed with antibodies to phospho IkB-a then stripped and re probed with antibodies to native
IkB-a.
E. Phospho p38 MAPK was probed at the indicated times. The blots were stripped and reprobed with antibodies to native p38 MAPK.
determined whether Leishmania promastigote infection of
macrophages results in the phosphorylation of p38
MAPK. Figure 2E shows that unlike in resting RAW 264.7
cells, where p38 MAPK is not phosphorylated, incubation
of macrophages with promastigotes of L. major results in
p38 phosphorylation within 15 min of infection. Comparable results were obtained when macrophages were
infected with L. pifanoi (not shown) and L. amazonensis
promastigotes. Infection had no effect on native p38
MAPK levels. These findings indicate that infection with
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 9, 84–96
Leishmania activate the PI3K/Akt pathway 89
Leishmania promastigotes results in the activation of p38
MAPK signalling.
Inhibition of PI3K/Akt signalling and not NFkB or p38
MAPK signalling reverses the Leishmania-induced
resistance to apoptosis
We next assessed what effect the inhibition of signalling
through the PI3K/Akt pathway, NFkB or p38 MAPK would
have on the resistance to apoptosis by infected cells.
Chemical inhibitors of these pathways were used. As discussed earlier, apoptosis was assessed by evaluating the
increase in caspase 3 activity in lysates of infected cells or
by determining the extent of DNA fragmentation after apoptosis induction. In contrast to macrophages infected for
4 h with L. major that have caspase 3 levels comparable
to those of uninduced cells, apoptosis induction in the
presence of the inhibitors of the PI3K/Akt pathway
(LY294002 or AKT inhibitor IV) resulted in significantly
higher caspase 3 activity (Fig. 3A). All three parasite
species used in these studies were unable to confer resistance to apoptosis in macrophages when apoptosis was
induced in the presence of inhibitors of the PI3K pathway.
However, when apoptosis was induced in infected
macrophages in the presence of the NFkB inhibitor,
wedelolactone or the p38 MAPK inhibitor SB202190, the
resistance to apoptosis conferred by Leishmania infection
was not reversed (Fig. 3A). The effect of signalling
pathway inhibition was also determined after 24 h
infections. We had previously observed that older infections were less efficient at resisting apoptosis (Fig. 1).
Nonetheless, as Fig. 3B shows, the resistance to apoptosis observed after 24 h infection with L. major parasites
was reversed in the presence of LY294002 and AKT IV
inhibitor and not wedelolactone or SB202190. It is important to note that the activity of these inhibitors had been
verified earlier (Fig. 2). Moreover, in the case of the NFkB
inhibitor, macrophage production of nitric oxide, which has
previously been shown to be dependent on NFkB signalling (Xie et al., 1994), was blocked by wedelolactone in
response to LPS stimulation (not shown). Based on the
results from the caspase 3 assays, it is evident that signalling through the PI3K/Akt pathway is more relevant
for resistance to chemically induced apoptosis in
Leishmania-infected cells than signalling through NFkB or
p38 MAPK.
Since all the studies thus far have evaluated Leishmania infection of macrophage cell lines in long-term culture,
we wanted to know whether in primary macrophages,
PI3K/Akt signalling plays a significant role in apoptosis
resistance as well. To address this issue, apoptosis was
induced in both infected and uninfected BMDMs with
campothecin in the presence or absence of LY294002 or
Akt inhibitor. Figure 3C shows that BMDMs infected for
4 h with either L. major or L. amazonensis are resistant to
apoptosis. Furthermore, this resistance to apoptosis is
reversed by treatment with either LY294002 or AKT IV
inhibitor. Although the caspase levels in BMDM cultures
where apoptosis induction was performed in the presence
of LY294002 were significantly higher than caspase levels
in infected cells without the inhibitor, this reversal in apoptosis resistance was not as robust as was the case with
the AKT IV inhibitor. This notwithstanding, we conclude
that PI3K/Akt signalling is important for apoptosis resistance in BMDMs as well.
To confirm the observed effects of the signalling
pathway inhibitors on caspase levels after inducing apoptosis in infected cells, DNA fragmentation was also
measured in the presence of the signalling pathway
inhibitors. The histograms in Fig. 4 show that induction of
apoptosis in L. major infected cells in the presence of
AKT inhibitor IV resulted in an increase in DNA fragmentation (Fig. 4C) as compared to when apoptosis was
induced in infected cells without inhibition (Fig. 4B). Inhibition of signalling through NFkB (Fig. 4D) slightly
reversed the resistance of infected cells to apoptosis
induction, while inhibition of signalling through p38
MAPK (Fig. 4E) had no affect on apoptosis resistance by
infected cells. Similar observations were made with
L. amazonensis and L. pifanoi (not shown). Taken
together, the results of these experiments show that by
activating and sustaining PI3K/Akt signalling, Leishmania
infection confers resistance to infected cells from potent
inducers of apoptosis.
Reduction of AKT levels by siRNA transfection confirms
the critical role of signalling through Akt in
parasite-induced protection from apoptosis
Akt is not the sole downstream signalling intermediate of
PI3K, so it is possible that treatment with an inhibitor like
LY294002 has a wider effect on signalling. To confirm a
central role for Akt signalling in parasite resistance to
apoptosis, we performed a targeted reduction of Akt levels
by introducing Akt-specific short interfering RNAs
(siRNA). Total cellular Akt protein levels are derived from
the expression of three isoforms (Akt 1,2,3) (Song et al.,
2005). It is not known if each isoform performs a unique
function or whether all three possess redundant activities.
A recent study has shown that a 30% reduction in Akt 1
levels has a measurable effect on Akt function (Pengal
et al., 2006). In the studies presented here, Akt levels
were suppressed by targeting all three isoforms of Akt. We
determined in previous experiments using cycloheximide
that Akt turnover occurs after 24 h (data not shown).
Figure 5A shows that 24 h after transfection of Akt specific
siRNA, there was ~60% reduction of Akt levels as compared to Akt levels in cells transfected with control siRNA.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 9, 84–96
90
A. Ruhland, N. Leal and P. E. Kima
A
9000
Caspase-3 Activity
8000
7000
6000
5000
4000
3000
2000
1000
L.major
No inhibitor
L.amazonensis
IKK inhibitor
Akt inhibitor
PI3K inhibitor
p38 inhibitor
B
Fig. 3. Engagement of the PI3K/Akt pathway
is required for the reduced caspase activation
in Leishmania infected macrophages.
A and B. RAW 267.4 macrophages were
infected with promastigotes for 4 h (A) or 24 h
(B). The cultures were washed prior to
addition of 2 mM campothecin alone or in the
presence of specific kinase inhibitors. NFkB
was inhibited with wedelolactone; p38 MAPK
was inhibited with SB202190; PI3K signalling
was inhibited with LY294002 or the AKT
inhibitor IV. Caspase 3 levels were
determined 7 h after induction with
campothecin.
C. Bone marrow-derived macrophages were
infected for 4 h with either L. major or
L. amazonensis promastigotes. These
cultures were then treated with campothecin
alone or in the presence of LY294002 or the
AKT IV inhibitor. Results are compiled from at
least three separate experiments and
significance was determined by the Student’s
t-test (P < 0.05).
Caspase-3 Activity
6000
5000
4000
3000
2000
1000
No inhibitor
IKK inhibitor
Akt inhibitor
PI3K inhibitor
p38 inhibitor
C
6000
Caspase-3 Activity
5000
4000
3000
2000
1000
Campothecin
–
+
Vehicle
+
L.major
+
No inhibitor
L.amazonensis
+
+
+
+
Akt inhibitor
Actin levels in these cell lysates verified that equivalent
protein amounts were loaded in each lane and also that
the siRNA did not exert a generalized suppression on
protein expression. Cells expressing reduced Akt levels
were infected for 4 h with promastigotes of L. major or
L. amazonensis and then analysed in caspase 3 assays
after induction of apoptosis. Figure 5B shows that infected
PI3K inhibitor
cells with lower Akt expression (Akt siRNA) were no
longer able to resist induction of apoptosis as compared
to cells transfected with control siRNA that are resistant
to apoptosis. The result of this experiment complements
the other observations described above that signalling
through Akt is necessary for parasite-induced resistance
to apoptosis.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 9, 84–96
Leishmania activate the PI3K/Akt pathway 91
Fig. 4. Signalling through Akt is required for protection from DNA fragmentation induced by actinomycin D treatment. RAW 267.4
macrophages were infected with L. major promastigotes at an moi of 10:1 for 4 h prior to cotreatment of a specific kinase inhibitor and 10 mM
actinomycin D. After an additional 7 h, cells were harvested and DNA fragmentation was assayed by TUNEL analysis. The marker for
histogram analysis was set to exclude background FITC fluorescence measured from resting macrophages. Cells that fluoresced above this
threshold channel number were considered apoptotic. Results are typical of infections with each promastigote described in above experiments.
A
B
Fig. 5. Infected cells expressing reduced Akt levels do not resist apoptosis. RAW 264.7 macrophages at logarithmic growth were transfected
with siRNAs specific to the three Akt isoforms. A control group was transfected with control siRNA. Twenty-four hours after transfection, cells
were recovered and their Akt levels assessed in Western blots (A). Other cells were infected for 4 h before apoptosis induction with
campothecin. Caspase 3 activation was measured in caspase 3 assays (B) and caspase levels in cells transfected with control siRNA were
compared to levels in cells transfected with Akt-specific siRNAs. Results are compiled from at least two experiments and significance was
determined by the Student’s t-test (P < 0.05).
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 9, 84–96
92
A. Ruhland, N. Leal and P. E. Kima
Fig. 6. Infection with Leishmania induces Akt-dependent
phosphorylation of Bad. At the indicated times cell lysates were
prepared from macrophages infected with L. major or
L. amazonensis promastigotes. Equal amounts (50 mg) were
resolved by SDS-PAGE and analysed by Western blotting with
antibodies to Bad Ser 136. The blots were stripped and re-probed
with b-actin. This experiments is representative of two experiments.
Infection induces the phosphorylation of BAD
It has been reported that Bad phosphorylation at Ser-136,
which results in the sequestration of Bad and a block in
cytochrome release from the mitochondria, is promoted
by the PI3K/Akt pathway (Datta et al., 1997). We therefore
determined the phosphorylation status of Bad in
Leishmania-infected cells. Lysates obtained at discrete
times from infected cells were assessed by Western blot
analysis for Bad phosphorylation. Figure 6 shows the
appearance of phospho-Bad 2 h after infection with
L. major or L. amazonensis promastigotes. Bad phosphorylation is sustained through the 24 h infection course.
These results further establish that signalling through Akt
plays an important role in infection- induced resistance to
apoptosis.
Discussion
Many intracellular organisms have been shown to confer
resistance to apoptosis to their host cell when apoptosis is
induced by a wide range of agents (Gao and Abu Kwaik,
2000; Heussler et al., 2001a). Although the importance of
this phenomenon is mostly speculative, the fact that a
wide range of organisms confer this property to their
mammalian host cell suggests some evolutionary
importance. Elucidation of the mechanism by which intracellular organisms achieve this phenomenon is the
subject of several recent reports (Maiti et al., 2001; Payne
et al., 2003; Yilmaz et al., 2004). Given that each intracellular organism has a unique interaction with its mammalian host cell, it should be expected that each of these
organisms would employ a different mechanism to
achieve the resistance to apoptosis.
In the studies described here, we confirm previous
observations that had shown that infection with the promastigote form of L. major and L. donovani induces
infected cells to resist apoptosis (Moore and Matlashewski, 1994; Aga et al., 2002; Akarid et al., 2004; Lisi et al.,
2005). We extend these observations and show that parasites of the L. mexicana complex, L. pifanoi and L. ama-
zonensis also induce resistance to apoptosis. As we
considered the mechanism that underlies parasiteinduced resistance to apoptosis we surmised that there
are at least two likely scenarios: (i) parasites elaborate
effectors that block signals emanating from apoptosis
inducers, (ii) parasites are able to engage endogenous
host signalling pathways that result in apoptosis inhibition.
In experiments to test the latter possibility we determined
that infection with Leishmania promastigotes engages signalling through NFkB, p38 MAPK and PI3K/Akt, which
have been shown to mediate apoptosis resistance in other
systems. Whereas inhibition of PI3K/Akt signalling overcame parasite-induced resistance to apoptosis, blockage
of NFkB signalling or p38 MAPK signalling did not affect
apoptosis resistance conferred by Leishmania parasites.
This thus identifies the activation of the PI3K/Akt pathway
as an important pathway engaged by Leishmania parasites to ensure the prolonged survival of their host cell.
PI3K are phosphoinositide kinases of the Class I subgroup, which upon interaction with membrane receptors
phosphorylate integral membrane inositol phospholipid
substrates to produce the second messengers PtdIns3,4,5-P3 and PtdIns-3,4-P2. These messengers recruit and
activate downstream kinases such as Akt, which interact
with these second messengers via a pleckstrin homology
(PH) domain (Alessi and Cohen, 1998). Given that PI3K is
mostly associated with interactions at the plasma membrane, it was initially surprising that signalling through this
pathway was required for apoptosis resistance in infections that were several hours old. So it was gratifying to
observe that Akt, which is downstream of PI3K, remains
active for many hours after the initiation of infection
(Figs 2 and 3). It is likely that the initial interactions of the
parasite with ligands (internalization receptor) on the host
cell surface are sufficient to sustain prolonged effects of
PI3K signalling. In support of this view, a recent study of
signalling following gC1q engagement showed that even
after a brief stimulation pulse of 30 min, the activated form
of Akt could be detected for over 6 h (Waggoner et al.,
2005). Alternatively, the parasitophorous vacuole that harbours the parasite might mediate interactions that sustain
PI3K signalling. There was a suggestive observation in
Trypanosoma cruzi infections, which showed using a PH
domain/GFP chimera as a probe for PI3K activation, that
the nascent T. cruzi parasitophorous vacuole can recruit
downstream kinases of PI3K (Woolsey et al., 2003). Even
though the mechanism of T. cruzi entry into nonphagocytic cells is different from that of Leishmania entry
into macrophages, the implication from that study is that
molecules on a nascent parasitophorous vacuole are able
to sustain PI3K/Akt signalling. Future studies should
provide insight into the likelihood that the Leishmania
parasitophorous vacuole membrane can initiate or sustain
PI3K/Akt signalling.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 9, 84–96
Leishmania activate the PI3K/Akt pathway 93
At this time it is not known which surface molecules on
the macrophage interact with Leishmania to activate PI3K
signalling. PI3K signalling can be activated following the
interactions with a wide range of surface molecules
including Fc receptors and integrins (Dib, 2000; Deane
and Fruman, 2004). It is known that both the complement
receptor 3 and Fc receptors are significant receptors that
mediate Leishmania internalization (Mosser and Edelson,
1985; Da Silva et al., 1989; Mosser, 1994; Kima et al.,
2000). A previous report detailing studies on neuronal
cells had found that wild-type L. major promastigotes
were not able to confer to these cells, the capacity to
resist apoptosis (Chuenkova et al., 2001). More precisely
it was observed that the L. major organisms did not activate the PI3K/Akt pathway in schwann cells unless they
were genetically modified to express T. cruzi salidases.
One interpretation of these results is that schwann cells,
unlike macrophages, do not express the molecule(s) that
wild-type Leishmania interact with on macrophages to
trigger engagement of the PI3K/Akt pathway.
On the parasite side, the fact that promastigotes of
L. major as well as those of the L. mexicana complex
behave similarly in these studies, suggests that these
parasites are using an invariant molecule on their surface
to mediate signalling through the PI3K/Akt pathway. This
is significant because previous studies have documented
differences in the interactions of different Leishmania
species with their mammalian hosts (McMahon-Pratt and
Alexander, 2004). Future studies of Leishmania’s ability to
activate signalling through the PI3K/Akt pathway should
hopefully bring to the fore common mechanisms of pathogenesis employed by Leishmania parasites.
We observed that p38 MAPK signalling is activated by
Leishmania parasite entry into macrophages. This point is
controversial because several investigators have reported
that signalling through p38 MAPK, like other signalling
pathways, is either avoided by Leishmania parasites or
actively suppressed (Olivier et al., 2005). Nonetheless, at
least two reports have shown the appearance of phosphorylated p38 MAPK soon after infection with Leishmania (Junghae and Raynes, 2002; Balaraman et al., 2005).
Although the basis for these different observations is not
known, it is likely that the times after infection at which p38
MAPK is evaluated might be critical. As is shown in Fig. 2,
we found that within 15 min of incubation with late stationary stage parasites, phosphorylated p38 MAPK is
detected in lysates from RAW 264.7 and B10R (not
shown) macrophages. Infections with L. major and the
L. mexicana parasites yielded comparable results. Even
though p38 MAPK signalling has been associated with the
regulation of apoptosis in other infection models (Park
et al., 2002; Zhang et al., 2005), inhibition of p38 MAPK
signalling during Leishmania infections had no effect on
the parasite-induced resistance to apoptosis.
The observation that NFkB is activated during Leishmania promastigote infection agrees with the observation
of others (Singh et al., 2004). Signalling through NFkB
was assessed by evaluating the phosphorylation of IkB
by IKK, which is a point of convergence of NFkB signalling from most stimuli that signal through NFkB (Ghosh
and Karin, 2002). It was interesting that each parasite
infection exhibited differences in the time course of IkB
phosphorylation (Fig. 2C). We presumed that the
expected loss of native IkB, which has been shown to
reflect dissociation of IkB from p50, must have occurred
within the first 10 min of parasite exposure with macrophages. However, inhibition of IkB phosphorylation under
conditions that block LPS-elicited production of nitric
oxide had limited or modest effect on parasite-induced
resistance to apoptosis (Figs 3 and 4). It is interesting
that inhibition of NFkB signalling during infection with
other protozoan parasites such as Theilera parva and
Toxoplasma, reverses parasite-induced resistance to
apoptosis (Heussler et al., 2002; Payne et al., 2003). In
fact even though both the PI3K/Akt pathway and NFkB
signalling are engaged in Theileria infections, inhibition
of PI3K/Akt does not affect parasite-induced resistance
to apoptosis (Heussler et al., 2001b). Such observations
underscore the differences in host–parasite interactions
to yield what appears to be a similar outcome. Along
these lines, the finding that PI3K signalling plays little
role in the long term survival of cells infected with Plasmodium berghei sporozoites but contributes to the establishment of the infection in hepatocytes is noteworthy
(Leiriao et al., 2005; van de Sand et al., 2005). Our
studies too showed that older infections can less efficiently resist apoptosis. This is intriguing because we can
detect an increase in Akt levels at those later times.
Future studies should clarify these observations.
Although there is evidence of cross talk between PI3K/
Akt and NFkB signalling pathways (Kane et al., 1999;
Tanaka et al., 2005), PI3K signalling that results in resistance to apoptosis in Leishmania, does not appear to be
linked to NFkB signalling.
We commenced the analysis of the events that occur
between PI3K/Akt signalling and the prevention of DNA
fragmentation. Akt is believed to modulate the activity of
proapoptotic molecules of the BCL-2 family, which play a
role in the release of cytochrome c from the mitochondria
(Song et al., 2005). We showed that the proapoptotic
molecule, Bad becomes phosphorylated during the
course of infection with Leishmania promastigotes
(Fig. 6). Phosphorylated Bad mediates cell survival
through its interactions with 14-3-3 proteins. As PI3K/Akt
signalling affects other cellular processes, we look
forward to evaluating how these other processes
might contribute to the pathogenesis of Leishmania
parasites.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 9, 84–96
94
A. Ruhland, N. Leal and P. E. Kima
Experimental procedures
Parasites and cell lines
Leishmania major LV39 (Rho/SU/59/P) and Leishmania pifanoi
promastigotes (MHOM/VE/57/LL1) obtained from the ATCC were
grown in Schneiders medium supplemented with 10% fetal calf
serum and 10 mg ml-1 gentamicin at 23°C. Leishmania amazonensis (MHOM/BR/77/LTB0016) promastigotes were maintained
in complete medium (Schneider’s Drosophila medium (Gibco
BRL, Grand Island, NY) supplemented with 10% heat-inactivated
fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY) and
10 mg ml-1 gentamicin at 23°C. Infectivity of parasites was maintained by periodic passage through BALB/c mice as reported
previously (Soong et al., 1996). All parasites were used in the late
stationary phase. The RAW 264.7 murine macrophage cell line
was cultured in RPMI supplemented with 10% fetal calf serum
and 100 units Penicillin/Streptomycin at 37°C under a 5% CO2
atmosphere. The B10R macrophage line (obtained from Dr
Olivier Martin, McGill University, Montreal, Canada) was cultured
in DMEM supplemented with 10% fetal calf serum and 100 units
Penicillin/Streptomycin at 37°C under a 5% CO2 atmosphere.
Bone marrow-derived macrophages were obtained from
BALB/c mouse femurs and plated directly into 100 mm tissue
culture plastic dishes (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) at a cell density of 5 ¥ 106 cells per dish. These
cells were cultured in complete medium supplemented with 20%
L929 cell-conditioned medium. Medium on these cells was
replaced on day 5. After 7 days, BMDM had reached confluency.
The cells were dislodged with dispase (BD Biosciences, San
Diego, CA), counted and replated in complete DMEM for studies
described below.
Western blots
Equal protein amounts of each lysate obtained after infection,
were loaded per lane of 12% SDS PAGE SDS gels and transferred to membranes after electrophoresis. Membranes were
blocked in Tris-buffered saline (TBS) supplemented with 5% carnation milk and 0.1% Tween before incubation overnight in
primary antibodies. The following antibodies were used: antiphospho Akt (Cell Signalling Technology, Danvers, MA); anti-Akt
(Biovision, Mountain View, CA); anti-phospho IkB, anti IkB, antiphospo p38 MAPK, anti-p38 MAPK (Santa Cruz Biotechnology,
Santa Cruz, CA) and anti-phospho-Bad (Ser136) (Cell Signalling
Technology); anti-b-actin (JLA20, Developmental Studies Hybridoma Bank, Iowa City, IA). After removal of primary antibodies
and washing, membranes were incubated in the appropriate
secondary antibodies conjugated to horse-radish peroxidase.
Washed blots were incubated with chemiluminescence (ECL,
Amersham) reagents. Antibody reactivity was visualized by exposure of blots to X-ray film. Some blots were stripped by incubation
in 62.5 mM Tris HCl pH 6.8 supplemented with 20 mM
2-mercaptoethanol and 2% SDS, for 30 min at 50°C. The blots
were then reprobed with other antibodies and processed as
described above.
Induction and blockage of apoptosis
Apoptosis was induced by adding campothecin (2 mM) or actinomycin D (10 mM) (Biovision) to 1 ¥ 106 RAW 264.7, B10R mac-
rophages or BMDMs that were seeded the day before in six well
plates. Cells from each well were recovered after 7 h for either
the assessment of caspase 3 activity or to evaluate the extent of
DNA fragmentation. To determine whether infection confers resistance to the induction of apoptosis, macrophages were incubated
with late stationary stage promastigotes at a 1:10 ratio (macrophage to parasite) for 4, 12 or 24 h. The cultures were washed to
remove uninternalized parasites before adding the apoptosis
inducers. To assess the involvement of signalling pathways in
infection-induced resistance, the PI3K pathway inhibitors,
LY294002 (10 mM) or the AKT inhibitor, AKT inhibitor IV (10 mM)
(EMD Biosciences, San Diego, CA) or the NFkB inhibitors IKK
inhibitor V or wedelolactone (EMD Biosciences) that target IkB
Kinase or the p38 MAPK inhibitor, SB202190 (EMD Biosciences)
were added at the time of apoptosis induction.
Caspase 3 activity assay
Following the induction of apoptosis of 1 ¥ 106 cells in six well
plates, cells were scraped and recovered by centrifugation. They
were lysed in cell lysis buffer supplied with the caspase 3 Fluorometric Assay Kit (BioVision). Protein concentration of lysates
was determined by the Bio-Rad protein assay. Equal amounts of
protein from each treatment were split into triplicates and used to
determine caspase 3 activity in a 96-well format following the
protocol supplied with the kit. Incubation was for 2 h at 37°C after
which the relative fluorescence was measured in the FLx800
fluorescence reader from Biotech (Winooski, VT) at 360/40 excitation and 528/20 emission. The assay is based on the detection
of the cleavage of AFC (7-amino-4-trifluoromethyl coumarin) from
DEVD-AFC by caspase 3.
Terminal deoxynucleotidyltransferase-mediated dUTP
nick end labelling (TUNEL) assays
Macrophages (1.5 ¥ 106) were plated in six well tissue culture plates overnight and then infected with late stationary
phase Leishmania promastigotes at a ratio of 10:1
(parasite : macrophage). After 4 h, free parasites were washed
off macrophage monolayers with PBS and apoptosis was
induced with either 10 mM actinomycin D or 2 mM campothecin
for 7 h. Cells were scraped and processed for bromodeoxyuridine
(BrdU) incorporation and propidium iodide staining according to
manufacturer’s protocol (Biovision). Briefly, cells were fixed in 1%
PFA and permeabilized in 70% ethanol. Incorporation of BrdU
was carried out at 37°C for 60 min followed by detection with
anti-BrdU-FITC antibody. Cells were then stained with propidium
iodide in running buffer supplemented with RNase A. Ten thousand cells were analysed on the FACSort flow cytometer (BD
Biosciences) at the UFL Core Flow Cytometry Laboratory. Cells
that showed higher FITC fluorescence than those of resting macrophages were considered apoptotic.
siRNA experiments
Small inhibitory RNAs specific to each of the Akt isoforms as well
as a control siRNA were purchased from Ambion (Austin, TX).
They were transfected into macrophages that had been recovered during logarithmic growth, using the nucleofection system
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 9, 84–96
Leishmania activate the PI3K/Akt pathway 95
(Amaxa, Gaithesburg, MD). Following transfection, cells were
plated in six well plates and incubated for 24 h before infecting
them or inducing apoptosis. Evaluation of Akt levels was performed by Western blotting and with anti-Akt antibody. Cells
transfected with control siRNA were compared to those transfected with Akt specific siRNA. Caspase 3 assays were performed as described above.
Statistics
Experiments were repeated at least three times, and probability
(P) was calculated using a Student t-test. P-values of < 0.05 were
considered statistically significant.
Acknowledgements
The authors would like to thank Dr Olivier Martin for providing us
with the B10R macrophage cell line. We also thank Dr Howard
Johnson for critically reading this manuscript. This work was
supported by NIH award #RO1 AIO48739 (P.E.K.).
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Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 9, 84–96