Microbial Pathogenesis xxx (2011) 1e9

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Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Acinetobacter baumannii-induced lung cell death: Role of inammation, oxidative

stress and cytosolic calcium
Younes Smani*, Fernando Docobo-Prez, Michael J. McConnell, Jernimo Pachn
Service of Infectious Diseases, Institute of Biomedicine of Sevilla (IBIS), University Hospital Virgen del Roco/CSIC/University of Sevilla, Av. Manuel Siurot s/n, 41013 Sevilla, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 16 December 2010
Received in revised form
21 January 2011
Accepted 24 January 2011
Available online xxx

A growing body of evidence supports the notion that susceptible Acinetobacter baumannii strain ATCC
19606 induces human epithelial cells death. However, most of the cellular and molecular mechanisms
associated with this cell death remain unknown, and also the degree of the cytotoxic effects of a clinical
panresistant strain compared with a susceptible strain has never been studied. Due to the role of
proinammatory cytokine release, oxidative stress and cytosolic calcium increase in the cell deathinduced by other Gram-negative bacteria, we investigated whether these intracellular targets were
involved in the cell death induced by clinical panresistant 113-16 and susceptible ATCC 19606 strains.
Data presented here show that 113-16 and ATCC 19606 induce time-dependent cell death of lung
epithelial cells involving a perturbation of cytosolic calcium homeostasis with subsequent calpain and
caspase-3 activation. Prevention of this cell death by TNF-a and interleukin-6 blockers and antioxidant
highlights the involvement of proinammatory cytokines and oxidative stress in this phenomenon.
These results demonstrate the involvement of calpain calcium-dependent in cell death induced by
A. baumannii and the impact of proinammatory cytokines and oxidative stress in this cell death; it is
noteworthy to stress that some mechanisms are less induced by the panresistant strain.
2011 Elsevier Ltd. All rights reserved.

Keywords:
Acinetobacter baumannii
Pneumonia
Panresistant
Cell death

1. Introduction
Acinetobacter baumannii is a Gram-negative non-fermentative
coccobacillus which has emerged over the last decades from an
organism with questionable pathogenecity to an infectious agent
with clinical importance [1]. Infections caused by A. baumannii have
become very difcult to treat due to the emergence of strains
resistant to all or almost all antimicrobials used clinically [2]. The
difculty of eradicating this microorganism has allowed it to
acquire a state of highly successful human pathogen [3] that is
involved in different serious infections including pneumonia,
bacteremia and meningitis.
Lung inammation is one of the hallmarks of pneumonia
induced by A. baumannii resulting in epithelial barrier destruction.
This lung inammation was observed previously by our group in
mouse and guinea pig pneumonia models caused by A. baumannii
[4,5]. Infection of human epithelial cells by A. baumannii was
associated with adherence and invasion of this pathogen into these
cells [6e8] and induction of cellular death [9]. Two reports have

* Corresponding author. Tel.: 34 955 01 36 51; fax: 34 955 01 23 77.

E-mail addresses: younes.smani.exts@juntadeandalucia.es, y_smani@hotmail.
com (Y. Smani).

indicated that A. baumannii induces the activation of caspase-9,

caspase-8 and caspase-3, and the liberation of apoptosis inducing
factor (AIF) and cytochrome c, both leading to caspase-dependent
pathways in epithelial cells [9,10]. Despite the evidence that
A. baumannii induces cell death, studies have never investigated
this cytotoxic effect with resistant clinical strains and compared it
with that produced by a susceptible strain.
Increasing evidence suggests that the cellular death induced by
A. baumannii involve activation of caspases, which could participate
in apoptosis through the initiation of intracellular pathways [9,10].
In these studies, the intracellular pathways were not completely
characterized and remained unclear. Indeed, some experimental
studies indicate that an outer membrane protein (Omp) 38 of
A. baumannii, also called OmpA-like, is trafcked to both mitochondria and the nucleus and induces eukaryotic cell death pathways [10e12], similar to what has been observed in in vitro models
of cytotoxicity with Omps of other Gram-negative bacteria [13,14].
One of these Omps, PorB, induces an increased activity of calpain
which is associated with impaired calcium (Ca2) homeostasis and
results in cell death [14]. However, it is likely that increased activity
of calpain associated with deregulated Ca2 homeostasis may be
involved in cell death-induced by A. baumannii. In addition, mitochondrial perturbation has been demonstrated in epithelial
cells co-incubated with the OmpA-like protein or with entire

0882-4010/$ e see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.micpath.2011.01.008

Please cite this article in press as: Smani Y, et al., Acinetobacter baumannii-induced lung cell death: Role of inammation, oxidative stress and
cytosolic calcium, Microbial Pathogenesis (2011), doi:10.1016/j.micpath.2011.01.008

Y. Smani et al. / Microbial Pathogenesis xxx (2011) 1e9

A. baumannii cells [10]. Moreover, calpain is activated by Ca2

liberated from mitochondria and endoplasmic reticulum [15,16]
which supports the hypothesis that A. baumannii can induce the
release of intracellular Ca2 and consequently the activation of
calpain. In addition, calpain or their proteolytic products have been
shown to act as effectors of cell death [17]. These observations
strongly support the involvement of calpain Ca2-dependent in the
cell death induced by A. baumannii.
During lung infection by bacteria, epithelium is one of the rst
targets and plays a critical role in the release of proinammatory
mediators which can kill host cells and damage host tissues [18]. In
lung epithelial cells, the tumor necrosis factor alpha (TNF-a)
receptor can be activated directly by Staphylococcus aureus [19]. In
the case of A. baumannii, puried OmpA-like elicits a type 1 T helper
(Th1)-mediated immune response [20] and upregulates inducible
nitric oxide synthase (iNOS) [21].
In addition, the overexpression of a variety of proinammatory
cytokines including TNF-a, interleukin 1 (IL-1) and interleukin 6
(IL-6) have been involved in cellular death [22e24]. Inhibiting
proinammatory signals can be protective during lung infections.
For example, interrupting both TNF-a and IL-1 signaling decreases
pulmonary edema and the loss of lung compliance that are often
found in mice with Escherichia coli pneumonia [25,26]. In this
regard, it was hypothetized that the increased release of proinammatory cytokines may play a role in the cell death-induced by
A. baumannii.
Accumulative evidence has put emphasis on the critical role of
oxidative stress in cell death (for review see Ref. [27]). Indeed, the
role of reactive oxygen species (ROS) production and the subsequent cell death induced by various bacteria has been established
[28,29]. However, ROS involvement in the cell death induced by
A. baumannii has not yet been characterized.
The aim of this study is to determine the different mediators
involved in the cell death produced by the panresistant strain. The
data presented here propose for the rst time the involvement of
calpain and Ca2 dependence in the cell death induced by both
strains and demonstrate the involvement of proinammatory
cytokines and oxidative stress in this cell death.
2. Materials and methods
2.1. Bacterial strains
The reference A. baumannii strain ATCC 19606, susceptible to all
antimicrobials, the clinical panresistant A. baumannii isolate 113-16
[30], and the non-pathogenic strain 77 were used in this study. All
the strains were grown in Mueller Hinton Broth (MHB) at 37  C for
20e24 h. The cultured bacterial strains were washed with phosphate-buffered saline (PBS) and resuspended in DMEM prior to use
in cell culture experiments.

100. Immediately before infection, A549 cells were washed three

times with prewarmed PBS and further incubated in DMEM without
FBS and antibiotics, and then A. baumannii strains was added gently
to A549 cells and incubated for 6, 10 and 24 h in a humidied, 5% CO2
at 37  C.
2.3. Cellular viability, apoptosis and necrosis
A549 cells were infected with A. baumannii (ATCC 19606, 113-16,
or 77) for the indicated times (6, 10 and 24 h). A. baumannii
cytotoxicity was initially assessed quantitatively by monitoring
mitochondrial reduction activity using the MTT assay (Sigma,
Spain) as described previously [31]. The simultaneous determination of live and dead cells was achieved with the LIVE/DEAD kit
according to the manufacturers instructions (Invitrogen, Spain).
To monitor apoptosis, cell nuclei were visualized using 40 ,6diamidino-2-phenylindole (DAPI). A549 cells, grown on glass
coverslips in 24 well plates, were washed with cold PBS, xed in
methanol for 8 min at 20  C, incubated for 10 min at room
temperature with DAPI (0.5 mg/mL), washed with PBS, mounted
with SlowFade Gold antiFade reagent (Invitrogen, Spain), and
visualized using a Leica uorescence microscope (DM-6000; Leica
Microsystems Wetzlar GmbH, Germany).
For necrosis determination, LDH activity in the supernatants
was determined by the cytotoxicity detection kit (Roche Diagnostic,
Spain) according to the manufacturers instructions.
2.4. Cytokine assay and effect on cellular viability
A549 cells were pretreated for 30 min with thalidomide (100 mg/
mL), an inhibitor of TNF-a and PD098059 (20 mg/mL), an inhibitor of
IL-6, (Sigma, Spain) and for 1 h with anti-human TNF-a (10 mg/mL)
and anti-human IL-6 neutralizing antibodies (10 mg/mL) (eBioscience, USA). After infection with A. baumannii (ATCC 19606 or
113-16) in the presence or absence of thalidomide (100 mg/mL) or
PD098059 (20 mg/mL), supernatants were removed and stored
at 80  C. TNF-a and IL-6 levels were measured using an ELISA kit
(R&D Systems) according to the manufacturers instructions. For
the assessment of cellular viability by an MTT assay, A549 cells were
pretreated with thalidomide (100 mg/mL), anti-human TNFa neutralizing antibody (10 mg/mL), PD098059 (20 mg/mL) and antihuman IL-6 neutralizing antibody (10 mg/mL), washed three times
with prewarmed PBS and infected with A. baumannii (ATCC 19606
or 113-16) or treated with 5 mg/mL of cycloheximide (CHX) (Sigma,
Spain), 20 ng/mL of human TNF-a (Invitrogen, Spain) alone or in
combination with 5 mg/mL of CHX, and 200 ng/mL of human IL-6
(Invitrogen, Spain).
2.5. Superoxide anion assay

2.2. Human cell culture and infection

The type II pneumocyte cell line A549 derived from a human lung
carcinoma were a gift from Dr. Felipe Fernandez-Cuenca and grown
in DMEM medium supplemented with 10% heat-inactivated FBS
(Gibco, Spain), vancomycin (50 mg/mL), gentamicin (20 mg/mL),
amphotericin B (0.25 mg/mL) (Gibco, Spain) and 1% HEPES in
a humidied, 5% CO2 at 37  C. A549 cells were routinely passaged
every 3e4 days. The cells were seeded 24 h in 96 well plates (for 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
and lactate dehydrogenase (LDH) assays), in 6 well plates (for
enzymatic activities and western blot experiments) and in 24 well
plates (for the rest of experiments) prior to infection with A. baumannii (ATCC 19606 or 113-16) at a multipilicty of infection (MOI) of

Superoxide anion (O2 .) production was assayed by spectrophotometric measurement of ferricytochrome c reduction as
described previously [31]. A549 cells were pretreated for 30 min
with an antioxidant, Trolox (1 mM) (Sigma, Spain). After infection
of A549 cells with A. baumannii (ATCC 19606 or 113-16) in the
presence or absence of Trolox (1 mM), the cells were washed three
times with prewarmed PBS and incubated with 0.5 mL of reaction
mixture consisting of 150 mM oxidized (Fe3) cytochrome c in
EDTA (100 mM) and sodium phosphate buffer (50 mM; pH 7.5) at
37  C for 1 h. After this incubation, the supernatants were collected
and used to quantify the amount of reduced cytochrome c by
absorbance at 550 nm. The O2 . release was calculated using the
ferricytochrome c extinction coefcient (21.1 mM.cm1).

Please cite this article in press as: Smani Y, et al., Acinetobacter baumannii-induced lung cell death: Role of inammation, oxidative stress and
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Y. Smani et al. / Microbial Pathogenesis xxx (2011) 1e9

2.6. Intracellular Ca2 measurement

Fluorescence was monitored using a Nikon TS-100 inverted
microscope equipped with a 20 uor objective (0.75 NA) as
described previously [32]. A549 cells plated on coverslips in 24 well
plates and mounted in a Teon chamber were incubated in DMEM
with 2 mM fura-2/AM (Invitrogen, Spain) for 30 min at 37  C, and
washed with PBS. Fluorescence images of 20e30 cells were recorded and analyzed with a digital uorescence imaging system (InCyt
Im2, Intracellular Imaging Inc., Imsol, UK) equipped with a lightsensitive CCD camera (Cooke PixelFly, ASI, Eugene, OR, USA).
Changes in intracellular Ca2 are represented as the ratio of fura-2
uorescence induced at an emission wavelength of 510 nm due to
excitation at 340 and 380 nm (ratio F340/F380). Experiments were
done in 0 Ca2 solution (in mM: 120 NaCl, 4.7 KCl, 4 MgCl2, 0.2
EGTA, 10 Hepes). The change in cytsolic Ca2 release was monitored
5 min before and 15 min after addition of A. baumannii onto A549
cells. This change was determinated by the Dratio calculated as the
difference between the peak ratio after addition of A. baumannii
(ATCC 19606 or 113-16) and its level right before A. baumannii
addition. Ca2 inux was determined from changes in fura-2/AM
uorescence after re-addition of Ca2 (2 mM) 5 min after addition
of A. baumannii onto A549 cells. Dratio was calculated as the
difference between the peak ratio after extracellular Ca2 was
added and its level right before Ca2 addition.
2.7. Measurement of calpain and caspase-3-like proteolytic
activities
The calpain and caspase-3 activities were measured by cleavage
of the substrates succinyl-Leu-Tyr-AMC and Ac-DEVD-AMC
(Bachem, Germany). Briey, A549 cells were seeded in 6 well plates
and cultured overnight. At 24 h after infection with A. baumannii
(ATCC 19606 or 113-16) in presence or absence of calpain and
caspase-3 inhibitors, 50 mM MDL28170 and 50 mM Ac-DEVD-CHO
(Bachem, Germany), A549 cells were collected, homogenized in
RIPA buffer supplemented with 1 mM PMSF and 10% cocktail of
protease inhibitors (Sigma) and centrifuged at 20 000 g for 15 min
at 4  C. The supernatant was removed and the amount of proteins
was determined using the Bio-Rad protein dye reagent (Bradford
method). The samples were stored at 80  C until later use. Fifty mg
of proteins were incubated for 2 h with 100 mM succinyl-Leu-TyrAMC and 100 mM Ac-DEVD-AMC substrates initially dissolved in
Me2SO. The cleavage of the calpain and caspase-3 substrates was
monitored by uorescence emission at 460 nm after excitation at
360 nm using a Fluorescence microplates reader (Berthold Technologies GmbH & Co, Germany).
2.8. Statistical analysis
Group data are presented as mean  S.D. The ANOVA test and
post-hoc tests (Tukey and Dunnet) were used to determine the
difference between means. The difference was considered signicant at P < 0.05.
3. Results
3.1. A. baumannii is cytotoxic to human lung epithelial cells
We rst investigated the cytotoxicity of A. baumannii panresistant strain 113-16 and susceptible strain ATCC 19606 using
A549 cells. Incubation of A549 cells with A. baumannii resulted in
a time and dose-dependent decrease in cell viability as monitored
using the MTT assay. The decrease in cellular viability upon
A. baumannii exposure was signicant after a 6 and 24 h incubation

with ATCC 19606 and 113-16, respectively. In the case of exposure

to the ATCC 19606 strain, the decrease in cell viability reached
levels of 68.89  6.44%, 59.88  10.99% and 39.5  0.43% (relative to
controls) after 6, 10 and 24 h of exposure. In the case of the 113-16
strain, the decrease in cell viability reached levels of 69.48  12.38%
(relative to controls) after 24 h of exposure (Fig. 1A). In contrast, the
non-pathogenic A. baumannii 77 showed a decrease in cell viability
to 89.35  2.44% after 24 h of exposure. Moreover, 24 h exposure of
A549 cells with A. baumannii (ATCC 19606 or 113-16) showed that
the decrease in cell viability was higher for ATCC 19606 strain than
113-16 strain: 39.5  0.43% vs. 69.48  12.38%, relative to controls.
Furthermore, a 24 h exposure of A549 cells to A. baumannii (ATCC
19606 or 113-16) at MOI from 0.001 to 100 induced only a signicant reduction in cellular viability for A. baumannii (ATCC 19606 or
113-16) at MOI of 100 (data not shown).
The cytotoxicity of A. baumannii was also investigated using a twocolor uorescence cell viability assay. In contrast to intense and
uniform green uorescence based on intracellular esterase activity
displayed by control A549 cells, cells exposed for 24 h to ATCC 19606
strains were stained mainly with ethidium homodimer-1, producing
a bright red uorescence indicative of dead cells than in 113-16 strain.
In contrast, little few dead cells were observed after infection of A549
cells with A. baumannii 77 (Fig. 1B). Additionally, morphological
examination of cellular nuclei stained with DAPI showed that A549
cells exposed to A. baumannii presented typical apoptotic
morphology, with condensation of chromatin and fragmentation of
the nuclei for ATCC 19606 and 113-16 strains (Fig. 1C). Indeed, A549
cells exposed to ATCC 19606 strain and 113-16 strain for 24 h
exhibited a strain-dependent increase in the number of apoptotic
nuclei in the case of ATCC 19606 strain, whereas the 77 strain and the
control cells presented no signicant apoptotic nuclei. Moreover,
while low levels (5.98  1.81%) of LDH liberation, a necrosis marker,
were detected in infected A549 cells with A. baumannii 77, the level of
LDH liberation strongly increased to 20.95  14.32% and
52.96  15.39% (relative to controls) induced by 113-16 strain and
ATCC 19606 strain, respectively (Fig.1D). Taken together, these results
showed that panresistant A. baumannii induces cell death of A549
cells but with low intensity when compared with a non-panresistant
A. baumannii. We therefore selected a range of exposure times corresponding to 24 h with both A. baumannii strains at MOI of 100 to
study molecular pathways and the associated intracellular events in
A. baumannii-induced cell death.
3.2. TNF-a and IL-6 mediates A. baumannii-induced cell death
We further tested the hypothesis that TNF-a and IL-6 might be
involved in A. baumannii-induced cell death by monitoring the
sensitivity of TNF-a and IL-6 liberation to specic inhibitors during
cell death. None of these tested inhibitors showed defect of growth
and adherence/invasion of ATCC 19606 and 113-16 strains to A549
cells, excluding the possibility of differential cell death due to
differential growth and adherence/invasion rate (data not shown).
The TNF-a assay showed that 113-16 strain induces the release of
TNF-a less than ATCC 19606 strain at 6 h of exposure with these
strains (51.82  31.56 pg/mL vs. 198.21  87.38 pg/mL), whereas at
24 h none of both strains induced a signicative release of TNFa (16.27  6.21 pg/mL vs. 19.36  10.86 pg/mL). Pretreatment with
thalidomide (100 mg/mL) decreased the liberation of TNF-a induced
by exposure of A549 cells with A. baumannii (ATCC 19606 or 11316) for 6 h (Fig. 2A). Interestingly, the preincubation of A549 cells
with thalidomide, a non specic TNF-a inhibitor, and anti-human
TNF-a (10 mg/mL) neutralizing antibody, a specic TNF-a neutralizer, prevented the cell death induced by both strains of A. baumannii as monitored by the MTT assay: 79.16  6.96% and
80.14  7.44% compared with 31.49  7.87% reduction relative

Please cite this article in press as: Smani Y, et al., Acinetobacter baumannii-induced lung cell death: Role of inammation, oxidative stress and
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Fig. 1. Apoptotic and necrotic cellular death induced by A. baumannii. A549 cells were cultured in the absence or presence of A. baumannii (113-16, ATCC 19606, or 77). A. baumannii
cytotoxicity was determined by monitoring the mitochondrial reduction activity using the MTT assay for 6, 10 or 24 h (A). A. baumannii cytotoxicity was next illustrated with
representative microscopic elds of the two-color uorescence cell viability assay (LIVE/DEAD viability/cytotoxicity kit) for 24 h (B). Cellular apoptosis at 24 h (white arrows) is
illustrated here with representative microscopic elds. Cell nuclei are stained with DAPI (blue) (C). Necrosis at 24 h was determined by following LDH release using the cytotoxicity
detection assay (D). The positive control was 10 mg/mL staurosporine (Str). Data are the means  S.D. of three different experiments, and are normalized to the control (A549 cells
without treatment, designated as 100%). P < 0.05: * between control and treated groups, y treated groups vs. time. (For interpretation of the references to colour in this gure
legend, the reader is referred to the web version of this article.)

to controls, for ATCC 19606 strain, and 79.75  6.28% and

84.02  15.96% compared with 59.44  5.78% reduction relative to
controls, for 113-16 strain (Fig. 2B). However, induction of cell death
by TNF-a in A549 cells required CHX treatment, an inhibitor of
eukaryotic protein synthesis (Fig. 2B).
In the case of IL-6, both A. baumannii strains induced
a progressive increase in IL-6 liberation between 6 and 24 h
(22.48  3.62 pg/mL and 35.31  6.8 pg/mL, for ATCC 19606 strain)
and (6.27  1.04 pg/mL and 16.2  1.72 pg/mL, for 113-16 strain),
respectively. Indeed, pretreatment of A549 cells with PD098059
(20 mg/mL) resulted in a signicant inhibition of IL-6 liberation at
24 h (Fig. 2C). Similarly, having demonstrated that TNF-a is
involved in the cell death induced by both A. baumannii strains, we
also examined the involvement of IL-6 in the cell death produced by
these strains. The presence of PD098059, a non specic IL-6
inhibitor, and anti-human IL-6 neutralizing antibody (10 mg/mL),
a specic IL-6 neutralizer, signicantly prevented the cell death
induced by these strains of A. baumannii: 85.39  9.17% and
80.19  11.03% compared with 31.49  7.87% reduction relative to
controls, for ATCC 19606 strain, and 86.77  12.96% and
93.19  8.68% compared with 59.44  5.78% reduction relative to
controls, for 113-16 strain (Fig. 2D). To conrm the A549 cells
susceptibility to IL-6, treated A549 cell with human IL-6 (200 ng/
mL) induce a z40% of cell death. Altogether, these data indicate
that panresistant strain of A. baumannii induces the release of
proinammatory cytokines less than non-panresistant strain of
A. baumannii and these proinammatory cytokines mediate in part
the cell death caused by both strains.

3.3. A. baumannii-induced cell death is oxidative stress-dependent

In previous reports, it was demonstrated that gram-negative
bacteria like Pseudomonas aeruginosa and Helicobacter pylori
induces cell death via an increase in the formation of ROS [29,33].
Firstly, we showed that exposure of A549 cells to A. baumannii
(ATCC 19606 or 113-16) for 24 h induced a higher increase in the
liberation of O2 . for ATCC 19606 strain than 113-16 strain (Fig. 3A).
Incubation of A549 cells with 1 mM Trolox, a cell-permeable and
water-soluble derivative of a-tocopherol, prior to A. baumannii
infection signicantly reduced the liberation of O2 . (Fig. 3A). To
demostrate the involvement of ROS in A. baumannii-induced cell
death, we thus investigated the effect of Trolox. Incubation of A549
cells with 1 mM Trolox prior to infection with A. baumannii (ATCC
19606 or 113-16) induced a persistent increase in cell survival as
monitored by the MTT assay: 76.03  7.95% compared with
51.92  7.8% reduction relative to controls, and 82.11  16%
compared with 71.66  12.5% reduction relative to controls,
respectively (Fig. 3B). Taken together, these results indicate that
panresistant A. baumannii induces an oxidative stress-dependent
cell death lower than non-panresistant A. baumannii.
3.4. A. baumannii induces perturbation of cellular Ca2
homeostasis
A possible mechanism by which A. baumannii induces cell death
might depend on the perturbation of cellular Ca2 homeostasis as
demonstrated with other Gram-negative bacteria [14]. Therefore,

Please cite this article in press as: Smani Y, et al., Acinetobacter baumannii-induced lung cell death: Role of inammation, oxidative stress and
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Y. Smani et al. / Microbial Pathogenesis xxx (2011) 1e9

Fig. 2. Proinammatory cytokine involvement in the cell death induced by A. baumannii. A549 cells were cultured in the absence or presence of A. baumannii (113-16 or ATCC
19606). TNF-a and IL-6 concentrations were determined at 6 or 24 h in the presence or absence of 100 mg/mL thalidomide and 20 mg/mL PD098059, respectively (A,C). A. baumannii
cytotoxicity was determined using the MTT assay at 24 h in the presence or absence of 100 mg/mL thalidomide and 10 mg/mL anti-human TNF-a neutralizing antibody (Ab) (B) and
20 mg/mL PD098059 and 10 mg/mL anti-human IL-6 neutralizing Ab (D). As positive control, A549 cells were treated with CHX (5 mg/mL), TNF-a (25 ng/mL) alone or in combination
with CHX (5 mg/mL), and IL-6 (100 and 200 ng/mL). Data are the means  S.D. of three different experiments. P < 0.05: * between control and treated groups, y between treated
groups, # between treated groups vs. time, $ between 113-16 or ATCC 19606 vs. 113-16 PD0098059 or ATCC 19606 PD0098059.

we studied the effect of exposure to A. baumannii on the cytosolic

Ca2 levels in A549 cells. Cytosolic Ca2 levels were rst visualized
using the uorescent probe Fura-2/AM and labeling of control cells
revealed basal uorescence levels. After adding A. baumannii (ATCC
19606 or 113-16), the release of intracellular Ca2 increased

immediately (Dratio 0.2  0.09 and 0.5  0.18, respectively) and

was higher for 113-16 strain (Fig. 4A). Interestingly, pretreatment
with thapsigargin (TG) (2 mM), a Sarco(Endoplasmic)Ca2 ATP-ase
(SERCA) inhibitor, or CCCP (carbonyl cyanide m-chlorophenylhydrazone) (50 mM), a mitochondria protonophore, signicantly

Fig. 3. Oxidative stress involvement in the cell death induced by A. baumannii. A549 cells were cultured in the absence or presence of A. baumannii (113-16 or ATCC 19606). O$
2
concentration was determined for 6 or 24 h in presence or absence of 1 mM Trolox (A). A. baumannii cytotoxicity was determined using the MTT assay for 24 h in the presence or
absence of 1 mM Trolox (B). Data are the means  S.D. of three different experiments. P < 0.05: * between control and treated groups, y between treated groups, $ between ATCC
19606 vs. ATCC 19606 Trolox.

Please cite this article in press as: Smani Y, et al., Acinetobacter baumannii-induced lung cell death: Role of inammation, oxidative stress and
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Fig. 4. Increase in cytosolic Ca2 levels induced by A. baumannii. A549 cells were co-incubated with A. baumannii (113-16 or ATCC 19606) for 5 min. Intracellular Ca2 release were
monitored in untreated or pretreated A549 cells with 2 mM TG or 50 mM CCCP for 15 min (A). Ca2 inux was monitored in untreated or pretreated A549 cells with 50 nM NiCl2 for
15 min (B). Data are the means  S.D. of three different experiments. P < 0.05: * between control and treated groups, y between treated groups.

reduced the release of intracellular Ca2 induced by both strains of

A. baumannii (Fig. 4A). Moreover, A. baumannii (ATCC 19606 or 11316) triggered Ca2 inux (Dratio 0.99  0.33 and 1.14  0.37,
respectively) that was signicantly abolished by pretreatment with
nickel chloride (NiCl2) (50 nM), a blocker of cationic channels
(Fig. 4B). Altogether, these data support the idea that A. baumannii
induces a cytosolic Ca2 increasing: i) intracellular Ca2 release
from two major reservoirs like endoplasmic reticulum and mitochondria and ii) extracellular Ca2 inux. These data suggest the
involvement of a perturbation of cellular Ca2 homeostasis in
panresistant A. baumannii-induced cell death.

3.5. A. baumannii-induced cell death is calpain and caspase-3dependent

We next sought to determine whether A. baumannii-induced
apoptotic cell death is associated with the activation of calpain,
a cysteine activated by Ca2, and caspase-3. Calpain and caspase-3
activation during the cell death process induced by A. baumannii
was investigated by analysis of their proteolytic activities. 113-16
strain induce a signicant increase of calpain activation compared
with ATCC 19606 strain; meanwhile ATCC 19606 strain induced
a signicant increase of caspase-3 activation compared with 113-16
strain. In the lysates of A549 cells pretreated with MDL28170,
a calpain inhibitor, or Ac-DEVD-CHO, a caspase-3 inhibitor, and
infected with A. baumannii (ATCC 19606 or 113-16) for 24 h, calpain
and caspase-3 activities increased to 118.45  5.18% and
167.57  29.56% relative to controls, for ATCC 19606 strain and
122.99  4.14% and 136.51  12.67% relative to controls, for 113-16
strain. Interestingly, the activation of the both proteases induced by
these strains was completely inhibited in the presence of their
inhibitors, MDL28170 and Ac-DEVD-CHO (Fig. 5A,C). We thus
studied the effect of calpain and caspase-3 on the cell death
induced by A. baumannii. Preincubation of A549 cells with 50 mM
MDL28170 and 50 mM Ac-DEVD-CHO prior to infection with
A. baumannii (ATCC 19606 or 113-16) induced a persistent increase
in cell survival as monitored by the MTT assay: 72.62  12.42% and
71.24  8.86% compared with 48.02  3.02% reduction relative to
controls, for ATCC 19606 strain and 73.21  6.17% and 75.96  8.86%
compared with 59.01  4.24% reduction relative to controls, for 11316 strain (Fig. 5B,D). Taken together, these results indicate that

panresistant A. baumannii induces the activation of calpain and

caspase-3 with different rates compared with the non-panresistant
A. baumannii. These proteases are involved in the cell death induced
by A. baumannii.

4. Discussion
A. baumannii pneumonia poses an increased threat to hospitalized patients, as reected in the rising number of nosocomial
pneumonia caused by this bacterial species and the incidence of
mortality among infected individuals [1]. A. baumannii pneumonia
infection is characterized by various disorders in the lung epithelium including inammation, bronchitis and edema [34]. Alterations of epithelial cell growth and enhanced programmed cell
death may play a role in A. baumannii infection. In the light of the
high antimicrobial resistance of A. baumannii, knowledge about the
mechanisms responsible for these changes in the epithelial cells
remains unclear.
The results presented in this study provide the rst evidence of
the cytotoxicity of a panresistant strain of A. baumannii, which is
less intense than that of the non-panresistant strain. Indeed, panresistant and non-panresistant strain-induced cell death involved
an early perturbation of Ca2 homeostasis with susbsequent calpain and caspase-3 activations. By demonstrating that cell death
was prevented by the presence of TNF-a and IL-6 inhibitors and
neutralizing antibodies and an antioxidant, we have conrmed the
involvement of proinammatory cytokines and ROS generation in
the cell death induced by these strains.
Given the importance of inammation and oxidative stress in cell
death, it is therefore not surprising that A. baumannii, which induces
inammation and oxidative stress, contributes to the death of lung
epithelial cells. This could explain the lung injury observed in
experimental murine models and patients infected with A. baumannii [4,5,35]. The host inammatory and oxidative stress as
primary mediators of cytotoxicity induced by various pathogens
have also been described in other studies [29,33,36]. In our study, we
have showed that both cytokines TNF-a and IL-6 were involved in
the cell death induced by A. baumannii. TNF-a is the major extrinsic
mediator of the apoptosis, which binds to its specic receptor and
initiates the activation of the caspase 8, 10 and 3 [27]. Moreover, IL-6
has been shown to induce apoptosis mediating the modulated

Please cite this article in press as: Smani Y, et al., Acinetobacter baumannii-induced lung cell death: Role of inammation, oxidative stress and
cytosolic calcium, Microbial Pathogenesis (2011), doi:10.1016/j.micpath.2011.01.008

Y. Smani et al. / Microbial Pathogenesis xxx (2011) 1e9

Fig. 5. Calpain and caspase-3 activation and involvement in the cell death induced by A. baumannii. A549 cells were cultured for 24 h in the absence or presence of A. baumannii
(113-16 or ATCC 19606). Calpain and caspase-3 activation was investigated by proteolytic activities of calpain and caspase-3 in the absence or presence of their inhibitors 50 mM
MDL28170 and 50 mM Ac-DEVD-CHO, respectively (A,C). A. baumannii cytotoxicity was determined using the MTT assay for 24 h in the presence or absence of 50 mM MDL28170 and
50 mM Ac-DEVD-CHO (B,D). Data are the means  S.D. of three different experiments. P < 0.05: * between control and treated groups, y between treated groups, # between ATCC
19606 and 113-16.

expression of pro- or anti-apoptotic factors involved in the activation

of the intrinsic pathway of apoptosis [37e39]. The present study
does not address the link between inammation and oxidative
stress, but showed independently their involvement in the cell death
induced by A. baumannii. It is knowed that oxidative stress mediates
inammation-induced DNA damage [40]. TNF-a was reported to
induce the increase of ROS production from mitochondria and
plasma-membrane NADPH oxidase [41,42]. The use of radical scavengers sensitizes TNF-a-mediated cytotoxicity [43].
It is of particular interest to note that the Acinetobacter sp.
OmpA-like protein regulates the expression of other cytokines like
IL-8 [44]. Campylobacter and P. aeruginosa, other Gram-negative
bacteria, induce the release of IL-8, IL-6 and TNF-a that requires the
activation of the TLR family in epithelial cells and in monocytes
[45,46]. Recently, it was demonstrated that the expression of TLR-2
in epithelial cells was increased by the A. baumannii OmpA-like
protein [21]. Chun and Prince have found that P. aeruginosa induced
the activation of TLR-2 resulting in increased Ca2 inux and the
activation of calpain, both involved in the alterations of epithelial
barrier integrity [47]. In our study, we demonstrated that A. baumannii-induced the release of TNF-a and IL-6 and increased the
Ca2 inux.
In this study, we have demonstrated that A. baumannii induces
a perturbation of Ca2 homeostasis. Several lines of evidence
strongly support a role for alteration of cellular Ca2 homeostasis in

the pathogenesis of other Gram-negative bacteria such as P. aeruginosa and Neisseria gonorrheae [13,14]. Interestingly, it has been
demonstrated that mitochondria are the main target of an
increasing number of bacterial proteins including the A. baumannii
OmpA-like protein, which is transferred to the epithelial cells
during infection [10,48]. Mitochondria dysfunction occurs after cell
exposure to A. baumannii inducing the release of AIF and cytochrome c, suggesting a breakdown of the mitochondrial membrane
potential (DJm) which promotes cell death [10,48]. These data
support our ndings, in which, we have provided the rst
biochemical evidence of the intracelluar Ca2 release from the
mitochondria and the endoplasmic reticulum. As shown in Fig. 4A,
pretreatment with CCCP, a mitochondria protonophore, decreased
the release of intracellular Ca2-induced by A. baumannii. Thus, this
microogranism would induce the release of Ca2 from mitochondria by mediating the breakdown of DJm leading to the release of
pro-apoptotic factors. Consistent with other studies on the regulation of Gram-negative bacteria on the Ca2 inux in epithelial
cells [13,14], we found that the exposure of A549 cells to A. baumannii increased Ca2 inux. This increase was abolished by preincubation of A549 cells with NiCl2, a blocker of cationic channels
such as Store Operaterd Channels (SOCs).
It has suggested that elevated concentrations of intracellular free
Ca2 correlate with cell death [49]. It is not surprising, therefore, that
destabilization of cellular Ca2 homeostasis by bacterial infection

Please cite this article in press as: Smani Y, et al., Acinetobacter baumannii-induced lung cell death: Role of inammation, oxidative stress and
cytosolic calcium, Microbial Pathogenesis (2011), doi:10.1016/j.micpath.2011.01.008

Y. Smani et al. / Microbial Pathogenesis xxx (2011) 1e9

might also be an important trigger of cell death via activation of an

array of cellular enzymes, including the calpain system [14]. Interestingly, in our study we have shown that exposure of A549 cells to
non-panresistant panresistant A. baumannii induces the activation of
calpain and caspase-3, both involved in the cell death induced by
this microorganism. Schmeck et al., 2004 have found that calpain
mediated Streptococcus pneumoniae-induced apoptosis in A549 cells
[50]. Recently, it was found that P. aeruginosa signicantly increased
the calpain activity induced by Ca2 inux in epithelial cells and
mediated by TLR-2 resulting in the alterations of the epithelial
barrier integrity [47]. These events also occurred with A. baumannii
as demonstrated by our and another study [21].
Most of observations and data reported in this study have
demonstrated that panresistance of A. baumannii does not provide
any signicant cytotoxicity advantage when compared with a nonpanresistant strain. We suggest that the cause of this difference may
be due to the reduced rate of the panresistant strain adherence to
A549 cells in comparison with the non-panresistant strain (Fig. 1S),
whereas no signicant difference in growth in the extracellular
medium of A549 cells between both strains was observed (Fig. 2S).
Also, some antimicrobial resistance mechanisms present in the
panresistant strain, as the change in the Omps expression could
explained the difference in the cytotoxicity induced by both strains
of A. baumannii. The panresistant strain is resistant to imipenem and
sulbactam and expressed less OmpA-like, Omp 33e36 kDa, CarO and
OprD than ATCC 19606 strain (unpublished data from our laboratory). As mentioned previously, OmpA-like was shown to be involved
in the death of human respiratory epithelial cells.
5. Conclusions

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]
[16]

[17]
[18]

In summary, the present study suggests that the degree of

cytotoxicity caused by panresistant A. baumannii is lesser than that
of susceptible A. baumannii. Our results reveal that both strains of
A. baumannii caused proinammatory cytokine liberation, oxidative stress and cytosolic Ca2 increase appearing to activate calpain
Ca2-dependent and caspase-3 involved in cell death. Identication
of these pathways may lead to a broader understanding of the
pathogenic effect of A. baumannii during pneumonia infection.
Acknowledgments
We would like to thank T. Smani and P. Prez-Romero for
valuable help with the manuscript and to J. Vila for kindly provide
the non-pathogenic A. baumannii 77 strain. Y. Smani is funded by
the Spanish Network for Research in Infectious Diseases (REIPI
RD06/0008, Instituto de Salud Carlos III-FEDER, Ministerio de
Ciencia e Innovacin). This study was supported by a research grant
from the Consejera de Salud of the Junta de Andaluca (288/2008).

[19]

[20]

[21]

[22]

[23]

[24]

[25]

Appendix. Supplementary material

[26]

Supplementary data related to this article can be found online at

doi:10.1016/j.micpath.2011.01.008.

[27]
[28]

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cytosolic calcium, Microbial Pathogenesis (2011), doi:10.1016/j.micpath.2011.01.008