ell

. Mital V

Acinetobacter baumanniiPneumoniaPanresistantCell death

ce sthelathred

m-negver thnecity

mouse and guinea pig pneumonia models caused by A. baumannii[4,5]. Infection of human epithelial cells by A. baumannii wasassociated with adherence and invasion of this pathogen into thesecells [6e8] and induction of cellular death [9]. Two reports have

ways [10e12], similar to what has been observed in in vitromodelsof cytotoxicity with Omps of other Gram-negative bacteria [13,14].One of these Omps, PorB, induces an increased activity of calpainwhich is associated with impaired calcium (Ca2) homeostasis andresults in cell death [14]. However, it is likely that increased activityof calpain associated with deregulated Ca2 homeostasis may beinvolved in cell death-induced by A. baumannii. In addition, mito-chondrial perturbation has been demonstrated in epithelialcells co-incubated with the OmpA-like protein or with entire

* 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.

Contents lists availab

Microbial Pa

els

Microbial Pathogenesis xxx (2011) 1e9com (Y. Smani).with clinical importance [1]. Infections caused by A. baumannii havebecome very difcult to treat due to the emergence of strainsresistant to all or almost all antimicrobials used clinically [2]. Thedifculty of eradicating this microorganism has allowed it toacquire a state of highly successful human pathogen [3] that isinvolved in different serious infections including pneumonia,bacteremia and meningitis.

Lung inammation is one of the hallmarks of pneumoniainduced by A. baumannii resulting in epithelial barrier destruction.This lung inammation was observed previously by our group in

this cytotoxic effect with resistant clinical strains and compared itwith that produced by a susceptible strain.

Increasing evidence suggests that the cellular death induced byA. baumannii involve activation of caspases, which could participatein apoptosis through the initiation of intracellular pathways [9,10].In these studies, the intracellular pathways were not completelycharacterized and remained unclear. Indeed, some experimentalstudies indicate that an outer membrane protein (Omp) 38 ofA. baumannii, also called OmpA-like, is trafcked to both mito-chondria and the nucleus and induces eukaryotic cell death path-1. Introduction

Acinetobacter baumannii is a Gracoccobacillus which has emerged oorganism with questionable pathoge0882-4010/$ e see front matter 2011 Elsevier Ltd.doi:10.1016/j.micpath.2011.01.008

Please cite this article in press as: Smani Y, ecytosolic calcium, Microbial Pathogenesis (2Data presented here show that 113-16 and ATCC 19606 induce time-dependent cell death of lungepithelial cells involving a perturbation of cytosolic calcium homeostasis with subsequent calpain andcaspase-3 activation. Prevention of this cell death by TNF-a and interleukin-6 blockers and antioxidanthighlights 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 isnoteworthy to stress that some mechanisms are less induced by the panresistant strain.

2011 Elsevier Ltd. All rights reserved.

ative non-fermentativee last decades from anto an infectious agent

indicated that A. baumannii induces the activation of caspase-9,caspase-8 and caspase-3, and the liberation of apoptosis inducingfactor (AIF) and cytochrome c, both leading to caspase-dependentpathways in epithelial cells [9,10]. Despite the evidence thatA. baumannii induces cell death, studies have never investigatedKeywords:induced by other Gram-negative bacteria, we investigated whether these intracellular targets wereinvolved in the cell death induced by clinical panresistant 113-16 and susceptible ATCC 19606 strains.Available online xxx proinammatory cytokine release, oxidative stress and cytosolic calcium increase in the cell death-Acinetobacter baumannii-induced lung cstress and cytosolic calcium

Younes Smani*, Fernando Docobo-Prez, Michael JService of Infectious Diseases, Institute of Biomedicine of Sevilla (IBIS), University Hosp

a r t i c l e i n f o

Article history:Received 16 December 2010Received in revised form21 January 2011Accepted 24 January 2011

a b s t r a c t

A growing body of eviden19606 induces human epiassociated with this cell depanresistant strain compa

journal homepage: www.All rights reserved.

t al., Acinetobacter baumanni011), doi:10.1016/j.micpath.2death: Role of inammation, oxidative

cConnell, Jernimo Pachnirgen del Roco/CSIC/University of Sevilla, Av. Manuel Siurot s/n, 41013 Sevilla, Spain

upports the notion that susceptible Acinetobacter baumannii strain ATCCial cells death. However, most of the cellular and molecular mechanismsremain unknown, and also the degree of the cytotoxic effects of a clinicalwith a susceptible strain has never been studied. Due to the role of

le at ScienceDirect

thogenesis

evier .com/locate/micpathi-induced lung cell death: Role of inammation, oxidative stress and011.01.008

tion of live and dead cells was achieved with the LIVE/DEAD kit

113-16) in the presence or absence of thalidomide (100 mg/mL) or

presence or absence of Trolox (1 mM), the cells were washed three

PathA. 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 therelease of intracellular Ca2 and consequently the activation ofcalpain. In addition, calpain or their proteolytic products have beenshown to act as effectors of cell death [17]. These observationsstrongly support the involvement of calpain Ca2-dependent in thecell death induced by A. baumannii.

During lung infection by bacteria, epithelium is one of the rsttargets and plays a critical role in the release of proinammatorymediators which can kill host cells and damage host tissues [18]. Inlung epithelial cells, the tumor necrosis factor alpha (TNF-a)receptor can be activated directly by Staphylococcus aureus [19]. Inthe case of A. baumannii, puried OmpA-like elicits a type 1 T helper(Th1)-mediated immune response [20] and upregulates induciblenitric oxide synthase (iNOS) [21].

In addition, the overexpression of a variety of proinammatorycytokines including TNF-a, interleukin 1 (IL-1) and interleukin 6(IL-6) have been involved in cellular death [22e24]. Inhibitingproinammatory signals can be protective during lung infections.For example, interrupting both TNF-a and IL-1 signaling decreasespulmonary edema and the loss of lung compliance that are oftenfound in mice with Escherichia coli pneumonia [25,26]. In thisregard, it was hypothetized that the increased release of proin-ammatory cytokines may play a role in the cell death-induced byA. baumannii.

Accumulative evidence has put emphasis on the critical role ofoxidative stress in cell death (for review see Ref. [27]). Indeed, therole of reactive oxygen species (ROS) production and the subse-quent cell death induced by various bacteria has been established[28,29]. However, ROS involvement in the cell death induced byA. baumannii has not yet been characterized.

The aim of this study is to determine the different mediatorsinvolved in the cell death produced by the panresistant strain. Thedata presented here propose for the rst time the involvement ofcalpain and Ca2 dependence in the cell death induced by bothstrains and demonstrate the involvement of proinammatorycytokines and oxidative stress in this cell death.

2. Materials and methods

2.1. Bacterial strains

The reference A. baumannii strain ATCC 19606, susceptible to allantimicrobials, the clinical panresistant A. baumannii isolate 113-16[30], and the non-pathogenic strain 77 were used in this study. Allthe strains were grown in Mueller Hinton Broth (MHB) at 37 C for20e24 h. The cultured bacterial strains were washed with phos-phate-buffered saline (PBS) and resuspended in DMEM prior to usein cell culture experiments.

2.2. Human cell culture and infection

The type II pneumocyte cell lineA549derived fromahuman lungcarcinomawere a gift fromDr. Felipe Fernandez-Cuenca and grownin 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 ina humidied, 5% CO2 at 37 C. A549 cells were routinely passagedevery 3e4 days. The cells were seeded 24 h in 96 well plates (for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT)and lactate dehydrogenase (LDH) assays), in 6 well plates (forenzymatic activities and western blot experiments) and in 24 wellplates (for the rest of experiments) prior to infection with A. bau-

Y. Smani et al. / Microbial2mannii (ATCC 19606 or 113-16) at amultipilicty of infection (MOI) of

Please cite this article in press as: Smani Y, et al., Acinetobacter baumannicytosolic calcium, Microbial Pathogenesis (2011), doi:10.1016/j.micpath.2times with prewarmed PBS and incubated with 0.5 mL of reactionmixture consisting of 150 mM oxidized (Fe3) cytochrome c inEDTA (100 mM) and sodium phosphate buffer (50 mM; pH 7.5) at37 C for 1 h. After this incubation, the supernatants were collectedand used to quantify the amount of reduced cytochrome c byabsorbance at 550 nm. The O . release was calculated using thePD098059 (20 mg/mL), supernatants were removed and storedat 80 C. TNF-a and IL-6 levels were measured using an ELISA kit(R&D Systems) according to the manufacturers instructions. Forthe assessment of cellular viability by anMTTassay, A549 cells werepretreated with thalidomide (100 mg/mL), anti-human TNF-a neutralizing antibody (10 mg/mL), PD098059 (20 mg/mL) and anti-human IL-6 neutralizing antibody (10 mg/mL), washed three timeswith prewarmed PBS and infected with A. baumannii (ATCC 19606or 113-16) or treated with 5 mg/mL of cycloheximide (CHX) (Sigma,Spain), 20 ng/mL of human TNF-a (Invitrogen, Spain) alone or incombination with 5 mg/mL of CHX, and 200 ng/mL of human IL-6(Invitrogen, Spain).

2.5. Superoxide anion assay

Superoxide anion (O2 .) production was assayed by spectro-photometric measurement of ferricytochrome c reduction asdescribed previously [31]. A549 cells were pretreated for 30 minwith an antioxidant, Trolox (1 mM) (Sigma, Spain). After infectionof A549 cells with A. baumannii (ATCC 19606 or 113-16) in theaccording to the manufacturers instructions (Invitrogen, Spain).To monitor apoptosis, cell nuclei were visualized using 40,6-

diamidino-2-phenylindole (DAPI). A549 cells, grown on glasscoverslips in 24 well plates, were washed with cold PBS, xed inmethanol for 8 min at 20 C, incubated for 10 min at roomtemperature with DAPI (0.5 mg/mL), washed with PBS, mountedwith SlowFade Gold antiFade reagent (Invitrogen, Spain), andvisualized using a Leica uorescence microscope (DM-6000; LeicaMicrosystems Wetzlar GmbH, Germany).

For necrosis determination, LDH activity in the supernatantswas 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 30minwith thalidomide (100 mg/mL), an inhibitor of TNF-a and PD098059 (20 mg/mL), an inhibitor ofIL-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) (eBio-science, USA). After infection with A. baumannii (ATCC 19606 or100. Immediately before infection, A549 cells were washed threetimeswith prewarmedPBS and further incubated inDMEMwithoutFBS and antibiotics, and then A. baumannii strains was added gentlyto A549 cells and incubated for 6,10 and 24h in a humidied, 5% CO2at 37 C.

2.3. Cellular viability, apoptosis and necrosis

A549 cells were infectedwith A. baumannii (ATCC 19606,113-16,or 77) for the indicated times (6, 10 and 24 h). A. baumanniicytotoxicity was initially assessed quantitatively by monitoringmitochondrial reduction activity using the MTT assay (Sigma,Spain) as described previously [31]. The simultaneous determina-

ogenesis xxx (2011) 1e92ferricytochrome c extinction coefcient (21.1 mM.cm1).

i-induced lung cell death: Role of inammation, oxidative stress and011.01.008

Path2.6. Intracellular Ca2 measurement

Fluorescence was monitored using a Nikon TS-100 invertedmicroscope equipped with a 20 uor objective (0.75 NA) asdescribed previously [32]. A549 cells plated on coverslips in 24wellplates and mounted in a Teon chamber were incubated in DMEMwith 2 mM fura-2/AM (Invitrogen, Spain) for 30 min at 37 C, andwashed with PBS. Fluorescence images of 20e30 cells were recor-ded and analyzed with a digital uorescence imaging system (InCytIm2, Intracellular Imaging Inc., Imsol, UK) equipped with a light-sensitive CCD camera (Cooke PixelFly, ASI, Eugene, OR, USA).Changes in intracellular Ca2 are represented as the ratio of fura-2uorescence induced at an emission wavelength of 510 nm due toexcitation at 340 and 380 nm (ratio F340/F380). Experiments weredone in 0 Ca2 solution (in mM: 120 NaCl, 4.7 KCl, 4 MgCl2, 0.2EGTA,10 Hepes). The change in cytsolic Ca2 release wasmonitored5 min before and 15 min after addition of A. baumannii onto A549cells. This change was determinated by the Dratio calculated as thedifference between the peak ratio after addition of A. baumannii(ATCC 19606 or 113-16) and its level right before A. baumanniiaddition. Ca2 inux was determined from changes in fura-2/AMuorescence after re-addition of Ca2 (2 mM) 5 min after additionof A. baumannii onto A549 cells. Dratio was calculated as thedifference between the peak ratio after extracellular Ca2 wasadded and its level right before Ca2 addition.

2.7. Measurement of calpain and caspase-3-like proteolyticactivities

The calpain and caspase-3 activities were measured by cleavageof the substrates succinyl-Leu-Tyr-AMC and Ac-DEVD-AMC(Bachem, Germany). Briey, A549 cells were seeded in 6 well platesand cultured overnight. At 24 h after infection with A. baumannii(ATCC 19606 or 113-16) in presence or absence of calpain andcaspase-3 inhibitors, 50 mM MDL28170 and 50 mM Ac-DEVD-CHO(Bachem, Germany), A549 cells were collected, homogenized inRIPA buffer supplemented with 1 mM PMSF and 10% cocktail ofprotease inhibitors (Sigma) and centrifuged at 20 000 g for 15 minat 4 C. The supernatant was removed and the amount of proteinswas determined using the Bio-Rad protein dye reagent (Bradfordmethod). The samples were stored at80 C until later use. Fifty mgof proteins were incubated for 2 h with 100 mM succinyl-Leu-Tyr-AMC and 100 mM Ac-DEVD-AMC substrates initially dissolved inMe2SO. The cleavage of the calpain and caspase-3 substrates wasmonitored by uorescence emission at 460 nm after excitation at360 nm using a Fluorescence microplates reader (Berthold Tech-nologies GmbH & Co, Germany).

2.8. Statistical analysis

Group data are presented as mean S.D. The ANOVA test andpost-hoc tests (Tukey and Dunnet) were used to determine thedifference between means. The difference was considered signi-cant 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 pan-resistant strain 113-16 and susceptible strain ATCC 19606 usingA549 cells. Incubation of A549 cells with A. baumannii resulted ina time and dose-dependent decrease in cell viability as monitoredusing the MTT assay. The decrease in cellular viability upon

Y. Smani et al. / MicrobialA. baumannii exposure was signicant after a 6 and 24 h incubation

Please cite this article in press as: Smani Y, et al., Acinetobacter baumannicytosolic calcium, Microbial Pathogenesis (2011), doi:10.1016/j.micpath.2with ATCC 19606 and 113-16, respectively. In the case of exposureto the ATCC 19606 strain, the decrease in cell viability reachedlevels of 68.89 6.44%, 59.88 10.99% and 39.5 0.43% (relative tocontrols) after 6, 10 and 24 h of exposure. In the case of the 113-16strain, the decrease in cell viability reached levels of 69.48 12.38%(relative to controls) after 24 h of exposure (Fig. 1A). In contrast, thenon-pathogenic A. baumannii 77 showed a decrease in cell viabilityto 89.35 2.44% after 24 h of exposure. Moreover, 24 h exposure ofA549 cells with A. baumannii (ATCC 19606 or 113-16) showed thatthe decrease in cell viability was higher for ATCC 19606 strain than113-16 strain: 39.5 0.43% vs. 69.48 12.38%, relative to controls.Furthermore, a 24 h exposure of A549 cells to A. baumannii (ATCC19606 or 113-16) at MOI from 0.001 to 100 induced only a signi-cant reduction in cellular viability for A. baumannii (ATCC 19606 or113-16) at MOI of 100 (data not shown).

The cytotoxicityofA. baumanniiwasalso investigatedusing a two-color uorescence cell viability assay. In contrast to intense anduniform green uorescence based on intracellular esterase activitydisplayed by control A549 cells, cells exposed for 24 h to ATCC 19606strains were stained mainly with ethidium homodimer-1, producinga bright reduorescence indicative of dead cells than in113-16 strain.In contrast, little few dead cellswere observed after infection of A549cells with A. baumannii 77 (Fig. 1B). Additionally, morphologicalexamination of cellular nuclei stained with DAPI showed that A549cells exposed to A. baumannii presented typical apoptoticmorphology, with condensation of chromatin and fragmentation ofthe nuclei for ATCC 19606 and 113-16 strains (Fig. 1C). Indeed, A549cells exposed to ATCC 19606 strain and 113-16 strain for 24 hexhibited a strain-dependent increase in the number of apoptoticnuclei in the case of ATCC 19606 strain,whereas the 77 strain and thecontrol cells presented no signicant apoptotic nuclei. Moreover,while low levels (5.98 1.81%) of LDH liberation, a necrosis marker,were detected in infectedA549 cellswithA. baumannii77, the level ofLDH liberation strongly increased to 20.95 14.32% and52.96 15.39% (relative to controls) induced by 113-16 strain andATCC19606 strain, respectively (Fig.1D). Taken together, these resultsshowed that panresistant A. baumannii induces cell death of A549cells but with low intensitywhen comparedwith a non-panresistantA. baumannii. We therefore selected a range of exposure times cor-responding to 24 h with both A. baumannii strains at MOI of 100 tostudy molecular pathways and the associated intracellular events inA. 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 beinvolved in A. baumannii-induced cell death by monitoring thesensitivity of TNF-a and IL-6 liberation to specic inhibitors duringcell death. None of these tested inhibitors showed defect of growthand adherence/invasion of ATCC 19606 and 113-16 strains to A549cells, excluding the possibility of differential cell death due todifferential growth and adherence/invasion rate (data not shown).The TNF-a assay showed that 113-16 strain induces the release ofTNF-a less than ATCC 19606 strain at 6 h of exposure with thesestrains (51.82 31.56 pg/mL vs. 198.21 87.38 pg/mL), whereas at24 h none of both strains induced a signicative release of TNF-a (16.27 6.21 pg/mL vs. 19.36 10.86 pg/mL). Pretreatment withthalidomide (100 mg/mL) decreased the liberation of TNF-a inducedby exposure of A549 cells with A. baumannii (ATCC 19606 or 113-16) for 6 h (Fig. 2A). Interestingly, the preincubation of A549 cellswith thalidomide, a non specic TNF-a inhibitor, and anti-humanTNF-a (10 mg/mL) neutralizing antibody, a specic TNF-a neutral-izer, prevented the cell death induced by both strains of A. bau-mannii as monitored by the MTT assay: 79.16 6.96% and

ogenesis xxx (2011) 1e9 380.14 7.44% compared with 31.49 7.87% reduction relative

i-induced lung cell death: Role of inammation, oxidative stress and011.01.008

PathY. Smani et al. / Microbial4to controls, for ATCC 19606 strain, and 79.75 6.28% and84.02 15.96% compared with 59.44 5.78% reduction relative tocontrols, for 113-16 strain (Fig. 2B). However, induction of cell deathby TNF-a in A549 cells required CHX treatment, an inhibitor ofeukaryotic protein synthesis (Fig. 2B).

In the case of IL-6, both A. baumannii strains induceda 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 at24 h (Fig. 2C). Similarly, having demonstrated that TNF-a isinvolved in the cell death induced by both A. baumannii strains, wealso examined the involvement of IL-6 in the cell death produced bythese strains. The presence of PD098059, a non specic IL-6inhibitor, and anti-human IL-6 neutralizing antibody (10 mg/mL),a specic IL-6 neutralizer, signicantly prevented the cell deathinduced by these strains of A. baumannii: 85.39 9.17% and80.19 11.03% compared with 31.49 7.87% reduction relative tocontrols, for ATCC 19606 strain, and 86.77 12.96% and93.19 8.68% compared with 59.44 5.78% reduction relative tocontrols, for 113-16 strain (Fig. 2D). To conrm the A549 cellssusceptibility to IL-6, treated A549 cell with human IL-6 (200 ng/mL) induce a z40% of cell death. Altogether, these data indicatethat panresistant strain of A. baumannii induces the release ofproinammatory cytokines less than non-panresistant strain ofA. baumannii and these proinammatory cytokines mediate in partthe cell death caused by both strains.

Fig. 1. Apoptotic and necrotic cellular death induced by A. baumannii. A549 cells were culturcytotoxicity was determined by monitoring the mitochondrial reduction activity using therepresentative microscopic elds of the two-color uorescence cell viability assay (LIVE/DEillustrated here with representative microscopic elds. Cell nuclei are stained with DAPI (bludetection assay (D). The positive control was 10 mg/mL staurosporine (Str). Data are the meawithout treatment, designated as 100%). P < 0.05: * between control and treated groups,legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Smani Y, et al., Acinetobacter baumannicytosolic calcium, Microbial Pathogenesis (2011), doi:10.1016/j.micpath.2ogenesis xxx (2011) 1e93.3. A. baumannii-induced cell death is oxidative stress-dependent

In previous reports, it was demonstrated that gram-negativebacteria like Pseudomonas aeruginosa and Helicobacter pyloriinduces 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 theliberation of O2 . for ATCC 19606 strain than 113-16 strain (Fig. 3A).Incubation of A549 cells with 1 mM Trolox, a cell-permeable andwater-soluble derivative of a-tocopherol, prior to A. baumanniiinfection signicantly reduced the liberation of O2 . (Fig. 3A). Todemostrate the involvement of ROS in A. baumannii-induced celldeath, we thus investigated the effect of Trolox. Incubation of A549cells with 1 mM Trolox prior to infection with A. baumannii (ATCC19606 or 113-16) induced a persistent increase in cell survival asmonitored by the MTT assay: 76.03 7.95% compared with51.92 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 thatpanresistant A. baumannii induces an oxidative stress-dependentcell death lower than non-panresistant A. baumannii.

3.4. A. baumannii induces perturbation of cellular Ca2

homeostasis

A possible mechanism bywhich A. baumannii induces cell deathmight depend on the perturbation of cellular Ca2 homeostasis asdemonstrated with other Gram-negative bacteria [14]. Therefore,

ed in the absence or presence of A. baumannii (113-16, ATCC 19606, or 77). A. baumanniiMTT assay for 6, 10 or 24 h (A). A. baumannii cytotoxicity was next illustrated withAD viability/cytotoxicity kit) for 24 h (B). Cellular apoptosis at 24 h (white arrows) ise) (C). Necrosis at 24 h was determined by following LDH release using the cytotoxicityns S.D. of three different experiments, and are normalized to the control (A549 cellsy treated groups vs. time. (For interpretation of the references to colour in this gure

i-induced lung cell death: Role of inammation, oxidative stress and011.01.008

A B

C D

Y. Smani et al. / Microbial Pathogenesis xxx (2011) 1e9 5we studied the effect of exposure to A. baumannii on the cytosolicCa2 levels in A549 cells. Cytosolic Ca2 levels were rst visualizedusing the uorescent probe Fura-2/AM and labeling of control cellsrevealed basal uorescence levels. After adding A. baumannii (ATCC19606 or 113-16), the release of intracellular Ca2 increased

A

Fig. 3. Oxidative stress involvement in the cell death induced by A. baumannii. A549 cells wconcentration was determined for 6 or 24 h in presence or absence of 1 mM Trolox (A). A. babsence of 1 mM Trolox (B). Data are the means S.D. of three different experiments. P < 019606 vs. ATCC 19606 Trolox.

Fig. 2. Proinammatory cytokine involvement in the cell death induced by A. baumannii. A19606). TNF-a and IL-6 concentrations were determined at 6 or 24 h in the presence or absencytotoxicity was determined using the MTT assay at 24 h in the presence or absence of 100 m20 mg/mL PD098059 and 10 mg/mL anti-human IL-6 neutralizing Ab (D). As positive control, Awith CHX (5 mg/mL), and IL-6 (100 and 200 ng/mL). Data are the means S.D. of three diffgroups, # between treated groups vs. time, $ between 113-16 or ATCC 19606 vs. 113-16 P

Please cite this article in press as: Smani Y, et al., Acinetobacter baumannicytosolic calcium, Microbial Pathogenesis (2011), doi:10.1016/j.micpath.2immediately (Dratio 0.2 0.09 and 0.5 0.18, respectively) andwas higher for 113-16 strain (Fig. 4A). Interestingly, pretreatmentwith thapsigargin (TG) (2 mM), a Sarco(Endoplasmic)Ca2 ATP-ase(SERCA) inhibitor, or CCCP (carbonyl cyanide m-chlorophenylhy-drazone) (50 mM), a mitochondria protonophore, signicantly

B

ere cultured in the absence or presence of A. baumannii (113-16 or ATCC 19606). O2$

aumannii cytotoxicity was determined using the MTT assay for 24 h in the presence or.05: * between control and treated groups, y between treated groups, $ between ATCC

549 cells were cultured in the absence or presence of A. baumannii (113-16 or ATCCce of 100 mg/mL thalidomide and 20 mg/mL PD098059, respectively (A,C). A. baumanniig/mL thalidomide and 10 mg/mL anti-human TNF-a neutralizing antibody (Ab) (B) and549 cells were treated with CHX (5 mg/mL), TNF-a (25 ng/mL) alone or in combination

erent experiments. P < 0.05: * between control and treated groups, y between treatedD0098059 or ATCC 19606 PD0098059.

i-induced lung cell death: Role of inammation, oxidative stress and011.01.008

nickel chloride (NiCl2) (50 nM), a blocker of cationic channels(Fig. 4B). Altogether, these data support the idea that A. baumannii

mortality among infected individuals [1]. A. baumannii pneumoniainfection is characterized by various disorders in the lung epithe-

ubat(A)een

Pathinduces a cytosolic Ca2 increasing: i) intracellular Ca2 releasefrom two major reservoirs like endoplasmic reticulum and mito-chondria and ii) extracellular Ca2 inux. These data suggest theinvolvement of a perturbation of cellular Ca2 homeostasis inpanresistant A. baumannii-induced cell death.

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

We next sought to determine whether A. baumannii-inducedreduced the release of intracellular Ca2 induced by both strains ofA. baumannii (Fig. 4A). Moreover, A. baumannii (ATCC 19606 or 113-16) triggered Ca2 inux (Dratio 0.99 0.33 and 1.14 0.37,respectively) that was signicantly abolished by pretreatment with

A

Fig. 4. Increase in cytosolic Ca2 levels induced by A. baumannii. A549 cells were co-incmonitored in untreated or pretreated A549 cells with 2 mM TG or 50 mM CCCP for 15 min15 min (B). Data are the means S.D. of three different experiments. P < 0.05: * betw

Y. Smani et al. / Microbial6apoptotic cell death is associated with the activation of calpain,a cysteine activated by Ca2, and caspase-3. Calpain and caspase-3activation during the cell death process induced by A. baumanniiwas investigated by analysis of their proteolytic activities. 113-16strain induce a signicant increase of calpain activation comparedwith ATCC 19606 strain; meanwhile ATCC 19606 strain induceda signicant increase of caspase-3 activation compared with 113-16strain. In the lysates of A549 cells pretreated with MDL28170,a calpain inhibitor, or Ac-DEVD-CHO, a caspase-3 inhibitor, andinfected with A. baumannii (ATCC 19606 or 113-16) for 24 h, calpainand caspase-3 activities increased to 118.45 5.18% and167.57 29.56% relative to controls, for ATCC 19606 strain and122.99 4.14% and 136.51 12.67% relative to controls, for 113-16strain. Interestingly, the activation of the both proteases induced bythese strains was completely inhibited in the presence of theirinhibitors, MDL28170 and Ac-DEVD-CHO (Fig. 5A,C). We thusstudied the effect of calpain and caspase-3 on the cell deathinduced by A. baumannii. Preincubation of A549 cells with 50 mMMDL28170 and 50 mM Ac-DEVD-CHO prior to infection withA. baumannii (ATCC 19606 or 113-16) induced a persistent increasein cell survival as monitored by the MTT assay: 72.62 12.42% and71.24 8.86% compared with 48.02 3.02% reduction relative tocontrols, for ATCC 19606 strain and 73.216.17% and 75.96 8.86%comparedwith 59.014.24% reduction relative to controls, for 113-16 strain (Fig. 5B,D). Taken together, these results indicate that

Please cite this article in press as: Smani Y, et al., Acinetobacter baumannicytosolic calcium, Microbial Pathogenesis (2011), doi:10.1016/j.micpath.2lium including inammation, bronchitis and edema [34]. Alter-ations of epithelial cell growth and enhanced programmed celldeath may play a role in A. baumannii infection. In the light of thehigh antimicrobial resistance of A. baumannii, knowledge about thepanresistant A. baumannii induces the activation of calpain andcaspase-3 with different rates compared with the non-panresistantA. baumannii. These proteases are involved in the cell death inducedby A. baumannii.

4. Discussion

A. baumannii pneumonia poses an increased threat to hospi-talized patients, as reected in the rising number of nosocomialpneumonia caused by this bacterial species and the incidence of

B

ed with A. baumannii (113-16 or ATCC 19606) for 5 min. Intracellular Ca2 release were. Ca2 inux was monitored in untreated or pretreated A549 cells with 50 nM NiCl2 forcontrol and treated groups, y between treated groups.

ogenesis xxx (2011) 1e9mechanisms responsible for these changes in the epithelial cellsremains unclear.

The results presented in this study provide the rst evidence ofthe cytotoxicity of a panresistant strain of A. baumannii, which isless intense than that of the non-panresistant strain. Indeed, pan-resistant and non-panresistant strain-induced cell death involvedan early perturbation of Ca2 homeostasis with susbsequent cal-pain and caspase-3 activations. By demonstrating that cell deathwas prevented by the presence of TNF-a and IL-6 inhibitors andneutralizing antibodies and an antioxidant, we have conrmed theinvolvement of proinammatory cytokines and ROS generation inthe cell death induced by these strains.

Given the importance of inammation and oxidative stress in celldeath, it is therefore not surprising that A. baumannii, which inducesinammation and oxidative stress, contributes to the death of lungepithelial cells. This could explain the lung injury observed inexperimental murine models and patients infected with A. bau-mannii [4,5,35]. The host inammatory and oxidative stress asprimary mediators of cytotoxicity induced by various pathogenshave also been described in other studies [29,33,36]. In our study, wehave showed that both cytokines TNF-a and IL-6 were involved inthe cell death induced by A. baumannii. TNF-a is the major extrinsicmediator of the apoptosis, which binds to its specic receptor andinitiates the activation of the caspase 8, 10 and 3 [27]. Moreover, IL-6has been shown to induce apoptosis mediating the modulated

i-induced lung cell death: Role of inammation, oxidative stress and011.01.008

PathA

B

Y. Smani et al. / Microbialexpression of pro- or anti-apoptotic factors involved in the activationof the intrinsic pathway of apoptosis [37e39]. The present studydoes not address the link between inammation and oxidativestress, but showed independently their involvement in the cell deathinduced by A. baumannii. It is knowed that oxidative stress mediatesinammation-induced DNA damage [40]. TNF-a was reported toinduce the increase of ROS production from mitochondria andplasma-membrane NADPH oxidase [41,42]. The use of radical scav-engers 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 likeIL-8 [44]. Campylobacter and P. aeruginosa, other Gram-negativebacteria, induce the release of IL-8, IL-6 and TNF-a that requires theactivation of the TLR family in epithelial cells and in monocytes[45,46]. Recently, it was demonstrated that the expression of TLR-2in epithelial cells was increased by the A. baumannii OmpA-likeprotein [21]. Chun and Prince have found that P. aeruginosa inducedthe activation of TLR-2 resulting in increased Ca2 inux and theactivation of calpain, both involved in the alterations of epithelialbarrier integrity [47]. In our study, we demonstrated that A. bau-mannii-induced the release of TNF-a and IL-6 and increased theCa2 inux.

In this study, we have demonstrated that A. baumannii inducesa perturbation of Ca2 homeostasis. Several lines of evidencestrongly support a role for alteration of cellular Ca2 homeostasis in

Fig. 5. Calpain and caspase-3 activation and involvement in the cell death induced by A. ba(113-16 or ATCC 19606). Calpain and caspase-3 activation was investigated by proteolytic aMDL28170 and 50 mM Ac-DEVD-CHO, respectively (A,C). A. baumannii cytotoxicity was deter50 mM Ac-DEVD-CHO (B,D). Data are the means S.D. of three different experiments. P < 019606 and 113-16.

Please cite this article in press as: Smani Y, et al., Acinetobacter baumannicytosolic calcium, Microbial Pathogenesis (2011), doi:10.1016/j.micpath.2C

D

ogenesis xxx (2011) 1e9 7the pathogenesis of other Gram-negative bacteria such as P. aeru-ginosa and Neisseria gonorrheae [13,14]. Interestingly, it has beendemonstrated that mitochondria are the main target of anincreasing number of bacterial proteins including the A. baumanniiOmpA-like protein, which is transferred to the epithelial cellsduring infection [10,48]. Mitochondria dysfunction occurs after cellexposure to A. baumannii inducing the release of AIF and cyto-chrome c, suggesting a breakdown of the mitochondrial membranepotential (DJm) which promotes cell death [10,48]. These datasupport our ndings, in which, we have provided the rstbiochemical evidence of the intracelluar Ca2 release from themitochondria and the endoplasmic reticulum. As shown in Fig. 4A,pretreatment with CCCP, a mitochondria protonophore, decreasedthe release of intracellular Ca2-induced by A. baumannii. Thus, thismicroogranism would induce the release of Ca2 from mitochon-dria by mediating the breakdown of DJm leading to the release ofpro-apoptotic factors. Consistent with other studies on the regu-lation of Gram-negative bacteria on the Ca2 inux in epithelialcells [13,14], we found that the exposure of A549 cells to A. bau-mannii increased Ca2 inux. This increase was abolished by pre-incubation of A549 cells with NiCl2, a blocker of cationic channelssuch as Store Operaterd Channels (SOCs).

It has suggested that elevated concentrations of intracellular freeCa2 correlate with cell death [49]. It is not surprising, therefore, thatdestabilization of cellular Ca2 homeostasis by bacterial infection

umannii. A549 cells were cultured for 24 h in the absence or presence of A. baumanniictivities of calpain and caspase-3 in the absence or presence of their inhibitors 50 mMmined using the MTT assay for 24 h in the presence or absence of 50 mMMDL28170 and.05: * between control and treated groups, y between treated groups, # between ATCC

i-induced lung cell death: Role of inammation, oxidative stress and011.01.008

Pathmight also be an important trigger of cell death via activation of anarray of cellular enzymes, including the calpain system [14]. Inter-estingly, in our study we have shown that exposure of A549 cells tonon-panresistant panresistant A. baumannii induces the activation ofcalpain and caspase-3, both involved in the cell death induced bythis microorganism. Schmeck et al., 2004 have found that calpainmediated Streptococcus pneumoniae-induced apoptosis in A549 cells[50]. Recently, it was found that P. aeruginosa signicantly increasedthe calpain activity induced by Ca2 inux in epithelial cells andmediated by TLR-2 resulting in the alterations of the epithelialbarrier integrity [47]. These events also occurred with A. baumanniias demonstrated by our and another study [21].

Most of observations and data reported in this study havedemonstrated that panresistance of A. baumannii does not provideany signicant cytotoxicity advantage when compared with a non-panresistant strain. We suggest that the cause of this difference maybe due to the reduced rate of the panresistant strain adherence toA549 cells in comparison with the non-panresistant strain (Fig. 1S),whereas no signicant difference in growth in the extracellularmedium of A549 cells between both strains was observed (Fig. 2S).Also, some antimicrobial resistance mechanisms present in thepanresistant strain, as the change in the Omps expression couldexplained the difference in the cytotoxicity induced by both strainsof A. baumannii. The panresistant strain is resistant to imipenem andsulbactam and expressed less OmpA-like, Omp 33e36 kDa, CarO andOprD than ATCC 19606 strain (unpublished data from our labora-tory). Asmentioned previously, OmpA-likewas shown to be involvedin the death of human respiratory epithelial cells.

5. Conclusions

In summary, the present study suggests that the degree ofcytotoxicity caused by panresistant A. baumannii is lesser than thatof susceptible A. baumannii. Our results reveal that both strains ofA. baumannii caused proinammatory cytokine liberation, oxida-tive stress and cytosolic Ca2 increase appearing to activate calpainCa2-dependent and caspase-3 involved in cell death. Identicationof these pathways may lead to a broader understanding of thepathogenic effect of A. baumannii during pneumonia infection.

Acknowledgments

We would like to thank T. Smani and P. Prez-Romero forvaluable help with the manuscript and to J. Vila for kindly providethe non-pathogenic A. baumannii 77 strain. Y. Smani is funded bythe Spanish Network for Research in Infectious Diseases (REIPIRD06/0008, Instituto de Salud Carlos III-FEDER, Ministerio deCiencia e Innovacin). This study was supported by a research grantfrom the Consejera de Salud of the Junta de Andaluca (288/2008).

Appendix. Supplementary material

Supplementary data related to this article can be found online atdoi:10.1016/j.micpath.2011.01.008.

References

[1] Munoz-Price LS, Weinstein RA. Acinetobacter infection. N Engl J Med2008;358:1271e81.

[2] Hsueh PR, Teng LJ, Chen CY, Chen WH, Yu CJ, Ho SW, et al. Pandrug-resistantAcinetobacter baumannii causing nosocomial infections in a university hospitalTaiwan. Emerg Infect Dis 2002;8:827e32.

[3] Perez F, Endimiani A, Bonomo RA. Why are we afraid of Acinetobacter bau-mannii? Expert Rev Anti Infect Ther 2008;6:269e71.

[4] Bernabeu-Wittel M, Pichardo C, Garca-Curiel A, Pachn-Ibez ME, Ibez-

Y. Smani et al. / Microbial8Martnez J, Jimnez-Mejias ME, et al. Pharmacokinetic / pharmacodynamicassessment of the in-vivo efcacy of imipenem alone or in combination with

Please cite this article in press as: Smani Y, et al., Acinetobacter baumannicytosolic calcium, Microbial Pathogenesis (2011), doi:10.1016/j.micpath.2amikacin for the treatment of experimental multiresistant Acinetobacterbaumannii pneumonia. Clin Microbiol Infect 2005;11:319e25.

[5] Rodrguez-Hernndez MJ, Pachn J, Pichardo C, Cuberos L, Ibez-Martinez J,Garcia-Curiel A, et al. Imipenem, doxcycline and amikacin in monotherapyand in combination in Acinetobacter baumannii experimental pneumonia.J Antimicrob Chemother 2000;45:493e501.

[6] Choi CH, Lee JS, Lee YC, Park TI, Lee JC. Acinetobacter baumannii invadesepithelial cells and outer membrane protein A mediates interactions withepithelial cells. BMC Microbiol 2008;8:216.

[7] Lee HW, Koh YM, Kim J, Lee JC, Lee YC, Seol SY, et al. Capacity of multidrug-resistant clinical isolates of Acinetobacter baumannii to form biolm andadhere to epithelial cell surfaces. Clin Microbiol Infect 2008;14:49e54.

[8] Lee JC, Koerten H, van den Broek P, Beekhuizen H, Wolterbeek R, van denBarselaar M, et al. Adherence of Acinetobacter baumannii strains to humanbronchial epithelial cells. Res Microbiol 2006;157:360e6.

[9] Lee JC, Oh JY, Kim KS, Jeong YW, Park JC, Cho JW. Apoptotic cell death inducedby Acinetobacter baumannii in epithelial cells through caspase-3 activation.Acta Pathol Microbiol Immunol Scand 2001;109:679e84.

[10] Choi CH, Lee EY, Lee YC, Park TI, Kim HJ, Lee SK, et al. Outer membrane protein38 of Acinetobacter baumannii localizes to the mitochondria and inducesapoptosis of epithelial cells. Cell Microbiol 2005;7:1127e38.

[11] Choi CH, Hyun SH, Kim J, Lee YC, Seol SY, Cho DT, et al. Nuclear translocationand DNAse I-like enzymatic activity of Acinetobacter baumannii outermembrane protein A. FEMS Microbiol Lett 2008;288:62e7.

[12] Choi CH, Hyun SH, Lee JY, Lee JS, Lee YS, Kim SA, et al. Acinetobacter baumanniiouter membrane protein A targets the nucleus and induces cytotoxicity. CellMicrobiol 2008;10:309e19.

[13] Buommino E, Morelli F, Metfora S, Rossano F, Perfetto B, Baroni A, et al. Porinfrom Pseudomonas aeruginosa induces apoptosis in an epithelial cell linederived from rat seminal vesicles. Infect Immun 1999;67:4794e800.

[14] Muller A, Gunther D, Dux F, Naumann M, Meyer TF, Rudel T. Neisserial porin(PorB) causes rapid calcium inux in target cells and induces apoptosis by theactivation of cysteine protease. EMBO J 1999;18:339e52.

[15] Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 2003;4:552e65.

[16] Whiteman M, Armstrong JS, Cheung NM, Siau JL, Rose P, Schantz JT, et al.Peroxynitrite mediates calcium-dependent mitochondrial dysfunction andcell death via activation of calpains. FASEB J 2004;18:1395e7.

[17] Goll DE, Thompson VF, Li H, Wei W, Cong J. The calpain system. Physiol Rev2003;83:731e801.

[18] Mizgerd JP. Acute lower respiratory tract infection. N Engl J Med 2008;358:716e27.

[19] Gomez MI, Lee A, Reddy B, Muir A, Soong G, Pitt A, et al. Staphylococcus aureusprotein A induces airway epithelial inammatory responses by activatingTNFR1. Nat Med 2004;10:842e8.

[20] Lee JS, Lee JC, Lee CM, Jung ID, Jeong YI, Seong EY, et al. Outer membraneprotein A of Acinetobacter baumannii induces differentiation of CD4 T cellstoward a TH1 polarizing phenotype through the activation of dendritic cells.Biochem Pharmacol 2007;74:86e97.

[21] Kim SA, Yoo SM, Hyun SH, Choi CH, Yang SY, Kim HJ, et al. Global geneexpression patterns and induction of innate immune response in humanlaryngeal epithelial cells in response to Acinetobacter baumannii outermembrane protein A. FEMS Immunol Med Microbiol 2008;54:45e52.

[22] Afford SC, Pongracz J, Stockley RA, Crocker J, Burnett D. The induction byhuman interleukin-6 of apoptosis in the promonocytic cell line U937 andhuman neutrophils. J Biol Chem 1992;267:21612e6.

[23] Allan SM, Parker LC, Collins B, Davies R, Luheshi GN, Rothwell NJ. Cortical celldeath induced by IL-1 is mediated via actions in the hypothalamus of the rat.Proc Natl Acad Sci U.S.A. 2000;97:5580e5.

[24] Yang XX, Hu ZP, Xu AL, Duan W, Zhu YZ, Huang M, et al. A mechanistic studyon reduced toxicity of irinotecan by coadministration thalidomide, a tumornecrosis factor-a inhibitor. J Pharmacol Exp Ther 2006;319:82e104.

[25] Mizgerd JP, Lupa MM, Hjoberg J, Vallone JC, Warren HB, Butler JP, et al. Rolesfor early response cytokines during Escherichia coli pneumonia revealed bymice with combined deciences of all signalling receptors for TNF and IL-1.Am J Physiol Lung Cell Mol Physiol 2004;286:L1302e10.

[26] Mizgerd JP, Spieker MR, Doerschuk CM. Early response cytokines and innateimmunity: essential roles for TNF receptor 1 and type I L1-1 receptor duringEscherichia coli pneumonia in mice. J Immunol 2001;166:4042e8.

[27] Kroemer G, Galluzzi L, Brenner C. Mitochondrial membrane permeabilizationin cell death. Physiol Rev 2007;87:99e163.

[28] Obst B, Wanger S, Sewing KF, Beil W. Helicobacter pylori causes DNA damagein gastric epithelial cells. Carcinogenesis 2000;21:1111e5.

[29] Saliba AM, de Assis MC, Nishi R, Raymond B, Marques EA, Lopes UG, et al.Implications of oxidative stress in the cytotoxicity of Pseudomonas aeruginosaExoU. Microbes Infect 2006;8:450e9.

[30] Valencia R, Arroyo LA, Conde M, Aldana JM, Torres MJ, Fernndez-Cuenca F,et al. Nosocomial outbreak of infection with pan-drug-resistant Acinetobacterbaumannii infection in a tertiary care university hospital. Infect Control HospEpidemiol 2009;30:257e63.

[31] Smani Y, Dominguez-Herrera J, Pachn J. Rifampin protects human lungepithelial cells against cytotoxicity induced by clinical multi and pandrug-resistant Acinetobacter baumannii. J Infect Dis 2011; doi: 10.1093/infdis/jiq159.

ogenesis xxx (2011) 1e9[32] Smani T, Domnguez-Rodrguez A, Hmadcha A, Caldern-Snchez E, Horrillo-Ledesma A, Ordez A. Role of Ca2-independent phospholipase A2 and

i-induced lung cell death: Role of inammation, oxidative stress and011.01.008

store-operated pathway in urocortin-induced vasodilatation of rat coronaryartery. Circ Res 2007;101:1194e203.

[33] Ding SZ, Minohara Y, Fan XJ, Wang J, Reyes VE, Patel J, et al. Helicobacter pyloriinfection induces oxidative stress and programmed cell death in humangastric epithelial cells. Infect Immun 2007;75:4030e9.

[34] Renckens R, Roelofs JJTH, Knapp S, de Vos AF, Florquin S, van der Poll T. Theacute-phase response and serum amyloid A inhibit the inammatory responseto Acinetobacter baumannii pneumonia. J Infect Dis 2006;193:187e95.

[35] Russo TA, Beanan JM, Olson R, MacDonal U, Luke NR, Gill SR, et al. Ratpneumonia and soft-tissue infection models for the study of Acinetobacterbaumannii biology. Infect Immun 2008;76:3577e86.

[36] Deghmane AE, Veckerl C, Giorgini D, Hong E, Ruckly C, Taha MK. Differentialmodulation of TNF-a-induced apoptosis by Neisseria meningitides. PLoSPathog 2009;5:e1000405.

[37] Usuda J, Okunaka T, Furukawa K, Tsuchida T, Kuroiwa Y, Ohe Y, et al. Increasedcytotoxic effects of photodynamic therapy in IL-6 gene transfected cells viaenhanced apoptosis. Int J Cancer 2001;93:475e80.

[38] Minami R, Muta K, Ilseung C, Abe Y, Nishimura J, Nawata H. Interleukin-6sensitizes multiple myeloma cell lines for apoptosis induced by interferon-alpha. Exp Hematol 2000;28:244e55.

[39] Oritani K, Tomiyama Y, Kincade PW, Aoyama K, Yokota T, Matsumura I, et al.Both Stat3-activation and Stat3-independent bcl-2 downregulation areimportant for interleukin-6-induced apoptosis of 1A9-M cells. Blood1999;93:1346e54.

[40] Suematsu N, Tsutsui H, Wen J, Kang D, Ikeuchi M, Ide T, et al. Oxidative stressmediates tumor necrosis factor-alpha-induced mitochondrial DNA damageand dysfunction in cardiac myocytes. Circulation 2003;107:1418e23.

[41] Hennet T, Richter C, Peterhans E. Tumour necrosis factor-a induces superoxideanion generation in mitochondria of L929 cells. Biochem J 1993;289:587e92.

[42] Meier B, Radeke HH, Selle S, Younes M, Sies H, Resch K, et al. Human bro-blasts release reactive oxygen species in response to interleukin-1 or tumournecrosis factor-a. Biochem J 1989;263:539e45.

[43] Shrivastava A, Aggarwal BB. Antioxidants differentially regulate activation ofnuclear factor-kappa B, activator protein-1, c-jun amino-terminal kinases, andapoptosis induced by tumor necrosis factor: evidence that JNK and NF-kBactivation are not linked to apoptosis. Antioxid Redox Signal 1999;1:181e91.

[44] Ofori-Darko E, Zavros Y, Rieder G, Tarl SA, Antwerp MV, Merchant JL. AnOmpA-like protein from Acinetobacter spp. stimulates gastrin and interleukin-8 promoters. Infect Immun 2000;68:3657e66.

[45] Zheng J, Meng J, Zhao S, Singh R, Song W. Campylobacter-induced interleukin-8 secretion in polarized human intestinal epithelial cells requires Campylo-bacter-secreted cytolethal distending toxin and Toll-like receptor-mediatedactivation of NF-kappaB. Infect Immun 2008;76:4498e508.

[46] Lagoumintzis G, Xaplanteri P, Dimitracopoulos G, Paliogianni F. TNF-alphainduction by Pseudomonas aeruginosa lipopolysaccharide or slime-glyco-lipoprotein in humana monocytes is regulated at the level of mitogen-acti-vated protein kinase activity: a distinct role of toll-like receptor 2 and 4. ScandJ Immunol 2007;67:193e203.

[47] Chun J, Prince A. TLR2-induced calpain cleavage of epithelial junctionalproteins facilitates leukocyte transmigration. Cell Host Microbe2009;5:47e58.

[48] Kozjac-Pavlovic V, Ross K, Rudel T. Import of bacterial pathogenicity factorsinto mitochondria. Curr Opin Microbiol 2008;11:9e14.

[49] Jones DP, McConkey DJ, Nicotera P, Orrenius S. Calcium-activated DNA frag-mentation in rat liver nuclei. J Biol Chem 1989;264:6398e403.

[50] Schmeck B, Gross R, Dje NGuessan P, Hocke AC, Hammerschmidt S,Mitchell TG, et al. Streptococcus pneumoniae-induced caspase 6-dependentapoptosis in lung epithelium. Infect Immun 2004;72:4940e7.

Y. Smani et al. / Microbial Pathogenesis xxx (2011) 1e9 9Please cite this article in press as: Smani Y, et al., Acinetobacter baumannicytosolic calcium, Microbial Pathogenesis (2011), doi:10.1016/j.micpath.2i-induced lung cell death: Role of inammation, oxidative stress and011.01.008

Acinetobacter baumannii-induced lung cell death: Role of inflammation, oxidative stress and cytosolic calciumIntroductionMaterials and methodsBacterial strainsHuman cell culture and infectionCellular viability, apoptosis and necrosisCytokine assay and effect on cellular viabilitySuperoxide anion assayIntracellular Ca2+ measurementMeasurement of calpain and caspase-3-like proteolytic activitiesStatistical analysis

ResultsA. baumannii is cytotoxic to human lung epithelial cellsTNF- and IL-6 mediates A. baumannii-induced cell deathA. baumannii-induced cell death is oxidative stress-dependentA. baumannii induces perturbation of cellular Ca2+ homeostasisA. baumannii-induced cell death is calpain and caspase-3-dependent

DiscussionConclusionsAcknowledgmentsSupplementary materialReferences