Pseudomonas aeruginosa ExoT Induces MitochondrialApoptosis in Target Host Cells in a Manner That Depends onIts GTPase-activating Protein (GAP) Domain Activity*

Received for publication, September 3, 2015, and in revised form, October 7, 2015 Published, JBC Papers in Press, October 8, 2015, DOI 10.1074/jbc.M115.689950

Stephen J. Wood‡1, Josef W. Goldufsky‡1, Daniella Bello‡, Sara Masood‡, and Sasha H. Shafikhani‡§¶2

From the ‡Department of Immunology/Microbiology, §Department of Internal Medicine, and ¶Cancer Center, Rush UniversityMedical Center, Chicago, Illinois 60612

Background: The GAP domain of ExoT induces apoptosis in epithelial cells, but the mechanism underlying GAP-inducedapoptosis remains unknown.Results: GAP domain activates JNK1/2, causes cytochrome c release, and activates caspase-9 and caspase-3.Conclusion: GAP domain of ExoT induces intrinsic apoptosis in epithelial cells.Significance: The GAP and the ADPRT domains make ExoT into a potent cytotoxin, capable of inducing different forms ofapoptosis.

Pseudomonas aeruginosa is the most common cause of hospi-tal-acquired pneumonia and a killer of immunocompromisedpatients. We and others have demonstrated that the type IIIsecretion system (T3SS) effector protein ExoT plays a pivotalrole in facilitating P. aeruginosa pathogenesis. ExoT possessesan N-terminal GTPase-activating protein (GAP) domain and aC-terminal ADP-ribosyltransferase (ADPRT) domain. Becauseit targets multiple non-overlapping cellular targets, ExoT per-forms several distinct virulence functions for P. aeruginosa,including induction of apoptosis in a variety of target host cells.Both the ADPRT and the GAP domain activities contribute toExoT-induced apoptosis. The ADPRT domain of ExoT inducesatypical anoikis by transforming an innocuous cellular protein,Crk, into a cytotoxin, which interferes with integrin survival sig-naling. However, the mechanism underlying the GAP-inducedapoptosis remains unknown. In this study, we demonstrate thatthe GAP domain activity is both necessary and sufficient toinduce mitochondrial (intrinsic) apoptosis. We show that intox-ication with GAP domain results in: (i) JNK1/2 activation; (ii)substantial increases in the mitochondrial levels of activatedpro-apoptotic proteins Bax and Bid, and to a lesser extent Bim;(iii) loss of mitochondrial membrane potential and cytochromec release; and (iv) activation of initiator caspase-9 and execu-tioner caspase-3. Further, GAP-induced apoptosis is partiallymediated by JNK1/2, but it is completely dependent oncaspase-9 activity. Together, the ADPRT and the GAP domainsmake ExoT into a highly versatile and potent cytotoxin, capableof inducing multiple forms of apoptosis in target host cells.

Pseudomonas aeruginosa is one of the most virulent oppor-tunistic pathogens known to man. Despite aggressive antibiotic

therapy, the fatality rate among individuals with P. aeruginosainfection is as high as 40% (1, 2). These figures have notimproved in decades, due to the high intrinsic resistance ofP. aeruginosa to many antibiotics and the emergence of multi-drug-resistant strains (2– 4). P. aeruginosa is the epitome of anopportunistic pathogen of humans. P. aeruginosa does notinfect uncompromised tissues; however, there is hardly any tis-sue that P. aeruginosa cannot infect if the tissue defenses arecompromised in some manner. A number of in vitro and ex vivostudies suggest that the injured epithelium (i.e. wound) is apreferred niche for P. aeruginosa infections (5– 8). Not surpris-ingly, P. aeruginosa has evolved multiple strategies, mediatedby various virulence factors, to inhibit wound healing and toexpand its favorite niche (2, 9 –13).

Prominent among the many cell-associated and secreted vir-ulence factors that facilitate P. aeruginosa pathogenesis duringinfection is a syringe-like structure known as the type 3 secre-tion system (T3SS).3 This highly conserved needle-like struc-ture functions as a conduit, allowing P. aeruginosa to directlytranslocate a set of virulence factors, known as the T3SSeffector proteins, into the target cell where they subvert hostmachinery and advance P. aeruginosa infections (14). T3SSeffectors play crucial roles in mediating P. aeruginosa wounddamage both in vitro and in vivo (9, 10). To date, four T3SSeffectors have been characterized in P. aeruginosa, namelyExoT, ExoS, ExoU, and ExoY (4). ExoT is the only T3SS effectorprotein that is encoded and expressed in all virulent P. aerugi-nosa clinical isolates (4, 15), suggesting a fundamental role forthis virulence factor in P. aeruginosa pathogenesis. Consistentwith this view, P. aeruginosa strains lacking ExoT exhibitreduced virulence and are impaired in dissemination in mice(10, 16, 17). Further highlighting the importance of ExoT in

* This work was supported by National Institutes of Health Grant R21AI110685-01 (to S. H. S.) and Grant 56613 from The Bears Care (to S. H. S.).The authors declare that they have no conflicts of interest with the con-tents of this article.

1 Both authors contributed equally to this work.1 To whom correspondence should be addressed. Tel.: 312-942-1368; Fax:

312-942-2808; E-mail: Sasha_Shafikhani@rush.edu.

3 The abbreviations used are: T3SS, type 3 secretion system; GAP, GTPase-activating protein; ADPRT, ADP-ribosyltransferase; IRES, internal ribosomalentry site; DMSO, dimethyl sulfoxide; IF, immunofluorescence; PI, pro-pidium iodide; MMP, mitochondrial membrane potential; M.O.I., multiplic-ity of infection; PCD, programed cell death; Z, benzyloxycarbonyl; fmk,fluoromethylketone.

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P. aeruginosa pathogenesis is a host defense mechanism involv-ing ubiquitin ligase Cbl-b, which specifically targets ExoT, butnot ExoS or ExoU, for proteasomal degradation (17).

ExoT is a bi-functional protein possessing an N-terminalGTPase-activating protein (GAP) domain, which inhibitsRhoA, Rac1, and Cdc42, small GTPases, and a C-terminal ADP-ribosyltransferase (ADPRT) domain, which targets Crk adaptorproteins and PGK1 glycolytic enzyme (18, 19). Due to its mul-tiple targets, ExoT performs a number of distinct virulencefunctions for P. aeruginosa. ExoT has been demonstrated toinhibit wound healing, alter actin cytoskeleton, function as ananti-internalization factor, cause cell rounding, inhibit cellmigration, block cell division by targeting cytokinesis at multi-ple steps, and induce potent apoptosis in a variety of trans-formed and non-transformed epithelial cells (10 –12, 18, 20,21). Both the ADPRT and the GAP domains contribute toExoT-induced apoptosis, although the ADPRT-induced cyto-toxicity predominates (12). The ADPRT domain activityinduces apoptosis in epithelial cells within 8.5 � 1.3 h of expo-sure, whereas the GAP domain induces apoptosis in HeLacells with slower kinetics within 16.2 � 1.3 h (12). These datasuggest that the GAP and the ADPRT domain activities tar-get different apoptotic pathways to trigger cell death ininfected host cells. Recently, we demonstrated that theADPRT domain of ExoT, by ADP-ribosylating Crk adaptorprotein, transforms this innocuous cellular protein into acytotoxin that induces atypical anoikis apoptosis by disrupt-ing focal adhesion sites and by interfering with the integrinsurvival signaling (13). However, the mechanism underlyingthe GAP-induced apoptosis remains unknown.

In this study, we demonstrate that the GAP domain activity isboth necessary and sufficient to induce the intrinsic pathway ofapoptosis. We show that GAP domain intoxication results inJNK activation and increases in the mitochondrial localizationof the pro-apoptotic proteins Bax and Bid, and to a lesser extentBim, which in turn cause mitochondrial membrane disruption,cytochrome c release, and caspase-9 and caspase-3 activation,culminating in apoptosis.

Experimental Procedures

Cell Culture and Reagents—HeLa cells were cultured in com-plete DMEM (Life Technologies) containing phenol red sup-plemented with 10% FCS, 1% penicillin/streptomycin, and 1%L-glutamine at 37 °C in the presence of 5% CO2. For transfec-tion experiments, 0.4 �g of plasmid DNA was used with Effect-ene (Qiagen) according to the manufacturer’s protocol. Thesources of antibodies (either mouse (Ms), or rabbit (Rb)) used inthese studies are as follows: cytochrome c (Cell Signaling Tech-nology; 12959; Ms); Bax (Cell Signaling Technology; 2774; Rb)for Western blot; Bax (Abcam; ab5714; Ms) for immunofluo-rescence; Bid (Cell Signaling Technology; 2002; Rb); Bim (CellSignaling Technology; 2933; Rb); Bcl-2 (Cell Signaling; 2876;Rb); Bcl-xL (Cell Signaling Technology; 2764; Rb); CoxIV (CellSignaling Technology; 11967; Ms); JNK (Cell Signaling Tech-nology; 9252; Rb); phospho-JNK (Cell Signaling Technology;4668; Rb); c-Jun (Cell Signaling Technology 9165; Rb); phos-pho-c-Jun (Cell Signaling Technology; 3270; Rb); GAPDH(Sigma Life Sciences; G9545; Rb); caspase-9 (Cell Signaling

Technology; 9508; Ms); active caspase-9 (Cell Signaling Tech-nology; 9505; Rb); caspase-3 (Cell Signaling Technology; 9668;Ms); and active caspase-3 (Cell Signaling Technology; 9661;Rb). For Western blots, HRP-linked anti-rabbit (Cell SignalingTechnology; 7074) or anti-mouse (Cell Signaling Technology;7076) IgG secondary antibodies were used.

SP600125 (JNK inhibitor), Z-LEHD-FMK (caspase-9 inhibi-tor), Z-DEVD-FMK (caspase-3 inhibitor), and Z-VAD (pan-caspase-inhibitor) were obtained from R&D Systems. Theseinhibitors were reconstituted in DMSO per the manufacturer’sinstructions, and then added to HeLa cells at 20 �M (forSP600125) or 60 �M (for caspase inhibitors) final concentra-tions, 2 h prior to infection or transfection. An equivalentamount of DMSO (vector) was added to control cells.

Cytotoxicity Measurement by Time-lapse Videomicroscopy—Cytotoxicity measurement by time-lapse videomicroscopy wasperformed as described previously (21, 22). Briefly, HeLa cellswere grown in DMEM without phenol red with (for transfec-tion studies) or without antibiotics (for infection studies) for24 h. These cells were then transfected with the indicatedexpression vectors as described (11) or infected with the indi-cated strains as described (13). 1 h after transfection or at thetime of infection, cells were given 7 �g/ml propidium iodide(Sigma) and then placed on an Axiovert Z1 microscope (Zeiss)fitted with a live-imaging culture box (Pecon) maintaining37 °C in the presence of 5% CO2. Time-lapse videos were takenusing AxioVision 4.2.8 software. Cytotoxicity was determinedfrom each frame, at 15-min interval, using the propidium iodide(PI) channel and analyzed in ImageJ v1.46 (National Institutesof Health) software by setting an appropriate threshold to iso-late PI-positive cells, and image stacks were then analyzed fortotal positive pixels per frame, as we described previously(12, 22).

Western Blot—Protein samples were assessed by Westernblot as described previously (22, 23). Briefly, cells were lysedfollowing infection with 1% Triton X-100 containing a proteaseinhibitor mixture (Roche Diagnostics), 100 mM PMSF, and 100mM Na3VO4. Lysates were mixed with 4� SDS loading bufferand loaded onto 8 –12% SDS-polyacrylamide gels, dependingon the size of the protein under study. After resolving, proteinswere transferred to PVDF membranes, which were blockedwith 5% milk, and probed overnight with primary antibody at4 °C. After washing, blots were probed with HRP-conjugatedsecondary antibody (Cell Signaling Technologies). Blots weredeveloped with ECL or ECL Plus reagent (GE Healthcare).Films were developed with an autoprocessor.

Construction of Expression Vectors for Transient Trans-fection—Restriction enzymes were purchased from New Eng-land Biolabs (Ipswich, MA). Primer sequences shown in lower-case below are not homologous to the chromosomal sequencesand contain the engineered restriction sites. The pIRES2-EGFPmammalian vector (Clontech) was modified as follows. Usinglong PCR (24, 25) and primers EGFP-f3 (5�-aaaactagtATGGT-GAGCAAGGGCGAGG-3�) and EGFP-r2 (5�-aaaagctagcGG-ATCTGACGGTTCAC-3�), which contain SpeI and NheI,respectively, the intervening sequences between the promoterand the EGFP-coding sequence including the internalribosomal entry site (IRES) were removed. Primers SS25-2 (5�-

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ccgctagcATGCATATTCAATCATCTCAGC-3�), with the en-gineered NheI site, and SS37 (5�-aacactagtTCACTTTACCTC-GCTCTCTAC-3�), with the engineered SpeI site, were used toamplify ExoT (G�), using pIRESKII-EGFP/T(2537), which con-tains GAP domain only (amino acids 1–232) as template (10),or to amplify ExoT (G�), using pIRESKII-EGFP/T(2537)(R-149K), which contains inactive GAP domain only with R149Kmutation in the GAP arginine finger motif (10), as the template,using a PCR program, as described (26). These PCR productswere then digested with NheI and SpeI and directionally clonedinto the modified pIRES2-GFP vector to generate pExoT(G�)and pExoT(G�), directly fused to GFP at their C termini.

Transient Transfection—For transient transfection studies,we used Effectene (Qiagen, Valencia, CA), using the vendor’sprotocol.

Immunofluorescence (IF) Microscopy—IF microscopy wascarried out as described previously (24). Briefly, coverslips weretreated with poly-L-lysine and 40 �g/ml human fibronectin(Millipore) before seeding cells. Cells were treated with 200 nM

MitoTracker Red CMXRos (Invitrogen) for 45 min prior tofixation and staining. After 24 h, cells were fixed with 10% ice-cold TCA for 10 min. Cells were permeabilized with 0.2% Tri-ton X-100 (Sigma) for 15 min at room temperature, and thenblocked with 3% FCS for 1 h at 37 °C before staining overnightwith primary antibody. Next, cells were washed three timeswith PBS before staining with conjugated secondary antibody,Alexa Fluor 594 or Alexa Fluor 488 (Life Technologies) for 1 hat 37 °C. The coverslip was mounted on DAPI containingVectaMount (Vector Laboratories). Cells were imaged withAxioVision 4.2.8 software using an Axiovert Z1 microscope(Zeiss) using a 63� objective.

Bacteria Strains and Plasmids—All bacterial strains andexpression vectors and their sources are indicated in Table 1.These isogenic strains were in PA103�exoU (�U) genetic back-ground. For infection studies, bacteria were cultured overnightin Luria-Bertani (LB) broth at 37 °C without shaking. In infec-tion studies, bacteria were added at a multiplicity of infection(M.O.I.) of 10.

Statistical Analysis—Two-tailed Student t-tests, one-wayanalysis of variance with Tukey’s post hoc test, and �-squaredanalyses were used to assess significance with p � 0.05 consid-ered significant. Analysis was carried out with Prism 6.0(GraphPad).

Results

ExoT-induced Mitochondrial Disruption Is due to Its GAPDomain Activity—Previously, we demonstrated that ExoTintoxication resulted in loss of mitochondrial membranepotential (MMP) and cytochrome c release in HeLa cells 5 hafter exposure to bacteria (12). However, it remained unclearwhether it was the activity of the ADPRT domain or the GAPdomain that was responsible for this virulence phenotype. Toaddress this question, we infected HeLa cells, at a multiplicity ofinfection (M.O.I.) of 10, with informative isogenic mutants ofPA103, a clinical isolate that expresses ExoU and ExoT (15, 27),including PA103�exoU/exoT(G�A�) (referred to as �U/T(G�A�)), which carries an in-frame deletion in the exoU genebut expresses ExoT with a mutant ADPRT but functionalGAP domain; PA103�exoU/exoT(G�A�) (referred to as�U/T(G�A�)), which carries an in-frame deletion in the exoUgene but expresses ExoT with a mutant GAP but functionalADPRT domain; PA103�exoU/�exoT (referred to as �U�T),which carries in-frame deletions in the exoU and the exoTgenes; or PA103 pscJ::gentR (T3SS mutant referred to as pscJ,which is unable to deliver T3SS effector proteins into hostcells). These strains have been described previously (9 –13)(Table 1). Five hours after infection, we assessed loss of MMP byIF microscopy, using MitoCapture, as described (12). MitoCap-ture is a cationic dye that accumulates and aggregates in intactmitochondria, resulting in punctate staining, but remains in thecytoplasm in its monomeric form upon disruption of the mito-chondrial membrane potential, exhibiting diffuse staining. As apositive control, uninfected HeLa cells were treated with camp-tothecin, a known inducer of the mitochondrial intrinsic path-way (28). As shown in Fig. 1A and B, HeLa cells infected withGAP-expressing �U/T(G�A�) exhibited significant loss ofMMP as manifested by diffuse cytoplasmic staining. In con-trast, untreated cells or cells infected with �U/T(G�A�),�U�T, or the T3SS mutant (pscJ) showed little evidence ofMMP loss and exhibited punctate mitochondrial staining.

To determine whether the GAP domain of ExoT was suffi-cient to disrupt MMP in the absence of other bacterial factors,we performed transient transfection of HeLa cells with a mam-malian expression vector (Table 1), harboring functional GAP(pExoT(G�A�)) or inactive GAP mutant (pExoT(G�A�)),both fused to GFP at the C terminus. Empty vector (pGFP) was

TABLE 1Strains and plasmids used in this study

Strains and plasmids Relevant characteristics Reference or source

StrainsPA103�U/T(G�A�) PA103�U (exoU deleted) with a point mutation in GAP (R149K) 12PA103�U/T(G�A�) PA103�U with point mutations in the ADPRT (EQE383–385AAA) domains of ExoT 12PA103pscJ::Tn5 Tn Gentr inserted into pscJ ; T3SS-defective 12

PlasmidspGFP pIRESK11-EGFP (Clontech) with bases 1870–1910 removed 11pExoT(G�A�)-GFP pIRES2-EGFP harboring full length ExoT with a functional GAP domain and a mutant ADPRT

domain, directly fused at its C terminus to EGFP11

pExoT(G�A�)-GFP pIRES2-EGFP harboring full length ExoT GAP and ADPRT double mutant, directly fused at itsC terminus to EGFP

11

pExoT(G�)-GFP pIRES2-EGFP harboring truncated ExoT containing only the GAP domain (amino acids 1–32),directly fused at its C terminus to EGFP

This study

pExoT(G�)-GFP pIRES2-EGFP harboring truncated ExoT containing only inactive GAP domain, with R149Kmutation in the GAP arginine finger motif (10), directly fused at its C terminus to EGFP

This study

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also included in these studies as the negative control. Cyto-chrome c release was probed by IF microscopy using an anti-body against cytochrome c (see “Experimental Procedures”). InHeLa cells transfected with pGFP or pExoT(G�A�), cyto-chrome c staining was punctate (Fig. 1C). This staining patternis consistent with subcellular localization of cytochrome c inmitochondria (29). In contrast, HeLa cells transfected withGAP-expressing pExoT(G�A�) exhibited diffuse cytoplasmicstaining (Fig. 1C, middle panel, indicated by arrow). This stain-ing pattern has been reported in cells undergoing intrinsic apo-ptosis (29). These data indicated that the GAP domain of ExoTwas responsible for ExoT-induced mitochondrial membranepermeabilization and also pointed to the mitochondrial (intrin-sic) pathway of apoptosis as the likely mechanism underlyingGAP-induced apoptosis.

GAP Intoxication Alters the Mitochondrial Contents ofPro- and Anti-apoptotic Bcl-2 Proteins—Bcl-2 family pro-teins regulate mitochondrial outer membrane permeabiliza-tion (reviewed in Ref. 30). Although pro-survival members (e.g.Bcl-2 and Bcl-xL) preserve the integrity of the mitochondrialouter membrane, the pro-apoptotic members (e.g. Bax, Bid, andBim) promote its permeabilization. Bcl-2, Bcl-xL, Bak, and Bimreside predominantly in the mitochondria, whereas Bax and

Bid are mainly in the cytosol of healthy cells (30, 31). In healthycells, Bcl-2 and Bcl-xL prevent Bax accumulation and oligomer-ization in the mitochondrial membrane, thus maintainingmitochondrial integrity and promoting survival (30). Whenintrinsic apoptosis is triggered, Bax becomes activated andundergoes a series of conformational changes that are neces-sary for its translocation to the mitochondrial membrane whereit oligomerizes into large complexes, which permeabilize themitochondrial outer membrane and cause the release of cyto-chrome c into the cytosol (30). Bid and Bim are BH3-only pro-teins, which cannot permeabilize the mitochondrial membraneon their own. Rather, they promote apoptosis by binding Bcl-2and Bcl-xL, freeing Bax to accumulate and oligomerize in themitochondrial outer membrane, leading to permeabilization ofmitochondrial outer membrane and the release of cytochromec (30).

To evaluate the impact of GAP intoxication on the dynamicsof pro- and anti-apoptotic proteins, we performed an infectionstudy where HeLa cells were infected with GAP-expressing�U/T(G�A�) or the T3SS mutant pscJ, or treated with PBS(Fig. 2, Mock). We evaluated the levels of Bax, Bid, and Bim(pro-apoptotic) and Bcl-2 and Bcl-2XL (anti-apoptotic) proteinsin the mitochondrial and cytosolic fractions by Western blot 5 h

FIGURE 1. The ExoT/GAP domain is primarily responsible for loss of mitochondrial membrane potential. A, HeLa cells were infected with the indicatedstrains at an M.O.I. 10. After 5 h, bacteria were removed, and medium containing MitoCapture stain was added to stain cells. Cells were imaged by IFmicroscopy without fixation. Representative images are shown. Punctate mitochondrial staining is apparent in uninfected cells, in cells infected with�U/T(G�A�) �U�T, and in T3SS mutant pscJ-infected cells. In contrast, cells treated with camptothecin (an inducer of intrinsic apoptosis) or infected withGAP-expressing �U/T(G�A�) exhibit diffuse cytoplasmic staining, consistent with disruption of the mitochondrial membrane potential. B, the fraction of cellswith diffuse staining from at least 18 random fields (6 fields/experiment, 3 independent experiments) for each sample is tabulated and shown as the mean �S.E. (*, p � 0.001). C, HeLa cells were transfected with an expression vector expressing ExoT with a functional GAP domain, pExoT(G�A�), or inactive ExoT,pExoT(G�A�), both fused to GFP at their C termini, or empty vector (pGFP). 20 h after transfection, cells were fixed and probed for cytochrome c (Cyt c) by IFmicroscopy. Note that expression of pExoT(G�A�) results in diffuse cytochrome c staining in contrast to the punctate staining pattern seen in cells transfectedwith pExoT(G�A�) or pGFP expression vectors.

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after infection. The results indicated that although the mito-chondrial levels of the anti-apoptotic Bcl-2 and Bcl-xL proteinsremained unchanged in the GAP-intoxicated cells, the pro-apo-ptotic proteins Bax and Bid became substantially enriched inthe mitochondrial membrane (Fig. 2A). Bim levels in the mito-chondrial membrane showed more modest effects in that itsthree isoforms, (BimEL, BimL, and BimS, which are all capa-ble of promoting apoptosis (32, 33)), were slightly increasedin the mitochondrial fraction, although Bim was substan-tially up-regulated in the cytosolic fraction. We further cor-roborated these data by IF microscopy, probing for Bax(green) and the mitochondrial marker, MitoTracker (red).Consistent with our Western blot analysis (Fig. 2A), Bax wassignificantly up-regulated and formed globular complexescolocalizing with MitoTracker in HeLa cells that wereinfected with GAP-expressing �U/T(G�A�) (Fig. 2, B and C,green panel, arrow points to a globular Bax structures in

mitochondria). These data indicated that GAP intoxicationresults in accumulation and activation of Bax, Bid, and to alesser extent Bim in mitochondria, where Bax oligomeriza-tion induces mitochondrial outer membrane permeabiliza-tion and initiates intrinsic apoptosis (31).

GAP-induced Apoptosis Is in Part Mediated by JNK—Bax,Bid, and Bim accumulation in the mitochondrial membraneand their activation are largely, but not completely, dependenton JNK (34, 35). Activated (phosphorylated) JNK (particularlyJNK1 and JNK2 isoforms) initiate apoptotic signaling eitherdirectly by activating and mobilizing Bax and Bid proteins tothe mitochondrial membrane or indirectly by activating spe-cific transcription factors such as c-Jun (through phosphoryla-tion), which in turn up-regulate the expression of pro-apoptoticproteins (34, 36 –38). We assessed the impact of GAP intoxica-tion on JNK1/2 and c-Jun expression and activation, using theaforementioned infection study. As indicated in Fig. 3A, infec-

FIGURE 2. ExoT/GAP intoxication increases pro-apoptotic proteins at the mitochondrial membrane. HeLa cells were treated with PBS (Mock) or infectedwith GAP-expressing �U/T(G�A�) or the T3SS mutant (pscJ) at an M.O.I. of 10. A, 5 h after infection, the cells were fractionated, and the cytoplasmic (Cyto) andmitochondrial (Mito) fractions were probed by Western blot for the pro-apoptotic proteins Bax, Bid, and Bim and the anti-apoptotic proteins Bcl-2 and Bcl-xL.GAPDH was used as loading control for cytoplasmic fraction, and CoxIV was used as loading control for mitochondrial fraction. Note that infection with�U/T(G�A�) results in substantial increases in Bax and Bid and to a lesser extent Bim in the mitochondrial membrane fraction. B and C, HeLa cells were infectedas in A. Five hours after infection, the cells were fixed and analyzed for Bax expression by determining the mean fluorescent intensity (MFI) and for subcellularlocalization of Bax (green) by IF microscopy. Representative images are shown in B, and the tabulated results from 16 random fields are shown in C. MitoTracker(red) was used as mitochondrial marker. (* indicates significance with p � 0.0001, �2 test relative to �U/T(G�A�). Note that in cells infected with �U/T(G�A�),Bax expression is increased. Bax also forms globular complexes in mitochondria (indicated by the arrow), as indicated by its colocalization with MitoTracker.Scale bar 10 �M.

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tion with GAP-expressing �U/T(G�A�) resulted in increasedexpression of JNK1/2 and c-Jun and their activation (phospho-JNK1/2 and phospho-c-Jun) at 5 h after infection. To furtherevaluate the role of JNK in mediating GAP-induced apoptosis,we repeated the aforementioned infection studies in the pres-ence or absence of JNK inhibitor SP600125 (added 2 h prior toinfection) and evaluated GAP-induced cytotoxicity by time-lapse videomicroscopy using PI uptake as a marker for celldeath, as we described previously (13, 22). JNK inhibition bySP600125 partially protected HeLa cells against the GAP-in-duced apoptosis (Fig. 3, B and C).

Caspase-9 Activation Is Required for GAP-mediated Apo-ptosis—Mitochondrial membrane disruption by Bax and thesubsequent release of cytochrome c result in activation (cleav-age) of the initiator caspase-9 through the apoptosome (39). Weconducted a time course infection study to evaluate the impactof GAP intoxication on caspase-9 activation. HeLa cells wereinfected with GAP-expressing �U/T(G�A�) or T3SS-defectivemutant pscJ, or treated with PBS (indicated by Mock). Cellswere harvested at 5, 10, and 15 h after infection and were eval-uated for caspase-9 activation by Western blotting. (These timepoints were chosen based on the kinetics of GAP-induced apo-

FIGURE 3. ExoT/GAP cytotoxicity is partially dependent on JNK. A, HeLa cells were treated with PBS (Mock) or infected with GAP-expressing �U/T(G�A�) orthe T3SS mutant pscJ at an M.O.I. of 10. Five hours after infection, the cytoplasmic fraction was probed by Western blot for activated/phosphorylated JNK(p-JNK1/2), total JNK (JNK1/2), or activated/phosphorylated c-Jun (p-c-Jun). GAPDH was used as loading control. Note that GAP intoxication results in substan-tial JNK and c-Jun activation in cytoplasm. B, HeLa cells were infected as in A with or without the JNK inhibitor SP600125 (SP), which was added 2 h prior toinfection. MFI, mean fluorescent intensity. C, cytotoxicity was assessed by time-lapse IF videomicroscopy, using PI uptake as a marker for death. Cytotoxicityanalysis was evaluated by measuring PI fluorescence at 15-min intervals (shown in B), and representative frames of the selected movies are shown in C. Notethat treatment with SP600125 partially protects HeLa cells from GAP-induced cytotoxicity. Protection reached significance between 15 and 20 h (p � 0.004,done in triplicates, one-way analysis of variance).

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ptosis, which occurs within 16.5 � 3.3 h (12).) As shown in Fig.4A, infection with GAP-expressing bacteria, �U/T(G�A�),resulted in substantial caspase-9 activation by 10 h after infec-tion, as manifested by p35 and p37 cleaved products ofcaspase-9. However, by 15 h after infection, no caspase-9(cleaved or un-cleaved) could be detected in HeLa cells infectedwith GAP-expressing �U/T(G�A�), likely reflecting the apo-ptotic environment of GAP-intoxicated cells at this time point.There was also some caspase-9 activation in response to infec-tion with the T3SS mutant strain pscJ, but this level of caspase-9activation was not sufficient to result in significant apoptosis(Fig. 3, B and C) (12). To determine whether the GAP domain ofExoT was sufficient to activate caspase-9 in the absence of otherbacterial factors, we transfected HeLa cells with mammalianexpression vectors harboring functional GAP (pExoT(G�A�))or inactive ExoT mutant (pExoT(G�A�)), both fused to GFP atthe C terminus, or the empty control vector (pGFP). Caspase-9activation was probed by IF microscopy using an antibodyagainst activated caspase-9. As can been seen in Fig. 4B, tran-sient transfection with pExoT(G�A�), but not withpExoT(G�A�) or pGFP empty vector, resulted in caspase-9activation.

Mitochondrial membrane permeabilization can also result incaspase-9-independent cell death (30). We next sought todetermine the dependence of GAP-induced cell death on

caspase-9 activation. To this end, HeLa cells were pretreatedeither with caspase-9-specific inhibitor Z-LEHD-FMK (40) orwith vehicle (indicated by Mock) for 2 h prior to infection withGAP-expressing �U/T(G�A�) or the T3SS-defective mutantstrain pscJ. Caspase-9 inhibition by Z-LEHD-FMK nearly com-pletely blocked the GAP-induced cytotoxicity, indicating thatGAP-induced apoptosis is mediated by caspase-9 initiatorcaspase (Fig. 5).

GAP-induced Apoptosis Is Mediated by Caspase-3—Acti-vated caspase-9 activates executioner caspases, particularlycaspase-3, which carry out cellular demise (39). We next eval-uated the impact of GAP intoxication on caspase-3 activation.HeLa cells were infected with GAP-expressing �U/T(G�A�)or T3SS-defective mutant pscJ, at M.O.I. of 10, or treated withPBS (indicated by Mock). Cells were harvested at 5, 10, and 15 hafter infection and were evaluated for caspase-3 activation byWestern blotting. Similar to caspase-9, infection with GAP-expressing �U/T(G�A�) also led to caspase-3 activation by10 h after infection, as manifested by p18 cleaved/active prod-uct of caspase-3 (Fig. 6A). Corroborating these data, transienttransfection with pExoT(G�A�), but not pExoT (G�A�) orpGFP control vector, also resulted in caspase-3 activation, indi-cating that GAP domain activity is sufficient to activate

FIGURE 4. ExoT/GAP is necessary and sufficient to activate caspase-9. A,HeLa cells were treated with PBS (Mock) or infected with GAP-expressing�U/T(G�A�) or the T3SS mutant PA103 pscJ::Tn5 (pscJ) at an M.O.I. of 10.Cells were probed by Western blot at the indicated time points for totalcaspase-9 or GAPDH, which was used as a loading control. Note that infectionwith �U/T(G�A�) results in caspase-9 activation (indicated by cleavage frag-ments p37 and p35). Pro-Cas9, pro-caspase-9. B, HeLa cells were transfectedwith an expression vector expressing ExoT with a functional GAP domain,pExoT(G�A�), or inactive ExoT, pExoT(G�A�), both fused to GFP at their Ctermini, or empty vector (pGFP). 20 h after transfection cells were fixed andprobed for activated caspase-9, using an antibody that binds activecaspase-9, by IF microscopy. Note that expression of GAP results in caspase-9activation.

FIGURE 5. ExoT/GAP cytotoxicity is dependent on caspase-9. HeLa cellswere infected with �U/T(G�A�) with or without the caspase-9 specific inhib-itor Z-LEHD-FMK (added at 60 �M final concentration), or the T3SS mutantpscJ at M.O.I. of 10. Cytotoxicity was assessed by time-lapse IF videomicros-copy, using PI uptake as a marker for cell death. Images were captured every15 min. A and B, representative frames are shown in A, where red indicates PIuptake and cytotoxicity, and the tabulated data are shown in B. (Experimentswere done in triplicates; p � 0.003 from 10 to 20 h, one-way analysis of vari-ance.) Note that caspase-9 inhibition by Z-LEHD-FMK significantly reducedGAP-induced cytotoxicity. MFI, mean fluorescent intensity.

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caspase-3 (Fig. 6B). Combined, these data indicate that GAPdomain activity is necessary and sufficient to induce the intrin-sic pathway of apoptosis.

To assess the dependence of GAP-induced apoptosis oncaspase-3, HeLa cells were pretreated either with caspase-3-specific inhibitor Z-DEVD-FMK (40) or with vehicle (indicatedby Mock) for 2 h prior to infection with GAP-expressing�U/T(G�A�) or the T3SS-defective mutant strain pscJ.Caspase-3 inhibition by Z-DEVD-FMK significantly protectedagainst the GAP-induced cytotoxicity, indicating that GAP-in-duced apoptosis is primarily mediated by caspase-3 execu-tioner caspase (Fig. 7).

GAP Domain Is Sufficient to Induce Apoptosis in HeLa Cells—So far our data indicated that the GAP domain function wasnecessary for ExoT to induce intrinsic apoptosis, but itremained unclear whether the GAP domain was sufficient onits own to cause apoptosis or whether it could only induce apo-ptosis as part of the full-length ExoT. To determine whether theisolated GAP domain was sufficient to induce apoptosis, weconstructed the pExoT(G�) mammalian expression vector,which expresses only the GAP domain (amino acids 1–232), as

defined (10), or pExoT(G�), which harbors the inactive GAPdomain, fused to GFP at their C termini (see “ExperimentalProcedures”). We transfected HeLa cells with these constructsand assessed cytotoxicity by PI staining and IF microscopy, aswe described previously (12). Similar to the GAP-induced apo-ptosis in the context of the full-length ExoT, ExoT(G�A�) (12),transfection with the isolated GAP domain, pExoT(G�), alsoresulted in Z-VAD-sensitive apoptosis in nearly 67% of trans-fected HeLa cells, as opposed to only 18.8% cytotoxicity thatoccurred in HeLa cells transfected with pExoT(G�) (Fig. 8, p �0.0001, n 64). Of note, the mean time to death (MTD) forpExoT (G�) was 17.1 � 0.97 h, which is nearly identical to theMTD for pExoT(G�A�), which we reported to be 16.2 � 1.3 h(12). We defined MTD as the time between the appearance ofGFP (green), an indicator of transfected gene expression, andthe time at which the cell membrane integrity was compro-mised resulting in the uptake of PI (red) (12). In our analyses, weonly included cells that could be followed for at least one MTDplus one standard deviation from the mean. Combined, thesedata indicate that the GAP domain of ExoT is sufficient toinduce apoptosis.

FIGURE 6. ExoT/GAP is necessary and sufficient to activate caspase-3. A,HeLa cells were treated with PBS (Mock) or infected with GAP-expressing�U/T(G�A�) or the T3SS mutant PA103 pscJ::Tn5 (pscJ) at an M.O.I. of 10.Cells were probed by Western blot at the indicated time points for totalcaspase-3 or GAPDH, which was used as a loading control. Note that infectionwith �U/T(G�A�) results in caspase-3 activation (indicated by cleavage frag-ment p18). Pro-Cas3, pro-caspase-3. B, HeLa cells were transfected with anexpression vector expressing ExoT with a functional GAP domain,pExoT(G�A�), or inactive ExoT, pExoT(G�A�), both fused to GFP at their Ctermini, or empty vector (pGFP). 20 h after transfection, cells were fixed andprobed for activated caspase-3, using an antibody that binds activecaspase-3, by IF microscopy. Note that expression of GAP results in caspase-3activation.

FIGURE 7. ExoT/GAP cytotoxicity is dependent on caspase-3. HeLa cellswere infected with �U/T(G�A�) with or without the caspase-3 specificinhibitor Z-DEVD-FMK (added at 60 �M final concentration), or the T3SSmutant pscJ at M.O.I. of 10. Cytotoxicity was assessed by time-lapse IFvideomicroscopy, using PI uptake as a marker for cell death. Images werecaptured every 15 min. A and B, representative frames are shown in A,where red indicates PI uptake, and cytotoxicity and the tabulated data areshown in B. (Experiments were done in triplicates; p � 0.003 from 10 to20 h, one-way analysis of variance.) Note that caspase-3 inhibition byZ-DEVD-FMK significantly reduced GAP-induced cytotoxicity. MFI, meanfluorescent intensity.

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Discussion

Previously, we reported that both the GAP and the ADPRTdomains of ExoT contribute to ExoT-induced apoptosis in epi-thelial cells (12). The ADPRT domain activity transforms Crkadaptor protein into a cytotoxin that induces atypical anoikisapoptosis by interfering with the integrin-survival signaling(13). However, the mechanism underlying the GAP-inducedapoptosis remained unknown. In this study, we demonstratethat the GAP domain of ExoT triggers the mitochondrialintrinsic pathway of apoptosis. Our data show that GAP intox-ication leads to: (i) activation and accumulation of Bax and Bid,and to a lesser extent Bim, in the mitochondrial membrane (Fig.2); (ii) loss of mitochondrial membrane potential and cyto-chrome c release (Fig. 1); and (iii) activation of the initiatorcaspase-9, which in turn activates the executioner caspase-3(Figs. 4 and 6), culminating in cell demise.

GAP-induced apoptosis is fully dependent on caspase-9activity, as caspase-9 inhibition by Z-LEHD-FMK almost com-

pletely blocks the GAP-induced apoptosis (Fig. 5). In contrast,GAP-induced apoptosis is only partially mediated by JNK activ-ity (Fig. 3). It is not surprising that JNK inhibition only partiallyprotected cells against the GAP-induced cell death, given thatfactors other than JNK can also activate the pro-apoptotic Bcl-2proteins, upstream of caspase-9 activation (34, 36 –38, 41– 43).

Given that the ADPRT domain of ExoT induces cell deathwith faster kinetics (12), what possible physiological role couldGAP-induced cytotoxicity play for ExoT or P. aeruginosa? Onepossibility is that in vivo, P. aeruginosa might encounter somehost cells that may be resistant to the ADPRT-induced apopto-sis. Under such scenarios, the GAP-induced apoptosis may becrucial in overcoming host survival mechanisms. Resistance toADPRT-induced apoptosis could occur if the target host cellreduces the expression of Crk adaptor protein, which we haveshown to be required for the ADPRT-induced apoptosis (13), orif the target host cell down-regulates the expression of FAS (a14-3-3 cellular protein), which has been shown to be requiredfor the ADPRT domain activity (19).

Although the GAP domains of ExoS and ExoT are highlyhomologous and have been shown to target RhoA, Rac1, andCdc42 small GTPases (44, 45), the GAP domain of ExoS has notbeen shown to contribute to the ExoS-induced apoptosis (46).This difference could be due to possible differences in the sub-cellular localizations of ExoS and ExoT within the target host.Another possibility is that the GAP domain of ExoS alsoinduces apoptosis in a manner that is similar to the GAPdomain of ExoT. However, because GAP-induced apoptosisoccurs with much slower kinetics (16 h (12)) than the ADPRTdomain of ExoS, which kills intoxicated cells within 5 h, thecontribution of GAP domain activity to ExoS-induced apopto-sis might have been overlooked. In the studies by Kaufman et al.(46), ExoS-induced cytotoxicity was assessed 5 h after infection.Interestingly, intoxication with ExoS also results in JNK activa-tion and cytochrome c release (47), which may be signs thatExoS/GAP-induced apoptosis may have been initiated in targethost cells. More studies are needed to address the role of GAP inthe ExoS-induced apoptosis.

How does ExoT/GAP induce intrinsic apoptosis? We pro-pose that GAP-induced apoptosis occurs as a consequence ofGAP inhibitory effect on RhoA (18, 45). Recent studies indicatethat RhoA promotes survival in various biological systems bystabilizing mitochondrial integrity. RhoA has been shown toup-regulate anti-apoptotic Bcl2 expression in T cells (48).RhoA has also been shown to activate ERK survival pathway inglomerular epithelial cells (49) and to enhance the expression ofErk/Bcl2 survival pathway in zebra fish (50).

Emerging data indicate that apoptotic programed cell death(PCD) and necrotic PCD are mutually exclusive and do notoccur concurrently in the same cell (51–53). However, it is notclear whether different apoptotic PCDs can occur simultane-ously within the same cell or whether their occurrence is alsomutually exclusive. Our data indicate that at least anoikis apo-ptotic PCD and intrinsic apoptotic PCD can occur concurrentlyin the same cell, when triggered by ExoT. Within 5 h of expo-sure, ExoT-intoxicated cells exhibit loss of focal adhesion anddisruption of integrin-mediated survival signaling, which aresigns of ADPRT-induced apoptosis (13). At the same time,

FIGURE 8. GAP domain is sufficient to induce apoptosis in HeLa cells. HeLacells were transiently transfected with mammalian expression vectorsexpressing either GAP domain of ExoT, pExoT(G�), in the presence or absenceof Z-VAD, or inactive GAP domain of ExoT, pExoT(G�), fused to GFP at the Ctermini. Cell death was analyzed by time-lapse videomicroscopy in the pres-ence of PI. Video images were captured every 15 min. A and B, representativeframes are shown in A, and the tabulated results, collected from multiplemovies, are shown in B. (The numbers above each column indicate the totalnumber of transfected cells that were scored; p � 0.0001, �-square test.) Notethat transfection with functional GAP domain of ExoT (pExoT(G�)) inducesapoptosis and that pretreatment with pan-caspase inhibitor Z-VAD or a nullmutation in the GAP domain (pExoT(G�)) interferes with GAP-inducedapoptosis.

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ExoT-intoxicated cells also exhibit mitochondrial outer mem-brane disruption and cytochrome c release (12), which we nowshow, in this study, to be dependent on the GAP domain activ-ity. Combined, the ADPRT and the GAP domains make ExoTinto a highly versatile and potent cytotoxin, capable of inducingdifferent forms of apoptosis in target host cells.

Author Contributions—S. H. S. conceived and coordinated the studyand wrote the paper. S. J. W. and J. W. G. coordinated the study andwrote the paper. S. J. W. and J. W. G. designed, performed, and ana-lyzed the experiments shown in Figures 1 thru 6. D. B. and S. M.provided technical assistance and contributed to the preparation ofthe figures. All authors reviewed the results and approved the finalversion of the manuscript.

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ShafikhaniStephen J. Wood, Josef W. Goldufsky, Daniella Bello, Sara Masood and Sasha H.

ActivityCells in a Manner That Depends on Its GTPase-activating Protein (GAP) Domain

ExoT Induces Mitochondrial Apoptosis in Target HostPseudomonas aeruginosa

doi: 10.1074/jbc.M115.689950 originally published online October 8, 20152015, 290:29063-29073.J. Biol. Chem. 

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