Targeting of Enteropathogenic Escherichia coli EspF to HostMitochondria Is Essential for Bacterial PathogenesisCRITICAL ROLE OF THE 16TH LEUCINE RESIDUE IN EspF*□S

Received for publication, October 12, 2004Published, JBC Papers in Press, November 8, 2004, DOI 10.1074/jbc.M411550200

Takeshi Nagai‡, Akio Abe§, and Chihiro Sasakawa‡¶

From the ‡Department of Microbiology and Immunity, Institute of Medical Science, University of Tokyo, 4-6-1,Shirokanedai, Minato-ku, Tokyo 108-8639, Japan and the §Laboratory of Bacterial Infection, Kitasato Institute for LifeScience, Kitasato University, 5-9-1, Shirokanedai, Minato-ku, Tokyo 108-8642, Japan

The attachment of enteropathogenic Escherichia coli(EPEC) to host cells and the induction of attaching andeffacing (A/E) lesions are prominent pathogenic features.EPEC infection also leads to host cell death and damageto the intestinal mucosa, which is partly dependent uponEspF, one of the effectors. In this study, we demonstratethat EspF is a mitochondrial import protein with a func-tional mitochondrial targeting signal (MTS), becauseEspF activity for importing into the mitochondria wasabrogated by MTS deletion mutants. Substitution of the16th leucine with glutamic acid (EspF(L16E)) completelyabolished EspF activity. Infection of HeLa cells with wildtype but not the espF mutant (�espF) decreased mitochon-drial membrane potential (��m), leading to cell death.The ��m decrease and cell death were restored in cellsinfected with �espF/pEspF but not �espF/pEspF(L16E),suggesting that the 16th leucine in the MTS is a criticalamino acid for EspF function. To demonstrate the impactof EspF in vivo, we exploited Citrobacter rodentium byinfecting C3H/HeJ mice with �espFCR, �espFCR/pEspFCR,or �espFCR/pEspF(L16E)CR. These results indicate thatEspF activity contributes to bacterial pathogenesis, asjudged by murine lethality and intestinal histopathology,and promotion of bacterial colonization of the intestinalmucosa.

Enteropathogenic Escherichia coli (EPEC)1 associated withsevere infantile diarrhea represents a major health problem indeveloping countries and is also responsible for occasional out-

breaks among infants in developed countries (1). EPEC attachesintimately to the intestinal epithelium and effaces brush bordermicrovilli, forming attaching/effacing (A/E) lesions. The genesrequired for A/E lesions to form are encoded on a pathogenicityisland termed the locus of enterocyte effacement (LEE) (2, 3). TheLEE encodes a type III secretion system (TTSS), translocators(EspA, EspB, and EspD), effectors (Tir, EspG, EspF, Map, andEspH), chaperones (CesAB, CesD, CesD2, CesF, and CesT), andregulators (Ler, GrlA, and GrlR) (2–6). These genes are highlyconserved among diarrheagenic enterohemorrhagic E. coli(EHEC) and Citrobacter rodentium. In addition, recent studieshave indicated that several genes outside the LEE encode effec-tors such as NleA/EspI (7, 8) and Cif (9), which act cooperativelywith LEE genes in pathogenesis.

EPEC attachment to the intestinal epithelium is accompa-nied by a number of cellular responses, including reorganiza-tion of host cell F-actin (2, 10), disruption of the intestinal cellbarrier (11), inflammatory reactions (11), and cell death (12–15). Importantly, these cellular responses may be a conse-quence of effector translocation into host cells. For example,Tir, which is translocated into the host cytoplasmic membrane,acts as the intimin receptor required for the intimate attach-ment involved in inducing actin pedestal structures by recruit-ing host proteins required for actin polymerization (16, 17).EspG and Orf3 both share significant amino acid homologywith Shigella VirA (18, 19) and stimulate microtubule destruc-tion, eventually leading to the stimulation of RhoA and Rac1(20). EspF may be involved in the disruption of tight junctionsand induction of cell death (see below). Map is reportedly tar-geted to the host mitochondria and interferes with membranepotential (21), as well as being involved in cytoskeletal rear-rangements in the formation of filopodia (22). EspH has beenindicated to promote pedestal formation (5). NleA/EspI hasrecently been identified as a novel effector targeted to the hostcell Golgi body using EHEC and C. rodentium, defects in whichaffect colonization of the mouse intestine and are involved inthe development of intestinal hyperplasia (7, 8).

One of the prominent pathogenic features of EPEC duringthe colonization of intestinal epithelial cells is the ability toinjure the epithelial barrier and eventually kill host cells, al-though the meaning of this biological paradoxical activity inbacterial colonization remains largely speculative. EspF seemsto play an important role in this form of cell damage (13).Indeed, studies indicate that though EspF is not involved inbacterial adherence to host cells, F-actin condensation, or ty-rosine phosphorylation (23), this protein plays some role in theinduction of host cell death (13) and decreasing transepithelialelectrical resistance via the destruction of tight junctions in theintestinal epithelium (24). Recently, Viswanathan et al. (25)

* This work was supported by a grant-in-aid for scientific research onPriority Areas from the Ministry of Education, Culture, Sports, Science,and Technology and CREST, Japan Science and Technology Corpora-tion. The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

□S The on-line version of this article (available at http://www.jbc.org)contains Supplemental Materials.

¶ To whom correspondence should be addressed. Tel.: 81-3-5449-5252; Fax: 81-3-5449-5405; E-mail: sasakawa@ims.u-tokyo.ac.jp.

1 The abbreviations used are: EPEC, enteropathogenic E. coli; LEE,locus of enterocyte effacement; ��m, mitochondrial membrane poten-tial; EGFP, enhanced green fluorescent protein; EH, ethidium homo-dimer; EspF, EPEC-secreted protein F; LDH, lactate dehydrogenase;mtHsp70, mitochondrial heat shock protein 70; MTS, mitochondrialtargeting signal; Rho123, rhodamine 123; TTSS, type III secretionsystem; WT, wild type; MnSOD, manganese superoxide dismutase;TUNEL, TdT-mediated dUTP nick-end labeling; Map, mitochondrial-associated protein; moi, multiplicity of infection; mAb, monoclonalantibody; Km, kanamycin; PBS, phosphate-buffered saline; cfu, colony-forming unit; DMEM, Dulbecco’s modified Eagle’s medium; TRITC,tetramethylrhodamine isothiocyanate; FITC, fluorescein isothiocyanate.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 4, Issue of January 28, pp. 2998–3011, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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indicated that EspF can interact with cytokeratin 18, and Nou-gayrede and Donnenberg (26) have shown that EspF can mi-grate into host mitochondria and induce host cell death. Im-portantly, recent studies with C. rodentium (strain DBS100)revealed that C57BL/6, NIH Swiss, or C3H/HeJ mice, infectedwith the espF mutant, was partly attenuated as compared withthat of wild-type C. rodentium (6, 8). Although these studieshave clearly demonstrated that EspF acts as a bacterial effec-tor, the biological relevance of each EspF activity in bacterialinfection of the intestinal mucosa remains unclear.

In this context, by creating a series of single amino acidsubstitutions in the mitochondrial targeting signal (MTS) ofEspF, we investigated the biological relevance of EspF activi-ties to migration into host mitochondria, as it pertains to bac-terially induced cell death, injury of the intestinal barrier, orbacterial colonization of the intestinal mucosa. One of the sin-gle amino acid substitutions at the 16th leucine in EspF abol-ished the ability to migrate into mitochondria, and this wasalso relevant to the EspF activity of initiating the mitochon-drial death pathway. The single amino acid-substituted EspFmutant was created using C. rodentium, and the cloned plas-mid was introduced into the espF mutant of C. rodentium inorder to evaluate the impact on the pathogenesis of bacterialinfection of the murine intestine. Our data provide for the firsttime convincing evidence that EspF activity contributes to in-jury of the intestinal mucosa including cell death and enhancesof bacterial colonization of the intestinal mucosa.

EXPERIMENTAL PROCEDURES

Bacterial Strains—Bacterial strains and plasmids used in this studyare listed in Table I. We created an EPEC (strain E2348/69) espFmutant by insertion of a kanamycin (Km)-resistant cassette gene. TwoDNA fragments (FL and FR) that flank the espF gene were amplifiedusing the chromosomal gene of EPEC as a template by PCR. FL wasamplified, using the primers FLP (5�-AACTGCAGGCACGTAACCAA-ACAGGGCTTAACGGC-3�) and FLS (5�-TCCCCCGGGTACAAGCTG-CCGCCCTAGTGTAGAAGC-3�) to obtain a 1.0-kbp fragment. RF wasamplified using the primers FRS (5�-TCCCCCGGGCTGCCTATGAG-CAATCGAAGAAAGGG-3�) and FRB (5�-CGGGATCCCTCTCCCGAC-GTTAACAAAACCCCTTC-3�) to obtain a 0.9-kbp fragment. The result-ing FL and FR fragments contained restriction sites for PstI-SmaI andSmaI-BamHI, which were used to clone PCR fragments into pBlue-Script II KS(�) (Stratagene) to generate pBS-FL and pBS-FR, respec-tively. The FL fragment obtained by digesting pBS-FL with PstI andSmaI was subcloned into pBS-FR to generate pBS-FLR. The Km-resis-tant cassette gene (aphA3) of pUC18K2 (27) was generated by digestionwith SmaI and was subcloned into the SmaI site of pBS-FLR togenerate pBS-FLAR. The FLAR fragment of pBS-FLAR digested withPstI and BamHI was subcloned into the temperature- and sucrose-sensitive suicide vector pCACTUS (28), and the resulting plasmid wasintroduced into EPEC by electroporation. The transformants were sele-cted as described previously (29). The selected espF mutant (�espF) wasdetermined by PCR and immunoblotting. The espF and map double

mutant (�espF-�map) were created using �map by the same methodas that used for the construction of �espF. The espF internal dele-tion mutant of C. rodentium (strain EX-33) was constructed usingpCACTUS, as described above, without aphA3.

Antibodies and Reagents—Anti-EspF polyclonal antibody was pre-pared as follows. The synthetic peptide of EspF (73CPSRPAPPPPTS-GQASGA89) was conjugated to KLH (keyhole limpet hemocyanin) andinjected into two male New Zealand white rabbits. Subsequently,polyclonal anti-EspF antibody was purified using epoxy-activatedSepharose beads (Amersham Biosciences) coupled with the EspF-(73–89) peptide.

Anti-mitochondrial heat shock protein 70 (mtHsp70) mAb (ALEXISBiochemicals), anti-cytochrome oxidase subunit II mAb (MolecularProbes), anti-cytochrome c mAb (BD Biosciences), anti-Tom20 (SantaCruz Biotechnology), anti-aldolase polyclonal antibody (Santa Cruz Bio-technology), rhodamine 123 (Molecular Probes), Mitotracker (MolecularProbes), ethidium homodimer (EH, Molecular Probes), and rhodamine-phalloidin (Molecular Probes) were obtained commercially. Anti-rabbitIgG-horseradish peroxidase, anti-mouse IgG-horseradish peroxidase,anti-rabbit IgG-FITC, and anti-mouse IgG-TRITC were purchasedfrom Sigma. Anti-intimin polyclonal antibody was used as describedpreviously (30).

Plasmid Constructions—pEspF was constructed by ligating theBamHI and XhoI fragment of the espF complementary gene (from �300bp to �116 bp of EPEC espF) into the BamHI and SalI sites of pBR322(ampicillin-resistant). pBREspFCR and pEspFCR were constructed byligating the BamHI and XhoI fragment of the espFCR complementarygene (from �150 bp to �174 bp of C. rodentium espF) into the BamHIand SalI sites of pBR322 and pSU18 (chloramphenicol (CP)-resistant)(31), respectively. The full-length or truncated pEGFP-EspFs andpEspF-EGFPs were constructed as follows. The various espF gene frag-ments amplified by PCR were digested with EcoRI and BamHI and thenligated into the corresponding sites of pEGFP-N3 or pEGFP-C2. Thepoint mutants on pEspF-EGFP, pEspF, and pEspFCR were created witha QuikChangeTM site-directed mutagenesis kit (Stratagene). pMnSOD-EGFP and pTom20-EGFP were constructed, respectively, by ligation ofMnSOD and tom20 fragments amplified by RT-PCR using total cDNAof HeLa cells with pEGFP-N3.

Conditions of Eukaryotic Cell Culture and Bacterial Culture—HeLaand COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM, Sigma) with 10% fetal calf serum (Sigma) at 37 °C in the pres-ence of 5% CO2. In the EPEC infection experiment, bacteria were grownovernight in LB broth containing 1% mannose at 37 °C with or withoutappropriate antibiotics (ABPC; 50 �g/ml or CP; 25 �g/ml). The bacteriawere diluted 1:10 into modified EPEC adherence medium (13) consistingof DMEM supplemented with 40 mM HEPES (at pH 7.4), 2% fetal calfserum, and 1% mannose with or without antibiotics and grown withshaking at 37 °C for 2 h. Cultured bacteria were added at a multiplicity ofinfection (moi) of 100 in the modified EPEC adherence medium.

Infection of Cultured Cells with EPECs—The state of infection was fol-lowed by the method of Crane et al. (13). HeLa cells (2 � 105 per well) weregrown (on coverslips in the experiment with immunofluorescent staining) on12-well cell culture plates in DMEM with 10% fetal calf serum for 16 h at37 °C in the presence of 5% CO2. Then, the culture medium was changed tomodified EPEC adherence medium prior to bacterial infection. The precul-tured bacteria as described above were added to HeLa cells and incubated at37 °C in the presence of 5% CO2. After 1 h of infection, the plate was washed

TABLE IStrains and plasmids in this study

Characteristics Reference andsource

StrainsE2348/69 EPEC wild type O127:H6 16�espF espF inframe-insertion mutant of E2348/69 This study�map map mutant of E2348/69 20�espF-�map �espF and �map double mutant of E2348/69 This studyEX33 Citrobacter rodentium wild type (previously called MPEC O115a,c:K(B)) 61, 62

�espFCR espF mutant of EX33 This studyPlasmids

pEspF Complementary espF of EPEC in pBR322 (middle-copy vector) This studypBREspFCR Complementary espF of C.rodentium in pBR322 (middle-copy vector) This studypEspFCR Complementary espF of C.rodentium in pSU18 (low-copy vector) This studypEGFP-N3 EGFP fusion protein expression vector ClonTechpEGFP-C2 EGFP fusion protein expression vector ClonTechpEspF-EGFP espF gene cloned into pEGFP-N3 This studypEGFP-EspF espF gene cloned into pEGFP-C2 This study

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with PBS three times and replaced with RPMI 1640 medium supplementedwith 40 mM HEPES (at pH 7.4), 1% mannose, and 0.1% bovine serumalbumin with or without antibiotics.

Immunofluorescent Staining and Immunoblotting—The cells on acoverslip were washed with PBS three times and immunostained withthe appropriate antibodies as described previously (32). The coverslipswere mounted on VectorShield (Vector Laboratory) for observing by aconfocal laser-scanning microscopy (MicroRadiance Plus, Bio-Rad). Im-munoblotting analysis was carried out as described previously (33).

Intracellular Expression of EspF-EGFP Fusion Proteins—COS-7cells (�50% confluent on 12-well plate) on coverslips were transfectedwith 1 �g/ml EGFP fusion protein expression vectors (pEspF-EGFPetc.) using FuGENE 6 (Roche Applied Science) according to the manu-facturer’s protocol in the absence or presence of 10 �M valinomycin(uncoupler of mitochondrial inner membrane potential). After 16 h ofincubation, the cells were treated with 100 nM MitoTracker (MolecularProbes) in DMEM (fetal calf serum-free) for 30 min in the absence orpresence of 10 �M valinomycin and then washed three times with PBS.After fixation with 4% paraformaldehyde in PBS, coverslips werewashed with PBS and then immunostained. They were observed byconfocal laser-scanning microscopy. To determine the ratio of EspF-EGFPs localized in mitochondria, 100 cells expressing EspF-EGFPswere counted (a cell whose mitochondrial EGFP fluorescent signal wasstronger than the cytosolic signal was counted as being localized inmitochondria). The counts were performed three times, independently.

Fractionation of Mitochondria from HeLa Cells Infected withEPEC—The mitochondrial fractionation was based on a method de-scribed previously (34, 35). HeLa cells (2 � 107) were infected with�espF/pEspF or �espF/pEspF(L16E) for 3 h and washed with PBS fourtimes. The following procedures were performed at 4 °C. Cells collectedwith a scraper were gently homogenized using a Microtube homoge-nizer (I.S.O) in buffer A (250 mM sucrose, 25 mM HEPES-KOH (atpH7.4), 25 mM KCl, 5 mM MgCl2, 0.5 mM EGTA, and 5 mM 4-(2-aminoethyl)-benzene sulfonyl fluoride hydrochloride (AEBSF). The ho-mogenate was centrifuged at 600 � g for 10 min, and the pellet wasused as the nuclei and unbroken cell fraction, while the supernatantwas centrifuged at 7000 � g for 10 min. The resulting pellet was thecrude mitochondrial fraction including bacteria, while the supernatantwas centrifuged at 100,000 � g for 1 h, yielding a pellet (microsomefraction) and another supernatant (cytosolic fraction). To obtain highlypurified mitochondria, the crude mitochondrial fraction was layered ona discontinuous sucrose gradient consisting of 4.5 ml of 1.6–1.0 M

sucrose from the bottom and centrifuged at 82,000 � g. After 200 min,the middle band was collected and was used as a mitochondrial fraction.

Measurement of Mitochondrial Inner Membrane Potential (��m)—HeLa cells preincubated with the ��m-sensitive dye rhodamine123(Rho123) at 2 �g/ml (36) in DMEM (fetal calf serum-free) were washedwith PBS three times and then infected with EPECs at an moi of 100 asdescribed above. After 6 h of infection, both detached (supernatant) andadherent (trypsinized) cells were collected and the non-adherent bacte-ria were removed by centrifugation (200 � g, for 10 min, at roomtemperature). The pellets were washed three times with PBS and thenresuspended in 500 �l of PBS. The intensity of ��m for at least 10,000cells was analyzed by FACSCaliburTM (BD Biosciences).

Time Course Imaging—HeLa cells preteated with 2 �g/ml of Rho123for 30 min were infected with wild-type EPEC (WT) at an moi of 100.After 1 h of infection, cells were washed and replaced with RPMI 1640supplemented with 40 mM HEPES (at pH7.4), 1% mannose, and 0.1%BSA, and 1 �g/ml of EH. Then, phase contrast and fluorescent images(Rho123 for 0.5 s, and EH for 0.2 s) were taken every 10 min for up to6.5 h at 37 °C in the presence of 5% CO2 by fluorescent microscopy(Axiovert 135-SENSYS, Zeiss).

LDH Assay—Cytotoxicity induced by EPEC infection was analyzedusing a CytoTox 96 cytotoxicity assay kit (Promega). The extent ofcytotoxicity was assayed by measuring the amount of cytosolic LDH(lactate dehydrogenase) released by cells, which represents loss of mem-brane integrity. The cytotoxicity was calculated as follows: experimen-tal LDH activity/total LDH activity � 100%.

Detection of Cytochrome c Release—The cytosolic fractions of HeLacells (4 � 106) infected with EPECs at an moi of 100 at various timepoints were obtained by a digitonin-based subcellular fractionationtechnique as described previously (37). For the detection of releasedcytochrome c, the equal amounts of cytosolic proteins were separated bySDS-PAGE and analyzed by immunoblotting with anti-cytochrome c,and anti-actin antibody as an internal standard control.

Detection of EspF Secreted by EPEC into the Culture Medium—The secretion of EspF was analyzed by immunoblot as describedpreviously (29).

Infection of Mice with C. rodentium—6-week old, female C3H/HeJmice (JC1, CLEA Japan) highly susceptible to C. rodentium (38), werehoused for a week in the animal facility of the Institute of MedicalScience, University of Tokyo in accordance with guidelines drafted bythe University of Tokyo. Wild-type C. rodentium (WTCR), �espFCR,complementary strain �espFCR/pEspFCR and �espFCR/pEspFCR (L16E)were cultured overnight in LB broth with or without 25 �g/ml of CP at37 °C. The respective cultures (2 ml) were centrifuged (1000 � g, 5 min)at room temperature, and after the supernatant had been discarded,bacteria were resuspended with 2 ml of LB broth. In order to examinethe survival assay, 200 �l of bacterial suspension (�2 � 108 cfu/head)were inoculated into ten mice by oral gavages. Survival was assesseddaily over the course of the infection for up to 20-days postinfection. Fordetermination of the numbers of adherent bacteria on the colon andcolonic weight, five mice were inoculated in the same manner. At 9-dayspostinfection, these mice were sacrificed, and 5.5 cm of distal colon fromthe rectum were cut vertically along the colon. These samples werewashed with PBS to remove fecal pellets. Weights were then deter-mined, and the specimens were homogenized in 5 ml of ice-cold PBSwith a Potter Elvehjem homogenizer (digital homogenizer, AS ONE).The homogenates were serially diluted with ice-cold PBS and plated onMacConkey agar plates with or without 25 �g/ml of CP. Colonies of C.rodentium, which were checked with PCR by amplifying the espFCR,were counted and the number of cfu per mouse was calculated. Forhistological analysis, colons infected with C. rodentium at 9-dayspostinfection, were fixed with 4% paraformaldehyde in PBS at 4 °Covernight and then frozen in tissue-freezing medium (Leica Jung).Frozen sections were cut with a cryostato (CM1900, Leica) and immu-nostained with anti-C. rodentium antiserum and rodamine-phalloidinby a method described previously (8). Immunostained samples wereexamined by fluorescent microscopy (Axioplan 2, Zeiss). TUNEL (TdT-mediated dUTP nick-end labeling) assay revealed dead cells to bestained by the DEAD-EndTM Fluorometric TUNEL System (Promega)and counterstained with rhodamine-phalloidin, DAPI (staining of nu-clei), and anti-C. rodentium antiserum. TUNEL-positive cells werecounted at least in five fields of view that included the proprial muscu-lar layer through the luminal side at �50 magnification and thenconverted to numbers per 1 mm2.

RESULTS

Subcellular Localization of EspF Secreted from EPEC—After3 h of infection of HeLa cells with WT EPEC or the espF mutant(�espF), the subcellular localization of EspF was examinedusing immunofluorescence microscopy with anti-EspF, anti-mitochondrial heat shock protein 70 (mtHsp70) antibody andTO-PRO3. As shown in Fig. 1A, the EspF signal (green) inHeLa cells merged with the mitochondrial signal (red) (Fig. 1A,panel j or o), in which the intensity profiles of EspF andmtHsp70 signals as scanned along the X-X� axis in panels l andm, were similar (Fig. 1B). Since the EspF signal mostly over-lapped with the bacterial signal, we subsequently scanned thesignals toward the Z-axis moving along to the X-X� to ensurethe special relationship among the three signals for bacteria,EspF, and mitochondria. As shown in Fig. 1C, the major EspFsignal was merged with that of the mitochondria. A similarsubcellular localization was observed in other mammalian celllines such as HEp-2, Caco-2, T84, and COS-7 infected withEPEC,2 supporting the notion that EspF secreted by EPEC isimported into host mitochondria.

EspF MTS Functions in Mitochondrial Import—To investi-gate whether or not the MTS exists in EspF, we constructedvarious truncated versions of EspF, and each was cloned intoeither pEGFP-N3 (for pEspF-EGPF derivatives) or pEGFP-C2(for pEGFP-EspF derivatives) (Fig. 2A). The resulting plasmidsintroduced into COS-7 cells were analyzed for cellular distri-butions of the signals for EspF-EGFP (EGFP, green) and Mi-toTracker, a mitochondrial specific marker (red), using immu-nofluorescence microscopy. As shown in Fig. 2, A and B, thefull-length EspF-EGFP, EspF-(1–72)-EGFP, EspF-(1–43)-EGFP, and EspF-(1–24)-EGFP were colocalized with mitochon-

2 T. Nagai and C. Sasakawa, unpublished data.

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dria, whereas EspF-(24–206)-EGPF, EspF-(134–206)-EGPF,EGPF-EspF-(134–206), and EGPF-EspF were dispersed in thecytoplasm, indicating that the N-terminal EspF sequence func-tions as the MTS.

EspF Migration into Mitochondria Is Dependent on Mito-chondrial Inner Membrane Potential—Because mitochondrialinner membrane potential (��m) has been shown to be re-quired for mitochondrial proteins (other than mitochondrialouter membrane proteins) to be imported into mitochondria(39), we examined whether or not EspF migration into mito-chondria would depend on ��m. COS-7 cell transfectants car-rying pEspF-EGFP, pMn-SOD-EGFP, or pTom20-EGFP wereinvestigated in the presence or absence of valinomycin (10 �M)for subcellular localization using immunofluorescence micros-copy. As can be seen in Fig. 3 (panels e, m, and u), becauseMitoTracker is incorporated into mitochondria in a manner

dependent on ��m, in valinomycin-treated cells the Mito-Tracker signal was not confined to the mitochondria insteadbeing distributed within the cytoplasm, confirming that the��m had disappeared. Tom20 could be imported into mitochon-dria regardless of valinomycin treatment (Fig. 3, panel v).Under these conditions, EspF, as well as Mn-SOD, which lo-calizes in the matrix, was unable to migrate into mitochondriaof cells treated with valinomycin (Fig. 3, panels f and n),strongly indicating that migration of EspF into mitochondriarequires ��m.

The 16th Leucine Residue of EspF Is Critical for Its Ability toMigrate into Host Mitochondria—N-terminal presequence ofthe mitochondrial import protein is involved in recognition bymitochondrial import machinery called TOM (translocase ofouter membrane), which has the potential to form positivelycharged amphiphilic helices (40). According to the helical wheel

FIG. 1. Subcellular localization of EspF secreted from EPEC into HeLa cells. A, HeLa cells were infected with the EPEC espF mutant(�espF) (panels a–e) or WT (panels f–o) for 3 h at an moi of 100. Then, the cells were fixed, permeabilized, and immunostained with antibodiesspecific for EspF, mitochondrial heat shock protein70 (mtHsp70) and TO-PRO3 (specific for bacteria and cell nuclei). The stained cells werevisualized by confocal laser scanning microscopy. High magnification views of cells infected with WT are shown in panels k–o (bar, 5 �m). B,signal intensity. The intensity of fluorescent signal of EspF (green) and mtHsp70 (red) on the X-X� line indicated in A are shown. Each patternof signal intensity was similar (arrowhead). C, cross-sections toward the Z-axis. Cross sections toward the Z-axis of the EspF signal (green),mtHsp70 signal (red), bacterial signal (blue), and merged images on the X-X� line are shown.

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structure of the 6–23 amino acids of EspF, one-half is rich inhydrophobic amino acids (see Fig. 3A, white circle), whereas theother side is rich in hydrophilic amino acids including twopositively charged amino acids such as Arg (Fig. 3A, gray andblack circles), thus forming a positively charged amphiphilicsecondary structure. Previous studies have indicated that Leuand Arg in the MTS are particularly important for import intomitochondria, and that point mutants at either of these aminoacids occasionally result in loss of the capacity to be imported(41, 42). Thus, we created a series of single amino acid substi-tuted mutants, along with the MTS based on EspF-EGFP (Fig.3B). These point mutants, designated EspF(L2E), EspF(L12E),EspF(G13E), EspF(R14Q), EspF(L16E), EspF(V17E), andEspF(R22Q) were investigated for their capacity to be trans-ported into mitochondria. We created EGFP-fused EspF mu-tants in pEGFP-N3, which were introduced into COS-7 cells bytransfection and investigated their localization in mitochon-

dria. The results showed capacity of EspF(L16E) to be almostcompletely abolished, whereas other point mutants such asEspF(R14Q), EspF(V17E), and EspF(R22Q) retained this abil-ity and others such as EspF(L2E), EspF(L12E), andEspF(G13E) had slightly less activity (Fig. 4, B and C). Becauseit has been suggested that the hydrophobic amino acids in MTSof the import proteins are important for interacting with themitochondrial receptor, and that the positively charged resi-dues involved in protein uptake into mitochondria depend on��m (43), we substituted the 14th and 22nd Arg residues withGln. The localization in mitochondria was investigated by thesame methods as those used for the point EspF mutants. In-terestingly, the import capacity of the resulting EspF(R14Q-R22Q)-EGFP was almost completely abolished, whileEspF(R14Q) and EspF(R22Q) retained this ability. Thus, the16th Leu in the MTS of EspF appears to be critical for inter-action with some putative mitochondrial receptor, whereas the

FIG. 2. Construction of chimericEspF-EGFP fusion proteins and in-tracellular localization in COS-7. A,construction of EspF-EGFP fusion pro-teins. Truncated EspF-EGFP fusion pro-teins were constructed in pEGFP-N3(EGFP was fused to the C terminus ofEspF) or pEGFP-C2 (EGFP was fused tothe N terminus of EspF). COS-7 cellstransfected with the resulting plasmidswere incubated for 16 h. After staining ofthe mitochondria with MitoTracker, a��m-sensitive mitochondrial dye, at 100nM for 30 min, the cells were fixed andthen examined for intracellular localiza-tion of the respective EspF-EGFP fusionproteins by confocal fluorescent micros-copy. B, expression of various EspF-EGFPfusion proteins in COS-7 cells. Fluores-cent images of COS-7 cells transfectedwith pEspF-EGFP (panels a–c), pEspF-(1–24)-EGFP (panels d–f), or pEspF-(24–206)-EGFP (panels g–i) are shown. Theleft panels are presented as MitoTracker(red), the middle panels as EGFP (green),and the right panels as merged images(yellow).

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14th or 22nd Arg may participate in EspF uptake bymitochondria.

To confirm the inability of EspF(L16E) to be imported intohost mitochondria during EPEC infection, HeLa cells wereinfected with espF complement strains (�espF/pEspF or �espF/pEspF(L16E)), and the subcellular localization of EspF wasanalyzed by immunofluorescence microscopy with anti-EspFand anti-mtHsp70 antibodies. As expected, the EspF signal(green) in HeLa cells infected with �espF/pEspF was confinedto the mitochondria (red), but the EspF(L16E) signal in HeLacells was dispersed in the cytoplasm (Fig. 5A). To further con-firm this, subcellular fractions of HeLa cells infected with�espF/pEspF or �espF/pEspF(L16E) were investigated for thelocalization of EspF or EspF(L16E). Using fractional centrifu-gation and sucrose density-gradient centrifugation (see “Exper-imental Procedures”), the locations of EspF (and EspF(L16E))in each of the subcellular fractions, the nuclei-unbroken cell,mitochondria, microsomes, and cytosol, were analyzed by im-munoblottings with anti-COX II (cytochrome oxidase subunitII) (mitochondrial marker), anti-Intimin (bacterial marker),

anti-aldolase (cytosolic marker), and anti-EspF antibodies, re-spectively. As shown in Fig. 5B, though EspF was mostly lo-calized in the mitochondria, EspF(L16E) was barely detectablein the mitochondrial fraction, instead being detected mostly inthe cytosol.

EspF Induces Cell Death and Can Reduce MitochondrialMembrane Potential—��m is necessary for producing ATP, amajor source of bioenergy, via oxidative phosphorylation, andplays a regulatory role in cell fate, “survival versus death” (44).Previous studies found that EPEC infection of cultured epithe-lial cells elicited both apoptosis and necrosis, in which the roleof EspF is important (13). Therefore, we investigated HeLa cellresponses to EPEC infection by focusing on the relationshipbetween cell death and ��m. HeLa cells pretreated with rho-damine123 (Rho123), a fluorescent dye sequestered by activemitochondria, were infected with EPEC for 30 min in thepresence of EH (1 �g/ml), a cell-impermeable fluorescent dye,allowing it to flow into the host cytosol and nucleus upondisruption of the cytoplasmic membrane (13). The time courseimaging of cell responses was analyzed using phase contrastand fluorescence microscopy up to 5.5-h postinfection. As seenin the series of photographs, the Rho123 signal (green: activemitochondria) within the cells gradually decreased up to 3.5-hpostinfection, after which the infected cell lapsed into a rapidcell burst without cell shrinkage (Fig. 6A, white arrowheads at2.5–3.5 h and yellow arrowheads at 3.5–5 h). Following the cellburst, an influx of EH into the nucleus resulted from the loss ofcytoplasmic membrane integrity (Fig. 6A, white arrowhead at3.5 h and yellow arrowhead at 4.5 h). These results thus sug-gested that the collapse of ��m directed by EPEC infectionleads to cell burst. (The videos of these phase (S1), fluorescent(S2), and merged (S3) images are published on the Journal ofBiological Chemistry website as Supplemental Data.) Thesecellular responses were not detectable when HeLa cells wereinfected with the �espF.2

Hence, we investigated whether or not a ��m reductionwould occur upon the import of EspF into mitochondria. To testthis, HeLa cells pretreated with Rho123 were infected withWT, �espF, or the espF complement strains. (The amount ofEspF secreted by these strains were almost the same (Fig. 6B).)HeLa cells at 6-h postinfection were collected and subjected toFACS flow cytometry in order to analyze the Rho123 intensityprofile (see “Experimental Procedures”). In this assay, a lowintensity Rho123 peak appeared in 99% of HeLa cells upontreatment with 0.05% Triton X-100 representing loss of ��m,whereas a high intensity Rho123 peak appeared in 99% ofuntreated HeLa cells, representing a high ��m level (Fig. 6C,top panel). As shown in Fig. 6C, upon infection with WT or�espF, a low intensity Rho123 peak was detected in 78.7 or18.5% of the cell population, respectively. Furthermore, whenHeLa cells were infected with �espF/pEspF or �espF/pEspF(L16E), low intensity Rho123 peaks were seen in 81.8 or23.3% of the cell population, respectively, indicating that thedissipation of ��m induced by EPEC depends upon the importof EspF into mitochondria. Kenny and Jepson (21) previouslyindicated that Map, one of the EPEC TTSS-mediated effectorssecreted, targets mitochondria and disrupts ��m. Therefore,we investigated �map and �espF-�map for their effects on��m. As shown in Fig. 6C (bottom panels), infection of HeLacells with �map and �espF-�map resulted in low intensityRho123 peaks in 58.5 and 8.9% of cells, respectively, suggestingthat, though the presence of Map affects ��m to some extent,the major factor leading to dissipation of ��m in this assay isEspF.

To further characterize the cell death induced by EspF dur-ing EPEC infection, we carried out an LDH assay to quantify

FIG. 3. Effect of valinomycin on EspF-EGFP migration into mi-tochondria. COS-7 cells were transfected with pEspF-EGFP, pMnSOD-EGFP (localized in the matrix), or pTom20-EGFP (localized in the mito-chondrial outer membrane) in the absence or presence of 10 �M

valinomycin (vali) and expressed for 24 h. Before fixation, the culturemedium was changed to DMEM containing 100 nM MitoTracker, in theabsence or presence of 10 �M valinomycin, and incubated for 30 min. Cellswere immunostained with anti-Tom20 and then observed by confocallaser scanning microscopy. Since MitoTracker import into mitochondria isdependent on ��m, cells in which ��m had been abolished by treatmentwith valinomycin were poorly stained throughout the cytosol (panels e, m,u). Tom20 localized in the mitochondrial outer membrane is stained as amitochondrial marker (panels c, g, k, o, s, w). Translocation of EspF-EGFPas well as MnSOD-EGFP but not that of Tom20-EGFP into mitochondriawas dependent on ��m (panels f, n, v).

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the amount of LDH released from the damaged cell cytoplasminto the medium. The amounts of LDH released from HeLacells into the medium at 2-, 4-, and 6-h postinfection weremeasured as described under “Experimental Procedures.” Asshown in Fig. 6D, upon WT infection of HeLa cells, cytotoxicityincreased markedly in a time course manner as compared withthat shown by untreated cells. However, the cytotoxicity wassignificantly reduced in HeLa cells infected with �espF. Simi-larly, upon infection of HeLa cells with �espF/pEspF, cytotox-

icity was high as compared with that of �espF/pEspF(L16E)infection, suggesting that EspF import into the mitochondria isinvolved in cell death. Since �espF and �espF/pEspF(L16E)infection of HeLa cells still elicited cell death to some extent, itis likely that some additional factor(s) associated with EspFparticipate in the induction of cell death. A similar cytotoxiceffect on HeLa cells by C. rodentium-borne EspF (EspFCR) wasobserved when we introduced pBREspFCR but notpBREspFCR(L16E) into the EPEC �espF (Fig. 6D). Further-

FIG. 4. MTS sequence of EspF, and the effect of point mutations on the MTS sequence. A, secondary structure of EspF (6–23). Thehelical wheel structure of EspF (6–23 amino acid) forms a positively charged amphiphilic structure (white circles: hydrophobic amino acids; graycircles, hydrophilic amino acids; and black circles, positively charged hydrophilic amino acids). Amino acids indicated by arrowheads are essentialfor translocation into mitochondria. B, single amino acid substitutions in the MTS of EspF. Various single amino acids were substituted inpEspF-EGFPs of the MTS, then transfected into COS-7 cells and expressed for 16 h. The percentages of EspF-EGFPs localized in mitochondriawere then calculated. Results are representative of three independent experiments. Data represent means � S.D. C, fluorescent images of COS-7cells transfected with pEspF(L16E)-EGFP (panels a–c) and pEspF(R14Q, R22Q)-EGFP (panels d–f).

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more, we examined the �map and �espF-�map for their capac-ities to induce cell death under the same conditions. As shownin Fig. 6D, the absence of Map from EPEC but not EspF had noappreciable effect on the EPEC-induced cytotoxicity in HeLacells. When the LDH assay was conducted using another cellline such as a colonic T84 cell monolayer, no significant differ-ences in the cytotoxity induced by WT and �espF infection wereobserved. However, when cytotoxicity on the T84 cell mono-layer was measured by counting the cells taking up EH, thecytotoxic pattern was similar to that of the LDH assay usingHeLa cells and was dependent on the ability of EspF to migrateinto mitochondria.2

Recent studies indicate that the pathway stimulating apop-totic cell death via the decrease in ��m is mediated by releaseof cytochrome c from the mitochondrial intermembrane spaceinto the cytoplasm; then a complex composed of caspase-9,Apaf-1 and dATP is formed, and activates caspase-3, -6, and -7which are required for apoptosis induction (45). Hence, weinvestigated whether or not cytochrome c is released frommitochondria in response to EPEC infection of epithelial cells.As shown in Fig. 6E, release of cytochrome c into the cytoplasmwas detected at 4 and 6 h after infecting HeLa cells with WTand �espF/pEspF but not �espF or �espF/pEspF(L16E) (Fig.6E). The results of this series of experiments indicate thatmigration of EspF into mitochondria during bacterial infectionleads to disappearance of ��m, which leads to the release ofcytochrome c from the mitochondria into the cytoplasm andultimately necrotic cell death.

The Status of EspF as a Virulence Factor—To establish thein vivo role of EspF in bacterial infection of the intestinalmucosa, we constructed a C. rodentium-borne espF non-polarmutant (�espFCR), and pEspFCR or pEspF(L16E)CR was intro-duced into �espFCR (called �espFCR/pEspFCR and �espFCR/pEspF(L16E)CR). As shown in Fig. 7A, the amounts of EspF

secreted by �espFCR/pEspFCR and �espFCR/pEspF(L16E)CR

were similar. EspFCR and EspF(L16E)CR were subsequentlyassessed for their ability to migrate into mitochondria by cre-ating pEspFCR-EGFP and pEspF(L16E)CR-EGFP. As expected,EspFCR- but not EspF(L16E)CR-EGFP could migrate into mi-tochondria.2 To investigate whether or not the import ofEspFCR into mitochondria affects bacterial pathogenesis, �2 �108 of wild-type C. rodentium (WTCR), �espFCR, �espFCR/pEspFCR, and �espFCR/pEspF(L16E)CR were orally adminis-tered via the stomach to 10 C3H/HeJ mice, and survival of themice was monitored up to 20 days after inoculation (Fig. 7B).Mice infected with WTCR showed 100% mortality up to day 12,whereas mice infected by �espFCR survived through day 12with 10% mortality after day 14. Similarly, mice infected with�espFCR/pEspFCR showed 80% mortality up to day 14, whereasthose infected with �espFCR/pEspF(L16E)CR exhibited 20%mortality by day 16 (Fig. 7B). Furthermore, C3H/HeJ mice (n 5) infected with C. rodentium strains were sacrificed on day 9and typical pathological features of the large intestine weremacroscopically observed (Fig. 7C). Although intestines fromthe mice inoculated with LB were healthy with solidified feces,those from mice infected with WTCR were edematous withoutsolidified feces and were typical findings of bacterially inducedintestinal colitis. The intestines from mice inoculated with�espFCR remained healthy with solidified feces, albeit someportions of the intestine were slightly swollen. The intestinesfrom mice inoculated with �espFCR/pEspFCR showed findingssimilar to those of specimens with WTCR. However, the intes-tines from mice infected with �espFCR/pEspF(L16E)CR barelyshowed such findings, though the content of solidified feces wasslightly less than with �espFCR. The mice infected with thesestrains were sacrificed on day 9 and were also investigated forcolonic weight, numbers of bacteria colonizing the colon, andintestinal mucosal layer thickness as described under “Exper-

FIG. 5. Intracellular distributionsof EspF or EspF(L16E) secreted byEPEC. A, HeLa cells were infected withespF complement strains, �espF/pEspF(panels a–d) or �espF/pEspF(L16E) (pan-els e–h), and the cells were immuno-stained by the same method as describedin the legend to Fig. 1. Shown are theEspF signals (panels a and e), mtHsp70signals (panels b and f), TO-PRO3 (panelsc and g) and the merged images (panels dand h). B, subcellular localization of EspFand EspF(L16E) using cell fractionation.HeLa cells infected with �espF/pEspF or�espF/pEspF(L16E) were fractionatedinto nuclei-unbroken cells, mitochondria,microsomes, and cytosol by the fractionalcentrifugation method (see under “Exper-imental Procedures.” These fractionswere immunoblotted with anti-EspF, anti-cytochrome oxidase subunit II (COX-II, mi-tochondria marker), anti-intimin (EPECmarker), or anti-aldolase (cytosol marker)antibodies.

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FIG. 6. Alteration of mitochondrial inner membrane potential and induction of cell death by infecting of HeLa cells with EPEC.A, time course imaging of HeLa cells infected with EPEC. HeLa cells pretreated with 2 �g/ml Rho123 were infected with WT at an moi of 100 inthe presence of 1 �g/ml of EH. At 1-h postinfection, the phase contrast, Rho123 signal (green) and EH signal (red) were examined using fluorescence

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imental Procedures.” These indices were significantly re-duced by infection with �espFCR as compared with WTCR.The same was true for infection with �espFCR/pEspF(L16E)CR or �espFCR/pEspFCR (Fig. 7, D–F). Bacteriapresent in the frozen distal colon sections on day 9 postinfec-tion were also stained using anti-C. rodentium antiserum andthen visualized using immunofluorescence microscopy. Phal-loidin (actin) staining was used to counterstain the tissue.High numbers of bacteria were visible over the epithelialsurfaces including intestinal crypts with WTCR or �espFCR/pEspFCR infection but not �espFCR or �espFCR/pEspF(L16E)CR infection (Fig. 7G). The bacteria were fre-quently visible in proximity to the lamina muscularismucosae in colonic sections (Fig. 7G, blue arrowheads),whereas large amounts of exfoliative epithelial tissue insidethe lumen were stained with anti-C. rodentium antiserum(Fig. 7G, white arrowheads). The intestinal tissue infectedwith WTCR or �espFCR/pEspFCR showed marked hyperplasia,whereas hyperplasia was significantly milder with �espFCR

or �espFCR/pEspF(L16E)CR infection. Thus, these resultsfrom this series of experiments further support the notionthat the ability of EspF to migrate into host mitochondria iscritical for bacterial colonization of the intestinal epitheliumand the initiation off disease processes.

TUNEL Staining in Vivo—The TUNEL assay involves label-ing of the 3�-hydroxyl DNA ends generated by DNA fragmen-tation during apoptosis by means of terminal deoxyribonucle-otidyl transferase (TdT) and labeled dUTP. However, severalreports suggest that non-apoptotic DNA fragmentation is la-beled also by the TUNEL assay (46). Since EspF can initiatethe mitochondrial death pathway, we visualized intestinal celldeath using TUNEL staining of the murine intestine infectedwith WTCR, �espFCR, �espFCR/pEspFCR, or �espFCR/pEspF(L16E)CR (Fig. 8). Frozen distal colonic sections on day-9postinfection were stained for dead cells by the TUNEL methodand counterstained with rhodamine-phalloidin, anti-C. roden-tium serum, and DAPI- and the TUNEL-positive cells werecounted. The numbers of TUNEL-positive cells per mm2 of asection showed cell death caused by infection with WTCR or�espFCR/pEspFCR to be decreased to one-fifth of that with�espFCR or �espFCR/pEspF(L16E)CR (Fig. 8A). Fig. 8B showsthe representative data from the TUNEL-positive cells in theinfected intestinal section. Based on the results of this series ofexperiments, we concluded that the ability of EspF to migrateinto mitochondria is biologically relevant to intestinal injuryincluding the cell death that results from bacterial infection.

DISCUSSION

In the present study, we have investigated the impact ofEspF as an EPEC effector in infection and obtained molecularevidences to support the concept that the capacity of EspF tomigrate into the host cell mitochondria is important for bacte-rial pathogenesis using mice orally infected with a C. roden-tium espF mutant.

Mitochondria are an important host target for many patho-gens determining the fate of infected host cells (47). For exam-

ple, human T-lymphotrophic virus type I protein p13II has anamphiphilic MTS and decreases mitochondrial membrane po-tential (48). PorB is the porin protein of Neisseria gonorrhoeaeand Neisseria meningitidis. Though N. gonorrhoeae PorB hasno typical MTS, it interacts with the mitochondrial outer mem-brane via binding to VDAC (voltage-dependent anion channel)protein, leading to apoptotic cell death (49). In contrast, thePorB of N. meningitidis protects cells from apoptosis (50). TheN-terminal cleavage product of VacA (p34) secreted from Heli-cobacter pylori has no MTS, but can be imported into mitochon-dria and trigger apoptotic cell death (51). SipB secreted fromSalmonella enterica serovar Typhimurium via TTSS can alsobe imported into mitochondria and lead to cell death via induc-tion of mitochondrial autophagy (52). Interestingly, althoughno genetic evidence has yet been obtained, Map secreted fromEPEC via the TTSS has a typical MTS in the N-terminal 42amino acids involved in import into mitochondria (22). Further-more, Tir, the intimin receptor, has also been indicated tomigrate into mitochondria and thereby lead to cell death (53).Together with those of a recent study indicating that EspF hasthe capacity to migrate into mitochondria (26), these findingsindicate that the delivery of various effectors into host mito-chondria from EPEC is likely to play some important roles inbacterial infection.

We demonstrated here that EspF can be imported into mi-tochondria, and that this protein import is crucial for EPECinfection. MTS involved in mitochondrial import of proteinsreportedly takes part in recognition by several mitochondrialouter membrane proteins such as Tom20, Tom22, and Tom70,followed by translocation into the matrix through a generalimport pore (40). Since the MTS of EspF is located at the Nterminus, EspF might initially be recognized by the Tom20-Tom22 complex instead of Tom70. A genetic and functionalstudy of Tom20 indicated that the MTS recognized by Tom20frequently possesses a motif composed of XX ( representsa hydrophobic amino acid, whereas X represents an arbitraryamino acid with a hydrophilic plus long side chain) (54). Thus,we looked for a putative consensus motif in the MTS of EspF,and found 5-ISNAA-9 and 13-GRQLV-17 to fit the motif. Thefact that EspF(L16E) (13-GRQEV-17) was poorly transportedinto mitochondria raised the possibility that the EspF MTSmight also be imported into mitochondria via an interactionwith Tom20.

As mentioned above, many pathogenic bacteria can kill hostcells by inducing cell death, although the type of cell deathvaries among target host cells, depending on cellular physio-logical or experimental conditions (55). Crane et al. (13) re-ported that EPEC infection of epithelial cells caused necrosis-like cell death, whereas ectopic expression of EspF in epithelialcells triggered apoptotic cell death (13). In this study, we dem-onstrated that import of EspF into mitochondria resulted in adecrease in ��m, accompanied by necrotic cell (see Fig. 6).Recent studies have shown Salmonella-induced macrophagenecrotic cell death to be caspase-1-dependent (56), while Bor-detella bronchiseptica-induced necrotic cell death in epithelial

microscopy every 30 min up to 5.5 h. Attached bacteria on HeLa cells are shown in the first panel (blue arrowhead) (bar; 10 �m). B, Western blotof EspF secreted by EPEC strains into DMEM. C, alteration of mitochondrial inner membrane potential (��m). HeLa cells pretreated with 2 �g/mlof Rho123 were infected with various EPEC strains. After 6 h of infection, the cells were collected and analyzed for Rho123 fluorescent intensityby FACS. As a control for ��m-disrupted cells, HeLa cells were incubated with 0.05% Triton X-100 for 30 min (top left panel). D, cytotoxicity ofEPEC to HeLa cells. HeLa cells were uninfected or infected with various EPEC strains. Cytotoxicity to HeLa cells was assayed by quantifying theamount of LDH released from cells into the culture supernatant at 2, 4, and 6-h postinfection, and cytotoxicity was calculated as described under“Experimental Procedures.” Results are representative of three independent experiments. Data are presented as means � S.D. E, release ofcytochrome c into the cytosol. HeLa cells were infected with EPECs as in D. After 2, 4, and 6 h of infection, the cytosolic proteins of infected HeLacells were obtained by a digitonin-based subcellular fractionation technique. These fractions were separated by SDS-PAGE and detected byimmunoblotting with anti-cytochrome c. The same blots were analyzed with anti-actin to control for protein loading. As a control for apoptosis,HeLa cells were incubated with 2 �M staurosporine (STS) for 6 h.

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FIG. 7. Studies of mice infected with C. rodentium. (C. rodentium EX33 strains were as follows, LB:a, WTCR:b, �espFCR:c, �espFCR/�EspFCR:d, �espFCR/pEspF(L16E)CR:e). A, Western blot of EspFCR secreted by C. rodentium strains into DMEM. B, survival curves. C3H/HeJ mice(n 10) were orally infected with 2 � 108 CFU of C. rodentium strains and monitored daily up to 20 days. Percentage of mice surviving from initialpopulation is shown. C–G, effect of EspFCR on colonic tissue. C3H/HeJ mice (n 5) were inoculated C. rodentium strains, as described in methodsfor B and sacrificed 9-days postinfection. Colons (from cecum to rectum) showing typical features of infection with the respective strains are shownin C. After weighting the colons (5.5 cm from rectum) without fecal pellets (shown in D), the specimens were homogenized and plated on MacConkeyagar plates. Colonies were then counted to determine the number of adherent bacteria in the mouse colon (shown in E, the y axis values arepresented on a log scale). Portions of these distal colons were fixed, frozen, cut into 7-�m sections, and immunostained with anti- C. rodentiumantiserum (green) and counterstained for actin with rhodamine-phalloidin (red). Thicknesses of mucosal layers were measured (shown in F).Typical immunofluorescent images are shown in G. Data are presented as means � S.D.

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cells is caspase-1-independent (57). Nevertheless, variousforms of necrotic cell death were prevented by adding glycine(inhibitor of necrosis caused by nonspecific ion fluxes through

the cytoplasmic membrane). Furthermore, EspF-induced celldeath was not inhibited by glycine but was inhibited by z-VAD-fmk (pancaspase inhibitor), although no cleavage of polyADP-

FIG. 8. TUNEL Assay. A, quantification of TUNEL-positive cells. After 9 days of infection with C.rodentium strains, C3H/HeJ mice weresacrificed and their distal colonic tissues were frozen and sectioned, as described in the legend to Fig. 7. Dead cells were stained by the Dead-Endfluorometric TUNEL system and TUNEL-positive cells were counted (see under “Experimental Procedures”). Data are presented as means � S.D.B, fluorescent images of colonic tissue stained by TUNEL. Sections stained by the TUNEL method (green: panels b, f, j, n, r) were counterstainedfor the actin cytoskeleton with rodamine-phalloidin (red: panels a, e, i, m, o), bacteria with anti-C.rodentium antiserum (yellow: panels a, e, i, m,o), and cell nuclei with DAPI (blue: panels c, g, k, o, s), and the sections were observed by fluorescent microscopy.

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ribose polymerase (PARP), a substrate for caspases, was ob-served.2 It has recently been shown that two different types ofcell death occur in Jurkat cells, with the type being determinedby the intracellular ATP concentration (58). The authors ob-served the concentration of intracellular ATP to act as a mo-lecular switch controlling the type of cell death. Indeed, theyreported that with a low concentration of intracellular ATP,necrotic cell death could be blocked by adding z-VADfmk with-out Lamin B, a substrate for caspases, being cleaved. Thesefeatures of necrotic cell death are similar to those of cell deathinduced by EPEC infection. Intriguingly, Crane et al. (13) re-ported that EspF has some capacity to reduce the intracellularATP concentration (13). If true, this raises the possibility thatnecrotic death of epithelial cells in response to EPEC infectionmay be triggered by reducing the ATP concentration via therelease of cytochrome c, which functions in respiration as a keymolecule, from mitochondria resulting from the import of EspFinto mitochondria.

Recent studies have indicated another biological activity ofEspF i.e. involvement in alteration of intestinal epithelial bar-rier function (24, 59). Dickman et al. (60) suggested that Rotavirus infection decreased metabolism and cellular ATP concen-trations, resulting in the destruction of cell-cell junctions.Thus, we are currently attempting to address whether EspFimport into the mitochondria causes destruction of cell-celljunctions via a decreased cellular ATP concentration.

Infection of the murine intestine with C. rodentium hasrecently been established as the most reliable model of EPECpathogenesis. The espFCR mutant created in C. rodentium(strain EX-33) in our study confirmed lower virulence, withoral administration to C3H/HeJ mice, than with WTCR. Impor-tantly, the ability of EspFCR to migrated into mitochondria asdetermined by EspF(L16E)CR was shown to be highly relevantto bacterial pathogenicity, as judged by the mouse mortalityrate, colon weight, and intestinal mucosal layer thickness. Fur-thermore, EspF activity was also required for promotion ofbacterial colonization. Though we have no other evidence asyet, we speculate that EspF activity might be needed for thepathogen to stimulate intestinal cell metabolism, thereby in-creasing the opportunity for bacteria to attach to the freshlyrenewed cell surface, possibly conferring some advantage overepithelial cells in a normal metabolic state. In fact, as shown inthe histopathological study of murine intestine infected withWTCR, it seems likely that the hyperplasia caused by the patho-gen increases opportunities for the bacteria to colonize theintestinal cryptae, as compared with the condition associatedwith the espF mutant. Since infection of mice with �espFCR or�espFCR/pEspF(L16E)CR still caused some intestinal hyperpla-sia as compared with the untreated intestine, bacterial effec-tors other than EspF, as described in the Introduction, mustalso be required for full bacterial virulence.

In summary, EspF secreted via the TTSS of EPEC targetshost mitochondria. The N-terminal 24 amino acids serve as amitochondrial targeting signal. In migration of EspF into hostmitochondria, Leu16 and Arg14,22 in the MTS are critical. As-sessment of mitochondrial membrane potential (��m) in in-fected epithelial cells indicated that EspF is required for loss of��m to be triggered by EPEC infection. Furthermore, EspF isassociated with the release of cytochrome c from mitochondriainto the cytoplasm, which leads to host cell death. Finally, thesignificance of the ability of EspF to migrate into mitochondriaduring bacterial infection was established for the first time bycreating the C. rodentium mutants, �espFCR, �espFCR/pEspFCR, and �espFCR/pEspF(L16E)CR, in the murine infec-tion model. Our findings thus provide clues to elucidating therole of EspF in initiation of the mitochondrial death pathway,

which appears to be an important mechanism by which EPECpromote colonization of the intestinal mucosa.

Acknowledgments—We thank Dr. Hiroyuki Abe, Dr. Ichiro Tatsuno,Dr. Toshihiko Suzuki, Dr. Sei Yoshida, Dr. Reiko Akakura,Dr. Michinaga Ogawa, and Dr. Takahito Toyotome (Institute of MedicalScience, University of Tokyo) for assistance with the experiments andcritical reading of the manuscript.

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Takeshi Nagai, Akio Abe and Chihiro SasakawaRESIDUE IN EspF

Essential for Bacterial Pathogenesis: CRITICAL ROLE OF THE 16TH LEUCINE EspF to Host Mitochondria IsEscherichia coliTargeting of Enteropathogenic

doi: 10.1074/jbc.M411550200 originally published online November 8, 20042005, 280:2998-3011.J. Biol. Chem. 

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