EspC translocation into epithelial cells byenteropathogenic Escherichia coli requiresa concerted participation of type V and IIIsecretion systems

Jorge E. Vidal and Fernando Navarro-García*Department of Cell Biology, Centro de Investigación yde Estudios Avanzados del IPN (CINVESTAV-IPN),Ap. Postal 14-740, 07000 México City, Mexico.

Summary

EspC is a non-locus of enterocyte effacement (LEE)-encoded autotransporter protein secreted by entero-pathogenic Escherichia coli (EPEC) that causes acytopathic effect on epithelial cells, including cytosk-eletal damage. EspC cytotoxicity depends on its inter-nalization and functional serine protease motif. Herewe show that during EPEC infection, EspC is secretedfrom the bacteria by the type V secretion system(T5SS) and then it is efficiently translocated into theepithelial cells through the type III secretion system(T3SS) translocon. By dissecting this mechanism, wefound that EspC internalization during EPEC–hostcell interaction occurs after 1 h, unlike purified EspC(8 h). LEE pathogenicity island is involved in specificEspC translocation as three espC-transformed at-taching and effacing (AE) pathogens translocatedEspC into the cells. A role for effectors and otherfactors involved in the intimate adherence encoded inLEE were discarded by using an exogenous EspCinternalization model. In this model, an isogenicEPEC DespC strain allows the efficient internalizationof purified EspC. Moreover, isogenic mutants in T3SSwere unable to translocate endogenous and exog-enous EspC into epithelial cells, as EspC–EspA inter-action is required. These data show for the first timethe efficient delivery of an autotransporter proteininside the epithelial cells by EPEC, through coopera-tion between T5SS and T3SS.

Introduction

Enteropathogenic Escherichia coli (EPEC) infection is aleading cause of infantile diarrhoea in developing coun-

tries which can be severe and lethal (Kaper et al., 2004).EPEC is the prototype of a family of pathogens that elicita histopathological lesion formed at the mucosal intestinalsurface that seems a pedestal-like structure, known asattaching and effacing (AE) lesion (Moon et al., 1983).The AE pathogens include enterohaemorrhagic E. coli(EHEC), rabbit EPEC (REPEC) and Citrobacter roden-tium, which infects mice. The genes responsible for theAE phenotype are located in a 35.6 kb pathogenicityisland (PAI) termed LEE (locus of enterocyte effacement)(McDaniel et al., 1995). LEE is organized into five poly-cistronic operons (LEE1 to LEE5). The LEE1, LEE2 andLEE3 operons encode the type III secretion system(T3SS) and a global regulator Ler (LEE-encodedregulator). LEE4 encodes T3SS-secreted proteins EspA,EspB and EspD (EPEC-secreted protein) that are alsocomponents of the translocation apparatus by which othereffector proteins are translocated into the cell. LEE5encodes intimin, Tir and the Tir-chaperone CesT (Elliottet al., 1998). Tir is injected by the translocation apparatusdirectly to the cell and is inserted in the membrane expos-ing an extracellular domain that is recognized by intimin(an EPEC membrane adhesin). Intimin–Tir interactionleads to intimate adherence and pedestal formationbeneath adherent bacteria (Kenny et al., 1997). Duringinfection, other effector proteins are translocated into thecell (EspF, EspG, EspH, Map and EspI), and they interferewith different aspects of the cell physiology (Elliott et al.,2001; McNamara et al., 2001). Notably, there are severalnewly identified non-LEE-encoded effectors in AE patho-gens that are translocated by the T3SS, including Cif,NleA/EspI, TccP/EspFU, EspJ, NleB, NleC and NleD(Marches et al., 2003; Campellone et al., 2004; Garmen-dia et al., 2004; Gruenheid et al., 2004; Mundy et al.,2004; Dahan et al., 2005; Marches et al., 2005; Kellyet al., 2006).

Thus, the mechanism of protein translocation into thecell plays a central role in EPEC virulence. Recently, wehave found that a 110 kDa protein, named EspC, causescytotoxic effects including cytoskeletal damage; theseeffects depend on the EspC internalization and on itsfunctional serine protease motif (Navarro-Garcia et al.,2004). EspC is a non-LEE-encoded protein that is

Received 21 February, 2008; revised 9 May, 2008; accepted 23 May,2008. *For correspondence. E-mail fnavarro@cell.cinvestav.mx;Tel. (+52) 55 5747 3990. Fax (+52) 55 5747 3393.

Cellular Microbiology (2008) 10(10), 1975–1986 doi:10.1111/j.1462-5822.2008.01181.xFirst published online 8 July 2008

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd

encoded in a second PAI of EPEC, and unlike proteinssecreted by the T3SS, EspC secretion is mediated by thetype V secretion system (T5SS). Proteins secreted by thissystem receive also the name of autotransporter proteinsbecause the pre-proteins promote their own secretionthrough the inner and outer membranes by using twoprocessing domains, the signal sequence (SS) and thetranslocation unit (TU) (Stein et al., 1996; Mellies et al.,2001; Henderson et al., 2004), to secrete the passengerdomain (PD). In the case of EspC, these domains com-prise from amino acids 1 to 53 (SS), 54 to 1030 (PD) and1031 to 1306 (TU). Interestingly, regulation of espC iscoupled to the global regulator Ler localized in LEE andthat controls virulence gene expression during EPECpathogenesis, including those encoding the T3SS-secreted Esp proteins, Tir and intimin (Mellies et al., 1999;Elliott et al., 2000). However, a deletion mutant in espC byallelic exchange seems to be indistinguishable from itsisogenic parent for adherence, invasion, actin rearrange-ment and Tir phosphorylation, events that are crucial forA/E lesion formation (Stein et al., 1996). Despite this, adirect role of EspC in EPEC pathogenesis and its relationwith other bacterial virulence factors during EPEC infec-tion have not been studied.

We have explored the EspC internalization mechanisminto epithelial cells, as this is a key event in inducing thecytoskeletal damage that might explain the cytopathiceffect caused by EPEC. We have shown that EspC, innon-physiological conditions (i.e. as a purified protein),is not efficiently internalized, because no receptor isinvolved in its uptake and no intracellular traffic isrequired. Additionally, the physiologically secreted EspCby EPEC, which is enhanced in tissue culture media andby cell contact, is efficiently internalized during EPEC andepithelial cell interaction (Vidal and Navarro-Garcia,2006). Here, we demonstrate that EspC translocation intoepithelial cells involves the T3SS translocon, strongly indi-cating cooperation between two secretion systems, T5SSand T3SS.

Results

Efficient EspC entry into epithelial cells depends onLEE-encoded factors

To investigate EspC internalization dependency duringthe EPEC infection, HEp-2 cells were infected withEPEC or other AE pathogens (carrying LEE locus) suchas EHEC and REPEC, which were transformed with theespC gene. Anti-EspC antibody detected EspC in thecytoplasmic fraction from cells infected for 4 h withEPEC, EHEC/espC or REPEC/espC but not in cellsinfected with wild-type EHEC, REPEC (as these strainsdo not have espC) or E. coli HB101, an avirulent labo-

ratory strain (Fig. 1A). These results suggest that EspCinternalization appears to be related to LEE-encodedfactors, thereby an isogenic EPEC mutant in escN,which is unable to secrete any of the T3SS-dependentsecreted proteins, was used to infect HEp-2 cells. After4 h of infection, the anti-EspC antibodies did not detecta clear EspC band in the cytoplasmic fraction ofEPECDescN-treated cells (Fig. 1B), despite that thismutant is able to secrete EspC when is grown inMinimum Essential Medium (MEM) culture medium(Fig. 1C, top) or during cell infection with EPECDescN(Fig. 1C, bottom). These data suggest that some of theEPEC factors encoded in LEE are required for the effi-cient internalization of EspC.

As EspC is an autotransporter protein, it must besecreted into the bacterial extracellular media; therefore,we used an exogenous EspC internalization model,which involved addition of purified EspC during epithelialcells infection by EPEC. To validate our model, HEp-2cells were incubated with various concentrations of puri-fied EspC for 4 h, and then cytoplasmic fractions wereobtained and probed by immunoblot using anti-EspCantibodies. Only a scarce EspC band was detected incells treated with 200 or 300 mg ml-1 EspC, whereasEspC was not detected in cells treated with 50 or

Fig. 1. LEE pathogenicity island is involved in efficient EspCtranslocation into epithelial cells. HEp-2 cells were infected withdifferent AE pathogens and the cytoplasmic fractions from theseinfected cells were analysed by immunoblot using anti-EspCantibodies.A. Infection with AE pathogens. HEp-2 cells were infected with thewild-type EPEC, EHEC or REPEC or with either of two last strainstransformed with espC (EHEC/espC or REPEC/espC).B. Infection with isogenic mutants. HEp-2 cells were infected withEPECDeae and EPECDescN for 4 h.C. Secretion of EspC by EPEC, EPECDeae or EPECDescN grownin MEM medium (top) or during infection of epithelial cells (bottom).Nitrocellulose membranes were also probed with anti-actinantibodies as protein loading control.

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© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 1975–1986

100 mg ml-1 EspC (Fig. S1A), indicating no contamina-tion of the cytoplasmic fraction by EspC. On the otherhand, to avoid purified EspC internalization by pinocyto-sis (Vidal and Navarro-Garcia, 2006), HEp-2 cells wereincubated with 100 mg ml-1 EspC for 6 and 8 h and thenthe cytoplasmic fraction was probed with the anti-EspCantibodies. No clear EspC band was observed after 6 h,while after 8 h a burly EspC band was detected by theanti-EspC antibodies (Fig. S1B). These results allow usto establish that treatment with 100 mg ml-1 EspC for 4 havoid cellular fraction contamination and internalizationby pinocytosis. Additionally, there was not detection ofother EPEC proteins, which are not translocated into theeukaryotic cell, such as EspA that was detected in thesupernatant, but not in the cytoplasmic fraction, ofEPEC-infected cells (Fig. S2B). By using this experimen-tal model, an stronger band of EspC was detected in thecytoplasmic fraction from HEp-2 cells co-incubated withexogenous EspC and wild-type EPEC than in cellstreated with EPEC but without exogenous EspC(Fig. 2A). These data indicate that EPEC is able to inter-nalize exogenous and endogenous EspC at the same

time until saturation; compare data of 6 h versus 8 h(Fig. 2A). To estimate how much EspC is being translo-cated inside the cells, we performed the following con-sideration: after 6 h of infection the amount of EspC bydensitometry is similar to that of actin (actin is the mostabundant intracellular protein in an eukaryotic cell), sug-gesting that EspC can reach an intracellular concentra-tion of about 0.2 mM; as a typical cytosolic concentrationof actin in non-muscle cells is 0.5 mM, but solubleG-actin concentration in the cytosol is about 0.2 mM. Inaddition, cytoplasmic EspC band is of about 260 ng from15 mg of protein from the cytoplasmic fraction run inthe sodium dodecyl sulfate-polyacrilamyde gels (SDS-PAGE), as compared with 300 ng of purified EspCprotein used as control (Fig. S2A). Interestingly, anEPEC isogenic mutant in espC (EPECDespC) wascapable of internalizing EspC into the cytoplasm ofHEp-2 cells if those cells were co-incubated with exog-enous EspC (Fig. 2A). The same results were obtainedwhen HEp-2 cells were co-incubated with EHEC andexogenous EspC (Fig. 2B), but not in cells co-incubatedwith EPECDescN and exogenous EspC (Fig. 2C). Allthese data indicate that EspC, which is secreted byT5SS, is then translocated to the cytosol by the action ofLEE-encoded factor(s).

EspC translocation is a specific event and independentof LEE-encoded effector proteins

To determine the specificity of EspC translocation byEPEC, HEp-2 cells were co-incubated with EPECDespCand exogenous Pic, another autotransporter protein(homology of 34.6%) secreted by EAEC, or GST-fodrin,an irrelevant protein, both of which have a similarmolecular weight (109 kDa) to EspC (110 kDa). Unlikethe cytoplasmic fraction from cells co-incubated withEPECDespC and exogenous EspC (Fig. S3B), neitherPic (Fig. S3A) nor GST-fodrin (Fig. S3B) was translo-cated to the cytoplasm by EPECDespC; even though Picand EPECDespC co-incubation was prolonged for 5 or6 h (Fig. S3A). These results strongly suggest that themechanism of translocation used by EPEC is specific forEspC.

As the effector proteins injected by EPEC have diverseeffects on the host cells (Dean et al., 2005), we testedtheir role on EspC translocation. HEp-2 cells wereinfected with EPECDespC for sufficient times for injectingthe effector proteins into the cytosol (1, 2 or 3 h). At theend of these incubation times, the bacteria were killedwith tetracycline. After removing killed bacteria, the cellswere incubated with exogenous EspC for 4 h. EspC wasnot detected in the cytoplasmic fraction from these cells(Fig. S3C), suggesting that the effector proteins may notparticipate in the EspC internalization.

Fig. 2. Bacteria encoding a functional LEE translocate exogenousand endogenous EspC into the cells. HEp-2 cells were infectedwith EPEC, their derivative mutants or EHEC and co-incubated withpurified EspC (100 mg ml-1) for 4, 6 or 8 h. Cytoplasmic fractionswere obtained and processed as in Fig. 1.A. Cells treated with EPEC or EPECDespC alone for 4 h orco-incubated with exogenous EspC for various times.B. Cells treated with EHEC alone for 4 h or co-incubated withexogenous EspC for various times. EPEC was used as a positivecontrol.C. Cells treated with EPECDescN alone for 4 h or co-incubatedwith exogenous EspC for various times. After 8 h of incubation withEPECDescN, exogenous EspC is internalized by pinocytosis.Nitrocellulose membranes were also probed with anti-actinantibodies as protein loading control.

An autotransporter is translocated by the T3SS 1977

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 1975–1986

EspC translocation into epithelial cells is independentof intimate adherence

We have shown that EspC translocation occurs over thepedestals formation (Vidal and Navarro-Garcia, 2006),suggesting a role for the intimate adherence. To search forthis possibility, HEp-2 cells were infected with EPECisogenic mutants unable to produce intimin or Tir(EPECDeae or EPECDtir). Neither of these mutants wasable to translocate EspC into the cells (Figs 1B and 3A),although both mutants secreted EspC to the extracellularmedium, and during epithelial cell infection (Figs 1C and3B). However, by using the HEp-2 cells model ofco-incubation between either EPECDeae or EPECDtir andexogenous EspC, we found that both mutants were ableto translocate EspC into the cytoplasmic fraction (Fig. 3Aand C).

Improving the EspC translocation by EPECDeae andEPECDtir for adding exogenous EspC suggests a dimin-ished efficiency undetectable by cellular fractionation dueto the reduced adherence of these mutants. To explorethis possibility, HEp-2 cells were infected with wild-type

EPEC, EPECDeae or EPECDescN for 3 h and then thecells were immunostained with anti-EspC antibodies(green) and the actin cytoskeleton with rhodamine-phalloidin (red). We searched for cells with adherent bac-teria by Normarsky microscopy, and in these cells, EspCwas detected in the cytoplasm of cells infected with EPEC(Fig. 4G–I) or EPECDeae (Fig. 4D–F), as observed in amiddle section by confocal microscopy. However, EspCwas not detected into the cells infected with EPECDescN(Fig. 4B), despite these bacteria being adherent to theepithelial cells (Fig. 4C) and EspC being secreted bythese bacteria (Fig. 4A).

To further demonstrate that the inefficiency ofEPECDeae and EPECDtir for EspC translocation wasdue to the diminished adherence of these mutants toepithelial cells, the multiplicities of infection (moi) ofEPECDtir were increased until a bacterial adhesionsimilar to wild-type EPEC was reached. As previouslyestablished, an moi of 10 was used for the wild type,while for the EPECDtir moi of 50 and 200 were used.The cells were stained with rhodamine-phalloidin (red)and immunostained with anti-EspC (green) and anti-EPEC outer membrane (blue). As shown in Fig. 5, with20 times more EPECDtir bacteria (moi = 200) it was pos-sible to reach an amount of adherent bacteria compa-rable to the wild type and in that condition also a similaramount of EspC was translocated to the cytoplasm(Fig. 5A–D versus Fig. 5I–L). However, with five timesmore EPECDtir, cells did not show as many attachedbacteria as the wild-type EPEC and only trace amountsof EspC were detected by confocal microscopy in middlesections (Fig. 5E–H). Only in EPEC-infected cells, thethree markers colocalized because EPEC displaysintimate adherence and forms pedestals. Thus, eventhough EspC translocation is favoured by intimateadherence, it is independent of this infection step.

T3SS translocon of EPEC allows EspC translocationinto epithelial cells

As EPECDescN, lacking the bacterial cytoplasmic trans-locator, is unable to translocate EspC, we then used othertwo mutants in the T3SS, lacking extracellular transloca-tors, EPECDescF and EPECDespA, as well as a mutant ina translocator involved in pore formation on the hostmembrane, EPECDespB. First of all, we verified thatthese mutants secreted EspC during epithelial cell infec-tion at similar levels to the wild-type EPEC (Figs 3B and6B), confirming that EspC secretion is not mediated byT3SS.

HEp-2 cells were infected with EPECDescF,EPECDespA, EPECDespB or with any of the two mutantsthat are able to translocate exogenous EspC,EPECDespC and EPECDtir. Infected cells were

Fig. 3. EspC translocation is independent of the intimateattachment. HEp-2 cells were treated and processed as in Fig. 2.A. Cytoplasmic fractions from cells treated with EPECDtir alone for4 h or co-incubated with exogenous EspC for various times. EPECwas used as positive control.B. Secretion of EspC by EPEC, EPECDescF, EPECDespA orEPECDtir during infection of epithelial cells.C. Cytoplasmic fractions from cells treated with EPECDeae alonefor 4 h or co-incubated with exogenous EspC for various times.Nitrocellulose membranes were also probed with anti-actinantibodies as protein loading control.

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© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 1975–1986

co-incubated with exogenous EspC for 6 h. As shownabove, EPECDespC and EPECDtir were able to translo-cate exogenous EspC into the cytoplasm (Fig. 6A).However, EspC was not detected in the cytoplasmic frac-

tions of cells infected with EPECDescF, EPECDespA orEPECDespB (Fig. 6A and B). These data indicate thatEPEC uses the T3SS translocon for EspC translocationinto the epithelial cells. To corroborate these results,

Fig. 4. Reduced adherence to the cells byintimate attachment in the mutants decreasesthe EspC translocation. HEp-2 cells wereinfected with EPEC, EPECDescN orEPECDeae. Treated cells wereimmunostained with anti-EspC antibodies andthe actin cytoskeleton was detected withrhodamine-phalloidin.A–C. Cells treated with EPECDescN.D–F. Cells treated with EPECDeae.G–I. Cells treated with EPEC.Images from optical sections by confocalmicroscopy are indicated: surface sections(A, D and G), middle sections (B, E and H).Adherent bacteria were localized byNormarsky microscopy (C, F and I). Whiteand blue arrows show EspC secreted overthe cell surface or inside the cellsrespectively. Black arrows show microcoloniesof adhered cells.

Fig. 5. Low EspC translocation by intimateadherence mutants is related to theirdefective initial adhesion. HEp-2 cells wereinfected with EPEC or EPECDtir for 3 h.A–D. Cells infected with EPEC with an moi of10.E–H. Cells infected with EPECDtir with an moiof 50.I–L. Cells infected with EPECDtir with an moiof 200.Treated cells were immunostained withanti-EspC antibodies (green; C, G and K;middle sections) and anti-EPEC membranes(blue; B, F and J; surface sections), andthe actin cytoskeleton was detected withrhodamine-phalloidin (red; A, E and I;projection of optical sections). (D), (H) and (L)are the merge of the three channels. Whitearrows show pedestal formation; yellowarrows show microcolonies of adherentbacteria.

An autotransporter is translocated by the T3SS 1979

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 1975–1986

HEp-2 cells were infected with a high moi (200) of T3SSmutants (EPECDescN or EPECDespA). In this system,both mutants were capable for adhering to epithelial cellsat similar levels to the wild-type EPEC used at an moi = 10(Fig. 6D, H and L). However, only the wild-type EPEC wascapable of translocating EspC into the cells (Fig. 6Eversus Fig. 6I and M). Furthermore, complementation ofT3SS mutants re-established EspC translocation. Thus,complementation of EPECDescF with a plasmid contain-ing escF gene allowed EspC detection in the cytoplasmfraction, but it was not detected in the cytoplasm fractionfrom the isogenic mutant in escF gene (Fig. S2C). Allthese data clearly indicate that EspC secreted by T5SS istranslocated into epithelial cells by using the T3SS trans-locon of EPEC.

EspC interacts with EspA

To understand how EspC could be interacting with theT3SS, we first infected HEp-2 cells with EPECDespCco-incubated with exogenous EspC. After the co-incubation, the cells were extensively washed, fixed,

but not permeabilized, and the bacterial membraneand EspC were detected by immunofluorescence byusing anti-EPEC membrane (red) and anti-EspC (green)antibodies. By optical sections under confocal microscopy(Fig. 7), it was possible to detect EspC (green) betweenthe eukaryotic plasma membrane (black background)(Fig. 7A) and the bacterial membrane (red) (Fig. 7B) andto show partial localization in between them, but predomi-nantly the red on the green fluorescence by merging bothchannels (Fig. 7C). To determine whether EspC is inter-acting with T3SS proteins on the bacterial membrane, weperformed immunoprecipitation assays from the mem-brane fraction of EPEC incubated with EspC. After 1 h ofco-incubation, EPEC membrane mixed with EspC wassubjected to immunoprecipitation with anti-EspC. ProteinA-bound immunocomplexes were separated by SDS-PAGE. Besides EspC, five main proteins were resolved bythe SDS-PAGE, which were of 80, 75, 37, 35 and 26 kDa(Fig. 7D). Interestingly, when membrane from the espAmutant was used in the immunoprecipitation experimentsthese proteins were not seen (Fig. 7D, last lane). The lastthree proteins co-immunoprecipitated with the anti-EspC

Fig. 6. EPEC uses T3SS translocon to allowthe EspC translocation.A and B. EPECDescF, EPECDespA andEPECDespB are unable to translocateexogenous EspC. HEp-2 cells were infectedwith EPECDespC, EPECDescF, EPECDespA,EPECDtir or EPECDespB and co-incubatedwith purified EspC (100 mg ml-1) for 6 h.Cytoplasmic fractions were obtained andprocessed as in Fig. 1.C–N. No EspC translocation occurred in cellsinfected with higher moi of EPECDescN orEPECDespA. HEp-2 cells were infectedwith EPEC with an moi of 10 (C–F) orEPECDescN (G–J) or EPECDespA (K–N) withan moi of 200, during 3 h. Treated cells wereimmunostained with anti-EspC antibodies(green; C, G and K; middle sections) andanti-EPEC membranes (blue; B, F and J;surface sections), and the actin cytoskeletonwas detected with rhodamine-phalloidin(red; A, E and I; projection of confocal opticalsections). (D), (H) and (L) are the merge ofthe three channels. White arrows pointpedestal formation; yellow arrows showmicrocolonies of adhered bacteria.EPECDespB-sec in (B) indicates secretedproteins during cells infection.

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© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 1975–1986

antibody from EPEC membranes suggesting that theymay be EspD, EspB and EspA. To test this, we performedoverlay experiments by separating purified EspC, EspB orEspA, as well as EPEC lysates with SDS-PAGE (Fig. 7E),

and probing nitrocellulose membranes containing theseseparated proteins with purified EspB and EspA, and theinteraction among these proteins were visualized by usinganti-EspB (Fig. 7F) and anti-EspA (Fig. 7G) antibodies.

Fig. 7. EspC interacts with the translocator protein EspA on the bacterial membrane.A–C. Detection of EspC in between eukaryotic plasma membrane and bacterial membrane. HEp-2 cells were infected with EPECDespCco-incubated with exogenous EspC for 1 h. After infection, cells were extensively washed, fixed, but not permeabilized, and immunostained byusing Anti-EPEC membrane (A) and anti-EspC (B). The localization of exogenous EspC outside of the cells and its interaction with thebacterial outer membrane was analysed for merging images (C) by confocal microscopy.D. Immunoprecipitation of EspC-interacting proteins on bacterial outer membrane. Purified outer membrane from wild-type EPEC orEPECDespA (*) were incubated with purified EspC for 1 h. The mixture was subjected to immunoprecipitation with anti-EspC. As controls inthe SDS-PAGE was run immunoprecipitated EspC alone, anti-EspC and protein A incubation, the protein removed from the mixture bywashing, as well as purified EspC and purified EPEC membrane fraction.E–G. Overlay assay among EspC, EspA and EspB. Purified EspA, EspB and EspC, as well as EPEC lysates were subjected to SDS-PAGEanalysis (E). These separated proteins were transferred to nitrocellulose membrane and incubated with either purified EspB or purified EspA.The EspB or EspA interactions were developed by immunoblot by using anti-EspB (F) or anti-EspA (G) antibodies.H–K. EspC translocation appears to occur through channel-like structures underneath the adhered bacteria. HEp-2 cells were infected withEPEC (H), EPECDespC (I) or EPECDespC plus purified EspC (J and K) for 3 h. Treated cells were immunostained with anti-EspC antibodies(green) and anti-EPEC membranes (blue), and the actin cytoskeleton was detected with rhodamine-phalloidin (red). Images show confocalvertical sections (yz) of infected cells. Arrowheads in (H) and (J) show EspC fluorescent marks forming channel-like structure. In (K), yellow,white and green arrows show bacteria, actin pedestal and EspC.

An autotransporter is translocated by the T3SS 1981

© 2008 The AuthorsJournal compilation © 2008 Blackwell Publishing Ltd, Cellular Microbiology, 10, 1975–1986

The anti-EspB antibody detected mainly the purified EspBand a 26 kDa band in EPEC lysates at similar molecularweight to purified EspA; this purified protein slightly inter-acted with purified EspB, as detected with the anti-EspBantibody (Fig. 7F). The anti-EspA antibody detectedmainly the purified EspA, and the EspA interacting withpurified EspB and EspC, as well as with proteins of 39 and110 kDa in EPEC lysates, which might be EspD andEspC. In fact this latter protein was at the same molecularweight as purified EspC, which strongly interacted withEspA (Fig. 7G).

To further investigate the mechanism of EspC translo-cation, HEp-2 cells were infected with EPEC or with eitherEPECDespC alone or co-incubated with exogenousEspC. Confocal microscopy analysis of cells stained withrhodamine-phalloidin and immunostained with anti-EspCshowed that EspC is observed inside the cells infectedwith either EPEC (Fig. 7H) or EPECDespC co-incubatedwith exogenous EspC (Fig. 7J), but not in those cellsinfected with EPECDespC alone (Fig. 7I). Interestingly,confocal sections in the plane yz of these cells showedEspC fluorescent marks coming from the extracellularregion (Fig. 7H and J, arrows): these fluorescent markswere channel-like passing the cell membrane andreached the cytoplasm, suggesting that they come fromadhering bacteria. Indeed, in similar experiments but nowmarking also the bacteria by using anti-EPEC membraneantibodies, EspC (green) seemed to emerge from thebacteria (blue) and was detected forming these channel-like structures just beneath the bacteria and the pedestals(red) (Fig. 7K). Remarkably, even though a high concen-tration of exogenous EspC is present, the EspC translo-cation appears to occur only through these channel-likestructures under the adhered bacteria. Similar resultswere observed in cells infected only with the wild-typeEPEC, suggesting the T3SS translocon is involved.

Discussion

We have demonstrated that EspC is efficiently translo-cated to the cytoplasm of cells in culture infected withEPEC (Vidal and Navarro-Garcia, 2006). This transloca-tion event is related to the LEE PAI as at least two otherAE pathogens allowed EspC translocation. EspC is rec-ognized in a specific manner for these pathogens to betranslocated into the epithelial cells, as irrelevant orhomologous proteins are not translocated by thesepathogens. Furthermore, EspC translocation is not anevent induced by effector proteins injected by EPEC intothe target cell. Intimate adherence between EPEC andepithelial cells favours the EspC translocation but is notessential for such event. Finally, EspC is translocated bythe T3SS translocon of EPEC, but unlike the LEE-encoded proteins or the recently reported, non-LEE-

encoded proteins that use this T3SS translocon to beinjected from the bacterial cytoplasm to the eukaryoticcytoplasm, EspC is secreted to the milieu and then isincorporated to the T3SS translocon to enter into the cellsthrough its interaction with EspA.

Recently, we found that EspC is efficiently internalizedduring the pedestal formation (Vidal and Navarro-Garcia,2006), suggesting that EspC translocation might berelated to this lesion and thereby, to the LEE PAI. By usingother two AE pathogens, which harbour LEE PAI but notespC gene, we found that these espC-transformed patho-gens displayed an efficient EspC translocation. Moreover,an isogenic mutant (EPECDescN), lacking a cytoplasmicATPase translocator protein that provides energy tosecrete proteins through the T3SS encoded in LEE(Gauthier et al., 2003), was unable to translocate EspCinto the cells. These data also suggest that any factorsecreted by the T3SS might be helping EspC transloca-tion, as EspC is an autotransporter protein secreted bythe T5SS (Stein et al., 1996; Mellies et al., 2001; Navarro-Garcia et al., 2004). To explore this controversy, we con-ceived an EspC translocation model by infecting cells withbacteria harbouring LEE and co-incubated with exog-enous EspC. This model and the use of an isogenicmutant in espC (EPECDespC) allowed us to establish thatEPEC is able to translocate exogenous and endogenousEspC, indicating that EspC is translocated after itssecretion.

This EspC translocation event assisted by LEE-encoded factor(s) was specific for EspC, as other proteinsincluding another serine protease autotransporter fromEAEC, called Pic, that is not internalized into the cellsas a purified protein (our unpublished data) were nottranslocated. This suggests that EspC translocationdoes not occur by increased membrane permeability orpinocytic event as a result of EPEC infection but insteadinvolves a somewhat precise mechanism. The LEE con-tains 41 genes and encodes a T3SS, a regulator (Ler), anadhesin (intimin) and its receptor (Tir) responsible forintimate attachment, several secreted proteins and theirchaperones (Frankel et al., 1998; Clarke et al., 2003).Interestingly, EspC is also regulated by Ler, despite espCbeing encoded by another PAI (Elliott et al., 2000; Mellieset al., 2001). Chaperones might not be involved in EspCtranslocation, as they are bacterial cytoplasmic proteinsand EspC is secreted by the T5SS. Thereby, secretedproteins and/or intimate attachment or the complete T3SStranslocon may be involved in EspC translocation. Thesecreted proteins consist of effectors (Tir, EspG, EspF,Map and EspH) as well as translocators (EspA, EspD andEspB). Five LEE-encoded effectors have been identified,which are involved in modulating host cytoskeleton,including paracellular permeability (Clarke et al., 2003; Tuet al., 2003; Matsuzawa et al., 2005). However, the effec-

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tors appear not to participate in the EspC internalizationas indicated in experiments injecting these effectors byusing EPECDespC, and exogenous EspC after killing thebacteria. Additionally, even though EspC translocationoccurs beneath adhered bacteria and over the pedestals(Vidal and Navarro-Garcia, 2006), the role of the intimateattachment in the EspC translocation was also discarded,as isogenic mutants in both proteins responsible for inti-mate attachment, intimin and Tir, were capable for EspCtranslocation into epithelial cells. However, it was neces-sary to overcome the reduced adherence observed inthese mutants (Shaw et al., 2005) by co-incubating themwith exogenous EspC or by increasing the moi at least 20times. Interestingly, by using these latter conditionsEPECDescN was still unable to translocate EspC, despitethose bacteria adhering to the epithelial cells and secret-ing EspC.

These last results lead us to suggest that the translo-cator proteins may participate in the EspC translocation,although these proteins (working as a T3SS translocon)only appear to translocate proteins from the bacterialcytoplasm to the eukaryotic cytosol. However, EspCtranslocation occurs when it has already been secretedto the intracellular milieu. Other findings also supportLEE–EspC association, including the fact that theLEE-encoded regulator (Ler) regulates the secretion oftranslocators as well as of EspC (Elliott et al., 2000). EspCis temporally the first protein secreted during the epithelialcell infection by EPEC (Kenny and Finlay, 1995), thus itmust be available for its translocation by the time thetranslocators are secreted. Remarkably, partially purifiedT3SS translocons, observed as injectosomes by electronmicroscopy, contain EspC as analysed by SDS-PAGE(Sekiya et al., 2001), even though this protein is secretedbefore the T3SS formation (Kenny and Finlay, 1995).Interestingly, EspC is separated from the injectosomesonly after (ultracentrifugation, sucrose cushion) a thirdpurification step on CsCl fractionation (Ogino et al., 2006).Supporting these isolated findings, we show here that thelack of the injectosome by using EPECDescN to infectcells prevented EspC translocation; also the lack of theneedle of the injectosome or of the filamentous extensionto the needle complex or translocators involved in poreformation on the membranes of the infected cells, byusing EPECDescF, EPECDespA or EPECDespB, respec-tively, also stopped EspC translocation. These data indi-cate that the T3SS translocon is required for allowingEspC translocation into epithelial cells, but also raise thequestion of how a milieu-secreted protein gains access tothe T3SS translocon to be injected into the host cells; isthe translocon a closed conduit? Furthermore, affinityexperiments using purified proteins showed that EspCclearly binds EspA, but not EspB (although EspC–EspDinteraction experiments were not performed). Additionally,

immunoprecipitation experiments from EPEC mem-branes incubated with EspC showed that EspCco-immunoprecipitates with EspA, and thereby with EspBand EspD (translocator proteins), as EspC does not inter-act with these proteins in membranes from EPECDespA.All these data suggest the T3SS might function asconduit/guiding filament by guiding proteins previouslysecreted from the bacteria but with affinity to the filament(EspA), as it was initially suggested for Pseudomonassyringae (Jin et al., 2001).

In summary, we demonstrated here that during EPECinfection, EspC is secreted by the T5SS and then it isefficiently translocated through the T3SS. This constitutestwo novel mechanisms of protein translocation into hostcells, which involves cooperation between two secretionsystems, T5SS and T3SS, as well as the uptake of aprotein from the culture/supernatant into the T3SS. Thiscooperation mechanism may be employed by otherpathogens encoding T3SS and autotransporter proteins,for instance, the T3SS and EspP of EHEC.

Experimental procedures

Bacterial strains and purification of recombinant protein

Characteristics of the strains used in this study are listed inTable S1. All strains were routinely grown in Luria–Bertani(LB) broth or MEM (without supplements) aerobically at 37°C.EPEC cultures were activated for 3 h as previously described(Rosenshine et al., 1996).

Strains HB101(pJLM174) or HB101(pPic1) were grown over-night in LB plus arabinose (0.2% w/v) and ampicillin (100 mg ml-1)or tetracycline (15 mg ml-1), respectively, at 37°C in shaking. Thesupernatants were obtained by centrifugation at 7000 g for15 min, filter sterilized through 0.22-mm-diameter filters (Corning,Cambridge, MA) and concentrated 100-fold in an ultrafree cen-trifugal filter device with a 100 kDa cut-off (Millipore, Bedford,MA). Recombinant proteins were filter sterilized again, aliquotedand quantified by the Bradford method (Bradford, 1976).

Tissue culture cells and cellular fractionation

The human epithelial cell line HEp-2 (ATCC-CCL23) was culturedin MEM supplemented with 10% fetal calf serum (FCS) (HyClone,Logan, UT), 1% non-essential amino acids, 5 mM L-glutamine,penicillin (100 U ml-1) and streptomycin (100 mg ml-1). Cells werenormally harvested with 10 mM EDTA and 0.25% trypsin (GibcoBRL, Grand Island, NY) in PBS (pH 7.4), re-suspended in theappropriated volume of supplemented DMEM and incubated at37°C in a humidified atmosphere of 5% CO2.

HEp-2 cells grown in 60 mm Petri dishes were infected withactivated cultures of EPEC or the indicated isogenic mutantalone or with either purified EspC or the indicated purified proteinduring the indicated time. EPEC-infected HEp-2 cells wereincubated in the presence of D-mannose (1%) and the appro-priated antibiotic. Cells were delicately washed three times withice-cold PBS, pH 7.4 and scraped in a buffer consisting of

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Tris-HCl (0.25 M), pH 7.5, phenylmethylsulfonyl fluoride (PMSF)(50 mg ml-1), aprotinin (0.5 mg ml-1) and EDTA (0.5 mM). Thencells were lysed by three repeating freeze–thaw cycles (5 minincubation in a dry ice-ethanol bath and 3 min incubation in athermoblock at 37°C) (Taylor et al., 1998). The cell lysates wereultracentrifuged at 100 000 g for 1 h at 4°C, and the supernatantfraction containing soluble cytoplasmic proteins was obtained.Pellets containing HEp-2 cell membranes, adherent bacteria,nuclei and cytoskeletal proteins were washed with cold PBS andre-suspended in PBS. Protein concentrations were estimated bythe method of Bradford. Equivalent volumes were boiled for7 min, analysed on SDS-PAGE and electrotransferred to nitro-cellulose membranes for Western blot analyses essentially aspreviously described (Vidal and Navarro-Garcia, 2006). Theidentity of the cellular fractions was confirmed with a mousemonoclonal anti-actin antibody (a gift from Manuel Hernández,CINVESTAV, Mexico) and a polyclonal rabbit anti-pan-cadherin(Zymed laboratories).

Confocal microscopy

HEp-2 cells were seeded into eight-well LabTek slides (VWR,Bridgeport, NJ) at a density of 3 ¥ 104 cells per well. Beforeinfection with activated EPEC cultures, cells were washed threetimes with warmed PBS (pH 7.4) and incubated at 37°C in DMEM(without supplements) during 30 min. Infections were carried outat the indicated time in the presence of D-mannose (1%)(Research Organics). Infected HEp-2 cells were washed withPBS, fixed with 2% formalin–PBS, washed, permeabilized by theaddition of 0.1% Triton X-100–PBS, and stained with 0.05 mg oftetramethyl rhodamine isothiocyanate (TRITC)-phalloidin ml-1

and with a polyclonal rabbit anti-EspC as previously described(Navarro-Garcia et al., 2004), followed by an anti-rabbitfluorescein-labelled antibody. When indicated, the bacteria weredetected by using an antibody against membrane proteins fromEPEC (a gift from Angel Manjarrez, UNAM, Mexico). Slides weremounted on Gelvatol, covered with glass coverslips and exam-ined under a Leica TCS SP2 confocal microscope.

Immunoprecipitation and overlay assays

Immunoprecipitation assays were performed as a previous report(Navarro-Garcia et al., 2007). Briefly, outer membrane from wild-type EPEC or EPECDespA, obtained by ultrasonic treatment andextraction in 1% sarcosyl according to the methodology previ-ously described by Klingman and Murphy (1994), was incubatedwith purified EspC for 1 h at 37°C. This mixture wasre-suspended in 1 ml of cold lysis buffer and 500 mg of proteinwas centrifuged, and the supernatants were used for immuno-precipitation experiments. Supernatants were incubated withanti-EspC (2 mg) antibodies. Then, protein A-agarose suspension(Roche Diagnostics, Mannheim, Germany) was added for 3 h at4°C. The complexes were collected by centrifugation and thesupernatant was removed. The pellet was washed five times andre-suspended in loading buffer, and the immunocomplexes wereresolved by SDS-PAGE.

Overlay assays were performed as a previous report(Canizalez-Roman and Navarro-Garcia, 2003). Four or twomicrograms of the purified proteins (EspA, EspB or EspC) orEPEC lysates were separated by 10% SDS-PAGE. These sepa-

rated proteins were then transferred to nitrocellulose membrane,which was blocked overnight in blocking buffer at 4°C. The mem-branes were then incubated for 1 h in interacting buffer witheither 5 mg ml-1 EspA or 5 mg ml-1 EspB (kindly donated by DrAngel Cataldi, Institute of Biotechnology, Argentina). The mem-branes were washed and then incubated for 1 h in blocking bufferwith rabbit polyclonal antiserum raised against EspA (1:3000dilution) or rabbit polyclonal antiserum raised against EspB(1:3000 dilution). These antibodies were kindly donated by JorgeGirón (The University of Arizona). Following another wash step,the strips were incubated for 1 h in blocking buffer with a goatanti-rabbit AP-conjugated antibody (1:10 000 dilution) (KPL,Gaithersburg, MD). Following a final wash, binding was detectedusing one-step NBT/BCIP substrate (Pierce, Rockford, IL).

Acknowledgements

We thank Dr James Kaper and Bruce McClane for reviewing themanuscript and their valuable advices. We thank James Kaper,Michael Donnenberg, James Nataro, Brett Finlay, Gad Frankel,Eric Oswald and Akio Abe for providing the E2348/69 derivativesand AE pathogens. We also thank Rocio Huerta and JazminHuerta for their technical help.

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Supplementary material

The following supplementary material is available for this articleonline:Table S1. Bacterial strains and plasmid used in this study.Fig. S1. Standardization of a model of exogenous EspCtranslocation.A. Concentration of purified EspC unable to contaminate thecytoplasmic fraction. HEp-2 cells were incubated with 50, 100,200 or 300 mg ml-1 EspC for 4 h. Cytoplasmic fractions of thesetreated cells were analysed by immunoblot by using anti-EspCantibodies. The first line shows cytoplasmic fraction obtained attime zero (addition of EspC, immediate cell washed and purifica-tion of the cytoplasmic fraction). The last line shows purifiedEspC used as positive reaction and molecular marker.B. Time-course of EspC internalization by pinocytosis detectedby cellular fractionation. HEp-2 cells were incubated with100 mg ml-1 EspC for 6 and 8 h. Untreated cells were used asnegative control.In both panels, nitrocellulose membranes were also probed withanti-actin antibodies as load control.

Fig. S2. Estimation of amount of translocated EspC, restorationof EspC translocation in complemented T3SS mutant and trans-location control of an EPEC protein that is not translocated to thecytoplasm.A. Densitometry measurement of EspC translocation versusintracellular actin in EPEC-infected cells, and purified EspC.B. Detection of EspA in supernatant and cytoplasm fractions fromEPEC-infected cells.C. Restoration of EspC translocation in EPECDescF comple-mented with a plasmid containing the escF gene.Fig. S3. EspC translocation is a specific event and effector pro-teins independent.A and B. Pic (A) and GST-fodrin (B) are not translocate by EPEC.HEp-2 cells were infected with EPECDespC and co-incubatedwith purified Pic (100 mg ml-1) for 4, 5 or 6 h or with GST-fodrin(100 mg ml-1) for 6 h. Cytoplasmic fractions of these treated cellswere analysed by immunoblot by using anti-Pic or anti-GSTantibodies. The first lines show cytoplasmic fraction from cellstreated only with EPECDespC for 4 h. Nitrocellulose membraneof (B) was also probed with anti-EspC antibodies; middle figure.The last lines show purified Pic or GST-fodrin used as positivereactions and molecular markers.C. Role of effectors on EspC translocation. HEp-2 cells wereinfected with EPECDespC for 1, 2 or 3 h and after this infectionthe bacteria were killed with tetracycline and then were incubatedwith purified EspC for 3 h. Cytoplasmic fraction was probed withanti-EspC antibodies.In all panels, nitrocellulose membranes were also probed withanti-actin antibodies as load control.

This material is available as part of the online article from:http://www.blackwell-synergy.com/doi/abs/10.1111/j.1462-5822.2008.01181.x

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