Yersinia enterocolitica YopP-induced Apoptosis of Macrophages Involves the Apoptotic Signaling Cascade Upstream of Bid*

Yersinia enterocoliticainduces apoptosis in macrophages by injecting the plasmid-encoded YopP (YopJ in other Yersinia species). Recently it was reported that YopP/J is a member of an ubiquitin-like protein cysteine protease family and that the catalytic core of YopP/J is required for its inhibition of the MAPK and NF-κB pathways. Here we analyzed the YopP/J-induced apoptotic signaling pathway. YopP-mediated cell death could be inhibited by addition of the zVAD caspase inhibitor, but not by DEVD or YVAD. Generation of truncated Bid (tBid) was the first apoptosis-related event that we observed. The subsequent translocation of tBid to the mitochondria induced the release of cytochrome c, leading to the activation of procaspase-9 and the executioner procaspases-3 and -7. Inhibition of the postmitochondrial executioner caspases-3 and -7 did not affect Bid cleavage. Bid cleavage could not be observed in ayopP-deficient Y. enterocolitica strain, showing that this event requires YopP. Disruption of the catalytic core of YopP abolished the rapid generation of tBid, thereby hampering induction of apoptosis by Y. enterocolitica. This finding supports the idea that YopP/J induces apoptosis by directly acting on cell death pathways, rather than being the mere consequence of gene induction inhibition in combination with microbial stimulation of the macrophage.

The abbreviations used are: TNF-␣, tumor necrosis factor-␣; cmk, chloromethylketone; fmk, fluoromethylketone; Ac-YVAD, acetyl-Tyr-Val-Ala-Asp; B-D, benzyloxycarbonyl-Asp(OMe); BLP(s), bacterial lipoprotein(s); FADD, Fas-associated death domain; FADD E , Fas-associated death domain C-terminal coupled to an E tag; IKK, IB␣ kinase kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MKK(s), MAPK kinase(s); m.o.i., multiplicity of infection; NF-B, nuclear factor B; TRADD E , TNF receptor-associated death domain C-terminal coupled to an E tag; TRADD, TNF receptor-associated death domain; TLR, toll-like receptor; zAAD, benzyloxycarbonyl-Ala-Ala-Asp; zDEVD, benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe); zVAD, benzyloxycarbonyl-Val-Ala-Asp(OMe); tBid, truncated Bid; WT, wild type; TUNEL, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling; SUMO-1, small ubiquitin-related modifier-1. proteolysis by interaction with adaptor proteins or by cleavage via upstream proteases in an intracellular cascade (12)(13)(14). Two main procaspase activation pathways during apoptosis have been proposed: (i) the extrinsic activation of initiator procaspases, triggered by the formation of a receptosome complex; and (ii) the intrinsic activation of initiator procaspases, initiated by the formation of an apoptosome complex. The extrinsic activation of initiator procaspases is initiated by the death domain-containing receptors of the TNF receptor superfamily (14,15). Binding of the ligand to the cell surface receptor leads to trimerization (16) of the receptors and subsequently oligomerization of their cytosolic death domains eventually leading to the recruitment of initiator procaspases, such as procaspase-8. This complex is called the death-inducing signaling complex (17). During this process caspase-8 can initiate cell death by directly cleaving the downstream executioner procaspases-3, -6, and -7 (13,18). In some cases, however, the death-inducing signaling complex does not generate sufficient caspase-8 levels to allow efficient proteolytic activation of the downstream executioner procaspases, and amplification of the caspase cascade by mitochondrial-derived factors is required to kill the cell (19). A molecular link connecting the death-inducing signaling complex activation and mitochondria is the caspase-8-mediated cleavage of Bid, a pro-apoptotic member of the Bcl-2 family. The C-terminal part of Bid (tBid) translocates to the mitochondria, where it induces the release of cytochrome c (20,21). Cytochrome c, together with dATP/ATP, binds the apoptotic protease activating factor-1 resulting in the formation of the apoptosome complex (22), which leads to recruitment and autoactivation of procaspase-9. In turn, caspase-9 activates procaspases-3, -6, and -7, thus initiating the caspase cascade (23). The consecutive activation of both initiator procaspases, procaspase-8 at the level of the death receptor complex and procaspase-9 at the postmitochondrial level, then leads to sufficient activation of the executioner procaspases resulting in cell demise. Intrinsic activation of procaspases, triggered by environmental insults, senescence, and developmental programs, involves the release of cytochrome c from the mitochondrial intermembrane space to the cytosol leading to assembly and activation of the apoptosome complex igniting the caspase cascade. The mechanism by which this cytochrome c release is induced is not yet clear.
In this report we describe that activation of procaspases is essential in Y. enterocolitica YopP-mediated cell death. Moreover, YopP-mediated apoptosis initiates the cell death pathway upstream of the cytosolic protein, Bid. Bid cleavage results in the subsequent release of cytochrome c from the mitochondria eventually leading to the activation of procaspase-9 and the executioner procaspases-3 and -7. The cleavage of Bid and the release of cytochrome c can both be inhibited by broad-spectrum caspase inhibitors, suggesting that YopP induces activation of upstream caspases, most likely caspase-8. Finally, we provide evidence that the protease activity of YopP is crucial for the induction of apoptosis in macrophages by Yersinia.
Plasmid pMSK13 is a mobilizable plasmid containing yopP E40WT (from the pYV plasmid of Y. enterocolitica E40). Plasmid pRB16 is a derivative of pMSK13 encoding yopP E40C172T in which the catalytic cysteine is replaced by a threonine. The mutant was engineered by site-directed mutagenesis as described by Kunkel et al. (27) using the mutation primer 5Ј-ACTAAAAATACCGGTTTCAGATGAGCT-3Ј, which introduces a PinA1 restriction site (underlined). Plasmids pMSK13 and pRB16 were used to complement the yopP knockout mutation. For reasons of clarity, strains E40(pMSK41)(pMSK13) and E40(pMSK41)(pRB16) will be referred to as YopP E40WTϩ and YopP E40C172Tϩ strains. Plasmid pGD2 was obtained by cloning yopP A127WT , the wild type yopP gene from strain A127, into the eukaryotic expression vector pEF6 Myc/His vector (Invitrogen, Carlsbad, CA) in frame with the Myc and His tags, using EcoRI and NotI sites. pCDNA1-TRADD E was described elsewhere (28). pCDNA1-FADD E was constructed in a similar way as pCDNA1-TRADD E by fusing the human FADD E . pEF1-Bid was made by cloning the reverse transcriptase polymerase chain reaction fragment of full-length Bid (from mouse lung mRNA) in the pEF1 V5/His vector (Invitrogen, Carlsbad, CA). pIKK2 FLAG , an expression vector for IKK2/IKK␤, was a generous gift from Drs. J. Schmitt and R. de Martin (University of Vienna, Vienna, Austria).
Bacteria were pregrown overnight in brain-heart infusion (Difco) before infection bacteria were diluted 1/20 in fresh brain-heart infusion and cultured under continuous shaking (110 rpm) for 120 min at room temperature; subsequently the bacteria were induced for Yop secretion by incubation for 30 min in a shaking water bath (110 rpm) at 37°C. Prior to infection bacteria were washed with RPMI 1640 (Life Technologies, Inc.).
Western Analysis of YopP, Cytochrome c, Caspases, and Bid-For analysis of YopP, cytochrome c release, and procaspase and Bid cleavage, macrophages were infected with Y. enterocolitica or were left untreated as a control. After 3 h, cells were harvested by centrifugation, washed with phosphate-buffered saline (PBS), resuspended in 0.002% digitonin (RBI, Natick, MA) PBS, and incubated for 3 min on ice. Cell membranes and organelles were pelleted by centrifugation (15,000 ϫ g for 5 min at 4°C). Subsequently, 50 g of cytosolic protein or an equivalent of 2.5 ϫ 10 4 cells were loaded per lane on 15% polyacrylamide gels. After electrophoresis, the gels were blotted onto nitrocellulose (BA 83; Schleicher & Schü ll, Dassel, Germany), which was then probed with a rabbit polyclonal anti-YopP antiserum, mouse monoclonal antibody to cytochrome c (Pharmingen, San Diego, CA), polyclonal antisera raised against recombinant murine caspases-3 or -7 (29), and polyclonal rabbit anti-murine caspase-9 antiserum (cell signaling technology; New England Biolabs Inc., Beverly, MA) or probed at 4°C with a polyclonal goat serum raised against Bid (R & D Systems, Abingdon, UK). The anti-YopP antiserum was raised against purified YopP produced in strain E40(pAB409)(pMSK13), affinity purified on nitrocellulose-bound YopP, and concentrated by passing through a protein G column (Mab kit; Amersham Pharmacia Biotech). Primary antibody binding was detected with horseradish peroxidase-conjugated goat anti-mouse IgG, goat anti-rabbit IgG, or rabbit anti-goat IgG (DAKO A/S, Glostrup, Danmark) for cytochrome c, caspases and YopP, and Bid, respectively, and visualized by enhanced chemiluminscence as described in the manufacturer's instructions (Roche Molecular Biochemicals).
Co-immunoprecipitation-HEK293T cells (1.5 ϫ 10 6 ) were seeded in 9-cm Petri dishes. The next day cells were transfected via the calcium phosphate precipitation method with 2 g of pCDNA1-TRADD E , pCDNA1-FADD E , pEF1-Bid, or pIKK2 FLAG (coding for IKK␤), in the absence or presence of 2 g of pEF6 M/H -YopP A127WT . Empty vector was added to a total amount of 5 g of DNA for each transfection. 24 h after transfection, cells were lysed in 50 mM Hepes, pH 7.6, 250 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM aprotinin, and 1 mM leupeptin, and lysates were incubated for 5 h with 2.4 g of anti-Myc-tagged antibody (Roche Diagnostics, Basel, Switzerland). Immune complexes were incubated for 1 h with protein A-Trisacryl beads, which were subsequently washed five times with lysis buffer. Coprecipitating proteins were separated by SDS polyacrylamide gel electrophoresis and analyzed by Western blotting with horseradish peroxidase-coupled anti-E-tagged antibody (Amersham Pharmacia Biotech), anti-Bid antibody followed by horseradish peroxidasecoupled anti-goat antibody (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-FLAG-tagged antibody (Sigma) followed by horseradish peroxidase-coupled anti-mouse antibody (Amersham Pharmacia Biotech), depending on the plasmid transfected. Protein bands were visualized using enhanced chemiluminscence as described in the manufacturer's instructions (Roche Molecular Biochemicals).

Caspases Mediate Yersinia YopP-induced Cell Death-
Previous results indicated that YopP/J is required for Yersinia to induce apoptosis in macrophages (7,8). To determine whether procaspase activation was involved in YopP/Jmediated apoptosis, J774A.1 macrophages were pretreated with various irreversible caspase inhibitors prior to infection with E40(pMSK41)(pMSK13), a Y. enterocolitica yopP knockout strain overproducing YopP E40WT (YopP E40WTϩ ). Ac-YVAD.cmk and zDEVD.fmk are specific inhibitors that bind to the active site of caspase-1 and caspases-3/-7, respectively, whereas B-D.fmk is a broad-spectrum caspase inhibitor, and zVAD.fmk is a rather specific inhibitor of the apoptosis-related initiator caspases, caspases-8 and -9 (30). zAAD, a granzyme B inhibitor, was used as a control. As shown in Fig. 1A, YopP-mediated apoptosis is characterized by the induction of membrane blebbing and nuclear condensation and fragmentation. Treatment of macrophages with the caspase inhibitors zDEVD.fmk or Ac-YVAD.cmk or the granzyme B inhibitor zAAD.cmk prior to Y. enterocolitica infection could not prevent YopP-mediated apoptotic cell death. Nevertheless, incubation with zDEVD.fmk resulted in the complete inhibition of intracellular DEVDase activity, as determined by a fluorogenic substrate assay (data not shown). In contrast, pretreatment of the cells with the caspase inhibitors zVAD.fmk and B-D.fmk completely prevented YopP-mediated cell death. Similar results were obtained when analyzing DNA fragmentation in these cultures by means of TUNEL staining (Fig. 1B). Incubation of the cells with the inhibitor alone did not alter their survival rate (data not shown). These results suggest that YopP/J kills macrophages by activating procaspases, but inhibition of caspase-1, -3, or -7 activity cannot prevent the execution of apoptosis.
Yersinia-induced Apoptosis Results in Early Bid Cleavage and Cytochrome c Release-To determine the molecular mechanism of Yersinia-mediated apoptosis we examined the intracellular events taking place by means of a time kinetics experiment evaluating procaspase activation, Bid cleavage, and cytochrome c release. J774A.1 macrophages were infected with the wild type Y. enterocolitica A127 (pYV127) strain or with the virulence plasmid-cured isogenic strain (pYV127 Ϫ ), and at different time intervals cytosolic fractions of these cultures were prepared. Non-infected cells served as a control. YopP could be detected in the macrophage cytosol in substantial amounts as soon as 40 min after co-incubation of cells with bacteria ( Fig.  2A). The first apoptosis-related intracellular event that we were able to monitor was Bid cleavage (Fig. 2B). Bid does not contain a transmembrane domain and is located in the cytosol. Cytosolic Bid was cleaved to a 13-kDa fragment (tBid), and the concentration of tBid gradually increased from 60 min on to 80 min after infection of the macrophages. Bid protein is a specific proximal substrate of caspase-8 in the signaling pathway to apoptosis (20,21). We were not able to analyze endogenous procaspase-8 expression and/or activation in J774A.1 cells because of the lack of anti-murine caspase-8 antibodies with sufficient detection efficiency. It has been described that the C-terminal fragment of Bid translocates from the cytosol to the mitochondrial membrane, a process that finally leads to the release of cytochrome c (20,21). Cytochrome c release became evident 20 min after the generation of tBid in the cell (Fig. 2B). The release of cytochrome c results in a rapid assembly of the apoptosome complex as witnessed by the processing of procaspase-9 to its enzymatically active p35/p37 subunits, eventually leading to the activation of the downstream executioner procaspases-3 and -7 (Fig. 2C). This activation of the apoptotic cascade finally resulted in the total demise of the macrophage resulting in the loss of the cell membrane integrity (Fig. 2D). None of the apoptotic events took place in non-infected cells or cells treated with the plasmid-cured strain (pYV127 Ϫ ). Taken together, our results indicate that Yersinia infection results in a rapid generation of tBid that will translocate to the mitochon- YopP Is Required for Bid Cleavage, Cytochrome c Release, and Procaspase Activation-To investigate which molecular events observed during Yersinia-induced apoptosis, viz. Bid cleavage, cytochrome c release, and procaspase activation, were YopP-dependent we infected J774A.1 macrophages with the E40(pMSK41) strain that is YopP-deficient (YopP Ϫ ) and with the E40(pMSK41)(pMSK13) strain that overproduces YopP E40WT (YopP E40WTϩ ). Our data demonstrate that Bid cleavage is YopP-dependent, because macrophages infected with the YopP Ϫ strain, expressing all other Yops, only contained the full-length Bid protein of 22 kDa as uninfected macrophages do, in contrast to the observed Bid cleavage when cells were infected with the YopP E40WTϩ strain (Fig. 3A). As expected the downstream effects of Bid cleavage, such as mitochondrial cytochrome c release (Fig. 3A) and subsequent pro-caspase-3 and -7 activation, also did not occur when the YopP Ϫ strain was used for infection (Fig. 3B). The absence of procaspase activation upon infection with the YopP Ϫ strain was confirmed by measuring the DEVDase activity in the respective cell lysates (Fig. 3C). In addition, cleavage of Bid and subsequent cytochrome c release could be prevented by pretreatment of the macrophages with the caspase inhibitor zVAD.fmk (Fig. 3A). To exclude the possibility that Bid cleavage is the result of a feedback loop, catalyzed by postmitochondrial procaspase-3 activation (32), we analyzed the levels of tBid in zDEVD.fmk pretreated and control macrophages infected with the YopP E40WTϩ strain. Hence, if Bid cleavage would indeed be the upstream trigger of the YopP-induced apoptotic signaling cascade as a result of initiator procaspase-8 activation, it should not be affected by pretreatment with the zDEVD.fmk inhibitor. As expected from the results obtained in the kinetics experiment (Fig. 2), caspase-3/-7 inhibition did not change the amount of tBid generated upon YopP E40WTϩ infec- , or anticaspase-9, -3, and -7 (C) antibodies. As a control for cell death, the percentage of cells that lost plasma membrane integrity (D), determined by propidium iodide (PI) uptake, was analyzed. tion (Fig. 4A). As a control we show that zDEVD.fmk addition prior to Yersinia infection prevented the generation of DEVDase activity (Fig. 4B). These data indicate that YopP in some way activates an upstream caspase activity, most likely caspase-8, resulting in the cleavage of Bid.

Disruption of the Catalytic Domain of YopP Abolishes Apoptosis Induction by Yersinia-
Recently it was shown that the secondary structure of YopP/J resembles that of an adenovirus protease (10). These authors identified YopP/J as a member of an ubiquitin-like protein cysteine protease family having SUMOylase activity and showed that the catalytic core of YopP/J is required for its inhibition of the MAPK and NF-B pathways. Alignment of the catalytic domains of Y. enterocolitica YopP A127 , 2 Y. pestis YopJ KIM5 , and Y. pseudotuberculosis YopJ YPIII with the human SUMO proteases SUSP1 and SENP1 (33,34) indicates that the residues of the catalytic triade (His, Asp/Glu, and Cys) are conserved in YopP/J (Fig. 5A). To analyze whether the YopP cysteine protease activity is required for induction of apoptosis, we mutated the conserved catalytic cysteine in YopP, and a YopP-deficient Y. enterocolitica strain was complemented with wild type YopP (YopP E40WTϩ ) or mutated YopP (YopP E40C172Tϩ ). Then macrophages were infected with the different Yersinia strains generated, and the effect of the YopP catalytic cysteine mutant on the apoptosis induction by YopP was analyzed. The Y. enterocolitica E40 strain is less virulent and therefore results in a slower onset of apoptosis as compared with the A127 strain. Mutation of the YopP catalytic cysteine abolished completely the ability of Y. enterocolitica to induce apoptosis in the infected macrophages (Fig. 5). Microscopic evaluation of the infected cultures indicated clear induction of apoptosis, characterized by the typical hallmarks as membrane blebbing and nuclear fragmentation, when the macrophages were co-incubated with the YopP E40WTϩ strain (Fig. 5B). In contrast, the YopP E40C172Tϩ -infected cells looked similar to YopP Ϫ -infected cultures, showing a non-apoptotic morphology. Importantly, we demonstrated that both the wild type and the mutant YopP were translocated equally well to the macrophages upon infection with the respective Yersinia strains (Fig. 5C), but only infection with the YopP E40WTϩ strain induced Bid processing (Fig. 5D). These observations were in agreement with the fact that cytosolic caspase activity was only generated in the YopP E40WTϩ -infected cultures (Fig. 5E). Overexpression of YopP A127WT and YopP A127C172T in HEK293T cells confirmed that the catalytic cysteine is required for inhibition of NF-B activation (data not shown), as shown previously (10).
These data indicate that YopP/J protease activity is required both for the inhibition of NF-B signaling and the induction of apoptosis by Yersinia.
YopP Does Not Interact with TRADD, FADD, or Bid-In cells in which clustering of death receptors causes weak procaspase-8 activation, the mammalian Bid protein is a specific proximal substrate of caspase-8 in the signaling pathway (19). YopP/J has been shown to bind to members of the MAPK family and to IKK␤ and to interfere with these signaling pathways. The mechanism by which YopP/J induces apoptosis is not yet completely clear, but our data suggest an implication of caspasedependent cleavage of Bid. A possible hypothesis could be that YopP/J clusters pro-apoptotic signaling molecules thereby starting the cell death machinery. For this reason we analyzed whether YopP could bind directly to pro-apoptotic signal transduction molecules such as TRADD, FADD, or Bid. Therefore, we overexpressed these molecules in HEK293T cells, together with YopP A127WT , and performed an immunoprecipitation experiment. FADD, TRADD, and Bid could not be co-immunoprecipitated with YopP A127WT (Fig. 6), indicating that YopP is not able to cluster these pro-apoptotic mediators thereby initiating an apoptotic cascade. As a positive control, we could co-immunoprecipitate IKK␤ with YopP A127WT (6). Additionally, YopP A127WT overexpression did not lead to Bid processing (data not shown), indicating that Bid is not a direct substrate for the YopP protease.

DISCUSSION
Previously it was shown that YopP/J is required for Yersiniainduced apoptosis in macrophages, but the exact mechanism of action remained unknown (7,8). Although the signals that generally lead to apoptosis are quite diverse, the activation of procaspases plays a central role in the initiation and execution phase of apoptosis. The caspase family comprises three groups, the inflammatory caspases (-1, -4, -5, and -11), the initiator caspases (2, -8, -9, and -10), and the executioner caspases (-3, -6, and -7). In this study we wanted to determine which signaling pathways are activated in YopP-mediated cell death. We demonstrated an early Bid cleavage after YopP was injected into the cells by the infecting bacteria. Bid cleavage clearly occurred before cytochrome c release, suggesting that the truncated Bid relocalizes to the outer mitochondrial membrane and initially causes the release of cytochrome c (20,21). Subsequent assembly of the cytochrome c⅐apoptotic protease activating factor-1⅐ATP⅐procaspase-9 complex then initiated the activation of procaspase-9 leading to postmitochondrial procaspase-3 and -7 activation (30,35). This seems to be a very rapid event, because cytochrome c release, procaspase-9 activation, and procaspase-3 and -7 activation all occur simultaneously. Because tBid generation becomes evident clearly before the release of cytochrome c, our data indicate that Bid cleavage is not the result of postmitochondrial caspase activity as described in other apoptotic systems (31). Inhibition of the executioner caspases-3 and -7 was not able to block YopP-mediated apoptosis. Nevertheless, pretreatment of cells with this inhibitor completely blocked the YopP-induced DEVDase activity, thus suggesting that the caspases-3 and -7 are dispensable for the onset of apoptosis and merely play an executing role in the late stage of apoptosis (36,37). Furthermore, Ac-YVAD.cmk, an

FIG. 5. The YopP catalytic cysteine is necessary for the induction of apoptosis during Y. enterocolitica infection of macrophages.
A, sequence alignment of the conserved putative catalytic domains of Y. enterocolitica YopP A127 , 2 Y. pestis YopJ KIM5 (Accession number NP047569), and Y. pseudotuberculosis YopJ YPIII (Accession number AAA68488) with the human SUMO proteases hSUSP1 (Accession number AF196304) and hSENP1 (Accession number AF149770) (33,34). Arrowheads indicate the amino acid residues of the catalytic triad (His, Asp/Glu, and Cys), and shading denotes identical amino acid residues. J774A.1 were infected with Y. enterocolitica YopP Ϫ , YopP E40WTϩ , and YopP E40C172Tϩ strains at a m.o.i. of 50. Non-infected (NI) J774A.1 cells were used as a control. B, light microscopic images were taken 3 h after infection to analyze apoptotic cell morphology. At different time intervals cytosolic fractions of J774A.1 cells were prepared by digitonin lysis and analyzed by polyacrylamide gel electrophoresis and Western blotting using anti-YopP (C) and anti-Bid (D) antibodies. Open and closed arrows indicate cleaved and uncleaved proteins, respectively. E, caspase activity in cell extracts was measured making use of the Ac-DEVD aminomethyl coumarin fluorogenic substrate, as described under "Experimental Procedures." inhibitor of caspase-1, also failed to block YopP-induced apoptosis despite the fact that procaspase-1 becomes activated upon Y. enterocolitica infection, 3 indicating that caspase-1 activity is not required for Yersinia-mediated macrophage apoptosis. This finding indicates that Yersinia activates a different cell death pathway in macrophages than Shigella or Salmonella, two pathogens that make use of a type III protein secretion mechanism similar to that of Yersinia sp. Shigella and Salmonella both produce similar apoptotic invasin proteins, named IpaB and SipB, respectively (38). During infection these proteins are translocated to the cell and bind to procaspase-1, resulting in its activation. In contrast to Yersinia-mediated apoptosis, pretreatment of macrophages with the Ac-YVAD-.cmk caspase-1 inhibitor prevented the induction of apoptosis by Shigella or Salmonella (39,40). Furthermore, caspase-1deficient macrophages are protected against Shigella-or Salmonella-induced cell death (41). When cells were pretreated with the caspase inhibitors zVAD.fmk and B-D.fmk, YopPmediated apoptosis could be completely blocked. In addition, YopP-induced Bid cleavage and the subsequent release of cytochrome c were completely blocked in the presence of zVAD-.fmk. Therefore it seems that YopP-mediated cell death is mainly caused by the activation of initiator procaspases acting upstream of caspases-3 and -7. Bid can be cleaved by different proteases such as caspase-3 (31) or -8 (20,21) or granzyme B (42). Although processing of endogenous procaspase-8 could not be analyzed because of the lack of anti-mouse caspase-8 antisera with sufficient detection efficiency, procaspase-8 activation may play a key role in YopP-mediated apoptosis because (a) YopP-induced procaspase-3 activation occurs only after Bid had been cleaved, and Ac-DEVD.fmk (a potent caspase-3 inhibitor) treatment could not prevent Bid cleavage; and (b) the granzyme B inhibitor zAAD.fmk did not affect the YopP-dependent apoptosis; in addition, granzyme B is normally not expressed in macrophages. The presently known activation mechanisms of pro-apoptotic initiator procaspases include two main pathways, the death domain receptor-initiated activation of procaspase-8 in the death-inducing signaling complex (43) and the apoptosome-mediated postmitochondrial activation of procaspase-8 by caspase-3 (32). Because the executioner procaspases became activated after Bid had been cleaved, and caspase-3/7 inhibition could not prevent the onset of the apoptotic program or the cleavage of Bid, it is less likely that procaspase-8 would be activated by the generation of postmi-tochondrial caspase activity. IpaB or SipB can bind directly to procaspase-1, thereby inducing procaspase-1 activation that is crucial for the induction of apoptosis by Shigella or Salmonella infections. Could YopP/J make use of a similar mechanism by binding to procaspase-8? We were not able to show a clear interaction between YopP and procaspase-8 in a yeast two hybrid experiment. 4 Another possibility might be that YopP/J would cluster intracellular pro-apoptotic adapter molecules involved in the activation of procaspase-8 such as TRADD or FADD, thereby mimicking the death-inducing signaling complex as formed by death receptors of the TNF-receptor superfamily. Upon receptor activation these receptors can recruit the adaptor molecules TRADD and/or FADD. Binding of FADD subsequently can recruit procaspase-8 or -10, and this event leads to proximity-induced autocleavage of these procaspases (44). Immunoprecipitation experiments with YopP could confirm that YopP is able to interact with IKK␤ (6) but did not provide evidence for binding of YopP with TRADD or FADD. Hence, the trigger that leads to YopP/J-induced procaspase activation required for Bid cleavage remains elusive. Given the observation that YopP is a protease we also considered whether Bid could be a direct substrate for YopP. Overexpression of YopP in HEK293T cells did not lead to processing of Bid or apoptosis. 5 Overexpression of wild type YopP, but not of YopP A127C172T , resulted in the inhibition of NF-B activation indicating that YopP has enzymatic activity upon overexpression in HEK293 cells.
YopP/J is not only responsible for the induction of apoptosis, but it also strongly interferes with macrophage signal transduction resulting in a blockade of two major cell signaling cascades; the MAPK and the NF-B pathway are both involved in transcriptional gene control (2)(3)(4)(5). Indeed, the production of proinflammatory cytokines would be disadvantageous for the extracellular lifestyle of Yersinia. It has been shown that YopP/J inhibits these pathways by interacting directly with IKK␤ and several MKKs, thereby preventing their activation (6). Recently it was suggested that the YopP/J-induced apoptosis of macrophages could merely result from its inhibition of NF-B activation in combination with lipopolysaccharide (LPS) (5,45). In contrast to macrophages, other cell types (fibroblasts and HeLa cells) do not undergo apoptotic cell death upon Yersinia infection. Nevertheless, YopP/J is translocated efficiently in HeLa cells (4), 6 resulting in inhibition of NF-B activation and interleukin-8 cytokine production (4,5). YopP/J exerts its effect on HeLa cells, but this does not lead to the induction of programmed cell death, suggesting the absence of a signaling pathway in this cell type when compared with macrophages. As shown for Yersinia and E. coli, LPS can trigger apoptosis when NF-B activation is inhibited (5,46). LPS binds the CD14 receptor, a receptor only present on monocytes and macrophages. The LPS signal is transduced across the plasma membrane by the toll-like receptor 4 (TLR-4), which associates with CD14 to form the LPS receptor complex (47).
Recently it was shown that another class of microbial surface molecules, the bacterial lipoproteins (BLPs), induced apoptosis in macrophages by binding TLR-2 (48). Furthermore, epithelial cell lines transfected with TLR-2 underwent bacterial lipoprotein-mediated apoptosis. Altogether these studies suggest that bacteria-mediated apoptosis requires the presence of Toll-like receptors at the cell surface. During Yersinia infection, LPS and/or BLPs and YopP might both be required to fulfill the process of apoptosis. In addition it was suggested that the BLP-stimulated TLR-2-triggered apoptosis occurs via a path-3 G. D. and G. C., unpublished results. 4 M. V. G. and P. V., unpublished results. 5 G. D. and G. C., unpublished results. 6 M.-P. S. and G. C., unpublished results. way involving MyD88, FADD, and caspase-8 (49). These data indicate that the early Bid cleavage observed in our experiments could well be because of activation of the initiator procaspase-8 by TLR-2 signaling. In this context, YopP/J would only be required to inhibit gene activation thereby preventing the up-regulation of anti-apoptotic proteins in the macrophage. This hypothesis would imply that infection of macrophages with the YopP E40C172Tϩ mutant strain would not affect the early Bid processing, already significant 20 min after YopP injection in the macrophage cytosol upon infection. Because we show here that Bid cleavage is impaired upon YopP E40C172Tϩ infection, we speculate that YopP/J induces apoptosis by a direct action on cell death signaling pathways.
Previous studies indicated that YopP/J interacts with MKKs and IKK␤ and prevents their phosphorylation. However, MKK SUMOylation was not observed, and data on IKK␤ SUMOylation are not available; hence the targets for YopP/J de-SUMOylation remain to be identified. MKKs could serve as shuttles to escort YopP/J to the signaling complex where it would affect critical SUMO-1-conjugated proteins. It has been observed that the NF-B signaling molecules such as IKK␤ can be recruited to the TNF⅐receptor signaling complex (50); a similar recruitment event could be valid for the Toll-like receptors. Hence, it should be considered that YopP/J may directly affect Toll-like receptor signaling in this way causing apoptotic signaling in macrophages.
Taken together, our data support a model in which the YopP/J protease activity targets both anti-and pro-apoptotic pathways in macrophages (Fig. 7). In our model YopP/J-mediated de-SUMOylation alters the Toll-like receptor signaling in a way allowing procaspase-8 activation leading to Bid processing. Truncated Bid then translocates to the mitochondria inducing the release of cytochrome c, resulting in the assembly of the apoptosome and the generation of activated caspase-9 and the executioner caspases-3 and -7. Identification of the YopP/J targets will be important to further elucidate the YopP/J signaling pathways.