Yersinia Phosphatase Induces Mitochondrially Dependent Apoptosis of T Cells*

To evade the immune system, the etiologic agent of plague, Yersinia pestis, injects an exceptionally active tyrosine phosphatase called YopH into host cells using a type III secretion system. We recently reported that YopH acutely inhibits T cell antigen receptor signaling by dephosphorylating the Lck tyrosine kinase. Here, we show that prolonged presence of YopH in primary T cells or Jurkat T leukemia cells causes apoptosis, detected by annexin V binding, mitochondrial breakdown, caspase activation, and internucleosomal fragmentation. YopH also causes cell death when expressed in HeLa cells, and this cell death was inhibited by YopH-specific small molecule inhibitors. Cell death induced by YopH was also prevented by caspase inhibition or co-expression of Bcl-xL. We conclude that YopH not only paralyzes T cells acutely, but also ensures that the cells will not recover to induce a protective immune response but instead undergo mitochondrially regulated programmed cell death.

To evade the immune system, the etiologic agent of plague, Yersinia pestis, injects an exceptionally active tyrosine phosphatase called YopH into host cells using a type III secretion system. We recently reported that YopH acutely inhibits T cell antigen receptor signaling by dephosphorylating the Lck tyrosine kinase. Here, we show that prolonged presence of YopH in primary T cells or Jurkat T leukemia cells causes apoptosis, detected by annexin V binding, mitochondrial breakdown, caspase activation, and internucleosomal fragmentation. YopH also causes cell death when expressed in HeLa cells, and this cell death was inhibited by YopHspecific small molecule inhibitors. Cell death induced by YopH was also prevented by caspase inhibition or coexpression of Bcl-xL. We conclude that YopH not only paralyzes T cells acutely, but also ensures that the cells will not recover to induce a protective immune response but instead undergo mitochondrially regulated programmed cell death.
In historical times, the plague-causing bacterium Yersinia pestis has caused social devastation on a scale unmatched by any other infectious disease, with over 200 million human deaths (1,2). Although this fatal bacterium is virtually eradicated and is effectively treated with antibiotic therapy, the rapidly fatal course of pneumonic plague, potential for secondary spread of infection, and developed capacity for massive aerosol distribution have made Y. pestis a feared biological weapon of terror (3). Thus, there exists a need for elucidation of the molecular mechanisms by which Y. pestis evades the immune system and causes the death of its host. In addition, targeting essential virulence factors therapeutically may prove useful in treating a potentially large outbreak of Y. pestis.
A 70-kb virulence plasmid, pYV, enables Y. pestis to survive and multiply in the lymphoid tissues of the host (4 -6). pYV encodes the Yop (Yersinia outer protein) virulon, a set of proteins that enable extracellular bacteria to paralyze cells of the immune system, disrupt their communications, or induce apoptosis (7). Most of the Yops fall into one of two groups. One group consists of intracellular effectors (YopE, YopH, YpkA/ YopO, YopP/YopJ, YopM, and YopT), whereas the others (YopB, YopD, and LcrV) form the secretion apparatus that is displayed at the bacterial surface (6,8). This type III secretion apparatus mediates the translocation of the intracellular effectors into the cytoplasm of eukaryotic host cells to directly interfere with host signal transduction pathways (6 -8).
YopH, a key Yop virulon protein, is a 468-amino-acid exceptionally active protein tyrosine phosphatase (9 -11). It has a C-terminal catalytic domain and a multifunctional N-terminal domain that binds tyrosine-phosphorylated proteins to target YopH to hot spots of signal transduction (12,13). Injection of YopH into phagocytic cell types results in disruption of focal adhesions and inhibition of integrin-mediated bacterial phagocytosis (14 -16), both of which are highly dependent on tyrosine phosphorylation (16). YopH also prevents integrin-mediated responses in affected phagocytes, such as tumor necrosis factor-␣ release and oxidative burst (17,18). The development of an adaptive immune response is also blocked through suppression of T and B lymphocyte activation in a YopH-dependent manner (19). Recently, we showed that YopH inhibits the activation of T lymphocytes by dephosphorylating the Lck tyrosine kinase at Tyr-394, resulting in a complete loss of its catalytic activity (20). Clearly, YopH is an essential effector protein involved in the virulence of Yersinia, and therapeutically targeting YopH may prove useful in combating a potential outbreak.
Here, we report that YopH induces apoptosis as measured by annexin V binding, loss of mitochondrial membrane potential, caspase activation, and internucleosomal DNA fragmentation. The pan-caspase inhibitor Boc-Asp-fmk (BAF) 1 blocks the increase in cell death and DNA degradation induced by YopH. Interestingly, co-expression of Bcl-xL with YopH prevents the induction of cell death, caspase activity, and DNA degradation. YopH also causes cell death when expressed in HeLa cells, and YopH-specific small molecule inhibitors blocked this effect but not cell death induced by other stimuli. These data are the first to show a new role for YopH: induction of mitochondrially dependent apoptosis. Taken together with previous findings, we conclude that the actions of YopH in T cells are two-fold: (a) initially acting to paralyze T cells, resulting in immediate inhibition of the adaptive immune response, and (b) actively causing apoptosis, thus efficiently blocking T cell recovery, resulting in long term inhibition of the immune response.

MATERIALS AND METHODS
Plasmids and Site-directed Mutagenesis-The expression plasmids for YopH and the catalytically inactive YopH-D356A in the pEF5HA * This work was supported by Grants AI53114 and AI55789 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. eukaryotic expression vector, which adds a 9-amino-acid hemagglutinin epitope to the N terminus of the insert, were as before (20). The cDNA encoding Bcl-xL and dominant negative caspase-9 were in the pcDNA3 expression vector.
Cells and Cell Treatments-Normal T lymphocytes were isolated from venous blood of healthy volunteers by Ficoll gradient centrifugation. Monocytes/macrophages were eliminated by adherence to plastic for 1 h at 37°C. Jurkat T leukemia cells were kept at logarithmic growth in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, non-essential amino acids, and 100 units/ml each of penicillin G and streptomycin. Transfections were done by electroporation as described previously (21)(22)(23). HeLaS cells were maintained at logarithmic growth in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM Lglutamine, and 100 units/ml each of penicillin G and streptomycin. HeLaS cells were transfected with Oligofectamine (Invitrogen) according to the manufacturer's protocol. ANT-YopH and ANT-YopH-D356A proteins were induced in transformed Escherichia coli and purified using Ni 2ϩ -nitrilotriacetic acid. Both proteins were of equal high purity. They were added at 6 M to cells in RPMI medium at 37°C. BAF (caspase inhibitor III, Calbiochem) was added at a concentration of 10 M immediately after transfection.
YopH Inhibitors-For Jurkat and HeLa cell treatments, LTY11, LTY9, and LTY61 were added at 36 or 48 M after transfection of YopH was completed.
Cell Death Assays-The percentage of cell death was determined by using trypan blue (Invitrogen) exclusion. 48 h after transfection, cells were diluted 50:50 (v/v) in 0.4% trypan blue in phosphate-buffered saline and viewed under a microscope, and total cell number as well as the number of trypan blue-positive (dead) cells were counted in four fields of view using a hemacytometer. The annexin V-FITC apoptosis detection kit (BioVision Research Products, Mountain View, CA) was used according to the manufacturer's protocol, and cells were analyzed on a BD Biosciences FACSort. Internucleosomal DNA fragmentation was determined using the nucleosome enzyme-linked immunosorbent assay (Oncogene Research Products, San Diego, CA) according to the manufacturer's protocol. Mitochondrial integrity assays were performed by staining 10 6 cells with 10 g/ml 5,5Ј,6,6Ј-tetrachloro-1,1Ј,3,3Ј-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) for 15 min at room temperature in the dark. The cells were washed in phosphate-buffered saline and subsequently analyzed on a BD Biosciences FACSort.
Caspase Assays-Caspase activity was measured as described previously (24,25). Briefly, 10 6 cells were lysed in 50 l of modified radioimmune precipitation buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 0.01 M sodium phosphate, pH 7.2, 2 mM EDTA, 50 mM sodium fluoride, 0.2 mM sodium vanadate) and left on ice for 15 min, and centrifugation was done at 14,000 rpm for 10 min. Protein concentration was determined using the Pierce BCA protein assay according to the manufacturer's protocol with bovine serum albumin as standard. Total caspase activity was measured using 100 M Ac-DEVD-AFC (Enzyme Systems Products, Aurora, OH) in caspase buffer (10 mM Pipes, pH 7.2, 100 mM NaCl, 10% sucrose, 0.1% Chaps, 1 mM EDTA, and 10 mM dithiothreitol). Amidolytic activity was measured on an f-max Molecular Device spectrofluorometer at 37°C (excitation, ϭ 405 nm; emission, ϭ 510 nm). A CaspACE™ FITC-VAD-fmk in situ marker (Promega, Madison, WI) was used to stain active caspases at a concentration of 10 M for 10 min in the dark, and the cells were then washed in phosphate-buffered saline and analyzed on a BD Biosciences FACSort.
Data Analysis and Statistics-Data collected by FACS were analyzed with FlowJo for Windows v5.0 (Tree Star Inc., Ashland, OR). Statistical analyses were performed with GraphPad Prism v4.0 for Windows (GraphPad Software Inc., San Diego, CA). All experiments were analyzed by two-way analysis of variance followed by Bonferroni posttests.

RESULTS
YopH Induces Apoptosis in Jurkat T Cells-In preliminary transfection experiments with Jurkat T cells and normal human T lymphocytes, we observed that the number of YopHpositive cells in many experiments was much lower than the number of cells expressing the catalytically inactive YopH-D356A or a control protein (e.g. GFP). This phenomenon was particularly evident at 48 or 72 h after transfection and was more prominent with normal human T lymphocytes when compared with Jurkat. The inequality of YopH-versus YopH-D356A-positive cell numbers between samples was abolished by the inclusion of Z-VAD-fmk, a polycaspase inhibitor, in the culture medium (data not shown). This observation led us to ask whether YopH expression results in caspase activation and subsequent apoptosis in T cells. To test this notion, we transfected Jurkat with the empty pEF5HA vector, wild-type YopH, or the catalytically inactive mutant YopH-D356A and analyzed the cells at 24 h after transfection by staining with FITCannexin V and propidium iodide. This method of analysis reveals two important aspects of cell death: (i) phosphatidylserine exposure on the extracellular face of the plasma membrane by FITC-annexin V binding and (ii) loss of plasma membrane integrity, resulting in entry of propidium iodide into the cells. YopH caused a significant increase in the percentage of cells double-positive for annexin V and propidium iodide (Fig. 1, A and C). This population of cells represents either late stage apoptosis or necrosis. YopH also increased single-positive cells for annexin V, which corresponds to apoptotic cells that have not yet lost outer membrane integrity but have lost phosphatidylserine asymmetry (Fig. 1, A and B). The cells expressing YopH first go through the annexin V single-positive stage and then proceed to the late stages of apoptosis and become positive for propidium iodide as well, thus accumulating in the double-positive population. Also, cells co-transfected with YopH and GFP and then analyzed by flow cytometry at 24 h (Table I) showed that fewer cells were alive in the YopHtransfected population, fewer GFP-expressing cells remained, and many more of the green fluorescent protein-positive cells were also annexin V-positive. The catalytically inactive (but substrate-binding) YopH-D356A mutant had a small (and not significant) effect when compared with vector controls in this and some other experiments. Thus, the catalytic activity of YopH is essential for its ability to induce apoptosis.
YopH-induced Cell Death Is Blocked by a Specific Small Molecule Inhibitor-To directly test whether the catalytic activity of YopH is essential for its ability to induce cell death, we transfected Jurkat cells with YopH and then treated the cells with the specific YopH inhibitors, LTY11 and LTY9, generated in our laboratory. 2 Both are membrane-permeable competitive small molecule inhibitors that specifically block the enzymatic activity of YopH with K i values of 143 and 208 nM, respectively, using the general tyrosine phosphatase substrate p-nitrophenyl phosphate in an in vitro assay. Jurkat cells were transfected with YopH and then kept in culture in the presence of LTY11 or LTY9, which largely (but not completely) abolished the ability of YopH to cause cell death (Fig. 1D). In the absence of YopH, these inhibitors did not affect cell death. We also used HeLa cells, which typically have higher transfection efficiencies and lower levels of background cell death when compared with Jurkat cells. Expression of YopH caused a highly significant increase in the amount of cell death when compared with empty vector (Fig. 1D)  a The percentage of live cells was determined by flow cytometry using forward and side scatter gating for live versus dead cells.
b The percentage of GFP-positive cells was determined by flow cytometry using untransfected cells as a negative control.
c The percentage of Annexin-positive cells was determined by flow cytometry using Alexa Fluor594-conjugated annexin V.
prevented by YopH inhibitors LTY11 and LTY61 (Fig. 1E). In contrast, cell death induced by etoposide was not affected at all by these YopH inhibitors. Together, all these results show that YopH is a potent activator of cell death in both T cells and the non-hematopoietic HeLa cell line. More importantly, treatment of cells with a specific small molecule inhibitor of YopH completely blocks the death induced by YopH. This finding provides a first proof of the concept that the essential virulence factor of Y. Pestis, YopH, may be a viable target for treatment of infection.
YopH Activates Caspases-To directly measure whether YopH expression resulted in caspase activation, Jurkat cells were transfected with empty vector, wild-type YopH, YopH-D356A, or PTEN (as positive control) and subjected to in vitro caspase activity assays 48 h later, which showed that YopH caused a significant 3-fold increase in caspase activity when compared with vector control cells ( Fig. 2A). Importantly, caspase activity in cells transfected with the catalytically inactive mutant of YopH was indistinguishable from vector-transfected control cells, whereas PTEN induced a 5-fold activation of caspases. We conclude that YopH activates caspases in a manner that is dependent upon its catalytic activity.
We have previously shown that the tyrosine kinase Lck is a target of YopH that accounts for its ability to inhibit T cell receptor signal transduction (20). To test whether Lck was important for the ability of YopH to induce caspase activation, we transfected the Lck-deficient Jurkat cell line JCaM1 with empty vector, wild-type YopH, or YopH-D356A and assayed in vitro caspase activity 48 h later. YopH did not cause caspase activation in this Lck-deficient cell line (Fig. 2B), whereas PTEN did. We conclude that YopH may well reduce T cell survival because it reduces basal Lck phosphorylation at Tyr-394 (20). If so, JCaM1 cells survive in the absence of Lck because additional mutations have uncoupled Lck from caspase activation or because of other alterations in the machinery for apoptosis. The induction of cell death in HeLa cells by YopH may be related to the dephosphorylation of other Src family kinases and perhaps other tyrosine kinases involved in survival of cells.
To better mimic the scenario in a lymph node of an infected patient, where the bacteria inject YopH protein directly into the cytoplasm of T lymphocytes, we incubated freshly isolated peripheral blood leukocytes (Ͼ80% T lymphocytes) with negative control buffer, the membrane-permeable fusion protein ANT-YopH (20), or etoposide as positive control for 24 h and then analyzed in vitro caspase activity. In normal T lymphocytes, ANT-YopH induced a very high level of caspase activity when compared with mock control (Fig. 2C). When compared with etoposide-treated cells, which typically have very high levels of caspase activity, ANT-YopH induced a higher increase in caspase activity. These data show that YopH is a very potent inducer of caspase activation in normal human T lymphocytes.
YopH Causes Loss of Mitochondrial Membrane Potential-An important event associated with the transduction of many apoptotic signals is the loss of mitochondrial membrane potential caused by translocation of pro-apoptotic Bcl-2 family members to the outer mitochondrial membrane. These pro- apoptotic molecules, such as Bax, form pores in the membrane, causing breakdown of the membrane potential and release of other proapoptotic molecules from the intermembrane space (26). Therefore, to test the hypothesis that YopH expression would result in loss of mitochondrial membrane potential, we transfected Jurkat cells with empty vector, YopH, or YopH-D356A and measured mitochondrial membrane potential 24 h later by using JC-1 staining. JC-1 is a lipophilic cation that forms orange fluorescent aggregates in intact mitochondria of healthy cells. In apoptotic cells where mitochondrial membrane potential has collapsed, JC-1 remains in the cytoplasm in its monomeric green fluorescent form. Cells are then easily distin-guished as having lost their mitochondrial membrane potential by FACS analysis. Fig. 3 reveals that YopH caused a significant decrease in the number of cells with intact mitochondrial membrane potential, whereas YopH-D356A did not (Fig. 3, A-C). These data indicate that expression of YopH results in mitochondrial membrane potential breakdown in a manner that is dependent upon its catalytic activity.
YopH-induced Cell Death Is Mitochondrially Mediated-Members of the Bcl-2 family of proteins are critical regulators of apoptosis via control of mitochondrial outer membrane characteristics. Bcl-xL, an anti-apoptotic member of this family, is able to counteract the effects of the pro-apoptotic members that are responsible for the mitochondrial membrane permeabilization and eventual cell death (27). To test whether YopH induction of caspase activity was mitochondrially mediated, we cotransfected Bcl-xL with YopH and determined the percentage of cells with activated caspases as measured by FACS analysis of FITC-VAD-fmk-stained cells. We found that YopH caused an increase in cells positive for active caspases (Fig. 4A). More importantly, expression of Bcl-xL blocked this increase. These data implicate mitochondria as a key regulator involved in the activation of caspases in response to YopH expression.
Next, we tested whether Bcl-xL could also prevent cell death induced by YopH. Although catalytically active YopH expression induced about a 2-fold increase in the percentage of dead cells, as measured by trypan blue exclusion (Fig. 4B), co-expression of Bcl-xL blocked this effect of YopH. We also demonstrate that caspase activity is required for YopH-induced cell death as the broad spectrum caspase inhibitor BAF completely blocked the effect of YopH. We conclude from these data that the cell death induced by catalytically active YopH involves an obligatory mitochondrial step and requires caspase activity.
YopH Induces Internucleosomal Fragmentation-A typical late stage characteristic of apoptosis is internucleosomal genomic DNA degradation (28). To evaluate the ability of YopH to cause this genomic DNA fragmentation pattern, we used a nucleosome enzyme-linked immunosorbent assay. We found that YopH causes internucleosomal fragmentation and that this effect again is dependent on its tyrosine phosphatase activity. The effect of YopH was also dependent upon caspase activation as BAF blocked the increase in DNA fragmentation due to YopH expression (Fig. 4C). DISCUSSION In T cells, apoptosis is most commonly initiated in response to death receptor ligation resulting in activation of caspase 8 (extrinsic pathway) or cellular stress followed by mitochondrial release of cytochrome c and subsequent activation of caspase 9 (intrinsic pathway) (26,29). However, activation of programmed cell death can be much more complex, and it is difficult to specifically implicate a single pathway responsible for the death of cells. Because of the multiple possible entry FIG. 4. YopH-induced cell death is blocked by Bcl-xL and BAF. Jurkat T cells were transiently transfected with 10 g of the indicated expression plasmids. A, the mean percentage of cells with active caspases was determined 48 h after transfection by staining with CaspACE, an irreversible FITC-conjugated caspase inhibitor, and detected using FACS. Cells with active caspases are positive for FITC in the FL-1 channel. B, 48 h later, the cells were diluted 50:50 (v/v) in 0.4% trypan blue, and total cell number was determined. Cells were counted as dead if they were positive for trypan blue. BAF was present at 10 M in the indicated sample. C, internucleosomal DNA fragmentation was determined at 48 h with a nucleosome enzyme-linked immunosorbent assay kit. Data are expressed as mean absorbance (Abs) at 450 nm Ϯ S.E. points into the apoptotic execution phase, it is important to delineate the pathway of apoptotic signaling events triggered by any specific stimulus. Here, we have demonstrated that the Yersinia phosphatase YopH causes annexin V binding, caspase activation, mitochondrial membrane potential loss, and internucleosomal DNA degradation in Jurkat cells. In addition, the Lck-deficient JCaM1 Jurkat cell line is not susceptible to YopH-induced caspase activation. Bcl-xL co-expression blocks the YopH-induced caspase activation, DNA degradation, and cell death. Caspase inhibition also inhibits the DNA degradation and cell death induced by YopH. Moreover, YopH expression in HeLa cells also causes cell death, and YopH-specific inhibitors block the cell death induced by YopH. These results demonstrate that YopH activates mitochondrially regulated apoptosis of Jurkat T cells.
We have previously shown that YopH targets Lck and thus reduces basal tyrosine phosphorylation as well as antigenstimulated tyrosine phosphorylation, efficiently inhibiting antigen-induced stimulation of T cells (20). Inactivation of Lck by YopH results in loss of phosphorylation of all downstream targets, a situation that is virtually indistinguishable from a broad and indiscriminate dephosphorylation of proteins by YopH. Since most phosphotyrosine-containing proteins are continuously dephosphorylated by efficient endogenous protein tyrosine phosphatases, a sharp decline in their rate of phosphorylation following Lck inactivation will result in a rapid clearance of total tyrosine phosphorylation. The global loss of tyrosine phosphorylation may lead to either the loss of inhibition of apoptotic mechanisms or the stimulation of a proapoptotic pathway. Here, we have shown that the induction of caspase activity by YopH is abolished in the Lck-deficient Jurkat cell line JCaM1. Lck activity may be needed for survival of normal Jurkat, and inhibition of Lck by YopH may result in the stimulation of apoptotic signal transduction. In this case, the transduction of the apoptotic signal is undoubtedly mediated by signals transmitted to mitochondria, integrated by Bcl-2 family members, and carried out by caspases.
Pathogenic bacteria have evolved numerous ways of evading the innate and adaptive immune systems (16). Y. pestis has adopted one of the most successful strategies, namely a type III secretion system that injects a set of tranquilizing proteins directly into the cytoplasm of macrophages and lymphocytes (4,8). As a result, the cells are unable to respond, and the bacteria can multiply unopposed by the normal mechanisms of host defense (6). We have previously implicated YopH as a key mediator of Y. pestis evasion of the immune response acting acutely to inhibit T cell activation (20). Here, we show that YopH induces programmed cell death in T cells. A similar induction apoptosis by another Yop virulon protein, YopJ, was reported in macrophages (30). Thus, it seems that several effector Yops may act to kill host cells, perhaps in a manner that involves synergistic actions, e.g. by YopH and YopJ. This may be critical in vivo, where the amounts of Yop proteins injected by bacteria into host cells must be relatively small. Nevertheless, the outcome is striking: acute inhibition of signal transduction in cells of both the innate and the adaptive immune systems followed by programmed cell death of these cells before they can recover to initiate host defense.