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ARTICLE |
CORRESPONDENCE Yueh-hsiu Chien: chien{at}stanford.edu
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The success of a pathogen in establishing an infection is dependent on its ability to avoid or resist host defense mechanisms. Although there is ample information on the mechanisms used by pathogenic bacteria to subvert the innate immune defense system, very little is known about how they affect the adaptive immune response (for review see reference 1). Yet, it is clear that the T cell response remains essential for the clearance of bacterial pathogens, including the pathogenic members of the genus Yersinia (2, 3). The genus Yersinia includes Yersinia pestis, the causative agent of plague, as well as Yersinia pseudotuberculosis and Yersinia enterocolitica. The latter two cause gastroenteritis and lymphadenitis and, in susceptible individuals, the infections have also been associated with the development of reactive arthritis (4).
Yersinia's virulence strategy is to modify host cell functions by injecting several effector proteins (the Yersinia outermembrane proteins, Yops) by a type III secretory pathway into the host cell upon contact (5). Studies of the in vitro effects of the Yops on macrophages and epithelial cell lines have been extensive (for reviews see references 5, 6). During the course of infection, Yersinia are also likely to encounter lymphocytes as the bacteria colonize and multiply extracellularly in the Peyer's patches, lymph nodes, and subsequently in the spleen and liver. T cells as well as macrophages are present in Yersinia-induced lesions in spleen and liver (2). We found that T cells transiently exposed to Yersinia are inhibited in their ability to be activated through the TCR and, thus, cannot mount subsequent effector functions (7). This is one of the first examples of a bacterial pathogen that directly inhibits T cell activation.
The virulence factor YopH, a potent phosphatase (810), was primarily responsible for this inhibitory effect (7). Surprisingly, YopE, YopT, and YpkA that are known to disrupt actin filaments leading to alterations in the cytoskeleton of both phagocytes and epithelial cells (5, 6) have no effects on T cells despite the fact that a redistribution and reorientation of the cytoskeleton occurs during lymphocyte activation. Furthermore, although the virulence factor YopJ was found to induce apoptosis in macrophages in vivo (11) and to suppress TNF- and IL-8 production in macrophages in vitro (5, 6), it has no effect on T cells or TCR-mediated cytokine secretion. Thus, it appears that T lymphocyte functions are specifically targeted by YopH.
YopH was shown in in vitro assays to block the oxidative burst of macrophages (12), to inhibit calcium signaling in neutrophils (13), to induce disassembly of focal adhesions in epithelial cells (14, 15), and to inhibit phagocytosis (16). These studies and our own previous investigation were all performed with a rather high multiplicity of infection (MOI) of >50. Yet, in vivo the bacteria to host cell ratio must be low, at least at the time of initial infection. Therefore, we set out to determine the efficacy and mechanism of YopH's inhibition of T cell activation.
Here, we report that YopH alters T cell function even at a level of just one bacterium per T cell. Consistent with such high efficiency, we show that the adaptor molecules linker for activation of T cells (LAT) and SH2-domaincontaining leukocyte protein of 76 kD (SLP-76) that are phosphorylated in response to TCR stimulation and are essential for T cell activation are primary targets of YopH. We find that although YopH has a broad targeting capability, it is quite specific in an infection where the bacteria/target cell ratio is low.
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Results |
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T cell blasts prepared from 5C.C7 ß TCR (specific for moth cytochrome c [MCC] peptide/I-Ek) transgenic mice (22) were exposed to wild-type Y. pseudotuberculosis at MOIs of 1, 2, 5, 10, 20, and 50 for 1 h and concurrently loaded with the calcium-sensitive dye indo-1 for 30 min. Their ability to flux calcium in response to CD3
cross-linking was analyzed by flow cytometry. Ratiometric analysis showed that the calcium flux of a T cell population exposed to an MOI of 1 was substantially reduced (Fig. 1 A). Incubations with higher MOIs resulted in a delayed response and a further reduction of the intracellular calcium flux levels. Consistent with our previous finding that YopH is the primary virulence factor affecting T cell responses (7), we found no inhibition of calcium flux when T cells were exposed to a Yersinia mutant expressing a catalytically inactive YopH, even at an MOI of 50 (Fig. 1 A). The catalytically inactive YopH stably traps its targets and thereby likely impairs downstream events. However, because only a limited mount of YopH is translocated into the host cell even at an MOI of 50, a large number of phosphorylated signaling proteins is not bound to the substrate trap (unpublished data).
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To evaluate the response of individual cells, the calcium flux of fura-2 (a calcium-sensitive dye) loaded 5C.C7 T cell blasts exposed to Yersinia at different MOIs and stimulated by murine CH27 B cells presenting the MCC peptide was analyzed using video fluorescence microscopy (Fig. 2). As seen before (Fig. 1), with increasing MOI, an increasing percentage of the cells could no longer flux calcium. Interestingly, a subset of exposed T cells (10% at an MOI of 1 and 50% at an MOI of 10) exhibited a "sparking" phenotype where the initial increase of intracellular calcium was almost at the level of stimulated unexposed T cells, but was followed by a rapid decline to the basal level in
5 min. In contrast, unexposed T cells (Fig. 2) or T cells incubated with a mutant strain of Y. pseudotuberculosis expressing inactive YopH (not depicted) maintained their elevated calcium concentrations for >10 min.
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Phosphorylated LAT and SLP-76 in activated T cells are primarily targeted by YopH
After T cell activation through the TCR, the TCR CD3 chains, antigen receptor-associated tyrosine kinases, such as Lck and ZAP-70, and adaptor/linker proteins, including LAT and SLP-76, are phosphorylated immediately. This precedes all downstream signaling events including calcium flux and the activation of PI3K. To understand how YopH inhibits TCR signaling, we followed up on our previous finding that, in activated Jurkat T cells exposed to Y. pseudotuberculosis at an MOI of 50, most of the TCR signalingassociated molecules were either not phosphorylated, or hypophosphorylated (7). To test whether YopH broadly targets most members of the TCR signaling complexes, or selectively few members of the complex whose lack of phosphorylation would lead to the hypophosphorylation of other members of the TCR signaling complex and the inability of inducing calcium flux and PI3K activity, we analyzed the tyrosine phosphorylation patterns of activated T cells exposed to wild-type Y. pseudotuberculosis at lower MOIs. Initial experiments were performed in Jurkat cells. T cell activation of primary murine 5C.C7 T cells and the human Jurkat T cells are similar in their sensitivity to Y. pseudotuberculosis infection (unpublished data). However, the biochemistry of antigen receptor signaling complexes is well characterized in Jurkat cells and mutant Jurkat lines defective in the TCR signaling pathway are available.
As shown in Fig. 3, even in activated Jurkat cells exposed to Y. pseudotuberculosis at an MOI of only 1.5, the overall tyrosine phosphorylation in response to TCR stimulation was reduced. The most substantial decrease was observed for phosphorylated LAT and SLP-76 (Fig. 3, A, lanes 2 and 4, and B). In contrast, Lck, Tyr394, CD3, and ZAP-70, which are phosphorylated earlier in the signaling cascade, and SLP-76associated phosphoprotein of 130 kD (SLAP-130; an adaptor protein binding to SLP-76, also known as Fyb, or ADAP) were not as affected. This trend continued in activated Jurkat cells exposed at an MOI of 5. After exposure at an MOI of 15, most TCR signaling proteins were uniformly hypophosphorylated, but Tyr394-phosphorylated Lck was still clearly detectable. The efficient dephosphorylation of TCR signaling proteins is consistent with the results that calcium flux and PI3K activity of T cells are efficiently altered by exposure to Y. pseudotuberculosis (Figs. 1 and 2). Recently, it was demonstrated that YopH inhibits the activity of recombinant Lck in vitro and that Tyr394-phosphorylated Lck is a target of YopH in in vitro immunoprecipitations with relatively high amounts of YopH (24). This has led to the hypothesis that YopH prevents T cell antigen receptor signaling by inhibiting Lck as the first step in the signaling cascade. Therefore, we analyzed Lck activity in Yersinia-exposed T cells. As shown in Fig. 3 C, no difference in the activity of Lck in T cells exposed to low MOIs of Yersinia (MOIs 1.5 and 5) or unexposed cells was observed, indicating that the hypophosphorylation of signaling molecules at these MOIs does not result from a reduction of Lck activity. Only at higher MOIs (15 and 50), we did find an inhibition of Lck activity. The results indicate that although YopH appears to have the ability to target multiple proteins in T cells at higher MOIs, LAT and SLP-76 or molecules responsible for their phosphorylation, but not Lck, are primary targets of YopH at a low bacteria to T cell ratio.
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Jurkat cells were exposed to wild-type, YopH-deficient (YopH), yopH-HA, and yopHC403A-HA Y. pseudotuberculosis, followed by activation with the CD3
-specific antibody OKT3. As shown in Fig. 4, wild-type as well as yopH-HAexposed Jurkat cells showed no observable phosphorylation in response to TCR stimulation (lanes 8 and 10), indicating that the HA-tag does not affect the activity of YopH. In contrast, exposure to
YopH or yopHC403A-HA Yersinia did not inhibit TCR signaling even at an MOI of 50 (lanes 4 and 6). Comparable amounts of YopH, YopH-HA, and YopHC403A-HA were present in the exposed T cells (Fig. 4, YopH).
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Lck, CD3, and ZAP-70 are phosphorylated earliest in the TCR-mediated signaling pathway. This leads to the phosphorylation of LAT and the recruitment of several proteins, including phospholipase C-
1, PI3K, and Grb2-like adaptor protein downstream of Shc (Gads). SLP-76 is constitutively associated with Gads and, thus, is recruited to the TCR signaling complex and subsequently phosphorylated. Phosphorylated SLP-76 attracts more proteins, including SLAP-130 and Vav, to the protein assembly, referred to as the LAT/SLP-76 signalosome (19, 29, 30).
As shown in Fig. 5, a significant amount of phosphorylated SLP-76, and in addition phosphorylated LAT and SLAP-130, the unphosphorylated Gads and two unidentified phosphorylated proteins of 50-kD and 65-kD size (lane 2) coprecipitated with YopHC403A-HA in lysates of activated Jurkat cells exposed to yopHC403A-HA at an MOI of 2. Phosphorylated Vav was present in the precipitate derived from T cells exposed at an MOI of 50 (unpublished data). Also a small amount of Lck was detected in YopHC403A precipitates from cells exposed to an MOI of 50. The precipitated 50- and 65-kD protein are likely to be part of the LAT/SLP-76 signalosome because phosphorylated proteins of 50- and 65-kD size also coprecipitated with LAT or SLP-76 from extracts of activated Jurkat cells (unpublished data).
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To verify that binding of YopHC403A-HA to the coprecipitating proteins occurred in the T cells and was not a result of YopHC403A-HA binding to host proteins in the lysate, we incubated Jurkat T cells with a mutant Yersinia strain that is incapable of translocating YopH into eukaryotic cells, an lcr mutant (31) expressing YopH-HA. The infection did not affect TCR-stimulated tyrosine phosphorylation (Fig. 6 A), and we were not able to precipitate YopH-HA from lysates of the exposed T cells, even though the lcr mutant synthesized similar amounts of the epitope-tagged YopH compared with the wild-type strain (Fig. 6, B and C). These results showed that, under the experimental conditions we used for T cell lysis, there was no concomitant lysis of Yersinia and that the precipitated YopH protein was that which had been injected into T cells.
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Because several phosphorylated proteins of the LAT/SLP-76 signalosome were found to precipitate with the substrate-trap YopH in exposed T cells, it was possible that the substrate trap YopH bound directly to every coprecipitated protein, or that it bound to one or a few of the proteins and precipitated the entire complex. To distinguish between these possibilities, we characterized YopH targets in Jurkat cells that were either SLP-76 deficient (J14; reference 32) or LAT deficient (J.CaM2; reference 33). In J14 cells, LAT and the 65-kD protein still coprecipitated with YopHC403A-HA (Fig. 7 A, lane 9). SLAP-130 and the 50-kD protein were barely detectable. This indicates that their association with YopHC403A-HA in wild-type T cells is mediated largely via SLP-76. In activated J.CaM2 cells, the lack of LAT expression resulted in a general reduction of phosphorylation level. Phosphorylated SLP-76 could not be detected in the T cell lysate. No phosphorylated proteins were found to coprecipitated with YopHC403A-HA (lane 7) after exposure to an MOI of 2. Because the signaling cascade from the T cell receptor is interrupted in these Jurkat mutants, we stimulated yopHC403A-HAexposed Jurkat cells (MOI 2) with per-vanadate to increase the phosphorylation of signaling molecules (Fig. 7 B). The Lck-deficient Jurkat line J.CaM1 (34) was also included in these experiments. In activated J.CaM1 cells, the overall phosphorylation level was greatly reduced compared with wild-type Jurkat cells. However, SLP-76, LAT, the 65-kD protein, and SLAP-130 still coprecipitated with YopHC403A, indicating that the presence of Lck is not required for the binding of those proteins to YopHC403A. In the LAT-deficient Jurkat line, SLP-76, SLAP-130, and the 50- and 65-kD proteins were detected, indicating that the binding of YopHC403A to these proteins is not dependent on LAT. In addition, we detected ZAP-70 coprecipitating with YopHC403A in these experiments, suggesting that artificial hyperphosphorylation of signaling molecules allows YopH to bind to additional proteins.
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Discussion |
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Our results are different from those reported by Alonso et al. (24), who found Lck, especially tyrosine 394 of Lck, to be the main target of YopH. The discrepancies are most likely due to differences in the amount of YopH used and the manner of its delivery into the cells. We constructed Yersinia strains that inject substrate trap YopH into cells to ensure that the mutant YopH is delivered by the same route and in similar quantity as translocated WT-YopH. Alonso et al. expressed YopH in transiently transfected T cells, treated T cells with membrane-permeable YopH, or added YopH to lysed cells. Furthermore, to identify targets of YopH, Alsonso et al. added 150 times as much substrate trap YopH to T cell lysates as is translocated by Yersinia at an MOI of 2. Indeed, at an MOI of 50, we also see Lck being targeted by YopH.
The ability to alter T cell signaling as seen in the sparking phenotypes of calcium flux and PI3K activity is consistent with our identification of LAT and SLP-76 as major targets of YopH. The sparking pattern of calcium flux is similar to the ones observed in T cells with a deletion of Itk, a Tec family kinase that associates with phosphorylated SLP-76 (36), or a mutation in LAT. In the Itk-deficient T cells, TCR stimulation results in a substantial reduction in IP3 generation and defects in IL-2 production. Jurkat cells expressing LAT Tyr132Phe or T cells from "knockin" mice homozygous for LAT Tyr136Phe (Tyr136 in murine LAT is equivalent to Tyr132 in human LAT) also are deficient in their ability to respond to TCR stimulation (37, 38). Partial dephosphorylation of LAT and SLP-76 might be conferred by very limited quantities of injected YopH and, thus, could profoundly alter TCR-mediated signaling and the subsequent responses in Yersinia infection. This suggests that, during a Yersinia infection, a wide range of T cellmediated responses may be affected. In addition, abnormal T cell signaling has been linked to the development of autoimmune diseases, particularly rheumatoid arthritis, in which the aberrant T cell signaling has been linked to severely impaired phosphorylation of LAT (39). One might speculate that the ability of Y. pseudotuberculosis to disrupt or alter T cell signaling could also play a role in triggering this disease.
When exposing T cells to Yersinia at low MOIs, wild-type as well as substrate trap YopH injected into T cells through the type III secretion system primarily targets LAT and SLP-76, but not Lck, CD3, or ZAP-70, even though the latter three molecules are phosphorylated earlier during T cell activation. The selectivity of YopH may in part result from the amino acid sequences of the target proteins. In vitro analysis has shown that the optimum peptide sequence for YopH catalysis is D/E D/E pYxxP (40). The amino-terminus of YopH that exhibits a substrate-binding domain that binds substrates directly in a phosphotyrosine-dependent manner (25) contributes to this selectivity. Both, SLP-76 and LAT, but not CD3
, ZAP-70, and Lck, feature tyrosine phosphorylation sites comprising the optimal motif for YopH's phosphatase activity. However, when T cell lysates were incubated with an amount of YopHC403A similar to the amount injected by Yersinia into T cells at an MOI of 2, CD3
and ZAP-70 were found to bind to substrate trap YopH, indicating that there may be additional unidentified targeting mechanisms that contribute to YopH specificity. An additional targeting mechanism might be provided by the proline-rich region of YopH that shows the consensus motif for binding to Src homology 3 domains (25, 41) and that might confer binding in a phosphorylation-independent manner. Studies in macrophages did not find this region of YopH to be important for the targeting of YopH (25, 42). However, Src homology 3 domains are present (e.g., in the small adaptor Gads), but we did not find any evidence that catalytically active YopH has the ability to bind to Gads (unpublished data).
The difference in the specificity of YopH when delivered into live cells compared with in vitro experiments suggests that the accessibility of signaling complexes in live cells plays an important role. Many signaling complexes in mammalian cells are organized into modules and preferential sequestration and localization of these modules allows for specificity and selectivity of activation. Furthermore, cellular compartmentalization and localization is important for the in vivo substrate specificity of phosphatases that have a broad substrate spectrum in vitro (43). Thus, delivery of YopH through the type III secretory pathway likely determines the interactions of YopH with the host cell signaling modules. In addition, specificity of YopH is also influenced by its concentration. In T cells exposed to higher MOIs, YopH targets additional proteins, including Lck. This indicates that, although YopH shows broad targeting capabilities, it is very specific at low bacteria to host cell ratios, a likely situation in infections in vivo.
Despite the observation that the action of Yop effectors shows no cell specificity among various adherent cells lines (44), YopH is the only Yersinia effector protein affecting T cells (7). The data presented here suggest that Yersinia may have developed a sophisticated mechanism to target effector proteins to special cellular compartments so that the effect of the few virulence factors delivered by a few bacteria can be maximized. This strategy may not be limited to Yersinia because type III secretion systems have been found in other pathogenic microorganisms, such as Salmonella, Shigella, and Chlamydia, that are also associated with reactive arthritis in humans (45). It is likely that these bacteria secrete molecules analogous to Yops that may alter host cell function and the immune response to the benefit of the microbe. For example, Salmonella translocates the effector protein, SptP, which has domains corresponding to those found in the Yersinia YopH and YopE (46).
In conclusion, we have identified a novel and efficient mechanism by which bacteria alter T cell responsiveness. These findings may have general applicability to the understanding of bacterial pathogenicity and the development of immune responses during infection.
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Materials and Methods |
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Wild-type Y. pseudotuberculosis YPIIIpIB1 (48), the YopH-deficient (YopH) strain YPIIIp502 (49), and newly generated mutant strains were grown in 2YT medium at 26°C and the appropriate antibiotics: 100 µg/ml ampicillin, and 32 µg/ml chloramphenicol. For T cell infections, overnight cultures were diluted to an OD578 of 0.1 into 2YT supplemented with 20 mM sodium oxalate and 20 mM magnesium chloride, grown for 2 h at 26°C, and shifted to 37°C for 2 h. Bacterial numbers were determined by OD578 = 109 bacteria/ml.
Y. pseudotuberculosis strains that secrete HA-tagged YopH or HA-tagged substrate trap YopH (designated as yopH-HA, yopHC403A-HA, and yopHD356A-HA, respectively), were generated as follows. The genes encoding YopH or YopHC403A were amplified including the promoter regions from YPIIIpIB1 or the substrate trap YopHC403A-expressing strain YPIIIpIB1C403A (9) and were inserted into pACYC184 (50). The HA-tag was introduced at the 3'-end of yopH and yopHC403A. The resulting plasmids pyopH-HA and pyopHC403A-HA were introduced into YPIIIp502. In addition, pyopH-HA was also introduced into the lcr mutant YPIIIpIB71 (31), yielding the strain lcr (pyopH-HA). pyopHD356A-HA was generated by site-directed mutagenesis of pyopH-HA using Quikchange (Stratagene) and introduced into YPIIIp502.
His-tagged YopHC403A (His-YopHC403A) as well as a glutathion S-transferase (GST) fusion protein of YopHC403A (GST-YopHC403A) were expressed in Escherichia coli. yopHC403A was inserted into pQE30 (QIAGEN) to produce His-YopHC403A. GST-YopHC403A was encoded by a pGEX-4T-1 (Amersham Biosciences) derivative containing yopHC403A.
Antibodies
A polyclonal antiserum to YopH was generated at Josman, LLC by immunizing rabbits with purified His-YopHC403A. The crude antiserum was affinity purified (51) using GST-YopHC403A fusion protein bound to glutathione sepharose (Amersham Biosciences). Antihuman CD3 (OKT3) was purified from hybridomas (American Type Culture Collection). In addition, antibodies against the following proteins and residues were used in this study: antiphosphotyrosine (4G10), LAT, Lck, Gads, ZAP-70, Vav, FAK (Upstate Biotechnology); CD3
, actin (Santa Cruz Biotechnology, Inc.); SLP-76, SLAP-130, paxillin, p130cas, PYK2 (Transduction Laboratories); phospho-Lck Tyr394 (detected by antiphospho-Src family [Tyr416] antibody; Cell Signaling), mouse CD3
(2C11)-biotin (BD Biosciences); HA-tag (HA.11; Covance). Antimouse IgG horseradish peroxidase (HRP) linked whole antibody and antirabbit IgG HRP linked F(ab')2 fragments were purchased from Amersham Biosciences.
T cell infection and activation
106 cells/ml Jurkat T cells or 2 x 106 cells/ml 5C.C7 T cell blasts were exposed to specific Y. pseudotuberculosis at a specified MOI for 1 h in prewarmed supplemented IMDM or RPMI 1640 without gentamycin in tissue culture plates at 37°C and 5% CO2. To facilitate contact between the bacteria and the T cells, the plates were spun for 3 min at 1,200 revolutions/min at the beginning of the infection. After the exposure, the T cells were collected in 1.5-ml tubes by centrifugation for 1.5 min (2 min for T cell blasts) at 1,600 revolutions/min (200 g) and washed four times with prewarmed PBS (37°C) to remove most of the unattached Yersinia.
Jurkat T cells were activated at 4 x 107 cells/ml in PBS at 37°C by adding OKT3 to a final concentration of 10 µg/ml. For the activation of T cell blasts, anti-CD3 2C11-biotin was preincubated with streptavidin and added at a final concentration of 10 µg/ml. For stimulation with per-vanadate (100 µM final concentration), ortho-vanadate was oxidized using H2O2, which was subsequently removed using catalase. After incubation for 2 min (vanadate 3 min), cells were spun down for 10 s at 4°C and lysed at 4 x 107 cells/ml (Jurkat) or 108 cells/ml (T cell blasts) in ice-cold TTS (25 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 10 mM ß-glycerolphosphate, 10 mM NaF, Complete protease inhibitors (Roche). Nuclei were removed by centrifugation for 10 min at 13,000 revolutions/min (16,000 g) at 4°C. Total lysates of 4 x 105 Jurkat cells or 2 x 106 T cell blasts were resolved by SDS-PAGE. Phosphotyrosine proteins were detected by immunoblot analysis using 4G10 antibody and the enhanced chemiluminescence system ECL (Amersham Biosciences). Blots were stripped according to the protocol provided by Amersham Biosciences and subjected to reprobing with appropriate antibodies. Results were quantified using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available at http://rsb.info.nih.gov/nih-image).
Immunoprecipitation of YopH-associated proteins
To identify YopHC403A/YopHD356A targets in exposed T cells, 3 x 107 Jurkat T cells were exposed to yopHC403A-HA or yopHD356A-HA at an MOI of 2, activated, and lysed as described in the previous paragraph. Extracts were precleared using protein G agarose (Sigma-Aldrich) and subsequently incubated for 2 h at 4°C with affinity purified anti-YopH antibodies or anti-HA monoclonal antibodies (final concentration of 10 µg/ml) and protein G agarose. The agarose was collected by centrifugation for 3 min at 16,000 g at 4°C, washed three times with TTS buffer, and boiled with reducing SDS sample buffer. The proteins were separated by SDS-PAGE and analyzed by Western blot analysis. For in vitro immunoprecipitations, Jurkat cells were activated and lysed, and YopHC403A, YopHD356A, or YopH were added to the precleared lysate. YopH was preincubated in lysis buffer with 1 mM vanadate for 15 min to inactivate it before adding it to the lysate. To match the amount of purified YopH proteins added to the in vitro experiments with the amount translocated by Yersinia, we compared the amounts of YopH injected into Jurkat T cells with a titration curve of YopH. We found that 20 ng of protein are translocated by Yersinia into 107 T cells exposed at an MOI of 2.
Lck kinase assay
Jurkat T cells were exposed to Yersinia as described before, stimulated with OKT3, and lysed in kinase assay buffer KAB (25 mM Hepes, pH 7.4, 150 mM KCl, 5 mM MnCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 1 mM Na3VO4). Lysates were precleared and incubated with 10 µg/ml anti-Lck monoclonal antibody and protein G agarose. The agarose was washed three times in KAB. Kinase activity was measured by ELISA as follows. The cytoplasmic domain of CD3 ([Biotin][AHX] GGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN) was used as a substrate for Lck and was bound on streptavidin-coated Immulon 4 plates. Precipitated Lck was added for 30 min at 32°C in KAB containing 20 µM ATP. Phosphorylation of the CD3
-peptides was quantified using antiphosphotyrosine antibody 4G10, antimouse IgG HRP conjugate and the HRP substrate OPD (Sigma-Aldrich) in comparison to a standard curve of phosphorylated CD3
-peptide (0.0512.5 ng/ml). Lck-deficient Jurkat cells (J.CaM1) were used as negative control.
Calcium flux and PI3K activity analysis
To analyze calcium flux by flow cytometry, 106 T cell blasts were exposed to specified Y. pseudotuberculosis strains and concurrently loaded with 2 µM indo-1, AM (Molecular Probes) for 30 min. T cells were collected by centrifugation at 1,600 revolutions/min (200 g) for 2 min in 1.5-ml tubes and washed twice with prewarmed antibiotic-free RPMI 1640 containing 25 mM Hepes, pH 7.4. Indo-1 loaded T cells were activated by the addition of 2C11-biotin antibody (preincubated with streptavidin) to a final concentration of 10 µg/ml. Calcium flux was analyzed at 37°C on a modified FACStar (FACS facility, Stanford University) by monitoring the ratio of 405/485 nm emission and time. As a positive control, 1 µM ionomycin (Sigma-Aldrich) was added to the T cells at the end of the measurement.
Calcium flux and the PI3K activity of T cells responding to antigenic stimulation were analyzed by single cell video microscopy using a Zeiss/Universal Imaging three-dimensional time-lapse system as described previously (21). 2 x 105 T cell blasts expressing the PH(AKT)-YFP were exposed to Yersinia and loaded with 1 µM fura-2, AM (Molecular Probes). After washing, the cells were resuspended in 200 µl prewarmed antibiotic-free RPMI 1640 supplemented with 25 mM Hepes, pH 7.4, and seeded into an eight-well glass-bottom chamber slide (Labtek/Nunc). 105 CH27 cells, preloaded with 1 µM MCC peptide for 1 h, were added to the T cells. Images were taken every 15 or 30 s for 15 min. At each time point, images of the two-dimensional differential interference contrast, the fura-2 fluorescence images (340/380 nm) and a z stack of YFP were recorded. Metamorph (Universal Imaging Corp.) was used for microscope control, data acquisition, and image analysis.
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Acknowledgments |
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This work was supported by funding from the National Institutes of Health and the Stanford Digestive Disease Center grant no. DK56339 (to Y.-h. Chien and S. Falkow).
The authors have no conflicting financial interests.
Submitted: 4 June 2004
Accepted: 22 November 2004
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