yopJ-dependent suppression of inducible cytokine expression
In screening Y. pseudotuberculosis strains deficient in various known plasmid-encoded virulence genes, we observed that HeLa cells infected with the yopJ strain (Galyov et al., 1994) released more IL-8 compared with cells infected with the isogenic wild-type strain (Table 1). HeLa cells infected with the yopJ strain containing a YopJ-encoding plasmid had IL-8 levels similar to those observed in wild-type-infected cells, even though the trans-complemented strain secreted much higher levels of YopJ than the wild-type strain (Fig. 1). The yopJ-dependent suppression of IL-8 expression was also observed in HeLa cells pretreated with cytochalasin D (not shown), which inhibits the uptake of bacteria into eukaryotic cells (Finlay and Falkow, 1988). To determine whether yopJ-dependent inhibition of IL-8 expression occurs in the absence of the other known Y. pseudotuberculosis Yop virulence proteins, we used the multiple Yop mutant (MYM) strain of Y. pseudotuberculosis containing deletions in yopM, yopE, yopK, ypkA and yopJ (Håkansson et al., 1996a). We observed more than a fourfold reduction in the amount of IL-8 secreted by HeLa cells infected with the MYM strain containing a YopJ-expressing plasmid compared with HeLa cells infected with the parental MYM strain (not shown). HeLa cells infected with the MYM strain containing a plasmid encoding YopM (Leung et al., 1990) secreted levels of IL-8 that were similar to the parental MYM strain, indicating that delivery of a Yop protein per se did not affect IL-8 secretion. These data indicate that yopJ encodes a protein that inhibits, independently of the other known Yop virulence proteins, the amount of IL-8 secreted by infected HeLa cells.
Table 1. . IL-8 secretion by Y. pseudotuberculosis-infected HeLa cells.aa. HeLa cells (2 × 106) were infected with the indicated strains of Y. pseudotuberculosis at a MOI of 30, and culture supernatants were removed at the indicated timepoints and assayed for IL-8 by ELISA.b. The values reported represent an average of two separate measurements and are representative of several independent experiments.c. Fold increase in IL-8 levels between the 2 h and 4 h measurements.
Figure 1. . YopJ secretion by Y. pseudotuberculosis. Cultures of the wild-type (lane 1), yopJ (YopJ−) (lane 2) and the yopJ strain trans-complemented with either wild-type or mutated YopJ-encoding plasmids (YopJ−/pYopJ and YopJ−/YopJDVE, lanes 3 and 4 respectively) under the control of the yopE promoter were induced for Yop expression for 4 h. The YopJ−/YopJDVE sample was diluted as indicated (lanes 5–7). Culture supernatants were TCA precipitated, fractionated by SDS–PAGE and transferred to a nitrocellulose membrane that was probed with YopJ-specific antisera.
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As the yopJ locus was essential for inhibiting the secretion of a cytokine that is normally expressed when eukaryotic cells interact with LPS-containing bacteria, it was important to determine whether the yopJ gene product affected the level of association between Y. pseudotuberculosis and eukaryotic cells. We used a double fluorescent antibody test (Persson et al., 1997) to distinguish intra- and extracellularly located bacteria after infection as well as to determine the total number of bacteria per cell for both the wild-type and the yopJ strains. There were no significant differences between the wild-type and yopJ strains in either the total number of bacteria per cell (11 ± 6 and 9 ± 3 respectively) or the percentage of cells that were located extracellularly (71.1% ± 15% and 65.0% ± 16% respectively). Thus, we conclude that the yopJ locus encodes a protein that does not affect the level of bacterial association or internalization by eukaryotic cells. Furthermore, we did not observe differences in the levels of YopE-mediated cytotoxicity in HeLa cells (Rosqvist et al., 1991) infected with either the wild-type or the yopJ strain (not shown), indicating that yopJ, unlike yopB (Håkansson et al., 1996b; see below), encodes a protein that is not involved in the delivery of other Yop proteins into eukaryotic cells.
We noted that YopJ contains a region that is similar to part of the SH2 domain of the eukaryotic signalling proteins p56lck, Syk and Shc (Koch et al., 1991). Within this region of similarity is a D/E-X-E motif that is also conserved in the YopJ-like proteins of Y. enterocolitica, Salmonella and Xanthomonas (Whalen et al., 1993; Hardt and Galán, 1997; Mills et al., 1997) (Fig. 2). To determine whether this domain was required for YopJ's anti-host activity, we replaced the DVE-encoding codons of yopJ with NAQ-encoding codons by site-directed mutagenesis to generate the D53N-V54A-E55Q YopJ mutant, referred to as YopJDVE, and tested whether the resulting YopJDVE protein was functionally active. The YopJDVE protein was secreted at similar levels to the wild-type plasmid-encoded YopJ protein (Fig. 1) but was completely unable to block IL-8 induction after infection (Table 1). These data may indicate that YopJ interacts directly with proteins involved in signalling pathways leading to IL-8 induction.
Figure 2. . Sequence comparison of YopJ (residues 44–60) with various SH2-containing eukaryotic signalling proteins (Altschul et al., 1990) and the YopJ-like proteins of Y. enterocolitica (YopP), Salmonella enterica (AvrA) and Xanthomonas campestris (AvrRxv). Residues indicated with an asterisk are conserved within the N-terminal region (motif I) of several SH2 domain-containing proteins. The residues in bold were replaced with NAQ to generate the D53N-V54A-E55Q YopJ mutant (designated YopJDVE).
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To assess whether the yopJ gene product affected steady-state IL-8 mRNA levels, total RNA was isolated from HeLa cells infected with various Y. pseudotuberculosis strains and analysed by reverse transcription–polymerase chain reaction (RT–PCR) (Fig. 3). IL-8 message levels were substantially higher in cells infected with the yopJ strain compared with either the wild-type or the trans-complemented yopJ strain. Increased IL-8 mRNA levels were also observed in cells infected with the yopB mutant strain. The yopB gene product has been shown to be required for the delivery of Yop virulence factors into infected eukaryotic cells (Håkansson et al., 1996b). Two other cytokines that have been reported to act as chemoattractants for neutrophils, IL-1α and melanoma growth-stimulating activity (MGSA) (Richmond et al., 1985; Dinarello, 1991), had similar patterns of expression to IL-8 in HeLa cells infected with the various Y. pseudotuberculosis strains (Fig. 3). HeLa cells pretreated with cytochalasin D had a similar pattern of IL-8 mRNA levels to untreated cells (not shown). As the relative changes in IL-8 message levels as a function of yopJ were similar to those observed for secreted IL-8 (Table 1), this suggests that YopJ suppress IL-8 induction in eukaryotic cells by a mechanism that prevents the accumulation of IL-8 mRNA.
Figure 3. . Message levels of various cytokines in HeLa cells infected with different strains of Y. pseudotuberculosis. Total RNA was isolated from infected cell cultures and analysed by RT–PCR using primers specific for the indicated cDNAs (see Experimental procedures). The PCR products were the expected size from spliced cDNA templates respectively. No correctly sized PCR products were observed if reverse transcriptase was omitted from the cDNA synthesis reaction (not shown).
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To test whether the previously reported Yersinia-mediated suppression of TNF-α expression was also dependent on yopJ (Beuscher et al., 1995), we measured the levels of TNF-α released by murine J774 cells after infection with various Y. pseudotuberculosis strains. Similar to the result shown for IL-8 (Table 1), we observed no increase in TNF-α levels in culture supernatants from J774 cells infected with strains possessing a wild-type yopJ (Table 2). In contrast, there were higher levels of TNF-α in the culture supernatant from cells infected with either the yopJ strain or the yopJ strain trans-complemented with the yopJDVE-containing plasmid. Surprisingly, cells infected with the YopJDVE-expressing strain released substantially more TNF-α than cells infected with the yopJ strain. YopJDVE-mediated hyperinduction of TNF-α secretion by J774 cells is in contrast to the effect observed in HeLa cells, in which the introduced mutation resulted in a null phenotype in terms of IL-8 secretion. These data show that a wild-type yopJ locus is required to suppress TNF-α induction and, furthermore, may indicate that inducible IL-8 and TNF-α expression are regulated by distinct mechanisms.
Table 2. . TNF-α secretion by Y. pseudotuberculosis-infected J774 cells.aa. J774 cells (106) were infected with the indicated strains of Y. pseudotuberculosis at a MOI of 30, and culture supernatants were removed at the indicated timepoints and assayed for TNF-α by ELISA.b. The values reported represent an average of two separate measurements and are representative of several independent experiments.c. Fold increase in TNF-α levels between the 0.5 h and 6 h measurements.
We tested whether the yopJ-dependent effect of both promoting apoptosis (Mills et al., 1997; Monack et al., 1997) and suppressing TNF-α expression (Table 2) occurred simultaneously in J774 cells. Six hours after the start of infection, TNF-α levels were ascertained in the cell culture media and, subsequently, cells were collected and measured for apoptotic cell death by the TUNEL assay followed by fluorescence-activated cell sorting (FACS) analysis. Uninfected cell cultures as well as cultures infected with the yopJ strain had comparable numbers of apoptotic-like cells, whereas there was a twofold increase in the number of apoptotic cells in cultures infected with the trans-complemented yopJ strain (Fig. 4). Similar to the experiment shown above (Table 2), there was a substantially higher level of TNF-α in culture supernatants of cells infected with the yopJ strain compared with the levels observed for either uninfected cells or cells infected with the trans-complemented yopJ strain. Strikingly, although supernatants from cultures infected with the yopJ strain containing a YopJDVE–encoding plasmid had greatly elevated levels of TNF-α, the number of apoptotic-like cells was relatively low. These data show that YopJ-expressing Yersinia blocks TNF-α induction and induces apoptosis with roughly the same kinetics. Furthermore, as TNF-α is known to induce apoptotic cell death (Robaye et al., 1991; Darnay and Aggarwal, 1997), these data suggest that the yopJ locus encodes a protein that uncouples apoptotic cell death from TNF-α levels.
Figure 4. . TNF-α levels and apoptotic cell death in macrophage cell cultures after infection with various Y. pseudotuberculosis strains. J774 cells (106) were infected with either the yopJ mutant strain (none) or the yopJ mutant trans-complemented with plasmids encoding either wild-type YopJ (pYopJ) or the YopJDVE mutant (pYopJDVE). Six hours after infection, the culture supernatants were assayed for TNF-α levels, and cells were fixed, permeabilized and incubated with TUNEL reaction components. The number of FITC-positive cells was determined by FACS analysis. Levels of TNF-α and TUNEL-positive cells are reported as a percentage of the levels observed in the YopJ–/pYopJDVE and YopJ–/pYopJ samples respectively.
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Yersinia-mediated inhibition of NF-κB activation is dependent on the yopJ locus
The IL-8 promoter region contains a number of binding sites specific for various families of transcription factors (Mukaida et al., 1989). The inducibility of IL-8 promoter activity, as well as the promoters of TNF-α, IL-1α and MGSA, have been shown to be dependent on their respective NF-κB binding sites (Collart et al., 1990; Anisowicz et al., 1991; Hiscott et al., 1993; Kunsch and Rosen, 1993; Matsusaka et al., 1993). To determine whether the activity of a NF-κB binding site-containing promoter was affected after infection with Yersinia, HeLa cells were transiently transfected with a luciferase reporter gene under the control of the thymidine kinase promoter (TKp) containing two upstream NF-κB binding sites and subsequently infected with various Y. pseudotuberculosis strains. Extracts prepared from HeLa cells infected with wild-type Y. pseudotuberculosis contained slightly higher levels of luciferase activity than extracts prepared from uninfected HeLa cells. In contrast, there was a 14-fold increase in luciferase activity in extracts prepared from HeLa cells infected with the yopJ mutant strain compared with the increase observed in wild-type-infected cell extracts, while there was only a 2.5-fold increase in luciferase activity in HeLa cell extracts from cultures infected with the trans-complemented yopJ strain (Fig. 5A). The yopJ-dependent suppression of NF-κB site-directed promoter activity most probably plays an important role in limiting inducible cytokine expression in Yersinia-infected eukaryotic cells.
Figure 5. . NF-κB activation in HeLa cells after infection with Y. pseudotuberculosis. A. HeLa cells (5 × 106) were transiently transfected with a NF-κB site-containing reporter gene (NF-κB-TKp-luc). Two days after transfection, cells were subcultured onto four separate dishes, washed free of antibiotic-containing media and infected the next day with the indicated strain of Y. pseudotuberculosis. Cells were collected 3 h after the onset of infection, and luciferase activity was measured in the resulting whole-cell extract. The data shown are representative of several independent experiments. B. NF-κB site-specific binding activity was measured in nuclear extracts prepared from HeLa cells 3 h after being infected with the indicated Y. pseudotuberculosis strains. The faster migrating signal is primarily comprised of p50, while the slower migrating signal is primarily p50/p65. USF-specific binding activity was measured in the same extracts to control for loading. C. Supershift analysis of the yopJ-infected nuclear extracts (shown in B) using p65-specific antisera. D. Protein levels of p65 in whole-cell extracts prepared from HeLa cells infected with the indicated Y. pseudotuberculosis strains.
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NF-κB site-binding transcription factors are retained in the cytoplasm of uninduced cells by IκB proteins. Upon the appropriate stimulus, IκB is phosphorylated and degraded, thereby unmasking NF-κB's nuclear localization signals and allowing NF-κB to translocate to the nucleus (reviewed in Verma et al., 1996). To test whether the low level of NF-κB-dependent promoter activity observed in wild-type Yersinia-infected HeLa cells (Fig. 5A) was correlated with a lack of NF-κB translocation to the nucleus, NF-κB site-binding activity was measured using a band shift assay in nuclear extracts prepared from HeLa cells infected with various Y. pseudotuberculosis strains. In our experimental system, there was a basal level of NF-κB binding activity observed in uninfected cells (Fig. 5B) that was identical to the pattern reported previously for this oligonucleotide probe (Shakhov et al., 1990). In nuclear extracts prepared from cells infected with wild-type Yersinia, there was a slight increase in the level of the slower migrating complex (p50/p65) and a small decrease in the level of the faster migrating complex (p50/p50) compared with the uninfected control samples. In contrast to these subtle changes, there was a severalfold increase in binding activity observed in nuclear extracts prepared from cells infected with the yopJ strain compared with the binding activity in the uninfected and wild-type-infected extracts. Binding activity in nuclear extracts prepared from cells infected with the trans-complemented yopJ strain was slightly higher compared with the binding activity in wild-type nuclear extracts but still substantially less than the binding activity in extracts prepared from the yopJ strain. The complexes observed in the yopJ-infected extracts could be supershifted by p65-specific antisera (Fig. 5C), indicating that p65 translocation occurs in the absence of YopJ. The fact that the suppression of IL-8 and TNF-α secretion, as well as IL-8, IL-1 and MGSA mRNA levels, were fully complemented in the trans-complemented yopJ strain (see Tables 1 and 2 and Fig. 3) but not in the reporter gene and band shift assays (Fig. 5A and B respectively) may indicate that the yopJ gene product affects other cellular processes in addition to NF-κB activation that limits inducible cytokine expression.
Differences in NF-κB site-binding activity between extracts were not attributable to differences in the levels of internalization among the various Yersinia strains, as a similar pattern of binding activity was observed in the presence of cytochalasin D (not shown). A similar pattern of NF-κB site-binding activity was also observed in the B cell line K46 infected with either wild-type or yopJ strains (not shown). No differences were detected in the level of the NF-κB transcription factor p65 in HeLa whole-cell extracts prepared from cultures infected with the various strains of Y. pseudotuberculosis (Fig. 5D), indicating that the observed lack of p65 translocation in the yopJ-infected cells was not caused by changes in the intracellular concentration of p65. Taken together, these data indicate that NF-κB-mediated signalling occurs in the absence of an intact yopJ gene and further suggests that YopJ functions to block the activation of this pathway during the infection process.
To test whether the effects on the NF-κB site-containing promoter described above were dependent on a phosphorylation-competent IκB, we co-transfected the NF-κB-TKp-luciferase reporter plasmid with plasmids encoding either a wild type or a dominant-negative mutant (containing substitutions at the S-32 and S-36 inducible phosphorylation sites; DiDonato et al., 1996) form of IκB under the control of a viral promoter. Following transfection, cells were infected with either the wild-type or the yopJ strain of Y. pseudotuberculosis. There was a 12-fold induction in luciferase activity in extracts prepared from wild-type IκB-transfected cells infected with the yopJ strain compared with extracts prepared from wild-type IκB-transfected cells infected with the wild-type Yersinia strain (Fig. 6A). These data indicate that yopJ-dependent suppression of NF-κB activation is not dependent on IκB being expressed by its native promoter. There was a fourfold increase in luciferase activity in extracts prepared from mutated-IκB transfected cells following infection with the yopJ strain compared to extracts prepared from mutated IκB-transfected cells infected with the wild-type strain. These data indicate that, in the absence of a functional yopJ locus, the majority of NF-κB activation is dependent on the inducible IκB phosphorylation sites.
Figure 6. . NF-κB activation in the presence of plasmid-encoded IκB proteins and endogenous IκB protein levels in HeLa cells after infection with Y. pseudotuberculosis. A. HeLa cells (5 × 106) were co-transfected with the NF-κB-TKp-luc reporter plasmid and plasmids encoding either a wild-type IκB (+ CMVpWtIκB) or dominant-negative IκB (+ CMVpMutIκB) under the control of the CMV promoter. Transfected cells were either uninfected (−) or infected with either the wild type or yopJ (YopJ−) Y. pseudotuberculosis strains and reporter gene expression was analysed as described in the legend to Fig. 5. The level of luciferase activity was essentially identical in wild-type Y. pseudotuberculosis-infected cells transfected with either the wild-type or mutated IκB-encoding plasmids. B. Whole-cell extracts were prepared from HeLa cells infected with either the wild-type or yopJ mutant (YopJ−) strains of Y. pseudotuberculosis, and IκB protein levels were determined by immunoblotting. The IκB-specific proteins shown are 35–37 kDa.
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As a more direct test of the role that IκB degradation plays in Y. pseudotuberculosis-infected cells in either the suppression of NF-κB activation (in the presence of YopJ) or NF-κB activation (in the absence of YopJ), we prepared whole-cell extracts from either uninfected cells or cells infected with either the wild-type or the yopJ strains and analysed the levels of IκB by immunoblotting. Upon the appropriate stimulus, the relative levels of phosphorylated IκB increase compared with the unphosphorylated IκB and can be visualized electrophoretically because of the relatively slower migration of phosphorylated IκB. In our experimental system, there was a basal level of IκB phosphorylation and turnover in uninfected HeLa cells (Fig. 6B). No differences in the relative levels of IκB phosphorylation were observed between uninfected and wild-type-infected cells. In HeLa cells infected with the yopJ strain, however, there was both an overall decrease in the total amount of IκB as well as an increase in the relative level of phosphorylated IκB compared with unphosphorylated IκB. This result, along with the reporter gene data (Fig. 6A), indicates that the yopJ locus is required to block the phosphorylation-mediated degradation of IκB and that this activity probably accounts for the inhibition of NF-κB activation after infection with YopJ-expressing Yersinia.
Delivery of a YopJ–Cya reporter protein into HeLa and J774 cells
The requirement of the yopJ-dependent effect on cytokine mRNA levels on yopB (Fig. 3), whose gene product has been demonstrated to be required for the delivery of other Yops into the cytoplasm of infected eukaryotic cells (Håkansson et al., 1996b), suggested that YopJ exerts its activity from within the eukaryotic cell. We constructed a reporter plasmid encoding the first 49 codons of yopJ fused to the adenylate cyclase-encoding domain of the cyclolysin gene of Bordetella pertussis (Sory and Cornelis, 1994) under the control of the YopE promoter. The activity of the resulting hybrid protein, YopJ49–Cya, requires calmodulin, an intracellular eukaryotic protein. Thus, delivery of YopJ49–Cya into eukaryotic cells can be monitored by measuring cAMP in extracts prepared from infected cells.
To determine whether the reporter protein was exported from the bacterial cell, intact cells of the Y. pseudotuberculosis multiple yop mutant strain (MYM) (Håkansson et al., 1996a) containing the YopJ49–Cya-encoding plasmid were assayed for adenylate cyclase activity in the presence of calmodulin. A low of level of cyclase activity was detected in YopJ49–Cya-containing MYM bacteria, while bacteria containing either YopE49–Cya- or YopB–Cya-encoding plasmids had relatively higher levels of cyclase activity (Table 3). The relative differences in the level of cyclase activity in the strains containing the three different reporter gene constructs mimics the differences observed in the levels of secretion of the respective full-length proteins; YopE and YopB are secreted to much higher levels into the culture supernatant compared with YopJ (Galyov et al., 1994).
Table 3. . Export of various Yop–Cya hybrid proteins by the Y. pseudotuberculosis MYM strain and their subsequent delivery into the cytoplasm of infected eukaryotic cells. a. Bacteria containing the indicated plasmids were collected after being induced for Yop expression, washed and resuspended in cyclase activity assay buffer containing calmodulin (Schesser et al., 1996).b. Cultured HeLa or J774 cells were infected with the indicated complemented MYM strain and, 4 h after the start of infection, cell cultures were washed and whole-cell lysates were prepared and measured for cAMP.
The subcellular location of YopJ49–Cya after infection was compared with YopE49–Cya and YopB–Cya hybrid proteins; YopE49–Cya has been shown to be both exported from the bacterial cell and delivered into the cytoplasm of infected eukaryotic cells (Sory et al., 1995; Schesser et al., 1996), while YopB–Cya has been shown to be exported from the bacterial cell but not delivered into eukaryotic cells (Håkansson et al., 1996b). Extracts prepared from both HeLa and J774 cells infected with the YopJ49–Cya-containing MYM strain had cAMP levels similar to the levels observed in extracts prepared from HeLa cells infected with the YopE49–Cya-containing MYM strain (Table 3). In contrast, cAMP was not detected in extracts prepared from HeLa cells infected with a YopB–Cya-containing MYM strain. These data show that YopJ49–Cya is similar to YopE49–Cya in terms of its delivery into the cytoplasm of eukaryotic cells during infection and further suggests that YopJ functions from within the eukaryotic cell to suppress the activity of signalling pathways that are required for inducible cytokine expression.