Pathogenic Yersinia resist uptake by eukaryotic cells by a mechanism involving the virulence protein YopH, a protein tyrosine phosphatase. We show that p130Cas and FAK are phosphorylated and recruited to peripheral focal complexes during bacterial uptake in HeLa cells. The inactive form of YopH interacts with the tyrosine phosphorylated forms of FAK and p130Cas and co‐localizes with these proteins in focal adhesions. On the other hand, the presence of active YopH results in inhibition of uptake, dephosphorylation of p130Cas and FAK, and disruption of peripheral focal complexes. We suggest that p130Cas and FAK are substrates for YopH and that the dephosphorylation of these proteins impairs the uptake of Yersinia pseudotuberculosis into HeLa cells.
Pathogenic Yersiniae infect both animals and humans and cause diseases ranging from mild gastroenteritis (Yersinia pseudotuberculosis and Y.enterocolitica) to bubonic plague (Y.pestis). The bacteria resist the non‐specific host defence and proliferate extracellularly in lymphatic tissues (Hanski et al., 1989; Simonet et al., 1990). This ability is linked to the presence of a common virulence plasmid that encodes a number of secreted proteins denoted Yersinia outer proteins (Yops), seven of which have been shown to be essential for virulence (Cornelis, 1992; Straley et al., 1993; Forsberg et al., 1994). Upon intimate contact with a eukaryotic target cell, the extracellularly located bacteria secrete Yops by the type III secretion machinery that is encoded by the virulence plasmid (Forsberg et al., 1994). The Yop effectors are then translocated through the plasma membrane into the interior of the target cell. Secretion and translocation of the Yops are mediated by a polarized mechanism that prevents secretion of Yops to the surrounding medium (Sory and Cornelis, 1994; Rosqvist et al., 1994; Persson et al., 1995; Sory et al., 1995). In contrast to extracellularly located bacteria, bacteria within a target cell do not express Yops (Rosqvist and Wolf‐Watz, 1986; Rosqvist et al., 1994; Persson et al., 1995).
YopH has the same signature sequence that is typical of eukaryotic protein tyrosine phosphatases (PTPases) EC 18.104.22.168 (Guan and Dixon, 1990). Moreover, the specific activity of YopH is the highest of all the PTPases described thus far (Zhang et al., 1992), and this activity is essential for the virulence of pathogenic Yersiniae (Bliska et al., 1991; Andersson et al., 1996). YopH has been shown to cause dephosphorylation of target cell proteins (Bliska et al., 1991, 1992; Hartland et al., 1994; Green et al., 1995) and to interrupt early phosphotyrosine signalling associated with the bacterial uptake process (Andersson et al., 1996). The ability of a cell surface attached bacterium to avoid being ingested implies that the mechanism involved is rapid and specific, which in turn suggests that the effect of YopH is exerted close to the site of interaction between the bacteria and the target cell (Forsberg et al., 1994). Y.pseudotuberculosis bacteria lacking the virulence plasmid are taken up by eukaryotic cells. This uptake is dependent on interactions between the bacterial protein invasin (chromosomally encoded) and β1‐integrins on the surface of the eukaryotic cell, and in the absence of invasin, the bacteria are not taken up at all (Rosenshine et al., 1992; Isberg and Tran van Nhieu, 1994; Fällman et al., 1995). Invasin stimulated uptake is associated with increased tyrosine kinase activity in the ingesting cell, and it has also been shown that the uptake is obstructed by the presence of tyrosine kinase inhibitors (Rosenshine et al., 1992; Fällman et al., 1995). Furthermore, upon β1‐integrin mediated interactions with Y.pseudotuberculosis strains that express Yops this uptake process is blocked (Persson et al., 1995), and it has been proposed that YopH acts by dephosphorylating proteins that are involved in regulation of the cellular cytoskeleton and thereby inhibiting integrin mediated uptake (Andersson et al., 1996). However, the specific role of YopH in this process has been difficult to evaluate, since other Yop effectors concomitantly expressed by this pathogen also influence the uptake process (Cornelis, 1992; Straley et al., 1993; Forsberg et al., 1994).
Integrins take part in adhesive interactions of eukaryotic cells by serving as a link between the extracellular matrix and the actin cytoskeleton at sites of close cell substratum contact (Lo and Chen, 1994). These sites, termed focal adhesions (FAs), are multi‐molecular complexes in which clustered integrins are associated with a large number of cytoplasmic derived molecules that exhibit structural (α‐actinin, vinculin, talin) and/or regulatory (focal adhesion kinase, Src family tyrosine kinases, paxillin and tensin) functions (Lo and Chen, 1994; Miyamoto et al., 1995b). The FA components participate in a variety of signalling events generated by ligation and clustering of integrins, for instance those mediating cytoskeletal rearrangement (Lo and Chen, 1994; Clark and Brugge, 1995). The precise mechanism underlying assembly of FAs is not known, although it has been shown to involve occupancy and clustering of integrins in association with tyrosine kinase activity (Burridge et al., 1992; Miyamoto et al., 1995a, 1995b). Rho family GTPases appear to play a regulatory role in the very early events of integrin clustering and subsequent FA assembly (Ridley and Hall, 1992; Hotchin and Hall, 1995; Nobes and Hall, 1995). Disassembly of FAs involves PTPase activity and occurs during processes that require cells to detach from the substratum and to reorganize their cytoskeleton (Dunlevy and Couchman, 1993; Nakamura et al., 1995).
One FA protein involved in the integrin mediated signalling cascade is the focal adhesion kinase (FAK). This kinase interacts with and phosphorylates several proteins and is therefore believed to function as a signal amplifier/transmitter. The mechanism for its activity, however, is not clear (Richardson and Parsons, 1995). The localization of FAK to FAs does not depend on integrin binding and tyrosine phosphorylation (Hildebrand et al., 1993), and the activity of the kinase is stimulated by a mechanism involving interactions with integrins (Richardson and Parsons, 1995). Polte and Hanks (1995) used the two hybrid screen technique to gain insight into the FAK signalling pathway and found that the Crk associated tyrosine kinase substrate p130Cas interacts with FAK and that a proline‐rich sequence of FAK serves as a binding site for the SH3 domain of p130Cas. Besides the SH3 domain, p130Cas contains several putative tyrosine phosphorylation sites that can serve as binding sites for SH2‐containing molecules (Sakai et al., 1994; Burnham et al., 1996). The role of p130Cas in FAK‐related signal transduction is not clear, although it was recently found that this protein is phosphorylated upon cellular adhesion to fibronectin, and has been found localized to focal adhesion, which suggests that p130Cas, like FAK, is involved in integrin mediated adhesion (Nojima et al., 1995; Petch et al., 1995; Vuori and Rouslahti, 1995; Harte et al., 1996; Vouri et al., 1996).
In the present study, we have examined the molecular mechanism by which YopH blocks β1‐integrin mediated uptake of bacteria by HeLa cells. We found that YopH, in infected cells, caused p130Cas and FAK to become dephosphorylated, which suggests that these proteins constitute native targets of YopH. We also observed that bacterial infection stimulated phosphorylation of p130Cas and FAK, and this was associated with recruitment of the former and accumulation of the latter in focal complexes located at the cell periphery.
YopH inhibits the uptake of bacteria by HeLa cells
Yersiniae pseudotuberculosis blocks its own uptake into eukaryotic cells and one bacterial key protein in this process is the PTPase YopH. Since the uptake process starts immediately upon contact between the pathogen and the target cell, YopH must instantly affect the uptake machinery of the cell. In accordance with this, it has been shown that YopH affects early phosphotyrosine signalling (Andersson et al., 1996). In general, PTPases exhibit low substrate specificity when studied in vitro but are strictly regulated in vivo, and it has been suggested that the targeting to specific intracellular locations is an important mechanism that regulates PTPase activity (Mauro and Dixon, 1994). Therefore, we have in this study chosen an in vivo infection model. We used a specifically engineered multiple Yop mutant (MYM) strain of Y.pseudotuberculosis (Håkansson et al., 1996), to examine the molecular mechanism of the PTPase YopH. This strain does not express YadA, YopH, YopE, YopM, YopK or YpkA, but it is still able to regulate, secrete and translocate Yops as the wild‐type Y.pseudotuberculosis strain does. A multicopy plasmid that encodes a given Yop can be introduced into MYM to study the behaviour of that particular Yop in the intracellular compartment of the target cell without interference from other Yop effectors (Håkansson et al., 1996). We examined the effects of the PTPase YopH by using the MYM strain and an isogenic pair of plasmid‐bearing MYM strains, i.e. MYM expressing the wild‐type YopH protein (MYMpyopH) and MYM expressing a catalytically inactive form of YopH (MYMpyopHC403A) (all strains used are described in Table I). The YopHC403A protein has a single amino acid substitution (Cys403→Ala) which totally abolishes the PTPase activity (Guan and Dixon, 1990). These three strains as well as the wild‐type strain were used to infect cultured HeLa cells, and the level of bacterial uptake into these cells was determined (Heesemann and Laufs, 1985; Rosqvist et al., 1988). When MYM or MYMpyopHC403A infected HeLa cells were analyzed, 84 or 75% of the cell associated bacteria were intracellularly located, while only 27% were intracellularly located when the MYMpyopH strain was studied (Figure 1A). The efficiency of the latter strain to resist being ingested by the HeLa cells mimic that of the wild‐type strain (Figure 1A). The uptake of the pathogen into eukaryotic cells is dependent on binding of the bacterial protein invasin to β1‐integrins present on the surface of the target cell (Isberg and Tran van Nhieu, 1994); consequently, an invasin mutant that lacks interactions with β1‐integrins was not ingested (data not shown). These results indicated that high expression of YopH alone was sufficient to impair β1‐integrin mediated bacterial uptake.
Uptake of bacteria by HeLa cells is associated with increased tyrosine phosphorylation
To evaluate the involvement of tyrosine kinase activity in bacterial uptake by HeLa cells, phosphotyrosine proteins were analyzed at different time points after infection of the cells with the MYM strain. The uptake of the bacteria was associated with a time dependent increase in the tyrosine phosphorylated state of, in particular, one protein class with a molecular weight of ∼120 kDa (Figure 1B). To explore the importance of invasin for stimulating the increased phosphorylation seen with the MYM strain, an isogenic pair of plasmid cured Y.pseudotuberculosis strains YP100 (invA) and YP100(pIRR1) (invA/pinvA+) was used. The invasin expressing strain caused an increased tyrosine phosphorylation of proteins in the 120 kDa range, similar to that seen with the MYM strain (Figure 1B). The invasin mutant, which was not taken up by the HeLa cells, did not induce any phosphotyrosine signal (Figure 1B). Thus, the presence of invasin was a prerequisite for the observed increase of tyrosine phosphorylation in the infected cells. Infection with the MYMpyopHC403A strain led to increased tyrosine phosphorylation of several proteins, with the predominant protein species being in the 120–130 kDa range (Figure 1C), and the increase in the tyrosine phosphorylated state of the 120–130 kDa proteins was much more pronounced than that caused by the MYM strain. One possible explanation for this could be that the presence of YopHC403A protected against dephosphorylation by endogenous PTPases, and this in turn suggested that the mutated form of YopH interacted with the proteins indicated.
Earlier studies have shown that YopH caused an overall dephosphorylation of cellular proteins (Bliska et al., 1991, 1992; Hartland et al., 1994). In contrast to this, we found that constitutively tyrosine phosphorylated proteins were only slightly affected after prolonged exposure to YopH, but the infection‐induced increase in tyrosine phosphorylation of the 120–130 kDa proteins was not detected (Figure 1C). This demonstrated that although YopH is an extremely potent PTPase, it was not unrestrained within the HeLa cells and the absence of the increased phosphorylation of the 120–130 kDa proteins suggested that YopH specifically interrupted the signal transduction involved in the uptake of the bacteria.
To determine if YopH could dephosphorylate the proteins showing increased tyrosine phosphorylation induced by the MYMpyopHC403A strain, HeLa cells were infected with the MYMpyopHC403A strain for 60 min (after exposure for that time, the 120–130 kDa proteins were highly phosphorylated) and then infected again but now with MYMpyopH. At different times after the second infection, the cellular content of phosphotyrosine proteins was determined. The presence of active YopH resulted in a rapid (detected within 5 min) dephosphorylation of the 120–130 kDa proteins (Figure 1C), suggesting that this protein class was a substrate of YopH.
YopHC403A associates with FAK and a highly phosphorylated form of p130Cas
To determine whether YopHC403A interacted with the 120–130 kDa phosphotyrosine proteins immunoprecipitations using anti‐YopH antisera were performed, employing the method described by Bliska et al. (1992). Immunoprecipitation of YopHC403A from infected cells resulted in a co‐precipitation of 120–130 kDa phosphotyrosine proteins (Figure 2A). Interestingly, the association between YopHC403A and these proteins could be detected already after 2 min of infection and no phosphotyrosine proteins of other molecular weights were found in the precipitate. Furthermore, immunoprecipitation of YopHC403A depleted the 120–130 kDa phosphotyrosine proteins from the lysate (Figure 2B), demonstrating that the majority of this protein class was associated with the catalytically inactive YopH.
β1‐integrin mediated adhesion stimulates tyrosine phosphorylation of two proteins, p130Cas and FAK, with molecular weights of 120–135 kDa (Nojima et al., 1995; Petch et al., 1995; Vuori and Rouslahti, 1995). This finding, and the fact that the uptake of Yersinia by HeLa cells is mediated by β1‐integrins (Isberg and Tran van Nhieu, 1994) suggested that the phosphotyrosine proteins associated with YopHC403A were p130Cas and/or FAK. Therefore, the YopH immunocomplexes were immunoblotted with anti‐p130Cas and anti‐FAK antibodies (Figure 2C). Both p130Cas and FAK were present in the YopH immunocomplex obtained from cells infected with the MYMpyopHC403A strain (Figure 2C). On the other hand, no tyrosine phosphorylated proteins were detected in YopH precipitates from cells infected with MYM or MYMpyopH (Figure 2C). The absence of FAK and p130Cas in the immunocomplex of PTPase‐active YopH indicated that the interaction of YopH with these proteins required them to be tyrosine phosphorylated.
To test the specificity of the association between YopHC403A, p130Cas and FAK, we examined whether other phosphotyrosine proteins of similar molecular weight (Cbl, vinculin, β1‐integrin and pp120) or proteins possibly involved in the regulation of p130Cas and FAK (Crk, Src, Abl and paxillin) were present in the precipitate. The tested proteins were found in the lysates before and after precipitation of YopHC403A, but none of these were detected in the precipitate (data not shown). The anti‐FAK antibody recognized a protein corresponding to the lower part of the 120–130 kDa YopHC403A immunocomplex, whereas the anti‐p130Cas antibody displayed a broad band with a migration correlating to the upper part of the 120–130 kDa protein class (Figure 2C). There are three forms of p130Cas: CasA, CasB and CasC. The former two arise from alternative splicing and CasC is a highly phosphorylated form of the protein, which can be discriminated from the other two forms by PAGE (Sakai et al., 1994; Polte and Hanks, 1995). Most of the total p130Cas protein (immunoprecipitated with anti‐p130Cas antibodies) had a lower molecular weight than the fraction of p130Cas associated with YopHC403A (Figure 2D), implying that YopHC403A interacted specifically with the highly phosphorylated form of the protein i.e. CasC.
The above findings indicated that the 120–130 kDa proteins that were phosphorylated during infection with MYM and MYMpyopHC403A (see Figure 1) were p130Cas and FAK. Immunoprecipitation of p130Cas or FAK revealed that these proteins were tyrosine phosphorylated upon infection of HeLa cells with the strains indicated above, and as expected, the MYMpyopHC403A strain caused a more pronounced tyrosine phosphorylation of the proteins (Figure 3). The opposite was found when the wild‐type YopH protein was studied: in this case, both p130Cas and FAK were totally dephosphorylated (Figure 3), showing that p130Cas and FAK were substrates of YopH. Moreover, these results did also suggest that these two proteins were involved in the β1‐integrin associated signalling that mediates uptake of bacteria, and that dephosphorylation of these proteins by YopH impaired the uptake process.
FAK and p130Cas are recruited to peripheral FAs upon infection
To analyse the subcellular localization of p130Cas after bacterial infection, HeLa cells were infected with either MYMpyopH or MYMpyopHC403A; samples were taken at different time points and further processed for visualization by confocal microscopy. The p130Cas protein was found distributed in the cytosol and enriched in the perinuclear region of uninfected cells (Figure 4A). After infection with MYMpyopHC403A, p130Cas was detected in distinct regions lining the edges of the HeLa cell; after 30 min of infection ∼60% of the peripheral HeLa cells exhibited this type of staining pattern, and after 60 min this was seen in all peripheral cells and no morphological changes of the infected cells were detected (Figure 4A). When the MYMpyopH strain was used to infect HeLa cells, p130Cas was not recruited to the distinct regions at the edges of the cells; instead, the cells became round in shape and were cytotoxically affected (Figure 4A). To ascertain whether the dotted staining pattern of p130Cas at the edges of the infected HeLa cell was due to enrichment of this protein in peripheral FAs, double immunofluorescence staining of p130Cas and FA component FAK was performed. The p130Cas protein was found to consistently co‐localize with FAK in distinct regions lining the edges of the MYMyopHC403A infected HeLa cells (Figure 4B). Although FAK was present in peripheral FAs before the infection (Figure 4B), it was evident that this protein also accumulated at the same sites as p130Cas in response to MYMyopHC403A exposure. These results suggested that p130Cas and FAK were recruited to peripheral FAs as a response to the MYMyopHC403A infection, and interestingly, this relocation correlated with the infection induced phosphorylation of FAK and p130Cas.
YopH disrupts FAs
The immunoprecipitation experiments showed that YopHC403A was associated with tyrosine phosphorylated forms of p130Cas and FAK, and that these two FA components were dephosphorylated by active YopH. Therefore, we explored the possibility that YopH could have an effect on the integrity of the FAs and the associated cytoskeleton in the target cell. HeLa cells, either not infected or infected for different periods of time with MYM, MYMyopHC403A or MYMyopH, were fixed and permeabilized, thereafter cellular F‐actin was stained with FITC conjugated phalloidin, and the FAs were visualized by indirect immunofluorescence using an antibody recognizing vinculin. Infection with the MYM or MYMpyopHC403A strains had no destructive effects on these structures and gave the same type of staining pattern as in the non‐infected control cells. The microfilament bundles were evenly distributed and the FA marker vinculin decorated the ends of the actin fibres facing the basal lateral side of the cell, even after 50 min of infection (Figure 5). Notably, a 4‐fold increase in the number of FAs was observed after 30 min of infection with these strains (data not shown). When the MYMpyopH strain was used to infect HeLa cells, a different staining pattern was observed. In this case, the F‐actin stress fibres were progressively disrupted with increasing time of infection, and after 50 min these structures were completely absent (Figure 5). The cortical actin, however, could still be seen as ring‐shaped structures lining the margin of the cytotoxically affected cells. Interestingly, the presence of active YopH resulted in a rapid disappearance of FA structures (Figure 5), and it is therefore likely that the YopH mediated change in the morphology of the infected HeLa cells was a consequence of a direct effect of YopH on the integrity of FAs.
The spatial localization of YopH and YopHC403A was investigated after infection of HeLa cells using either MYMpyopH or MYMpyopHC403A strains. YopHC403A was enriched at focal adhesion sites lining the edges of the cells and co‐localized with the FA proteins paxillin and vinculin at these sites (Figure 6). Consistent with the biochemical data, immunofluorescence staining revealed that YopHC403A co‐localized with p130Cas at focal adhesions (Figure 6). The YopHC403A protein was also seen in the cytosol of the infected cells, this phenomenon increased with time and seemed to be a result of ‘overloading’ (Figure 6). By contrast, when the strain expressing active YopH was used to infect HeLa cells, YopH was evenly distributed in the cytosol together with the main pool of FA marker proteins, and no obvious co‐localization between YopH and the FA proteins could be detected (Figure 6). The p130Cas protein was only detected in the cytosol whereas paxillin and vinculin were also found in retraction fibres, distinct from native FA structures (Figure 6).
The observed enrichment of YopHC403A at FAs indicated the target site of this enzyme. Thus, the active form of YopH present in a high local concentration at this intracellular location should be able to compete with and displace YopHC403A. With that in mind, a reinfection experiment was performed in which the HeLa cells were infected with the MYMpyopHC403A strain for 60 min and then infected again but now with the MYMpyopH strain. The active PTPase YopH released YopHC403A from FAs in a time dependent fashion (compare Figure 7 with 1C), after 60 min FAs were disrupted; and consequently, no YopH antigen was found associated with either FAs or with the vinculin and paxillin containing retraction fibres.
Our study focused on one specific interaction between the bacteria and the eukaryotic target cell: the invasin stimulated, β1‐integrin mediated uptake of Y.pseudotuberculosis by HeLa cells. This model system limits the number of potential signalling pathways involved in bacterial uptake, to encompass possibly only one. To explore the virulence determinant YopH without any interference of other Yops, we used a specifically engineered bacterial strain to introduce this PTPase alone into the interior milieu of the target cell. By using this minimalistic approach, we were able to identify two eukaryotic proteins, p130Cas and FAK, that were phosphorylated on tyrosine in response to interaction with the bacteria and that were recognized and dephosphorylated by YopH. Moreover, this activity of YopH resulted in disruption of FA structures and this was associated with impaired ability of the target cell to ingest the pathogen. This conclusion is based on results obtained by using three different experimental approaches: bacterial genetics, biochemistry and confocal microscopy studies. Infection of HeLa cells with a YopH mutant strain resulted in a rapid increase in tyrosine phosphorylation of FAK and p130Cas. This phosphorylation was not observed in the presence of PTPase‐active YopH. In addition, active YopH rapidly dephosphorylated infection induced phosphorylated forms of FAK and p130Cas, and this was observed already 5 min after addition of the YopH expressing strain. Although YopH has a broad substrate specificity upon prolonged incubations (Bliska et al., 1991, 1992; Hartland et al., 1994), these rapid events were only observed for FAK and p130Cas. In accordance, phosphorylated forms of FAK and p130Cas were found to bind to the catalytically inactive mutant form of YopH (YopHC403A). This interaction was observed within 2 min after infection, and no other phosphotyrosine proteins were detected in the immunocomplex. Interestingly, at this very early time point, FAK and p130Cas constituted a minor class of tyrosine phosphorylated proteins in the HeLa cells, indicating a high specificity of YopH for these proteins. Prolonged incubation resulted in elevated amounts of FAK and p130Cas in the immunocomplex, and this paralleled the infection induced phosphorylation of these proteins (see Figures 1 and 2). Moreover, the precipitation of YopH depleted the HeLa cell lysate of the majority of tyrosine phosphorylated FAK and p130Cas, further strengthening the hypothesis that phosphorylated FAK and p130Cas constitute targets for YopH. These biochemical results were confirmed by confocal microscopy, where we could demonstrate that the inactive form of YopH co‐localized with FAK and p130Cas in peripheral FAs in situ. We could also show that PTPase active YopH disrupted FAK and p130Cas containing focal adhesion, paralleling the results obtained from the biochemical results (see Figures 1C and 7).
The uptake of the pathogen into eukaryotic cells is dependent on binding of the bacterial protein invasin to β1‐integrins present on the surface of the target cell (Isberg and Tran van Nhieu, 1994). The interaction between the bacteria and the target cell results in occupancy and clustering of the β1‐integrin receptors, a condition that has been shown to generate a phosphotyrosine signal that is essential for linkage of β1‐integrins to F‐actin via FAs (Miyamoto et al., 1995b). It is likely that bacterial uptake stimulated by invasin binding to β1‐integrins generates an assembly of FA components, similar to the assembly seen in experiments with β1‐integrin ligand coated beads (Plopper and Ingber, 1993; Miyamoto et al., 1995a,b; Plopper et al., 1995). In line with this, the invasin stimulated uptake requires tyrosine kinase activity and is blocked by tyrosine kinase inhibitors (Rosenshine et al., 1992; Andersson et al., 1996). Therefore, it can be assumed that YopH counteracts the phosphotyrosine signal that is involved in the uptake of Yersinia mediated by β1‐integrin receptors.
FAK is located in FAs and it has been implicated in the regulation of these multi‐component structures (Richardson and Parsons, 1995, 1996). We found that infection with strains lacking YopH activity caused an increased tyrosine phosphorylation of FAK and an accumulation of this protein in peripheral FAs. A similar observation was recently presented by Sasakawa and co‐workers who showed that increased tyrosine phosphorylation of FAK was involved in β1‐integrin mediated uptake of Shigella flexneri by CHO cells (Watarai et al., 1996). These two findings suggest a role of FAK in β1‐integrin mediated bacterial uptake. It is known that FAK is tyrosine phosphorylated upon β1‐integrin clustering, and interestingly, in a recent study it was suggested that tyrosine phosphorylation of FAK is important in regulating the formation of new FAs (Richardson and Parsons, 1996). The role of FAK in FA assembly has been questioned, since FAK deficient cells were shown to contain FAs (Ilic et al., 1995). These kinds of results are, however, difficult to interpret due to functional redundancy and the FAK−/− cells did in fact exhibit defects in migration, suggesting a role of FAK in FA remodelling during cell motility (Ilic et al., 1996). Gilmore and Romer (1996) used a GST–C‐terminal fusion of FAK that were loaded into cells and found similar effects on cellular migration. Consequently, although the exact role of FAK in FA turnover is unclear, it is, based on the data presented herein, reasonable to suggest that the observed dephosphorylation of FAK by YopH could interfere with remodelling and/or assembly of FA complexes involved in β1‐integrin mediated internalization of the bacteria.
Phosphorylation of p130Cas causes a relocalization of this protein from the cytosolic fraction to the membrane fraction (Sakai et al., 1994). It is likely that the membrane association reflects the presence of phosphorylated p130Cas at FAs, an assumption supported by the recent findings that p130Cas interacts with the FA component FAK (Polte and Hanks, 1995; Harte et al., 1996). This is further strengthened by our finding that upon infection both FAK and p130Cas were recruited to peripheral FAs that contained YopHC403A. Interestingly, this relocation correlated with the infection induced phosphorylation of both FAK and p130Cas. The pathogen binds to the β1‐integrin exposed at the edges of a cell (Rosqvist et al., 1991; Young et al., 1992), i.e. in the region of peripheral FAs in which p130Cas and FAK were accumulated. Importantly, at early time points the bacteria are in close contact with FAs at the edges of the infected HeLa cells. Enrichment of FA protein‐containing structures in the cell periphery represents FAs under formation (Nobes and Hall, 1995; Richardson and Parsons, 1996), and inhibition of FAK tyrosine phosphorylation results in an impaired formation of such structures (Richardson and Parsons, 1996). Consequently, the observed co‐localization of YopHC403A and the p130Cas and FAK proteins at this site supports our hypothesis that the YopH PTPase exerts its activity in the vicinity of the bacterium and that the site of action is FAs under formation. Moreover, no p130Cas was present in FAs facing the basal lateral side of the cell, and, in contrast to the FA component vinculin, p130Cas did not associate with FAs in uninfected cells. This, and the fact that p130Cas was recruited to this site of action, suggests that this protein plays a specific role in FA formation that is important for the uptake process.
In contrast to YopHC403A, which was enriched in target cell FAs, active YopH was found evenly distributed in the cytosol, HeLa cells exposed to active YopH detached peripherally from the underlying substratum and became round in shape and the F‐actin stress fibres and FAs in these cells were disrupted. Due to the observed interactions of YopHC403A with FA proteins, it is reasonable to believe that FAs are the primary site for the YopH PTPase activity. Active YopH could release YopHC403A from FAs which indicates that active YopH dephosphorylated the molecular targets. The involvement of endogenous PTPases in the regulation of FAs and associated stress fibres has been suggested in several studies. Treatment of cells with PTPase inhibitors has been found to increase the number of FAs and to protect FAs and stress fibres from cytochalasin D mediated disruption (Nobes et al., 1994; Defilippi et al., 1995). In addition, the transmembranous PTPase LAR (leukocyte common antigen related), which is a homologue of YopH, is present in FAs, where it is associated with the proximal (disassembled) ends of the adhesion complex (Serra‐Pagès et al., 1995). This further indicates the involvement of such enzyme activity in regulation of these structures (Serra‐Pagès et al., 1995). Together, all of these findings strongly support a direct effect of YopH on FAs. This is reinforced by our observation that YopH specifically dephosphorylated the FA associated proteins p130Cas and FAK, and it is possible that dephosphorylation of these two proteins leads to disassembly of FAs.
Since the presence of YopH causes inhibition of phagocytic uptake of Yersinia by cells of the non‐specific immune defence (Rosqvist et al., 1988; Fällman et al., 1995; Andersson et al., 1996) it is tempting to speculate about the molecular mechanism that mediates inhibition in this system. The uptake of non‐opsonized Yersinia by phagocytes resembles that in HeLa cells with respect to the requirement of the presence of invasin on the bacterial surface (Fällman et al., 1995). It is therefore likely that YopH mediated dephosphorylation of FAK, p130Cas or related proteins thereof is of importance for phagocytic inhibition. Interestingly, upon exposure to macrophages YopH causes a very rapid dephosphorylation of phosphotyrosine proteins in the 120 kDa range. The majority of the tyrosine phosphorylated proteins are located in the detergent insoluble fraction, suggesting it to be cytoskeletally associated. In accordance with this, a 120 kDa phosphotyrosine protein co‐precipitates with an inactive form of YopH (Bliska et al., 1992) (unpublished data). In addition, YopH also inhibits other kinds of phagocytic uptake like those mediated by complement receptors (β2‐integrin receptors; Ruckdeschel et al., 1996) and Fc receptors (Fällman et al., 1995). Noteworthy in this context is that also these types of receptors mediate phosphorylation of FAK or p130Cas upon stimulation (Haimovich et al., 1996; Petruzelli et al., 1996). It will be of particular importance to further elucidate the role of FAK, p130Cas or related proteins in phagocytic cells.
Material and methods
The following antibodies were used: anti‐Crk mAb (1:2000), anti‐pp120 mAb (1:2000), anti‐paxillin mAb (1:50), anti‐p130Cas mAb (1:20; Transduction Laboratories, Lexington, KY), anti‐Src mAb (1:2000), anti‐FAK mAb 2A7 (Upstate Biotechnology, Lake Placid, NY), anti‐β1‐integrin K20 (1:2000; Immunotech, Marseille‐Cedex, France), anti‐Abl mAb 24‐11(1:2000), anti‐Cbl mAb C15 (1:1000), rabbit polyclonal anti‐FAK antibodies C‐20 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), anti‐vinculin mAb VIN‐11‐5 (1:50; Sigma Chemical Company, St Louis, MO), rabbit polyclonal anti‐p130Cas antibodies (1:2000; H. Hirai) and goat polyclonal anti‐YopH antibodies that recognized YopH and YopHC403A equally well. The YopH antisera were affinity purified as described previously (Persson et al., 1995) and the rabbit polyclonal anti‐FAK antibodies were affinity purified against the GST–FAK fusion protein (amino acids 903–1052; 902; Santa Cruz Biotechnology). The following secondary antibodies were used: FITC conjugated donkey anti‐goat antibodies (1:100), FITC conjugated donkey anti‐rabbit antibodies (1:100), Lissamine conjugated donkey anti‐mouse antibodies (1:100) and Lissamine conjugated donkey anti‐rabbit antibodies (1:100; Jackson Immuno Research Laboratories Inc., West Grove, PA). Horseradish peroxidase conjugated antibodies: recombinant anti‐phosphotyrosine antibody RC20 (1:2500; Transduction Laboratories, Lexington, KY), sheep anti‐mouse or donkey anti‐rabbit antibodies (1:4000; Amersham Sweden AB, Solna, Sweden). The dilutions given were used for Western blotting or immunostaining. To increase the sensitivity of the secondary antibodies used in immunofluorescence staining, the antiseras were run through six cycles of subtractive immunoabsorption to HeLa cells, as described in detail elsewhere (Persson et al., 1995).
The Y.pseudotuberculosis strains and plasmids used in this study are listed in Table I. The plasmids were isolated using standard methods (Sambrook et al., 1989) and introduced into the multiple mutant strain by electroporation (Conchas and Carniel, 1990) using a Gene Pulser apparatus (Bio‐Rad Laboratories, Richmond, CA).
Cultivation and infection of HeLa cells
HeLa cells were cultured in Leibovitz L‐15 medium containing 10% heat inactivated fetal calf serum (FCS) and 100 IU/ml penicillin at 37°C in a humidified atmosphere. For detection of bacterial uptake or immunofluorescent staining, HeLa cells were seeded on sterile cover slips (12 mm; 1×105 cells/cm2) placed in 24‐well tissue culture plates 2 days prior to infection. On day 2, the HeLa cultures were washed free from penicillin, and 1 ml Leibovitz L‐15 medium containing 10% heat‐inactivated FCS was added. The Y.pseudotuberculosis strains used to infect HeLa cells were grown overnight at 26°C in Luria broth (LB) medium. The suspension was diluted 200 times in Leibovitz L–15 medium containing 10% heat inactivated FCS without antibiotics and grown for 1 h at 37°C. Thereafter, 0.1 ml bacteria suspension was added to the HeLa cells (2×106 bacteria/well). To facilitate contact between bacteria and cells, the infected cell cultures were centrifuged for 3 min at 400 g and then incubated at 37°C.
For immunoprecipitation and Western blot experiments, HeLa cells grown to semi‐confluence in 280 cm2 cell culture bottles (1–3×107/cells) were washed twice with 37°C PBS and incubated in penicillin‐free culture medium for 2 h. The cells were infected by replacing the medium with 4 ml bacterial suspension that gave a calculated bacteria:HeLa cell ratio of 100:1.
Detection of bacterial uptake by HeLa cells
HeLa cells grown on cover slips were infected with different strains of Y.pseudotuberculosis, and the intra‐ and extracellular locations of bacteria associated with target cells were distinguished by a double fluorescent antibody test described elsewhere (Heesemann and Laufs, 1985; Rosqvist et al., 1988). The specimens were examined in a fluorescent microscope (Zeiss Axioskop 50) with epifluorescence illumination and a 100/1.4 plan‐apochromate oil immersion lens (final magnification ×1000). In each experiment, 80 HeLa cells were counted in randomly selected fields without previewing. Extracellularly located bacteria were detected with a rhodamine filter system, and the total number of cell associated (intracellular and extracellular) bacteria was determined with a fluorescein filter system.
Cell lysis and immunoprecipitation
Infections were terminated by removing the bacteria, washing the HeLa cells with 37°C phosphate buffered saline (PBS) pH 7.4 and then adding 4 ml ice‐cold immunoprecipitation buffer pH 7.4 (50 mM Tris–HCl, 1% NP‐40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM Na3VO4, 1 mM NaF and Complete™ protease inhibitor cocktail (Boehringer Mannheim Scandinavia AB, Bromma, Sweden). The cells were lysed for 30 min at 4°C and the lysates were clarified by centrifugation at 14 000 g for 10 min. For detection of total cellular phosphotyrosine proteins, aliquots of the clarified lysates were heated in an equal volume of 2× sample buffer for 5 min at 90°C and subjected to Western blot. For immunoprecipitations, the clarified lysates were precleared by incubation for 1 h at 4°C with protein G–Sepharose beads (Pharmacia Biotech Norden, Sollentuna, Sweden) precoupled to preimmune goat IgG or normal mouse IgG. The beads were then pelleted by a brief centrifugation, and the supernatants were incubated for 3 h at 4°C with protein G–Sepharose beads (20 μl beads/ml lysate) precoupled to goat anti‐YopH IgG or anti‐p130Cas mAb or anti‐FAK (mAb 2A7). The immunocomplexes were washed three times in immunoprecipitation buffer, heated in 2× sample buffer for 5 min at 90°C, and subjected to Western blot analysis.
Proteins were separated by 7.5% SDS–PAGE according to Laemmli (1970), and then electrotransferred to Immobilon‐P (Millipore Corp., Bedford, MA). For detection of phosphotyrosine proteins, the membranes were incubated with the horse radish peroxidase conjugated recombinant anti‐phosphotyrosine antibody RC20 as recommended by the manufacturer (Transduction Laboratories). For detection of specific proteins the membranes were blocked in Tris buffered saline containing 5% milk powder and 0.1% Tween 20 and then incubated with primary antibodies. Thereafter the membranes were washed and incubated with horseradish peroxidase conjugated secondary antibodies. The proteins were detected using a ECL chemoluminescence kit (Amersham Sweden AB, Solna, Sweden).
Bacterial infections were terminated by washing the cell monolayers twice in PBS. The cells were fixed in 2% paraformaldehyde, permeabilized with 0.5% Triton X‐100 and further processed for indirect immunofluorescence labelling. To visualize microfilaments and the integrity of FAs in the target cell, the fixed and permeabilized cells were incubated with FITC conjugated Phalloidin (0.5 ng/ml; Sigma Chemical Company, St Louis, MO) for 30 min and then overlaid with the anti‐vinculin mAb for 1 h, followed by 1 h with purified Lissamine conjugated donkey anti‐mouse secondary antibodies. In double labelling experiments, the cells were incubated with primary mAbs against FA proteins and then with the Lissamine conjugated secondary antibodies. The specimens were then incubated with goat anti‐YopH antibodies or anti‐FAK antibodies, and then with the FITC conjugated secondary antibodies. All incubations were performed at 37°C, and the specimens were mounted in a medium containing Cityflour (Cityflour Limited, London, UK).
Fluorescence confocal microscopy and image processing
The fluorescence images of infected HeLa cells were obtained with a confocal laser scanning microscope equipped with dual detectors and an argon‐krypton (Ar/Kr) laser for simultaneous scanning of two fluorochromes (Multiprobe 2001, Molecular Dynamics, Sunnyvale, CA). The same section was scanned simultaneously for Lissamine (excitation 568 nm) and FITC (excitation 488 nm) tagged markers. In each experiment, laser power and gain were set by using cells labelled with either fluorochrome alone, so that there was no crossover of signals from green to red or red to green channels. The sections with the image size of 1024×1024 were scanned with 0.07 μm pixel size, 0.3 μm step size and the pinhole setting was 100 μm. To determine possible co‐localization between FA proteins and YopH/YopHC403A in the infected cell, the images obtained were scrutinized with the ImageSpace™ version 3.10 software (Molecular Dynamics). Pixels with intensity values >100 in both the green and red channels were considered to co‐localize. The co‐localizing pixels were visualized as a light yellow colour on top of the companion sections (red and green channel); the companion sections were overlaid in some of the pictures.
We thank Drs H.Semb, C.Sentman and S.Tuck for helpful discussions and critical reading and P.Ödman for linguistic revision of the manuscript. This work was supported by the Swedish Medical Research Council, the Swedish Natural Science Research Council, the King Gustaf Vth 80 year Foundation, the Swedish Rheumatism Association, the Magnus Bergvalls Foundation and the Medical Faculty Research Foundation at Umeå University. C.P. and N.C. were recipients of grants from the Kempe foundation of the University of Umeå. We thank Dr Hisamaru Hirai for anti‐p130Cas antibodies.
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