TyeA of Yersinia pseudotuberculosis is involved in regulation of Yop expression and is required for polarized translocation of Yop effectors

Authors

  • Lena Sundberg,

    1. Department of Medical Countermeasures, Division of NBC-Defence, Swedish Defence Research Agency, S-901 82 Umeå, Sweden.
    2. Department of Molecular Biology, Umeå University, S-901 87 Umeå, Sweden.
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  • Åke Forsberg

    Corresponding author
    1. Department of Medical Countermeasures, Division of NBC-Defence, Swedish Defence Research Agency, S-901 82 Umeå, Sweden.
    2. Department of Molecular Biology, Umeå University, S-901 87 Umeå, Sweden.
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*For correspondence. E-mail ake.forsberg@ foi.se; Tel. (+46) 90 106660; Fax (+46) 90 106800.

Summary

Type III secretion-dependent translocation of Yop (Yersinia outer proteins) effector proteins into host cells is an essential virulence mechanism common to the pathogenic Yersinia species. One unique feature of this mechanism is the polarized secretion of Yops, i.e. Yops are only secreted at the site of contact with the host cell and not to the surrounding medium. In vitro, secretion occurs in Ca2+-depleted media, a condition believed to somehow mimic cell contact. Three proteins, YopN, LcrG and TyeA have been suggested to control secretion and mutating any of these genes results in constitutive secretion. In addition, in Y. enterocolitica TyeA has been implied to be specifically required for delivery of a subset of Yop effectors into infected cells. In this work we have investigated the role of TyeA in secretion and translocation of Yop effectors by Y. pseudotuberculosis. An in frame deletion mutant of tyeA was found to be temperature-sensitive for growth and this phenotype correlated to a lowered expression of the negative regulatory element LcrQ. In medium containing Ca2+, Yop expression was somewhat elevated compared to the wild-type strain and low levels of Yop secretion was also seen. Somewhat surprisingly, expression and secretion of Yops was lower than for the wild-type strain when the tyeA mutant was grown in Ca2+-depleted medium. Translocation of YopE, YopH, YopJ and YopM into infected HeLa cells was significantly lower in comparison with the isogenic wild-type strain and Yop proteins could also be recovered in the tissue culture medium. This indicated that the tyeA mutant had lost the ability to translocate Yop proteins by a polarized mechanism. In order to exclude that the defect in translocation seen in the tyeA mutant was a result of lowered expression/secretion of Yops, a double lcrQ/tyeA mutant was constructed. This strain was de-repressed for Yop expression and secretion but was still impaired for translocation of both YopE and YopM. In addition, the low level of YopE translocation in the tyeA mutant was independent of the YopE chaperone YerA/SycE. TyeA was found to localize to the cytoplasm of the bacterium and we were unable to find any evidence that TyeA was secreted or surface located. From our studies in Y. pseudotuberculosis we conclude that TyeA is involved in regulation of Yop expression and required for polarized delivery of Yop effectors in general and is not as suggested in Y. enterocolitica directly required for translocation of a subset of Yop effectors.

Introduction

Type III secretion systems (TTSS) are common to many Gram-negative pathogens and constitute an important virulence mechanism in several of these pathogens (Schesser et al., 2000) as well as for flagellar secretion and assembly (Aizawa, 2001). For the pathogenic Yersinia spp. the TTSS is responsible for translocation of virulence effectors denoted Yops (Yersinia outer proteins). These Yop effectors have been shown to mediate resistance to phagocytosis (Rosqvist et al., 1988; 1990) as well as prevention of an inflammatory response (Palmer et al., 1998; Schesser et al., 1998). It was early established that TTS is essential for virulence in a systemic mouse infection model (Straley and Bowmer, 1986; Bölin et al., 1988; Forsberg and Wolf-Watz, 1988; Mulder et al., 1989). This finding is in agreement with the observation that Yersinia predominantly replicates extracellulary in animal infection models (Hanski et al., 1989; Simonet et al., 1990).

The TTSS of Yersinia is encoded by a virulence plasmid common to the three pathogenic species, Y. pestis, Y. pseudotuberculosis and Y. enterocolitica (Portnoy et al., 1984). More than 20 proteins encoded by ysc (Yop secretion) genes constitute the secretion system (Cornelis et al., 1998; Perry et al., 1998; Deng et al., 2002; Parkhill et al., 2002) required for secretion across the bacterial membranes. In S. typhimurium (Kubori et al., 1998) and S. flexneri (Tamano et al., 2000) the secretion system has been visualized as an organelle resembling the basal body of flagella with a needle-like structure replacing the hook and flagellar filament.

To accomplish delivery of Yop effectors across the eukaryotic cell membrane at least three of the secreted proteins, YopB, YopD and LcrV, known as translocators are required (Rosqvist et al., 1994; Sory and Cornelis, 1994; Håkansson et al., 1996; Fields et al., 1999; Holmström et al., 2001). The presence of these proteins also correlates to lytic activity on infected erythrocytes, epithelial cells and macrophages. Therefore, it has been suggested that translocation of proteins through the TTSS conduit is linked to a pore in the target cell membrane (Håkansson et al., 1996; Neyt and Cornelis, 1999; Tardy et al., 1999; Holmström et al., 2001). The process of translocation is contact dependent and occurs without secretion to the surrounding medium during infection of eukaryotic cells (Rosqvist et al., 1994). Translocation has therefore been described as polarized, i.e. secretion takes place only at the zone of contact between the pathogen and the host cell. The inducing signal for both expression and secretion of Yops in vitro is 37°C and depletion of calcium from the growth media (Straley and Bowmer, 1986; Forsberg et al., 1987) which is believed to somehow mimic the cell contact in vivo (Pettersson et al., 1996). Three proteins, YopN, LcrG and TyeA, are involved in preventing secretion in vitro in Ca2+-containing media and to the surrounding culture media during infection of eukaryotic cells. Strains mutated for any of these genes display de-repressed Yop expression and secretion in vitro, i.e. high levels of expression and secretion are seen irrespective of the Ca2+ concentration (Forsberg et al., 1991; Nilles et al., 1997; Day and Plano, 1998; Iriarte et al., 1998; Cheng and Schneewind, 2000; Matson and Nilles, 2001). Infection of eukaryotic cells with yopN, lcrG or tyeA mutants result in significant leakage of Yops also to the culture medium, which means that secretion is no longer polarized (Cheng and Schneewind, 2000; Matson and Nilles, 2001). YopN is secreted via the TTSS and has been suggested to somehow sense cell contact, and serve as an outer gate, promoting opening of the secretion channel after interacting with the host cell (Forsberg et al., 1991). However, to date no direct evidence for the interaction of YopN with any host surface component has been obtained. In one study, LcrG of Y. enterocolitica has been suggested to have an extracellular role and bind to proteoglycans on host cells (Boyd et al., 1998). However, because of its mainly intracellular localization LcrG has been proposed to act as an inner gate of the TTSS and has a profound influence on Yop expression acting as a negative element. The secretion block has been postulated to be relieved when the levels of LcrV increases to lower the amount of free LcrG (Nilles et al., 1997; Matson and Nilles, 2001).

The actual delivery of Yop effectors across the host cell membrane does not appear to directly require neither YopN nor LcrG (Fields et al., 1999; Pettersson et al., 1999; Matson and Nilles, 2001). The lower levels of translocation rather seem to be the result of leakage of Yop proteins to the surrounding medium. For TyeA however, the situation is somewhat different. By using cya-fusions to different yop genes in Y. enterocolitica Cornelis and co-workers presented evidence that TyeA was required for translocation of YopE and YopH but not for delivery of YopM, YopO (YpkA), YopP (YopJ) and YopT (Iriarte et al., 1998; Iriarte and Cornelis, 1998). These investigators also concluded that TyeA similar to YopN localized to the bacterial surface, which was in agreement with the suggested role in translocation of a subset of Yop effectors. Using a different experimental approach, Olaf Schneewind's group has also addressed the role of TyeA in Yop effector translocation by Y. enterocolitica. Similar to Iriarte et al. (1998) they observed constitutive secretion of Yops in Ca2+-containing media and they also found leakage of Yops to the surrounding medium of infected cells. They, however, concluded that the tyeA mutant was still capable to deliver all effector Yops including YopE and YopH across the host cell membrane. This indicates a role for TyeA in polarization of Yop translocation rather than being directly involved and absolutely required for translocation of a subset of Yops. The latter investigators were also unable to verify the surface localization of TyeA and concluded that TyeA was an intracellular protein acting as a negative regulator of the TTS pathway (Cheng and Schneewind, 2000).

The somewhat contradictory results obtained regarding the role of TyeA in the TTSS of Y. enterocolitica prompted us to investigate TyeA in Y. pseudotuberculosis, as this had not been addressed previously. We employed YopE-mediated cytotoxicity, YopJ-mediated killing of macrophages and immunofluorescence to monitor intracellular delivery of several Yop proteins. We found that TyeA is mainly an intracellular protein involved in regulating Yop expression and that TyeA is required for polarized Yop effector translocation. However, the actual translocation of Yop proteins across the host cell membrane does not appear to require TyeA.

Results

TyeA is involved in regulation of Yop expression and secretion

To facilitate studies of TyeA in Y. pseudotuberculosis we constructed an in frame deletion of the region encoding amino acids 19–59 of TyeA and the resulting strain was denoted YPIII(pIB801). The tyeA mutant strain was found to be temperature sensitive (TS) for growth, i.e. unable to grow at 37°C irrespective of the Ca2+-concentration. The tyeA mutant strain was grown in different media and Yop expression was analysed by Western blot. Interestingly, the tyeA mutant showed lower expression (data not shown) and secretion (Fig. 1) of Yops compared to the wild-type strain in medium lacking Ca2+ whereas in medium containing 2.5 mM Ca2+ expression was somewhat higher than for the wild-type (data not shown) strain. In the latter case low levels of Yop secretion were also observed (Fig. 1). Trans-complementation of the tyeA mutant with a clone expressing TyeA under the control of the IPTG inducible tac-promoter restored the growth phenotype (data not shown) and Yop expression/secretion levels to those of the isogenic wild-type strain (Fig. 1). Thus, the phenotype of the tyeA mutant in Y. pseudotuberculosis is somewhat different from the mutants described in Y. enterocolitica where Yop expression as well as secretion was fully de-repressed at 37°C irrespective of the Ca2+-concentration (Iriarte et al., 1998; Cheng and Schneewind, 2000). TyeA has been shown to interact with YopN but similar to what was found in previous studies TyeA had no major influence on YopN secretion per se (data not shown, Iriarte et al., 1998; Cheng and Schneewind, 2000).

Figure 1.

Yop secretion is impaired in a tyeA mutant. The different strains were grown in medium containing or lacking Ca2+ at 26°C for 1 h and then for 3 h at 37°C. The bacteria were harvested by centrifugation and the culture supernatants were TCA precipitated, samples were separated by SDS-PAGE and subjected to ECL Western blot using anti-total Yop antibodies.

TyeA localizes to the bacterial cytoplasm.

Previous work in Y. enterocolitica has given somewhat contradictory results regarding the subcellular localization of TyeA (Iriarte et al., 1998; Cheng and Schneewind, 2000). We initially attempted to analyse the localization of TyeA using antibodies raised against a GST-TyeA fusion protein but this failed because of the low affinity of these antibodies to TyeA (data not shown). We therefore used another strategy where TyeA was tagged in the C-terminal end with the FlagTM-epitope. Plasmid pLS69 expressing TyeA-Flag was introduced into both the wild-type strain YPIII(pIB102) and the tyeA mutant strain YPIII(pIB801) and expression of Yop proteins and TyeA was monitored using anti-Yop antiserum and a monoclonal antibody directed against the Flag epitope respectively. The construct expressing TyeA-Flag complemented the tyeA mutant equally well as the construct expressing unmodified TyeA with respect to growth phenotype as well as Yop expression and secretion (data not shown). No TyeA could however, be detected in the culture supernatants of the strains expressing TyeA-Flag (Fig. 2A). In order to investigate if TyeA-Flag was surface located the bacterial samples were extracted with Xylene as described previously (Iriarte et al., 1998). However, no TyeA-Flag could be detected in the Xylene-extracts of the bacterial pellets (Fig. 2B).

Figure 2.

TyeA localizes inside the bacteria. Expression of TyeA-Flag was induced at 37°C by addition of IPTG during 2 h in Ca2+-depleted medium.

A. Bacterial pellets were harvested by centrifugation and dissolved in SDS-sample buffer and the culture supernatants were TCA- precipitated. Bacterial and supernatant samples were separated by SDS-PAGE and subjected to ECL Western blot using Flag-antibodies.

B. Bacteria were treated with p-Xylene for 5 min and bacterial pellets and Xylene-fractions were collected, separated by SDS-PAGE and subjected to ECL Western blot using anti-Flag antibodies.

From this we conclude that the majority of the biologically active TyeA-Flag protein resides in the cytoplasm of the bacterium.

The tyeA mutant shows reduced LcrQ expression

The tyeA mutants as well as lcrG and yopN mutants show a temperature sensitive (TS) phenotype, i.e. growth is restricted at 37°C irrespective of the Ca2+-concentration of the medium. As YopN expression and secretion is not affected in the tyeA mutant the growth phenotype of the tyeA is probably not due to an indirect effect on YopN. One major negative regulator of Yop expression is LcrQ (Rimpiläinen et al., 1992), which during low Ca2+-conditions is rapidly exported to allow induction of the yop regulon (Pettersson et al., 1996). Because lcrQ mutants are TS we decided to analyse LcrQ expression in the tyeA mutant. First the total amount of LcrQ expressed (both bacteria associated and secreted) was analysed and interestingly we found that LcrQ expression was much lower in the tyeA mutant compared to the wild-type strain (Fig. 3A). Trans-complementation of the mutant with TyeA, expressed under the control of the inducible tac-promoter, restored LcrQ expression levels to those of the wild-type strain YPIII(pIB102). Next the localization of LcrQ in the tyeA mutant was analysed and compared to the wild-type strain and a yopN mutant. As previously shown, LcrQ was intracellular when the wild-type strain was grown in Ca2+-containing medium (Fig. 3B), whereas secretion of LcrQ was essentially complete during growth in Ca2+-depleted media (Fig. 3C). For the yopN mutant strain, total levels of LcrQ appeared to be similar to those of the wild-type strain, however, in this case extensive secretion of LcrQ was seen irrespective of the Ca2+-concentration (Fig. 3C). In the tyeA mutant the low levels of LcrQ appeared to be equally distributed between the bacteria and the culture supernatant both in the presence and in the absence of Ca2+ (Fig. 3B and C).

Figure 3.

A tyeA mutant shows lowered LcrQ expression. The different strains were grown in medium containing or lacking Ca2+ for 3 h at 37°C and 100 µl of the total cultures was dissolved in 100 µl SDS-sample buffer. The bacteria were harvested by centrifugation and dissolved in SDS-sample buffer whereas to the culture supernatants 1 volume of SDS-sample buffer was added. Samples were separated by SDS-PAGE and subjected to ECL Western blot using anti-LcrQ antibodies. A; whole cultures, B; bacterial fractions, C; culture supernatants.

Thus, it is possible that the intermediate phenotype, with respect to Yop expression of the tyeA mutant, is the result of the low levels of LcrQ present inside the bacteria under these growth conditions and that LcrQ secretion is incomplete. Our findings regarding the low overall expression of LcrQ can explain the TS phenotype of the tyeA mutant and also indicate that there may be a link between TyeA and LcrQ in the complex regulation of Yop expression.

Loss of TyeA results in non-polarized secretion during infection of HeLa cells

Previous studies of TyeA in Y. enterocolitica showed that loss of TyeA resulted in Yop secretion to the culture medium during infection of host cells (Cheng and Schneewind, 2000). Therefore, we wished to verify if this was also the case for the tyeA mutant of Y. pseudotuberculosis. When the culture medium of HeLa cells infected with different strains of Y. pseudotuberculosis was analysed we could see that the wild-type strain YPIII(pIB102) did not secrete detectable amounts of Yop proteins to the culture medium (Fig. 4). As expected a yopN mutant secreted significant amounts of Yops to the culture medium. The tyeA mutant also secreted Yop proteins to the culture medium, but in this case the amounts secreted were significantly lower for all the Yops investigated (Fig. 4). Trans-complementation of the tyeA mutant with plasmid pLS23 fully restored the phenotype to that of the wild-type strain, i.e. no secretion of Yops to the culture medium. Thus, both in vitro and during cell infections the tyeA mutant of Y. pseudotuberculosis secretes Yop proteins during normally non-permissive conditions. However, the level of secretion is significantly lower compared to what was shown previously for tyeA mutants of Y. enterocolitica where the levels were comparable to those of a yopN mutant (Iriarte et al., 1998; Cheng and Schneewind, 2000).

Figure 4.

A tyeA mutant is defective in polarized Yop secretion during infection of HeLa cells. The different bacterial strains were used to infect HeLa cells for 3 h. The tissue culture medium was collected and the culture supernatant fractions were dissolved in SDS-sample buffer after harvesting of the bacteria by centrifugation. Samples were separated by SDS-PAGE and subjected to ECL Western blot using different Yop antibodies.

The tyeA mutant is delayed for cytotoxicity on infected HeLa cells

In previous work the levels of intracellular delivery by a tyeA mutant of Y. enterocolitica were monitored using either Cya-fusions to Yop proteins or by a fractionation assay (Iriarte et al., 1998; Cheng and Schneewind, 2000). As these methods gave contradictory results we decided to monitor the ability of the tyeA mutant to induce a cytotoxic response on infected HeLa cells. Cytotoxicity is mediated by YopE, a cytotoxin that inactivates RhoGTPases via a RhoGAP activity (Black and Bliska, 2000; Von Pawel-Rammingen et al., 2000) leading to actin microfilament rearrangements. This activity results in a characteristic rounding and subsequent detachment of the HeLa cells, which can be monitored by microscopy (Rosqvist et al., 1991; 1994). The wild-type strain showed a full cytotoxic effect on the HeLa cells after two hours of infection, whereas the yopN mutant was somewhat delayed with HeLa cell rounding seen after three hours of infection (Fig. 5A). Interestingly, the tyeA mutant also caused a cytotoxic response on the infected cells, which became evident after about four hours of infection (Fig. 5A). Complementation of the tyeA mutant with plasmid pLS23 fully restored cytotoxicity to the level of the wild-type strain. This indicates that the tyeA mutant is able to translocate YopE into infected host cells but the delayed cytotoxicity shows that the intracellular delivery of YopE is inefficient. The results were also verified by immunofluorescence staining of the infected HeLa cells using anti-YopE antiserum. Significant but low levels of YopE staining could be seen in cells infected with the tyeA mutant, in contrast to the yopD mutant where no intracellular staining for YopE could be detected (Fig. 5B). After prolonged infection, for four and a half hours, when the cytotoxic response was evident the levels of YopE staining were higher also in the tyeA mutant. As expected from the cytotoxicity assay cells infected with the yopN mutant or the wild-type strain showed higher levels of YopE staining compared to the tyeA mutant (Fig. 5B).

Figure 5.

The tyeA mutant shows a delayed cytotoxicity on infected HeLa cells. HeLa cells were infected with different strains of Y. pseudotuberculosis as described in Experimental procedures and the morphology of the cells was monitored by microscopy.

A. Phase-contrast microscopy after 3 h of infection (unless otherwise indicated).

B. Immunofluorescence staining of infected HeLa cells using anti-YopE antibodies after 3 h of infection (unless otherwise indicated).

Thus, a tyeA mutant displays lower levels of non-polarized secretion compared to a yopN mutant but is still able to translocate low levels of YopE into HeLa cells.

A tyeA mutant is highly impaired in phagocytosis resistance

YopE and YopH act in concert to prevent bacterial uptake by macrophages (Rosqvist et al., 1988; 1991). In the initial characterization of tyeA in Y. enterocolitica it was concluded that TyeA was specifically required for intracellular delivery of both YopE and YopH (Iriarte et al., 1998). As YopH is a major antiphagocytic protein in Y. pseudotuberculosis (Bölin et al., 1988) we decided to study the ability of the tyeA mutant to prevent uptake by J774 cells. For the wild-type strain YPIII(pIB102) 85 ± 4.3%[±1 SD (standard deviation)] of the cell associated bacteria were extracellular while only 30 ± 2.7% (±1 SD) of the tyeA mutants strain remained extracellular. This number is only slightly higher than what was found for a double yopE/yopH mutant for which 20 ± 5.4% (±1 SD) of the infecting bacteria were found to be extracellular. Trans-complementation of the tyeA mutant with plasmid pLS23 restored phagocytosis resistance levels with 80 ± 2.5% (±1 SD) of the bacteria remaining extracellular.

From these experiments where the biological activity of both YopE and YopH is measured it seems that loss of TyeA in Y. pseudotuberculosis results in impaired delivery of both YopE and YopH supporting the initial findings of Cornelis and co-workers (Iriarte et al., 1998).

The tyeA mutant mediates a delayed YopJ mediated killing of infected J774 cells

YopE and YopH have in common that secretion is facilitated by chaperones specific for the individual Yop. In order to elucidate the role of TyeA in translocation of Yop effectors in Y. pseudotuberculosis it was therefore important to include Yops, which are known not to depend on specific chaperons for secretion, like YopJ (YopP in Y. enterocolitica) and YopM. YopJ has been shown to induce cell death of infected macrophages by an apoptotic mechanism (Monack et al., 1997). To investigate if the tyeA mutant in Y. pseudotuberculosis was affected in the translocation of YopJ, we infected macrophages for five hours and determined the viability of the infected J774 cells. The tyeA mutant induced lower levels of killing of J774 cells compared to the wild-type strain and the trans-complemented strain. In contrast, the yopJ mutant strain YPIII(pIB232) and the translocation mutant (yopD) YPIII(pIB621) induced only low levels of cell death and were comparable to the uninfected control (Fig. 6). If the infection time was prolonged to eight hours both the tyeA mutant strain and the isogenic wild-type strain induced cell death in essentially 100% of the infected cells (data not shown).

Figure 6.

The tyeA mutant induces cell death in infected macrophages. The macrophage-like cell line J774 was infected with different strains of Y. pseudotuberculosis for 8 h. The relative numbers of living and dead cells were recorded by staining with calcein and EtBr respectively. The fraction of dead cells is indicated for cells infected with the respective strain.

The tyeA mutant is impaired for translocation of YopM

YopM is another effector, which does not appear to require a chaperone for secretion and translocation. We therefore wanted to investigate the role of TyeA in intracellular targeting of YopM by Y. pseudotuberculosis. In order to facilitate intracellular identification of YopM a plasmid encoding YopM under the control of its native promoter (pBK43) was introduced in different mutants of Y. pseudotuberculosis. Somewhat surprisingly, translocation of YopM was highly impaired in the tyeA mutant and intracellular levels of YopM were barely detectable after four hours of infection, whereas the intercellular levels of YopM in the yopN mutant and the wild-type strain were high (Fig. 7).

Figure 7.

The tyeA mutant is defective for translocation of YopM into HeLa cells. HeLa cells were infected with different strains of Y. pseudotuberculosis expressing YopM from pBK43 for 4 h. YopM was detected by immunofluorescence using an affinity purified specific YopM antibody.

Therefore, it appears that translocation of all the Yop effectors investigated was impaired in the tyeA mutant of Y. pseudotuberculosis.

Translocation of YopE is chaperone independent in the tyeA mutant

Efficient secretion/translocation of YopE requires the specific chaperone YerA (SycE) (Wattiau and Cornelis, 1993; Frithz-Lindsten et al., 1995). In order to analyse the relative importance of YerA and TyeA in translocation of YopE a double yerA/tyeA mutant of Y. pseudotuberculosis, YPIII(803), was constructed. Interestingly, Yop secretion levels of the yerA/tyeA mutant were higher compared to the tyeA mutant and similar to the levels of the wild-type strain (Fig. 8A). In contrast to the wild-type strain the yerA/tyeA mutant strain secreted high amounts of Yops also when grown in the presence of Ca2+ (Fig. 8A). The level of YopE secretion was however, lower compared to the wild-type strain as expected for a strain lacking YerA. Trans-complementation of the yerA/tyeA mutant YPIII(803) with plasmid pLS63 expressing TyeA resulted in a strain with similar Yop secretion profile as the wild-type strain YPIII(pIB102) for all Yops, except YopE, i.e. no secretion in the presence of Ca2+ and high levels of secretion of Yops in medium lacking Ca2+ (Fig. 8A). The yerA/tyeA mutant was also analysed for secretion into the culture medium during infection of HeLa cells and was found to maintain the phenotype of the tyeA mutant showing non-polarized Yop secretion. Secretion of YopE to the culture medium was found to be slightly lower compared to that of the tyeA mutant strain, whereas the release of YopM to the culture medium was higher (Fig. 8B). The yerA/tyeA mutant was found to induce a delayed cytotoxic effect on infected HeLa cells and rounding of the cells was evident after around 4 h of infection, which is similar to the effect caused by the tyeA as well as the yerA mutant (data not shown).

Figure 8.

Figure 8.

Secretion of Yop proteins is increased in a yerA/tyeA mutant.

A. The different strains were grown in medium containing or lacking Ca2+ at 26°C for one hour and then for 3 h at 37°C. The bacteria were harvested by centrifugation and the culture supernatants were TCA precipitated and the samples were separated by SDS-PAGE and subjected to ECL Western blot using anti-total Yop antibodies.

B. The different bacterial strains were used to infect HeLa cells for 3 h. The tissue culture medium was collected and the culture supernatant fractions were dissolved in SDS-sample buffer after harvesting of the bacteria by centrifugation. Samples were separated by SDS-PAGE and subjected to ECL Western blot using different Yop antibodies.

Figure 8.

Figure 8.

Secretion of Yop proteins is increased in a yerA/tyeA mutant.

A. The different strains were grown in medium containing or lacking Ca2+ at 26°C for one hour and then for 3 h at 37°C. The bacteria were harvested by centrifugation and the culture supernatants were TCA precipitated and the samples were separated by SDS-PAGE and subjected to ECL Western blot using anti-total Yop antibodies.

B. The different bacterial strains were used to infect HeLa cells for 3 h. The tissue culture medium was collected and the culture supernatant fractions were dissolved in SDS-sample buffer after harvesting of the bacteria by centrifugation. Samples were separated by SDS-PAGE and subjected to ECL Western blot using different Yop antibodies.

From this we can conclude that the low levels of YopE translocation in the tyeA mutant appear to be independent of its chaperone YerA.

An lcrQ/tyeA mutant is fully secretion competent but non-polarized for Yop translocation

The tyeA mutant of Y. pseudotuberculosis, in contrast to Y. enterocolitica (Iriarte et al., 1998; Cheng and Schneewind, 2000) displayed lower secretion and expression of Yops during inducing conditions in vitro. Therefore, it was not possible to exclude that the defect in translocation seen in the tyeA mutant mainly was a consequence of the lowered Yop expression/secretion. In order to uncouple Yop expression and secretion in the tyeA mutant a double lcrQ/tyeA mutant denoted YPIII(pIB804) was constructed. As expected the level of Yop secretion was high irrespective of the Ca2+ concentration of the culture medium and the level of secretion was comparable to that of the wild-type strain during full induction in Ca2+-depleted media (Fig. 9A). Complementation of the lcrQ/tyeA mutant with the plasmid pLS63 expressing TyeA resulted in much lowered Yop secretion when this strain was grown in the presence of Ca2+ and the phenotype of this strain was similar to that of the lcrQ mutant (Fig. 9A).

Figure 9.

Figure 9.

Yop secretion is elevated in a double lcrQtyeA mutant.

A. The different strains were grown in medium containing or lacking Ca2+ at 26°C for 1 h and then for 3 h at 37°C. The bacteria were harvested by centrifugation and the culture supernatants were TCA precipitated, samples were separated by SDS-PAGE and subjected to ECL Western blot using anti-total Yop antibodies.

B. The different bacterial strains were used to infect HeLa cells for 3 h. The tissue culture medium was collected and the culture supernatant fractions were dissolved in SDS-sample buffer after harvesting of the bacteria by centrifugation. Samples were separated by SDS-PAGE and subjected to ECL Western blot using different Yop antibodies.

Figure 9.

Figure 9.

Yop secretion is elevated in a double lcrQtyeA mutant.

A. The different strains were grown in medium containing or lacking Ca2+ at 26°C for 1 h and then for 3 h at 37°C. The bacteria were harvested by centrifugation and the culture supernatants were TCA precipitated, samples were separated by SDS-PAGE and subjected to ECL Western blot using anti-total Yop antibodies.

B. The different bacterial strains were used to infect HeLa cells for 3 h. The tissue culture medium was collected and the culture supernatant fractions were dissolved in SDS-sample buffer after harvesting of the bacteria by centrifugation. Samples were separated by SDS-PAGE and subjected to ECL Western blot using different Yop antibodies.

Next, the lcrQ/tyeA mutant was analysed for secretion of Yop proteins during infection of HeLa cells. Similar to the tyeA mutant the lcrQ/tyeA mutant also secreted YopE and YopM to the tissue culture medium (Fig. 9B) whereas the lcrQ mutant similar to the wild type did not secrete Yops to the medium (data not shown). Complementation of both mutants with pLS63 expressing TyeA resulted in blockage of secretion to the culture medium (Fig. 9B).

Finally, the translocation efficiency regarding YopE and YopM of the lcrQ/tyeA mutant was investigated by the HeLa cell infection assay. Intracellular delivery of YopE was first analysed by monitoring the effect on cell morphology induced by the different infecting strains. The lcrQ/tyeA mutant induced a more rapid rounding up of the HeLa cells compared to the tyeA mutant and the effect was almost complete after three hours of infection compared to four and a half hours for the single tyeA mutant (Fig. 10A). The wild-type strain, the lcrQ mutant strain and the lcrQ/tyeA mutant trans-complemented with pLS63 all induced a cytotoxic response after about two hours of infection, which is similar to the wild-type strain (Fig. 10A). YopE translocation was also analysed by immunofluorescence staining of intracellulary located YopE after four hours of infection. The results were essentially the same as those obtained by monitoring the cytotoxic effect. The lcrQ/tyeA mutant showed lower levels of staining compared to the wild-type strain, the lcrQ mutant strain and the pLS63 trans-complemented lcrQ/tyeA strain. The amount of intracellular YopE was however, significantly higher in the lcrQ/tyeA strain compared to the tyeA mutant (Fig. 10B). Intracellular levels of YopM were also analysed for HeLa cells infected four hours with strains expressing YopM from pBK43. Cells infected with the wild-type strain and the lcrQ mutant strain showed the most intense staining for YopM whereas only low amounts of YopM were seen inside cells infected with the tyeA mutant strain (Fig. 11). The lcrQ/tyeA strain translocated more YopM into the HeLa cells compared to the tyeA mutant strain but the amount of intracellular YopM was significantly lower compared to the amount inside cells infected with either the wild-type strain or the lcrQ mutant strain.

Figure 10.

The lcrQ/tyeA mutant is delayed for cytotoxicity on infected HeLa cells.

A. HeLa cells were infected with pre-induced Yersinia and the morphology of the cells was followed by microscopy. The figure displays the morphology after 3 h of infection.

B. Immunofluorescence staining of infected HeLa cells using anti-YopE antibodies after 4 h of infection.

Figure 11.

Translocation of YopM is impaired in the double lcrQ/tyeA mutant. HeLa cells were infected with different strains of Y. pseudotuberculosis expressing YopM from pBK43 for 4 h. YopM was detected by immunofluorescence using an affinity purified specific YopM antibody.

From this we conclude that TyeA appears to be involved in regulation of Yop expression and secretion possibly via LcrQ. Similar to YopN and LcrG, TyeA is required for polarized delivery of Yop effectors into infected host cells. This highlights the importance and the efficiency of the mechanism by which Yop effectors are targeted into host cells. The loss of polarized secretion and delivery is most likely the main reason for the lowered translocation seen in the absence of TyeA.

Discussion

The virulence plasmid encoded TTSS of pathogenic Yersinia spp. constitutes a major virulence mechanism that enables these pathogens to resist phagocytosis and prevent an early inflammatory response. A number of proteins have structural roles in the secretion system to promote secretion across the two bacterial membranes or are part of what can be described as the ‘translocon’ which function in the actual translocation of Yop effectors across the host cell membrane.

TyeA, which was first identified as a surface located protein in Y. enterocolitica was suggested to be part of this translocon, with a specific role in translocation for only a subset of Yop effectors, YopE and YopH, while intracellular targeting of the other effectors YopM, YopO (YpkA), YopP (YopJ) and YopT was TyeA independent (Iriarte et al., 1998). In a subsequent study, Cheng and Schneewind (2000) presented evidence that TyeA of Y. enterocolitica was not required for translocation of any of the effector Yops per se. They observed that levels of intracellular targeting of effector proteins were lower for a tyeA mutant and suggested that this was caused by uncontrolled secretion of Yops into the surrounding medium. In addition, Cheng and Schneewind were unable to show that TyeA localizes to the bacterial surface and concluded that TyeA was mainly found in the cytoplasm, a finding that is compatible with a role for TyeA in regulation and secretion control.

In this work we have investigated the role of TyeA in expression/secretion and translocation of Yop effectors by Y. pseudotuberculosis. In agreement with previous studies in Y. enterocolitica and Y. pestis we found that TyeA is involved in regulating Yop secretion (Day and Plano, 1998; Iriarte et al., 1998; Cheng and Schneewind, 2000). Loss of TyeA results in uncontrolled secretion into the surrounding medium during infection of cells, which during in vitro growth is seen as secretion irrespective of the Ca2+ concentration of the medium. We also show that translocation of Yop effectors per se appears to be TyeA independent and that the lower levels of intracellular targeting seen in tyeA mutants is mainly the result of the lack of polarized translocation that is associated with these mutants.

The role of TyeA in translocation of Yop effectors has been studied in Y. enterocolitica previously with somewhat conflicting results as discussed above. The obvious question in relation to the previously published work is the reason behind these apparently contradictory results. The major difference between the studies is the methods employed to study intracellular delivery of the Yersinia effector proteins. In this work we have used two different methods to verify intracellular localization of Yops. One method relies on the biological activity of some of the Yop proteins. YopE, YopH and YopJ all have activities, which give a distinct effect on the host cells once translocated. We also employed immunofluorescence staining of infected cells to analyse intracellular delivery of Yop effectors. Here it is essential to perform the cell infections in the presence of cytochalasin D to ensure that the infecting bacteria remain extracellular as internalized bacteria, which are secretion competent, could contribute to the intracellular staining via surface located Yops. Where it was possible to use both methods, to study delivery of an individual Yop effector, the results regarding intracellular delivery were the same for both methods. Our results are in agreement with those of Cheng and Schneewind (2000). In their study a fractionation method involving detergent lysis of the infected cells to distinguish soluble (intracellular) and insoluble proteins (bacteria-associated) was used. This method has been shown to be prone to give false positive results regarding intracellular delivery unless a protease treatment step is included to remove surface located proteins on bacteria adhering to the cell surface prior to the fractionation (Nordfelth and Wolf-Watz, 2001). Therefore, the fractionation assay as conducted in the study of Cheng and Schneewind is not alone sufficient to verify intracellular delivery of Yop effectors by the TyeA mutant. In the same study the intracellular targeting of YopE was also investigated by immunofluorescence. However, the experiments were not stated to be conducted in the presence of cytochalasin D to prevent bacterial uptake. Because of impaired delivery of YopE the tyeA mutant, in contrast to the wild-type strain, is likely to be mainly intracellular. It is possible that there is a contribution of YopE staining, from internalized bacteria expressing YopE on the surface prior to infection of the HeLa cells, and that this staining appears as intracellular in the immunofluorescence analysis.

Iriarte et al. (1998) used an elegant method based on cya-fusions to Yop effector genes where the intracellular adenylate cyclase activity is used as a measure of the intracellular delivery. This method gave results, which suggested that neither YopE, nor YopH was delivered into the host cells cytosol as the level of intracellular adenylate cyclase activity for the tyeA mutant was as low as for the translocation mutant yopB. Similarly, using cya-fusions, an lcrG mutant of Y. enterocolitica also appeared negative for translocation of Yops (Sarker et al., 1998). However, other studies have revealed that the levels of Yop translocation in lcrG mutants are similar to those of yopN mutants (Fields et al., 1999; DeBord et al., 2001; Matson and Nilles, 2001; our own unpublished results). Therefore, it is possible that using cya-fusions to monitor Yop translocation in some cases strongly under-estimates the level of translocation. Iriarte et al. (1998) also used a cell infection assay where macrophages were infected with a strain over-expressing YopE and found that after 3 h of infection YopE could not be detected inside macrophages infected with the tyeA mutant strain. It is likely that a prolonged infection had been required to allow identification of intracellulary localized YopE. In contrast to what was found for YopE and YopH, the study of Iriarte et al. (1998) revealed that the other Yop effectors, which are normally delivered in lower amounts, were TyeA independent for translocation. This is somewhat unexpected as the tyeA mutant was found to release also these Yop proteins to the culture medium and one would expect this to lower the translocation levels for these Yops. One difference in these experiments was that they were mainly conducted in a yop poly mutant derivative of Y. enterocolitica, which secrete and deliver high amounts of individual Yop proteins expressed from high copy number plasmids but have also recently been shown to have a significant lytic activity on Y. pseudotuberculosis infected macrophages (Viboud and Bliska, 2001). It is possible that this could result in overestimation of intracellular delivery when using adenylate cyclase fusions to monitor translocation. However, it cannot be excluded that there are some, differences in how Yop effectors are delivered in the different Yersinia species.

Loss of TyeA results in a temperature sensitive phenotype in Y. enterocolitica and Y. pestis and this phenotype is also associated with high levels of Yop expression and secretion in vitro, both in presence and absence of Ca2+. The levels of Yop expression and secretion seen are similar to that of the wild-type strain during induction in Ca2+-depleted media (Day and Plano, 1998; Iriarte et al., 1998; Cheng and Schneewind, 2000). The corresponding tyeA mutant in Y. pseudotuberculosis described in this study was also temperature sensitive for growth at 37°C, whereas the Yop expression/secretion profile was intermediate compared to the tyeA mutants described in the other Yersinia species. Yop expression/secretion in the presence of Ca2+ was higher compared to the isogenic wild-type strain but lower than the expression/secretion levels of the wild-type strain in Ca2+-depleted media. Secretion/expression of Yops of the tyeA mutant in medium lacking Ca2+ was lower than for the wild-type strain. Thus, the phenotype of the tyeA mutant was not completely de-repressed for Yop expression/secretion as seen for the tyeA mutants described in Y. enterocolitica and Y. pestis (Day and Plano, 1998; Iriarte et al., 1998; Cheng and Schneewind, 2000). The tyeA mutant constructed in this study is an in frame deletion extending from amino acid 19–59, which is the same deletion as the in the mutant described by Iriarte et al. (1998). This, together with the fact that the tyeA mutant was readily complemented with a clone expressing TyeA under control of the tac-promoter, makes it unlikely that the observed difference resulted from polar effects on genes downstream of tyeA.

The temperature sensitive (TS) phenotype of the tyeA mutant means that the bacterium enters growth restriction and is unable to grow at 37°C. LcrQ is a key regulator in the induction of Yop expression and secretion but the expression of LcrQ is not influenced by Ca2+ concentration. Instead, depletion of Ca2+ results in rapid secretion of LcrQ and giving subsequent induction of Yop expression and secretion (Rimpiläinen et al., 1992; Pettersson et al., 1996). For other TS mutants like yopN LcrQ is rapidly secreted also during growth in Ca2+-containing medium, which allows high levels of Yop expression and secretion under theses growth conditions (Fig. 3). The rapid secretion of LcrQ is well in line with the growth phenotype of the yopN mutant as lcrQ mutants are temperature sensitive for growth at 37°C as well. One possible explanation for the intermediate phenotype of the tyeA mutant is that the LcrQ expression levels were low in the bacterial fraction and that secretion of LcrQ appeared to be incomplete both in presence and absence of Ca2+. The low levels of LcrQ are unusual and have not been observed in any other mutant investigated so far and suggest a link between TyeA and LcrQ in the regulation of Yop expression.

The lower secretion levels of the tyeA mutant made it difficult to conclude if TyeA was really important for translocation as the effect seen could be attributed to the lower levels of Yop secretion seen for the tyeA mutant. However, by constructing a double lcrQ/tyeA mutant it was possible to conclude that TyeA per se was not required for secretion as derepression of Yop expression by loss of the negative element LcrQ resulted in a strain which was constitutively secreting high amounts of Yop proteins. Importantly, the lcrQ/tyeA mutant strain retained its impaired phenotype in intracellular delivery of all Yop effectors we investigated, which allowed us to conclude that the polarization of Yop effectors by Y. pseudotuberculosis indeed requires TyeA.

The TTSS of pathogenic Yersinia species is unusually complex both at the level of gene expression as well as at the level of secretion/translocation control. In addition to TyeA, LcrG and YopN are also required for preventing non-productive secretion to the external milieu. Loss of these factors also affects expression of the yop regulon. Some Yop effectors mediate phagocytosis resistance (Rosqvist et al., 1988; 1990; Grosdent et al., 2002) of which YopH has been shown to be able to dephosphorylate target proteins inside host cells within just a few minutes after cell contact (Andersson et al., 1996). This rapid delivery is chaperone-dependent and important to prevent phagocytosis. In this work we found that the tyeA mutant of Y. pseudotuberculosis was almost as impaired in blocking phagocytosis as a double yopE/yopH mutant. This could indicate that this strain is unable to rapidly deliver YopH and YopE, which then highly impairs the ability to prevent uptake. TyeA might also be involved in a process similar to, or act in concert with, Yop effector chaperones to introduce a hierarchy whereby these effectors are delivered first by a rapid mechanism to allow phagocytosis blockage. Support for such a role of the chaperones was recently presented (Boyd et al., 2000). However, it seems likely that translocation of all Yop effectors is inefficient as a result of the non-polarized secretion observed during infection of host cells.

Based on our findings we suggest that TyeA is required to ensure controlled and efficient translocation of Yop effectors and that the main role could be to mediate rapid delivery of those effectors which are essential at initial stages of infection to promote the extracellular infection of pathogenic Yersinia species.

Experimental procedures

Bacterial strains and growth conditions

The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were grown in Luria–Bertani broth (LB) and Yersinia strains in the rich medium, brain–heart infusion (BHI), which was supplemented either with 2.5 mM CaCl2 or 5 mM EGTA, 20 mM MgCl2, 0.1% Triton X-100. For solid media blood-agar-base (BAB, Merck) or Yersinia selective agar base (YSAB, Difco) plates were used. The antibiotics (medium/plates) kanamycin (20/50 µg ml−1), chloramphenicol (15/20 µg ml−1) and carbenicillin (50/100 µg ml−1) were used where appropriate.

Table 1. . Bacterial strains and plasmids.
Strains and plasmidsRelevant genotypeSource or reference
E. coli
 S17–1λpirRP4-2 Tc::Mu-Km::Tn7 (λpir)De Lorenzo and Timmis (1994)
Y. pseudotuberculosis
 YPIII(pIB102)wild type, yadA::Tn5Bölin and Wolf-Watz (1984)
 YPIII(pIB801)tyeAThis study
 YPIII(pIB803)yerA/tyeAThis study
 YPIII(pIB804)lcrQ/tyeAThis study
 YPIII(pIB251)yopE/yopHRosqvist et al. (1990)
 YPIII(pIB522yopEForsberg and Wolf-Watz (1990)
 YPIII(pIB82)yopNForsberg et al. (1991)
 YPIII(pIB604)yopBHåkansson et al. (1996)
 YPIII(pIB621)yopDFrancis and Wolf-Watz (1998)
 YPIII(pIB232)yopJSchesser et al. (1998)
 YPIII(pIB417)yerAFrithz-Lindsten, unpublished
 YPIII(pIB26)lcrQPettersson et al., (1996)
Plasmids
 pMMB66EHptac expression vector, CbrFürste et al. (1986)
 pDM4Suicide vector with sacB, CmrMilton et al. (1996)
 pGEXptac, GST-fusion, CbrGuan and Dixon (1991)
 pUC19C-terminal His-tag fusion, AprYanisch-Perron et al. (1985)
 pBK43pyopM, pUC19 with yopM-His-tagKihlberg, unpublished
 pUW1plcrQ, pMMB66EH with lcrQFrithz-Lindsten, unpublished
 pLS61pDM4 with ΔtyeAThis study
 pLS63ptyeA, pMMB66EH with tyeAThis study
 pLS69ptyeA-flag, pMMB66EH with tyeA-flagThis study

DNA methods

Preparations of plasmid DNA, restriction enzyme digests, ligations and transformations into E. coli were performed essentially as described by Sambrook et al. (1989). DNA fragments were purified from agarose gels using GeneClean kit (Bio 101) according to the manufacturer's instructions.

Construction of a tyeA in frame deletion mutant and double mutants

A double PCR was performed with the primers TyeA1 + TyeA2 and TyeA3 + TyeA4, respectively (Table 2) using plasmid pIB102 as template. TyeA1 was tailed with a site for XbaI and TyeA4 was tailed with a SacI site. This gave one fragment from the upstream and one from the downstream region of tyeA. The 5′-ends of the primers TyeA2 and TyeA3 contain overlapping sequences and a second PCR with the two gained fragments as templates and the primers TyeA1 and TyeA4 gave a fragment with the flanking side of the gene. After digestion with XbaI and SacI the fragment was cloned into the vector pDM4 (Table 1) and the resulting plasmid was termed pLS61. The plasmid was transformed into E. coli S17–1λpir and clones were selected on plates containing chloramphenicol and the resulting transformants were verified by PCR. Escherichia coli containing the plasmid was then used to conjugate the recipient Yersinia strain YPIII(pIB102). Clones where the plasmids integrated by a single recombinant event were selected on YSAB-plates containing 50 µg ml−1 kanamycin and 20 µg ml−1 chloramphenicol. The insertion was verified by PCR using the primers from the second PCR.

Table 2. . Primer sequences.
PrimerSequencebp
  1. Nucleotides in bold letters correspond to restriction enzyme recognition sites. Nucleotides in italics are identical to the positions given in the column bp. The sequences are derived from GenBank Accession No X51833.

TyeA15′-AGC TCT AGAGCT CTG GTC AGC ATG-3′1484 –1498
TyeA25′-ATC GCT AAA GCG CTT GTC AAC CAG TGC-3′1919 –02
TyeA35′-GAC AAG CGC TTT AGC GAT GAG GAG CAA CG-3′2040 –2059
TyeA45′-GAC GAG CTCCTA ACC ACC CCG CT-3′2388 –2370
TyeA75′-CTG ACT CTA GAC ATG GCG TAC GAC CTT TCT-3′1863 –1880
TyeA85′-CTG ACT GAG CTCTTC ATA CTT TGT GCA ACA GG-3′2865 –2846
TyeA115′-GCT CAG AAT TCG ATG GCG TAC GAC CTT TCT-3′1863 –1880
TyaA125′-CTG ACT CTG CAGTCA ATC CAA CTC ACT CAA TTC-3′2141 –2127
TyeA135′-CTG ACT CTG CAG TTA TTT ATC GTCATC ATC ATC
TTT ATA ATC TCA ATC CAA CTC ACT CAA TTC-3′
2141 –2127

The resulting strain was then subjected to a sucrose selection and mutants were verified by PCR using primers TyeA1 and TyeA4. The resulting mutant was denoted YPIII(pIB801).

The same procedure was used to mutate tyeA in the yerA mutant YPIII(pIB417) and the lcrQ mutant (YPIIIpIB26) background. These strains were denoted YPIII(pIB803) and YPIII(pIB804) respectively.

For trans-complementation studies the tyeA gene was amplified with PCR using the primers TyeA11 + TyeA12 tailed with sites for EcoRI and PstI, respectively, and cloned into the vector pMMB66EH under the IPTG-inducible tac-promoter in the strain E. coli S17-1-λpir. After conjugation into the mutant strain on YSAB-plates containing 100 µg ml−1 carbenicillin and 50 µg ml−1 kanamycin the plasmid (pLS63) was verified by a plasmid mini preparation and with PCR using the same primer pair. Plasmid pUW1 expressing LcrQ was constructed in the same way as pLS63, and was used to trans-complement the double lcrQtyeA mutant (E. Frithz-Lindsten, unpublished).

Sucrose selection

Overnight cultures of strains grown in Luria broth media containing 50 µg ml−1 kanamycin were diluted 1 : 10, 1 : 100 and 1 : 1000 and plated on blood-agar-base (BAB) plates containing 5% sucrose and 50 µg ml−1 kanamycin. Resulting colonies were patched on both kanamycin/chloramphenicol and kanamycin/sucrose plates. Colonies that were not growing on kanamycin/chloramphenicol plates were verified by PCR.

Analysis of growth phenotype

The growth phenotype of strains was tested by a MOX-test in the following way; Overnight cultures, grown in Luria broth containing appropriate antibiotics, were diluted 106 and 107 times. Each dilution was spread both on BAB plates supplemented with 20 mM Na oxalate, 20 mM MgCl2, 0.2% glucose (MOX plates) or plates containing 2.5 mM Ca2+. Duplicate plates were prepared; one was incubated at 37°C and one at 26°C for two days.

Analysis of yop gene expression and protein secretion

Overnight cultures grown in BHI, plus or minus calcium, with appropriate antibiotics were diluted 10-fold in the same media, grown for one hour at 26°C and then shifted to 37°C for three hours. The OD600 were measured and a sample of the whole bacterial culture was dissolved 1 : 1 in sodium dodecyl sulphate (SDS) sample buffer while the bacteria were harvested by centrifugation and dissolved in 400 µl SDS sample buffer.

The culture supernatants were filtered through a 0.45-µm filter and the proteins were precipitated with 10% trichloroacetic acid for two hours on ice and pelleted by centrifugation. The pellet was dissolved in 500 µl 2% SDS at 37°C, precipitated with acetone and pelleted again by centrifugation. The secreted proteins were finally dissolved in 200 µl SDS-sample buffer.

After boiling at 95°C for 5 min the samples, the volumes loaded were calculated as ratio to the OD600, were separated on a SDS-PAGE gel and the proteins were transferred by electroblotting to Immobilon-P Transfer Membranes (Millipore) using Trans-Blot Semi-Dry transfer cell (Bio-Rad) and buffers as described by Laemmli (1970). Membranes were blocked for one hour in Tris-saline (TBS) with 5% non-fat dry milk. The proteins were detected by selected antibodies and probed with a mouse or rabbit secondary antibody conjugated to horseradish peroxidase (Amersham Pharmacia Biotech) and visualized by enhanced chemiluminescence (ECL) Western blot (Amersham Pharmacia Biotech.). The primary antibodies used were: anti-total Yop antibodies and anti-YopE antibodies (Forsberg et al., 1987), anti-YopD antibodies (obtained from S. Håkansson) anti-YopM antibodies (obtained from E.E. Galyov) and anti-YopH antibodies (Persson et al., 1995)

Construction of a Flag-tagged TyeA protein

For trans-complementation studies the tyeA gene was amplified with PCR using the primers TyeA11 + TyeA13 tailed with sites for EcoRI and PstI and TyeA13 did also include the FlagTM-epitope. The fragment was cloned into the vector pMMB66EH in the strain E. coli S17-1 λpir. After conjugation into the mutant strain on YSAB-plates containing 100 µg ml−1 carbenicillin and 50 µg ml−1 kanamycin the plasmid (pLS63) was verified by a plasmid mini preparation and with PCR using the same primer pair.

Xylene extraction

Bacterial overnight cultures grown at 26°C were diluted 1 : 10 in 3 ml fresh BHI media with or without calcium. The cultures were grown at 26°C for one hour prior to a shift to 37°C for three hours. Four hundred microlitres of culture was mixed with 200 µl p-Xylene and was vortexed for five minutes. The solution was centrifuged for five minutes at 6000 g and the upper-layer was discarded while the middle-layer was precipitated with four volumes acetone at − 20°C for one hour, centrifuged for 10 min and then dissolved in SDS-sample buffer. The pellet was also dissolved in SDS-sample buffer and samples were separated on a SDS-PAGE gel and an ECL Western blot was made (as described above) with anti-Flag M2 monoclonal mouse antibodies (Sigma). As controls, antibodies were used against the cytoplasmic YerA and the surface located YopN protein.

Cloning of yop genes

A PCR was made to amplify the yopM gene. This gene fragment was cloned into the pUC19 vector in frame with a Histidine-tag (B-M. Kihlberg, unpublished) and electroporated into the respective recipient strain.

Analysis of Yop secretion during HeLa infection

HeLa cells (1.5 × 105) were grown in the defined medium RPMI supplemented with 10% fetal calf serum and 3 µg ml−1 gentamicin in a 24 well plate. Bacteria were grown in BHI medium and respective antibiotics overnight at 26°C. The overnight cultures were diluted 1 : 10 in RPMI and grown for one hour at 26°C prior a shift to 37°C for one hour. Strains containing plasmid for trans-complementation was induced with 0.4 mM IPTG upon the temperature shift and during the infection. During this time the HeLa cells were washed three times with RPMI and fresh medium supplemented with 0.5 µg ml−1 cytochalacin D was added. The HeLa cells were then infected with 4 × 106 bacteria for three hours. The medium was transferred into an Eppendorf tube and the non-adherent bacteria were harvested by centrifugation and the culture supernatant was dissolved 1 : 1 in 2 × SDS-sample buffer. The cells were then scraped off in 400 µl 0.1% Triton X-100 (which will lyse the cells but not the adherent bacteria) into an Eppendorf tube and centrifuged for 10 min at 4°C. A viable count was performed on the collected bacteria to ensure that equal amounts of the culture supernatant was loaded onto the SDS-PAGE gel and then subjected to ECL Western blot, as described above, using selected antibodies.

Cytotoxicity on HeLa cells

HeLa cells (1.5 × 105) were grown in the defined medium RPMI supplemented with 10% fetal calf serum and 3 µg ml−1 gentamicin in a 24-well plate. Bacteria were grown in BHI medium and respective antibiotics over night at 26°C. The overnight cultures were diluted 1 : 10 in RPMI and grown for one hour at 26°C prior to a shift to 37°C for one hour. Strains containing plasmid for trans-complementation were induced with 0.4 mM IPTG upon the temperature shift and during the infection. The HeLa cells were then infected with 4 × 105 bacteria for up to five hours. During the infection the morphology of the HeLa cells was studied under a microscope.

Immunofluorescence staining of infected HeLa cells

HeLa cells (0.5 × 105) were grown in the defined medium RPMI supplemented with 10% fetal calf serum and 3 µg ml−1 gentamicin on an 8-well slide. Bacteria were grown in LB medium supplemented with respective antibiotics over night at 26°C. The overnight cultures were diluted 1 : 10 in RPMI and grown for one hour at 26°C prior to a shift to 37°C for an additional hour. Strains containing plasmid for trans-complementation were induced with 0.4 mM IPTG upon the temperature shift and during the infection. The HeLa cells were washed three times with RPMI and fresh medium, supplemented with 0.5 µg ml−1 cytochalacin D, was added half an hour prior to infection and then they were infected with 4 × 106 bacteria for up to five hours. After infection the cells were washed three times with PBS, fixated with 2% formalin (diluted in PBS from a 20% stock solution: 20 g paraformaldehyde in 100 ml 60°C water plus Na2OH until the solution was clear) for 10 min and washed again. Then PBS with 0.1 M glycine was added for 10 min and they were washed three times with PBS and permeabilized in PBS with 0.15% saponin for 10 min. After washing again the specific antibodies were added in a 1 : 100 dilution in PBS containing 0.15% saponin, 2% BSA and 0.1 M glycine at 37°C for 30 min. The cells were then washed again three times with PBS and an Alexa Fluor 488 labelled secondary antibody (Molecular probes) diluted 1 : 1000 in PBS containing 0.15% saponin, 2% BSA and 0.1 M glycine was added and incubated at 37°C for 30 min. The cells were then washed three times with PBS and mounted with DAKO fluorescent mounting medium under a coverslip.

Phagocytosis inhibition assay

Macrophages (0.5 × 105) were seeded onto a slide with eight chambers in the defined medium DMEM supplemented with 10% fetal calf serum and 3 µg ml−1 gentamicin. Bacterial cultures were grown over night at 26°C in Luria broth containing the appropriate antibiotics.

The bacterial overnight cultures were diluted to an OD600 of 0.01 in DMEM and grown at 37°C for three hours. During this time the macrophages were washed with PBS and grown in 0.5 µl DMEM until infection. The macrophages were then infected with 4 × 106 bacteria for two hours. After the infection time the macrophages were washed with PBS and fixated with 2% formalin (diluted in PBS from a 20% stock solution: 20 g paraformaldehyde in 100 ml 60°C water plus Na2OH until clear solution). After additional washes PBS with 0.1 M glycine was added for 10 min and the cells were washed again. Then total-Yersinia antibodies diluted in PBS were added and incubated at 37°C for 30 min. After washing again with PBS an Alexa Fluor 568 (red, Molecular probes) labelled antibody diluted 1 : 1000 in PBS was added and incubated for an additional 30 min After this incubation the cells were permeabilized in PBS with 0.15% saponin for 10 min. The cells were washed with PBS and incubated again with the total-Yersinia antibodies but now diluted in PBS containing 0.15% saponin, 2% BSA and 0.1 M glycine. An Alexa Fluor 488 (green, Molecular probes) labelled secondary antibody was now used diluted in the same solution and after incubation and washing of the cells the fluorescent bacteria were observed under a microscope.

Macrophage viability assay

Macrophages (5 × 105) were seeded in a 12-well plate in the defined medium DMEM supplemented with 10% fetal calf serum and 3 µg ml−1 gentamicin. Bacteria were grown over night at 26°C in Luria broth containing appropriate antibiotics. The overnight cultures were diluted 100 µl to 2.5 ml DMEM and grown for one hour at 26°C prior a shift to 37°C for an additional two hour incubation. The macrophages were washed three times in DMEM and continued to grow in 1 ml DMEM until infection. The OD600 of the bacterial cultures were measured and macrophages were infected with 4 × 105 bacteria for five hours. The macrophages were scraped off and stained with an ethidium homodimer and calcein according to the manufacture (Live/dead, viability/cytotoxicity kit, Molecular probes). The per cent living and dead cells were counted under a microscope.

Acknowledgements

We thank Dr Elisabet Frithz-Lindsten, Dr Britt-Marie Kihlberg and Margaretha Lundquist and for supplying unpublished mutant strains and constructs for use in this study. We also would like to thank Dr Eduard Galyov and Dr Cathrine Persson for kindly providing antibodies used in this study. Marléne Lundström is acknowledged for skilled assistance in cell infection experiments. This study was supported by a grant from Swedish Research Council, and student stipends from the Wallenberg-foundation and Försvarsförbundet.

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