Type III-mediated translocation of Yop effectors is an essential virulence mechanism of pathogenic YersiniaLcrV is the only protein secreted by the type III secretion system that induces protective immunity. LcrV also plays a significant role in the regulation of Yop expression and secretion. The role of LcrV in the virulence process has, however, remained elusive on account of its pleiotropic effects. Here, we show that anti-LcrV antibodies can block the delivery of Yop effectors into the target cell cytosol. This argues strongly for a critical role of LcrV in the Yop translocation process. Additional evidence supporting this role was obtained by genetic analysis. LcrV was found to be present on the bacterial surface before the establishment of bacteria target cell contact. These findings suggest that LcrV serves an important role in the initiation of the translocation process and provides one possible explanation for the mechanism of LcrV-induced protective immunity.
V (LcrV) and W were the first antigens to be associated with virulence of Yersinia pestis (Burrows and Bacon, 1956; 1958). These investigators showed that only strains expressing V and W could induce protective immunity to plague in the mouse infection model. Lawton et al. (1963) raised antibodies against purified preparations of V and W and demonstrated that only anti-V antibodies provided protection against plague in mice. More recent studies (Une and Brubaker, 1984; Motin et al., 1994; Hill et al., 1997) have verified that polyclonal antibodies raised against highly purified LcrV as well as a monoclonal antibody that recognizes the central domain of LcrV conferred passive protection against experimental infections with Y. pestis and Y. pseudotuberculosis. It has been established that active immunization using LcrV as an antigen can provide high levels of protection against up to 107 lethal doses in experimental bubonic and pneumonic plague (Leary et al., 1995; Anderson et al., 1996). These studies argue for a role of LcrV in the virulence of Yersinia, and it has been proposed that LcrV acts by suppressing the inflammatory response early during infection. This is supported by experiments in which the injection of LcrV in mice suppressed the expression of TNF-α and IFN-γ and promoted the survival of LcrV-negative strains of Y. pestis as well as the survival of Salmonella and Listeria (Nakajima et al., 1995). LcrV has also been shown to delay allograft rejection and increase the tolerance to lipopolysaccharides (LPS) in mice (Motin et al., 1997; Nedialkov et al., 1997). In addition, Welkos et al. (1998) showed that LcrV inhibits neutrophil chemotaxis in vitro as well as in vivo.
One essential virulence function mediated by the type III targeting system is the ability of Yersinia to resist uptake by phagocytes (Rosqvist et al., 1988; 1990). This activity is dependent on two of the translocated effectors, the YopE cytotoxin and the YopH protein tyrosine phosphatase, which act in concert to block bacterial uptake (Rosqvist et al., 1990; Bliska et al., 1991). YopH has been shown to act downstream of the phagocytic receptor, probably targeting the focal adhesions (Black and Bliska, 1997; Persson et al., 1997). As a consequence, YopH can also mediate resistance to the uptake of IgG-opsonized bacteria via Fc receptors (Fällman et al., 1995). This powerful antiphagocytic mechanism is probably the main reason why neither effector proteins nor proteins localized to the zone of contact between the bacterium and the target cell (YopB, YopD, YopK, YopN) can induce protective immunity to Yersinia infections (Benner et al., 1998; Leary et al., 1999). Another possibility is that none of these proteins are accessible to antibodies with the potential to block the function of these essential virulence determinants. The unique ability of anti-LcrV antibodies to provide protection against Yersinia infections prompted us to investigate whether the antibodies could act by blocking an essential virulence function involving LcrV.
In this work, we show that LcrV is involved in translocation and that antibodies to LcrV can block this process. Based on our findings, we propose that the passive protection conferred by anti-LcrV antibodies is mediated by a specific inhibition of the translocation of Yop effectors. We also show that LcrV is exposed on the bacterial surface before contact between the pathogen and the target cell. Based on our findings, we suggest that LcrV functions in the initiation of the translocation process.
Anti-LcrV antibodies block translocation of Yop effectors
Previous work has established that LcrV is unique among the antigens secreted by the type III system in that it can provide protective immunity to Y. pestis infections (Lawton et al., 1963; Motin et al., 1994; Leary et al., 1995; 1999; Benner et al., 1998). Together with the fact that Yersinia can also block Fc receptor-mediated phagocytosis by professional phagocytes (Fällman et al., 1995), this suggested to us that one mechanism by which anti-LcrV antibodies could mediate protection was via the interference of translocation of Yop effectors. To test this hypothesis, we investigated whether anti-LcrV antibodies could interfere with the translocation of Yop effectors. A sensitive assay for translocation is to monitor the cytotoxic effect caused by translocation of the YopE cytotoxin, which is manifested as a typical rounding up of the infected cells. Interestingly, cells infected with wild-type Y. pseudotuberculosis treated with a monospecific polyclonal anti-LcrV antiserum displayed a dramatically decreased cytotoxicity compared with cells treated with the preimmune serum (Fig. 1). Antisera directed to essential components of the type III delivery system or to effector Yops (YopB, YopD, YopE, YopH, YopK, YopM and YopN) were all unable to block YopE-mediated cytotoxicity on HeLa cells infected with wild-type Y. pseudotuberculosis (Fig. 1; data not shown). In addition, a number of monoclonal antibodies (mAbs) directed to LcrV of Y. pestis were also evaluated in this system. Only the protective antibody mAb 7.3 (Fig. 2) affected the cytotoxicity, while the other non-protective mAbs had no effect on the YopE-mediated cytotoxicity of Y. pestis (data not shown). Yop translocation requires intimate bacteria–cell contact. Therefore, it was important to exclude the possibility that the anti-LcrV antiserum interfered with the bacterial binding to the HeLa cell surface and thereby indirectly affected the translocation process. We first measured whether anti-LcrV had an effect on the bacteria–HeLa cell binding by counting the number of cell-associated bacteria using an immunofluorescence method described earlier (Rosqvist et al., 1988). The addition of anti-LcrV antiserum had no effect on the number of cell-associated bacteria. Using a multiplicity of infection of 5, on average, 1.5–2 bacteria per HeLa cell were bound irrespective of the presence or absence of anti-LcrV antiserum. A lcrV whole-gene deletion mutant can be transcomplemented by the homologous protein PcrV of Pseudomonas aeruginosa (see below). The cytotoxic effect induced by the strain YPIII(pIB19, pLJ33), lcrV(pPtacpcrV+) on HeLa cells was not inhibited by the addition of anti-LcrV antiserum. As PcrV is not recognized by the anti-LcrV antibodies, this result demonstrates that the anti-LcrV antibodies inhibit the cytotoxicity via a specific interaction with LcrV. These results indicate that LcrV is involved in the delivery of Yop effectors into the target cell.
LcrV is involved in translocation
To provide genetic evidence for a role of LcrV in translocation, we constructed an in frame deletion mutant of lcrV in which codons 10–313 were removed, thereby essentially deleting the entire lcrV gene (Fig. 2). Like previously described lcrV mutants (Bergman et al., 1991; Skrzypek and Straley, 1995), the resulting mutant strain, YPIII(pIB19), was found to be downregulated for Yop expression (Fig. 3) and calcium independent for growth at 37°C (Table 1). In contrast to other calcium-independent mutants, the lcrV mutant secreted considerable amounts of all Yops, albeit in reduced amounts compared with the wild-type strain, when grown in calcium-depleted medium at 37°C. The introduction of pTB7 (Bergman et al., 1991), which expressed lcrV under the control of the tac promoter, restored the wild-type phenotype with respect to growth phenotype (Table 1) as well as Yop expression and secretion (Fig. 3).
Table 1. . HeLa cell cytotoxicity and growth phenotypes of different strains of Y. pseudotuberculosis. a. Strains unable to grow at 37°C without the addition of Ca2+ are defined as calcium dependent (CD), which is the wild-type phenotype of Y. pseudotuberculosis. Calcium-independent (CI) mutants are able to grow at 37°C, irrespective of the Ca2+ concentrations. If the strain is unable to grow at 37°C, it is defined as temperature sensitive (TS).
Interestingly, we found that the lcrV mutant, YPIII(pIB19), was unable to induce a cytotoxic effect on infected HeLa cells even after prolonged incubation (Table 1). This was found to result from inactivation of lcrV, as the transcomplemented strain, YPIII(pIB19, pTB7), induced a strong cytotoxic effect similar to that of the wild-type strain (Table 1). One could argue that the inability of YPIII(pIB19) to cause cytotoxicity on eukaryotic cells was the result of the lower levels of Yop expression by the lcrV mutant compared with the wild-type strain. It has been shown recently that a double yopN, lcrV mutant of Y. pestis was derepressed for Yop expression and secretion (Skrzypek and Straley, 1995). Therefore, we constructed a strain, YPIII(pIB82V), in which the mutant allele of the lcrV gene (Δ10-313) was introduced in a yopN background. Also in Y. pseudotuberculosis, YopN was found to be epistatic to LcrV, as the yopN, lcrV double mutant displayed constitutive Yop secretion at 37°C, with only a minor reduction in Yop secretion levels compared with the yopN mutant strain (Fig. 4). However, in contrast to the yopN mutant strain, the yopN, lcrV double mutant strain, YPIII(pIB82V), was non-cytotoxic for infected HeLa cells even after prolonged incubation (Table 1). The introduction of pTB7 (Ptac lcrV +) in YPIII(pIB82V) restored the ability to induce a cytotoxic effect on infected HeLa cells (Table 1), indicating that LcrV is important for translocation. However, one potential problem with this interpretation is the fact that the yopN, lcrV double mutant showed a reduction in the level of Yop secretion including the translocators YopB and YopD. Therefore, we conducted a genetic analysis of lcrV with the idea of isolating mutations that discriminated between Yop regulation and translocation.
It has become evident recently that the type III secretion machinery and the translocation systems of Yersinia spp. and P. aeruginosa are closely related (Yahr et al., 1996; 1997). Recent studies have shown that the type III secretion system and the translocation of virulence effectors are functionally conserved between these pathogens (Frithz-Lindsten et al., 1997; 1998). The protein corresponding to LcrV in P. aeruginosa, PcrV, is 41% identical to LcrV at the amino acid level, with a region of high homology localized in the central part of the protein, where 11 out of 12 amino acid residues are identical. In addition, the 55 C-terminal amino acids show 67% identity (Fig. 2). Analogous to the functional complementation by PopB/PopD of yopB/yopD mutants (Frithz-Lindsten et al., 1998), we found that pcrV + of P. aeruginosa (cloned under the control of the tac promoter of pLJ33) could complement the Y. pseudotuberculosis lcrV mutant, YPIII(pIB19). Strain YPIII(pIB19, pLJ33), lcrV(pPtacpcrV+), was calcium dependent for growth at 37°C (Table 1), secreted wild-type levels of Yops in medium devoid of calcium (Fig. 5) and induced cytotoxicity on HeLa cells (Table 1). These findings indicate that LcrV and PcrV are very similar with respect to their function in spite of the relatively low levels of homology at the amino acid level. Based on this, we argued that the highly conserved regions could be important for the function of the proteins. Therefore, a series of mutations were made in lcrV, based on the homologies between the two proteins. First, stepwise C-terminal deletions of 10 amino acids were made to produce the truncated proteins V316, V306 and V296 (Fig. 2). In addition, the highly conserved domain in the central part of lcrV was deleted, resulting in the protein VΔ160–171. The mutated forms of lcrV were cloned under the control of the tac promoter using the vector pMMB66HE and introduced into the lcrV mutant strain, YPIII(pIB19). However, none of the constructs could complement the lcrV mutant with respect to calcium phenotype, expression/secretion of Yops or cytotoxicity (Table 1; data not shown). In contrast, an in frame deletion mutant strain denoted YPIII(pIB119), in which codons 218–234 of lcrV were removed, displayed a wild-type phenotype (Fig. 5, Table 1[link]); this region showed high divergence between LcrV and PcrV as well as the LcrV proteins of the different Yersinia species. This indicated that the regions of LcrV and PcrV showing high homology are also of importance for the function of the proteins.
As none of the truncated forms of LcrV could restore wild-type expression/secretion of Yops, the constructs were introduced in the yopN, lcrV double mutant in an attempt to discriminate between the effects on regulation and translocation. Introduction of the truncated forms of LcrV resulted in levels of LcrV and Yop expression/secretion in vitro similar to the full-length LcrV (Fig. 6A). Importantly, we also verified that the level of Yop expression was similar during infection of HeLa cells for all these constructs (Fig. 6B). However, in contrast to the wild-type protein, none of the LcrV truncates could complement the translocation defect of the yopN, lcrV double mutant (Table 1). The yopN, lcrV double mutant showed a slight reduction in secretion of Yops (Figs 4 and 6A) and, therefore, we were concerned that the effect on translocation of the lcrV mutation was caused by an indirect effect of the lcrV mutation on the secretion of the Yops. However, as complementation of the lcrV mutation by the LcrV truncates results in the same level of expression and secretion of Yops, including YopB and YopD, as the strain transcomplemented by the full-length LcrV (Fig. 6), we can conclude that the effects on translocation are mediated by LcrV per se. In addition, the regions conserved between LcrV and PcrV are important for translocation. These findings were reinforced further by the fact that YopH was also translocated into HeLa cells in a LcrV-dependent manner, as shown by immunofluorescence staining followed by confocal laser scanning microscopy (CSLM) analysis (Fig. 7). We therefore conclude that LcrV is indeed involved in the translocation process.
LcrV is surface localized before target cell contact
As LcrV can be found not only intracellularly, but also in the culture medium (Straley, 1988; Bergman et al., 1991), we asked whether LcrV was translocated into the cytosol of the target cell. HeLa cells infected with wild-type Y. pseudotuberculosis were analysed by immunofluorescence staining followed by CLSM using monospecific anti-LcrV antibodies. LcrV was found to be associated with the extracellular bacteria, appearing as distinct foci on the bacterial surface, while no specific LcrV staining could be detected in the HeLa cell cytosol (data not shown). Given that translocation of one effector protein can be increased by removing the genes encoding the other translocated Yops (Håkansson et al., 1996a), we used a genetically engineered multiple yop mutant strain MYM lacking yopH, yopE, yopM, yopK and ypkA (Persson et al., 1997) to investigate whether LcrV translocation could be detected. When the spatial localization of LcrV was analysed after infection of HeLa cells with the MYM strain, no LcrV could be detected in the cytosol of the target cell (data not shown). As observed for the wild-type strain, LcrV was only found in discrete foci in the vicinity of the bacteria (Fig. 8). Interestingly, most bacteria exhibited a similar staining pattern irrespective of whether they were associated with the HeLa cells or not. This finding is distinct from the staining pattern observed for all the other Yops studied so far. In these cases, Yops could only be detected after association of the bacteria with the target cells. To investigate the localization of LcrV further, different bacterial strains grown in calcium-containing media were analysed by CLSM using anti-LcrV antibodies. A secretion mutant (yscS ), as well as a lcrV mutant (YPIII(pIB19)) displayed no LcrV staining, whereas the wild-type strain showed a staining pattern identical to that of bacteria associated with HeLa cells (Fig. 8). Importantly, as expected for bacteria grown in non-inducing conditions (+Ca2+), the yscS mutant expressed LcrV at the same level as the wild-type strain, as verified by Western blot analysis (Fig. 8). As shown above, PcrV of P. aeruginosa can functionally transcomplement an lcrV mutant with respect to the regulation, secretion and translocation of Yops (Fig. 6; Table 2[link]). However, PcrV is not recognized by the LcrV antiserum. As the lcrV mutant expressing PcrV, YPIII(pIB19,pLJ33), shows a wild-type phenotype, it served as the optimal negative control for the LcrV stainings. As expected, this strain was negative for surface-located LcrV (Fig. 8), demonstrating that the surface staining was specific for LcrV. The surface localization of LcrV was strengthened further by the fact that the addition of purified LcrV before the addition of anti-LcrV antiserum totally abolished the immunofluorescence signal (data not shown). When CLSM was used, the LcrV staining appeared as 10–20 discrete spots per bacteria (Fig. 8). Increasing the resolution by using immunogold labelling electron microscopy gave a similar result, i.e. clusters of immunogold particles were found at discrete areas of the bacterial surface. On average 153 ± 57 gold particles per bacteria were observed for the wild-type strain, whereas only 10 ± 5 gold particles per bacteria were seen for the negative control strain (YPIII(pIB19, pLJ33)) (Fig. 9A). In the LcrV-expressing strain, the gold particles were typically found in clusters (around 10–20 clusters per bacterium) most easily seen at the edges of the bacteria. Similar clusters were rarely found (less than one per 10 bacteria) on the negative control strain. These results showed that LcrV was localized to the bacterial surface and suggested to us that LcrV could form or be part of a structure on the bacterial surface. Therefore, the wild-type strain YPIII(pIB102) was analysed by thin-section immunogold labelling electron microscopy using anti-LcrV antiserum. As expected from the results obtained using CLSM, only a few clusters of gold particles were associated with the bacteria when the thin sections (50 nm) were analysed, and rarely were more than two clusters per bacterial section observed. No specific labelling was observed for the lcrV mutant strain, YPIII(pIB19), nor for the lcrV mutant strain transcomplemented by PcrV, YPIII(pIB19, pLJ33) (data not shown). Specific LcrV staining could usually be seen as one cluster per bacterium per thin section (Fig. 9B). Assuming that each bacteria could be divided into 20–40 sections and that we rarely found more than two clusters per bacterial section, one can estimate that one bacterium could maximally harbour 40–80 LcrV clusters. As many sections contained one or no cluster, this number is probably overestimated. The number of LcrV clusters observed by this method is thus comparable with the number of LcrV foci estimated from the CLSM analysis (Fig. 8) as well as from the electron microscopy analysis of whole bacteria (Fig. 9A). From these results, we conclude that LcrV is localized in discrete foci that are present on the bacterial surface before contact between the pathogen and the target cell has been established. However, the thin-section EM did not provide results supporting the hypothesis that LcrV is part of an organized surface structure.
One central virulence mechanism of Yersinia is to deliver effector proteins into the cytosol of the target cell via the contact-dependent type III secretion system. This process requires a number of discrete steps including attachment, secretion, translocation and targeting of the different Yop effectors. Inactivation of proteins required at any level of this process results in avirulence. Given that anti-LcrV antibodies mediate protection against Yersinia infections, the neutralizing effect of anti-LcrV antibodies could involve any of the steps mentioned above. To elucidate the step at which anti-LcrV antibodies act, we used the HeLa cell infection model and were able to show that anti-LcrV antibodies blocked YopE-mediated cytotoxicity. The antibodies did not inhibit attachment of the bacteria to the eukaryotic cells. Importantly, the anti-LcrV antibodies could not block cytotoxicity mediated by a lcrV mutant strain expressing PcrV of P. aeruginosa, which can functionally complement LcrV. Thus, the anti-LcrV antibodies obstructed the translocation process. Interestingly, one monoclonal antibody (mAb 7.3), which provides passive protection in experimental animal infections, has been identified (Hill et al., 1997). This mAb also affected the cytotoxic response, while other non-protective mAbs did not. Thus, there is a strong correlation between protective immunity and inhibition of Yop translocation. This finding argues that the LcrV-mediated humoral immunity against Yersinia infection can be accomplished by neutralization of the translocation process. LcrV apparently exhibits two virulence functions, translocation of Yop effectors and immunosuppression. Therefore, it cannot be excluded that protection could also be provided by interference of the immunosuppressing activities of LcrV, as suggested by Brubaker and co-workers (Nakajima et al., 1995; Motin et al., 1997).
These results suggested to us that LcrV is actually involved in the translocation process per se. To investigate this further, we constructed a non-polar in frame deletion mutant of lcrV. This mutant was indeed non-cytotoxic, but was also found to be downregulated for Yop expression and/or secretion, as shown previously for other lcrV mutants (Bergman et al., 1991; Price et al., 1991; Skrzypek and Straley, 1995). Therefore, these results did not allow us to conclude that LcrV was involved in translocation. To uncouple Yop expression/secretion from translocation, the effect of LcrV was studied in a yopN mutant background. yopN mutants are constitutive for Yop expression and secretion, and Straley and colleagues had shown earlier that a yopN (lcrE ) mutation is epistatic to a mutation in lcrV (Skrzypek and Straley, 1995). Thus, a double yopN, lcrV mutant is derepressed for Yop expression and secretion. We confirmed these results and could show further that such a mutant was non-cytotoxic, thus unable to translocate YopE. The block in translocation was general, as the translocation of YopH was also blocked, as shown by immunofluorescence and CLSM. Introduction of lcrV in trans restored translocation fully, demonstrating the importance of LcrV in this process.
PopB and PopD of P. aeruginosa can functionally complement yopB and yopD mutants of Y. pseudotuberculosis (Frithz-Lindsten et al., 1998). This is somewhat unexpected, as the corresponding proteins of the two species are only about 40% identical. In an attempt to identify functionally important domains of LcrV, we investigated whether the LcrV homologue of P. aeruginosa, PcrV, could complement the lcrV mutant, YPIII(pIB19), and this was indeed found to be the case. PcrV and LcrV display an overall identity of 41% but, interestingly, two regions, amino acids 160–171 and the 55 C-terminal amino acids, show significantly greater similarity (92% and 67% identity respectively). Based on this information, several deletions in lcrV were constructed. Deletions in either of these regions abolished lcrV function with respect to expression, secretion and translocation, i.e. the phenotypes of these mutants were indistinguishable from that of the lcrV null mutant. By analysing the phenotypes of these mutations in a yopN mutant background (with constitutive Yop expression and secretion), it was possible to show that these highly conserved domains were important for translocation of Yop effectors. The fact that the two regions identified as essential for LcrV function affect both Yop expression/secretion as well as translocation highlights the close link between these processes.
When we analysed the localization of LcrV during infection of HeLa cells, we were unable to detect any LcrV in the cytosol of the cells; the majority of the LcrV staining was found at the zone of contact between the bacteria and the eukaryotic cell. Using fractionation of infected cells, Nilles et al. (1998) suggested that LcrV was translocated into the cytosol of cells. Our results do not exclude the possibility that small amounts of LcrV are translocated but suggest that the majority of LcrV is localized in discrete areas of the bacterial surface. However, we could not show that LcrV was part of an organized surface structure. Knutton et al. (1998) showed recently that one of the proteins secreted by the type III system of enteropathogenic Escherichia coli, EspA, forms a fibrillar organelle on the bacterial surface, bridging the space between the pathogen and the target cell. Similar to LcrV, EspA was found to be important for translocation (Knutton et al., 1998). In another study, Kubori et al. (1998) isolated structures spanning the bacterial envelope and extending out from the bacterial surface that contained components of the type III secretion system encoded by SPI1 of Salmonella typhimurium. These structures showed similarities to the basal bodies of flagella. The part of the structure facing outwards from the bacteria was needle-like in appearance. Thus, these three pathogens have surface-localized proteins involved in the translocation of virulence effector proteins. Although the mechanism of translocation is similar and functionally conserved (Rosqvist et al., 1995), the structural appearance of these proteins seems to be different.
LcrV is present on the bacterial surface before target cell contact. This suggests that LcrV is required during the early stages of translocation. Purified LcrV has been shown to inhibit the chemotactic migration of neutrophils (Welkos et al., 1998) and to suppress IFN-γ and TNF-α induction (Nakajima et al., 1995), indicating the possibility that LcrV can interact with eukaryotic cell receptors. Thus, one potential role of LcrV could be to sense contact with a target cell receptor to initiate the translocation process. It is possible that the role of LcrV in translocation as well as in immunosuppression is mediated via a common receptor interaction. There are several molecular mechanisms that might explain how LcrV promotes translocation. One possibility is that LcrV and Yop proteins are positioned at the target cell surface by a continuous polymerization process and are internalized once the structures contact the eukaryotic cell membrane. Another conceivable mechanism is that LcrV forms an organelle, which is hollow, allowing other proteins to be channelled through this structure. Alternatively, as proposed by Nilles et al. (1998), LcrV could initiate translocation by mediating the correct positioning of another component(s) required for the process. These authors also observed that translocation was affected by mutating lcrV of Y. pestis and suggested that LcrV had an indirect role in the translocation process by affecting the deployment of the translocator protein YopB. Thus, it is possible that the role of the surface-localized LcrV is to conduit YopB to the target cell membrane.
In conclusion, we have shown that LcrV is surface localized and has an important role in the delivery of Yop effectors into the cytosol of the target cell. This virulence mechanism can be neutralized by antiserum directed to LcrV, thereby providing one possible explanation as to how anti-LcrV antibodies can provide full protection against subsequent Yersinia infections. Thus, increased understanding of the molecular mechanism of virulence may enable the development of novel treatments against infectious agents.
Bacterial strains, plasmids, growth conditions and DNA methods
The bacterial strains and plasmids used in this study are listed in Table 2. E. coli strains were grown in Luria broth (LB) or on Luria agar (LA) plates. Yersinia strains were grown in LB or in brain–heart infusion (BHI; Oxoid). BHI was supplemented with either 2.5 mM CaCl2 (BHI +) or 5 mM EGTA and 20 mM MgCl2 (BHI −). For solid media, Yersinia selective agar base (YSA; Difco), blood agar base (BAB; Oxoid) or LA was used. Where appropriate, antibiotics were used at the following concentrations: kanamycin (50 μg ml−1), chloramphenicol (20 μg ml−1) and carbenicillin/ampicillin (100 μg ml−1). Preparation of plasmid DNA, restriction enzyme digests, ligations and transformations into E. coli were performed essentially as described by Sambrook et al. (1989). Plasmids were introduced into Yersinia by conjugation as described by Rimpiläinen et al. (1992). DNA fragments were purified from agarose gels using Geneclean (Bio 101) according to the manufacturer's instructions.
Construction of plasmids and mutants
In frame deletions in lcrV were constructed by allelic exchange using the method described by Milton et al. (1996), which involves integration and excision of a suicide plasmid carrying upstream and downstream DNA of the fragment to be deleted. The near full-gene deletion mutant in which codons 10–313 of lcrV were removed was constructed as follows: the primer pairs LcrV1 and LcrV2 and LcrV3 and LcrV4 (Table 3) were used in polymerase chain reaction (PCR) with YPIII(pIB102) as the template DNA. The resulting amplified fragments U1 (primers LcrV1 and LcrV2) and D1 (primers LcrV3 and LcrV4), corresponding to about 400 bp upstream and 182 bp downstream of the lcrV gene, respectively, were purified by preparative agarose gel electrophoresis. The LcrV2 and LcrV3 primers were designed to contain complementary sequences, and a second overlapping PCR using primers LcrV1 and LcrV4 and the purified U1 and D1 fragments as the template DNA was performed. A 582 bp fragment with the desired deletion was obtained. Primers LcrV1 and LcrV4 contained sites for XbaI and SacI, respectively, and, after restriction enzyme digestion and purification, this 582 bp fragment was cloned into the suicide vector pDM4 (Milton et al., 1996), yielding plasmid pAH70. The plasmid was transformed into E. coli S17-1λpir, from which it was introduced by conjugation into the recipient Yersinia strain YPIII(pIB102). Clones with pAH70 integrated into pIB102 by a single recombination event were selected on plates containing chloramphenicol and kanamycin. The resulting co-integrate strain was verified by PCR, after which it was subjected to sucrose selection as described by Milton et al. (1996). This procedure selected for a double cross-over event in which the integrated suicide plasmid, encoding the sacB gene product, was excised from the virulence plasmid. Four chloramphenicol-sensitive clones carrying the desired deletion were identified by PCR, and one of these was denoted YPIII(pIB19). Analogously, the lcrV mutant allele was also introduced into the yopN mutant of Y. pseudotuberculosis, YPIII(pIB82), resulting in a yopN, lcrV double mutant denoted YPIII(pIB82V).
Table 3. . Primers used in this study. a. Nucleotides in bold correspond to restriction enzyme sites. Nucleotides in italic are identical to positions from the Y. pseudotuberculosis sequence (lcrGVH ) given to the right (accession no. M57893).
The method described above was also used for constructing the in frame deletion mutant YPIII(pIB119). Overlapping PCR using the U2 and D2 fragments (generated by primer pairs V1 + V2 and V3 + V4 with pIB102 as the template DNA) resulted in an 800 bp fragment that was cloned into pDM4 yielding pAH69. Conjugation of this plasmid into YPIII(pIB102) followed by allelic exchange resulted in the strain YPIII(pIB119), which is devoid of codons 218–234 of the lcrV gene. YPIII(pIB19), YPIII(pIB82V) and YPIII(pIB119) were verified by restriction analysis and Western blot analysis using an anti-LcrV antiserum.
Plasmid pLJ33 expressing pcrV + under the control of the tac promoter of pMMB66EH was constructed as follows. The pMS9 plasmid described by Frithz-Lindsten et al. (1998) contains a 5.3 kb fragment of the P. aeruginosa chromosome encoding the PcrR, PcrG, PcrV, PcrH, PopB and PopD proteins. A 2.8 kb EcoRI–Bgl II fragment of pMS9, containing pcrV with flanking genes, was cloned into the vector pMMB66EH. The new plasmid was digested with EcoRI and SphI, after which blunt ends were generated, and the larger fragment was subjected to preparative gel electrophoresis and religated. The resulting plasmid, pLJ33, contains only the very 3′ end of the pcrG gene, the entire pcrV gene and the 5′ end of pcrH. pLJ33 was electroporated into the E. coli strain S17-1λpir, from where it was introduced by conjugation into YPIII(pIB19).
Three C-terminally truncated variants of LcrV with stepwise deletions of 10 amino acids were constructed as follows. BamHI-tailed primers V316, V306 and V296 were used in PCR together with the primer V1 and YPIII(pIB102) as template. The three fragments were digested with BamHI and XhoI (internal site of the fragments) and purified by preparative gel electrophoresis. The fragments encoding truncated forms of the C-terminus of LcrV were cloned into pTB7 digested with the same enzymes, resulting in plasmids pV316, pV306 and pV296. Electroporation of the plasmids into S17-1λpir was followed by conjugation into YPIII(pIB19) and YPIII(pIB82V). Another derivative of pTB7 in which codons 160–171 were removed was constructed. Using YPIII(pIB102) as the template DNA, fragments U3 (primers G and VΔ160) and D3 (primers VΔ171 and VF) were generated and, again, overlapping PCR was used to obtain a 1200 bp fragment containing the desired deletion. The mutated region of lcrV was introduced into the pTB7 plasmid by digesting the plasmid and the fragment with ClaI and XhoI and replacing the wild-type DNA with the mutated form. The resulting plasmid, pVΔ160–171, was electroporated into S17-1λpir from which it was conjugated into YPIII(pIB19) and YPIII(pIB82V).
The plating frequencies of the different mutant strains under high- and low-Ca2+ conditions at 37°C were determined, and the subsequent phenotypes were defined as in Table 1 and as described previously by Bergman et al. (1991).
Yop expression and analysis
Overnight cultures of Yersinia strains grown at 26°C were diluted (1:20) in fresh media (BHI + or BHI −) containing 0.1% Triton X-100, grown for 30 min at 26°C, then shifted to 37°C and grown for an additional 3 h. After measuring the OD600, the cells were harvested, and the culture supernatant was collected and filtered (0.45 μm pore size; Sartorius). The proteins from the supernatant were precipitated with trichloroacetic acid (TCA) as described previously (Forsberg et al., 1987). The samples from the supernatant and the whole bacteria were dissolved in SDS sample buffer, and the volume of the samples was adjusted in accordance with the OD600 values of the bacterial cultures. The proteins were separated by SDS–PAGE and analysed by Coomassie blue staining or ECL Western blot analysis according to the manufacturer's instructions (Amersham).
Cultivation and infection of HeLa cells
The Y. pseudotuberculosis strains used to infect HeLa cells were grown overnight at 26°C in LB. Overnight culture (30 μl) was added to 3 ml of modified Eagle medium (MEM) with 10% heat-inactivated fetal calf serum without any antibiotics. The inoculated MEM cultures were grown for 30 min at 26°C followed by incubation at 37°C for 2 h before infection. Cultivation and infection of HeLa cells has been described in detail elsewhere (Rosqvist et al., 1990). The HeLa cells were seeded (1.0 × 105 per well) in a 24-well tissue culture plate in MEM with 10% heat-inactivated fetal calf serum and 100 IU ml−1 penicillin at 37°C in a 5% CO2 humidified atmosphere. For immunofluorescence studies, the HeLa cells were grown on 12 mm coverslips placed in a 24-well culture plate. Before infection, the HeLa cells were washed extensively, and MEM with 10% heat-inactivated fetal calf serum without any antibiotics was added. In experiments using cytochalasin D (0.5 μg ml−1 concentration), the drug was added 30 min before infection. After infection, the HeLa cells were centrifuged for 5 min at 400 × g to facilitate contact between the bacteria and the HeLa cells, followed by continued incubation at 37°C.
The HeLa cells were infected at a multiplicity of infection (MOI) of about 50 bacteria per cell with various strains of Y. pseudotuberculosis or with Y. pestis EV76p(pAMS2). When the neutralizing effects of various antisera were analysed, the antisera (polyclonal whole serum, Western blot titre 1:4000) at different concentrations (1:100, 1:50, 1:25, 1:10) were added to the bacterial cultures 5 min before infection, and the MOI was reduced to about two bacteria per cell. The infected cells were observed every hour by phase-contrast microscopy. A changed morphology, as visualized by rounding up of the HeLa cells (see Fig. 1) indicated a cytotoxic response.
Detection of bacterial adhesion to HeLa cells
HeLa cells grown on coverslips (≈1.0 × 105) were infected with different strains of Y. pseudotuberculosis (about 0.5 × 106 bacteria per well). Two hours after infection, the cells were washed twice in PBS and fixed in 2% paraformaldehyde for 10 min. The cells were then permeabilized with 0.5% Triton X-100 in a buffer containing 1 mM EGTA, 4% polyethylene glycol 6000 and 100 mM piperazine-N,N′-bis(2-ethanesulphonic acid), pH 6.9, and processed further for indirect immunofluorescence labelling (for details, see Rosqvist et al., 1991) using rabbit anti-Yersinia antibodies, followed by fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibodies. The specimens were examined using fluorescence microscopy. In each experiment, 200 HeLa cells were counted in randomly selected fields without previewing.
HeLa cells grown on coverslips (≈1.0 × 105) were infected with different strains of Y. pseudotuberculosis (about 0.5 × 107 bacteria per well). At various times after infection, the cell monolayers were washed twice in PBS and stained with wheat germ agglutinin (WGA) conjugated to Texas red (Molecular Probes) (10 μg ml−1 for 10 min at 24°C); the WGA-stained cells were fixed in 2% paraformaldehyde for 10 min. Unless indicated, the cells were then permeabilized with 0.5% Triton X-100 in a buffer containing 1 mM EGTA, 4% polyethylene glycol 6000 and 100 mM piperazine-N,N ′-bis(2-ethanesulphonic acid), pH 6.9, and processed further for indirect immunofluorescence labelling (for details, see Rosqvist et al., 1991) using affinity-purified rabbit anti-LcrV antibodies or affinity-purified goat anti-YopH antibodies, followed by FITC-conjugated anti-rabbit antibodies or FITC-conjugated anti-goat antibodies. The specimens were finally mounted in a mounting medium containing Citifluor as an antifading agent. The specimens were analysed using a CLSM equipped with dual detectors and an argon–krypton (Ar/Kr) laser for simultaneous scanning of two different fluorochromes (Multiprobe 2001; Molecular Dynamics). Laser power and gain were set by using cells labelled with either fluorochrome alone so that there was no cross-over of green to red or red to green channel. Sets of fluorescent images were acquired simultaneously for Texas red (the HeLa cell membrane) and fluorescein-tagged (LcrV or YopH) markers using a ×60/1.3 plan-apochromate Nikon oil immersion lens. Companion images (10 sections with image size 512 × 512) were scanned with either 0.28 μm or 0.07 μm pixel size and 0.3 μm step size and a pinhole setting of 50 μm.
For immunolabelling of bacteria, overnight cultures of wild-type Y. pseudotuberculosis, YPIII(pIB102), or the PcrV-expressing lcrV mutant strain, YPIII(pIB19,pLJ33), grown at 26°C were diluted (1:20) in fresh media (BHI +), grown for 30 min at 26°C, then shifted to 37°C and grown for an additional 2 h. The cells were washed twice in PBS, fixed for 10 min in 0.01% glutaraldehyde, washed again and then applied to grids by placing the grids face down on drops of bacterial suspension for 5 min. After washing, the grids were placed on drops of affinity-purified anti-LcrV serum for 2 h at room temperature, washed again and placed on drops of 6 nm gold-conjugated anti-rabbit antibodies (1:50 dilution; Jackson ImmunoResearch Laboratories) for 2 h at room temperature. After washing in distilled water, the grids were air dried. For the thin-section EM, HeLa cells grown in 35 mm Petri dishes were infected with different strains of Y. pseudotuberculosis (about 5 × 107 bacteria per dish). Eighty minutes after infection, the cell monolayers were washed twice in PBS, fixed for 10 min in 0.1% glutaraldehyde, washed again and then incubated with affinity-purified anti-LcrV serum for 2 h at room temperature. After washing, the samples were incubated with gold-conjugated anti-rabbit antibodies (Jackson ImmunoResearch Laboratories) for 12 h at 4°C. After thorough washing, cells were fixed in 3% buffered glutaraldehyde, post-fixed in osmium tetroxide, dehydrated in a graded ethanol series and embedded in Agar 100 resin (Agar Scientific). Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined in a JEOL 100CX transmission electron microscope at 80 kV.
†These authors have contributed equally to this work
We thank Anna-Lena Ström and Lars Jacobsson for excellent technical assistance. This work was supported by grants from the Swedish Medical Research Council (H.W.-W. and Å.F.), the Swedish Natural Science Research Council (H.W.-W.), Magnus Bergvall Foundation (Å.F.), the J. C. Kempe Memorial Foundation (A.H. and J.P.) and the Swedish Foundation for Strategic Research (H.W.-W.).