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Biochimie et Biophysique des Systèmes Intégrés, CEA-Grenoble, UMR CNRS, Grenoble
Centre de Recherches du Service de Santé des Armées, La Tronche, France
Biozentrum der Universitt Basel, Switzerland
Pathogenic Yersinia species and Pseudomonas aeruginosa share a similar type III secretion/translocation system. The translocation system consists of 3 secreted proteins, YopB/PopB, YopD/PopD, and LcrV/PcrV; the latter is known to be a protective antigen. In an in vitro assay, the translocation system causes the lysis of erythrocytes infected with wild-type (wt) P. aeruginosa. wt Y. enterocolitica is not hemolytic, but a multiknockout mutant deprived of all the effectors and of YopN (HOPEMN) is hemolytic. In the presence of antibodies against PcrV and Y. pestis LcrV, the hemolytic activity of P. aeruginosa was inhibited. Similarly, the hemolytic activity of HOPEMN was inhibited in the presence of anti-LcrV antibodies. The assembly of the translocon, composed of PopB/D and YopB/D proteins, was disturbed in immunoprotected erythrocyte membranes, mimicking the phenotypes of V knockout mutants. Thus, protective antibodies against the V antigens of Yersinia species and P. aeruginosa act at the level of the formation of the translocon pore in membranes of infected host cells by blocking the function of LcrV/PcrV. The hemolysis assay could be adapted for high-throughput screening of anti-infectious compounds that specifically target the type III translocon.
Injectisomes are complex nanomachines that allow pathogenic or symbiotic bacteria to inject proteins across the membrane of eukaryotic host cells. They consist of a basal body that contains a type III secretion (T3S) apparatus and a stiff needle that can be extended by a flexible pilus or filament [1, 2]. Upon contact with a eukaryotic cell membrane, the injectisome secretes, presumably in a sequential manner, a set of proteins called "translocators" and "intracellular effectors" [3, 4]. The translocators are a group of, generally, 3 proteins that are absolutely required for the proper delivery of the effectors across the host cell membrane [312].
Pathogenic members of the genus Yersiniafor example, Y. pestis (the causative agent of plague) and the enteric pathogens Y. enterocolitica and Y. pseudotuberculosisshare a common injectisome called "Ysc" and a common set of translocators and effectors called "Yops." The translocators are called "YopB," "YopD," and "LcrV." The latter is a bona fide Yop; it has a different name because it was discovered as a soluble protective antigen linked to virulence (hence, it is named "V antigen") >40 years ago [13, 14], long before the concept of T3S had been shaped [15]. Interestingly, the opportunistic pathogen Pseudomonas aeruginosa, which is evolutionarily remote from enterobacteriaceae, has a very similar injectisome called "Psc" and a set of similar translocators called "PopB," "PopD," and "PcrV" [1618]. Both sets of translocators have been shown to form pores in erythrocytes (red blood cells ) [5, 9], in eukaryotic cell membranes [19, 20], and in liposomes [21]. Formation of pores is readily detectable with wild-type (wt) P. aeruginosa but not with wt Y. enterocolitica. Only Y. enterocolitica mutants that are devoid of the effectors or of the control protein YopN form readily detectable pores [5, 11, 20]. An interpretation of this observation is that translocated Yop effectors obstruct the translocation channel [5, 20]. However, it has also been shown that the Rho-GAP activity of YopE prevents membrane damage in cells infected with wt bacteria [22]. When YopN is missing, secretion is contact independent and effectors are not efficiently translocated [11]. The structure of the translocation pore, the stage during which the translocation pore is inserted, and how it is connected to the needle of the injectisome are not clear yet. It is tempting to consider the translocation pore as an integral part of the injectisome, but, unlike the needle, it could be assembled only after contact with a target cell. It has been shown that PopB and PopD are able to oligomerize in vitro and that their interaction with lipids promotes the formation of ringlike structures with external and internal diameters of 80 and 40 , respectively [23]. Although PcrV is unable to interact with lipids either in vitro or in vivo [9, 23], it is required for proper assembly of the PopB/D translocon in membranes of infected erythrocytes [9].
Y. pestis LcrV possesses a highly protective antigenic character, and antibody therapy against bubonic and pneumonic plague has been shown to be effective when tested in animal models of disease [2426]. Similarly, in animal models, active immunization with PcrV or passive immunization with anti-V antibodies provides a high level of protection against lethal P. aeruginosa infections [7, 27]. Furthermore, administration of anti-PcrV F(ab)2 in a P. aeruginosaprovoked sepsis model reduced the inflammatory response and bacteremia levels [28]. Although the mechanism of action is not known, even in vitro, anti-V antibodies prevent the cytotoxicity of bacteria toward cultured cells [7, 29, 30].
In the present study, we examined the action of anti-V antibodies on formation of the translocation pore in P. aeruginosa and Y. enterocolitica hemolysis assays. It has already been shown with P. aeruginosa that a high level of hemolysis can be obtained at a low MOI (MOI, 1) within 1 h of coincubation. Another advantage of this model is that RBCs are not a limiting factor, and their membranes can be isolated after infection and analyzed for translocon proteins without being contaminated with infecting bacteria [9, 31]. In the present study, we applied the RBC assay to Yersinia species by using poly-Yop effector mutants of Y. enterocolitica [20]. We demonstrate that the antibodies against the V antigens, through their direct interaction with V proteins, inhibit bacteria-induced hemolysis by acting at the level of type III translocon assembly in host cell membranes, for both pathogenic species.
MATERIALS AND METHODS
Bacterial strains and growth conditions.
The bacterial strains used in the present study are listed in table 1. The P. aeruginosa strain used was the cytotoxic cystic fibrosis isolate CHA [19, 32]. P. aeruginosa were grown either on Pseudomonas isolation agar (Difco) plates or in liquid Luria broth (LB) medium at 37°C with agitation. All the mutants of Y. enterocolitica used were derived from the strain E40, a strain from serotype O:9 [4, 11]. Y. enterocolitica strains were inoculated to an OD600 of 0.1 and cultivated in brain-heart infusion broth (ICN) for 3 h at 37°C. In vitro, secretion of Y. enterocolitica was triggered by sodium oxalate [11].
Production and purification of antibodies.
The production and purification of the rabbit-derived anti-PcrV, anti-PopB, and anti-PopD polyclonal antibodies (PAbs) have been described elsewhere [9, 23]. MAb166 [27] was provided by D. W. Frank (Medical College of Wisconsin). The LcrV coding sequence was obtained from genomic DNA of the Y. pestis strain EV76 (gift from E. Carniel, Pasteur Institute, Paris) by polymerase chain reaction (PCR) using the primers 5-GATAAGAATTCGAGCCTACGAACAAAACCCA-3 and 5-AAGGATCGTCGACTTACATAATTACCTCGTGTCA-3. The PCR product was digested with EcoRI and SalI and cloned into the expression plasmid pGEX-6P-2 (Amersham Biosciences). A soluble glutathione-S-transferase (GST)LcrV fusion was produced in Escherichia coli and purified on GSTrap (Amersham Biosciences). The GST-LcrV protein fusion bound on the column was cleaved with PreScission Protease (Amersham), resulting in the elution of purified LcrV. Three CD1 mice were immunized with 100-L intramuscular injections of 10 g of purified LcrV in PBS containing 2% alhydrogel (Superfos Biosector) and 10 g of CpG oligonucleotide [33] as adjuvant. In each mouse, after 2 immunizations separated by a 2-week interval, the ELISA titer (the highest dilution giving twice the optical density obtained with naive CD1 serum) of anti-LcrV serum antibodies was >1 : 50,000. For production of anti-LcrV PAbs, the best-responding mouse was given an intraperitoneal (ip) booster injection of the same respective amounts of LcrV, alhydrogel, and CpG, followed 5 days later by a final ip injection containing LcrV, CpG, and 5 × 105 TG180 sarcoma cells. The ascitis fluid was collected 1 week later, and IgG was purified on protein A (Mab Trap; Amersham). The BCA protein assay kit (Pierce) was used to determine the concentration of purified antibodies.
Hemolysis assays and immunoprotection experiments.
Hemolysis assays were performed as described elsewhere [9]. Briefly, sheep RBCs (Eurobio) were washed 3 times in PBS (pH 7.4) (150 mmol/L NaCl) and resuspended in RPMI 1640 medium (Sigma) at 5 × 108 RBCs/mL at 4°C. Bacteria were grown in LB to an OD600 of 1.0, centrifuged, and resuspended in RPMI 1640 at 5 × 108 bacteria/mL. Hemolysis assays were initiated by mixing 100 L of RBCs and 100 L of bacteria in round-bottom 96-well plates, which were then centrifuged at 2000 g for 10 min and incubated for 1 h at 37°C. The release of hemoglobin was measured, and the percentage of hemolysis was calculated as described elsewhere [31]. Immunoprotection experiments were performed by incubating bacteria for 45 min with different concentrations of antibodies at room temperature, before the standard hemolytic assay. Negative controls included corresponding preimmune rabbit serum. In titration experiments, a constant concentration of 5 ng/L PAbs was added to bacteria, then increasing amounts of the recombinant PcrV or LcrV were incubated with the bacteria-PAb mixture for 45 min at room temperature.
RBC membrane isolation.
RBCs were resuspended in Tris-saline (30 mmol/L Tris and 150 mmol/L NaCl [pH 7.5]) at 1 × 1010 cells/mL. Before the infection, 3 × 109 bacteria were incubated with 180 g of PAbs in 700 L of Tris-saline buffer for 45 min at room temperature. Then, hemolytic reactions were prepared in 50-mL conical tubes. The mixture of bacteria and antibodies was mixed with 300 L of RBCs at 1 × 1010 cells/mL and 1 mL of a 2× protease inhibitor cocktail (Complete; Roche). Samples were centrifuged at 2000 g for 10 min at room temperature and incubated for 1 h at 37°C. Hemolysis was assessed spectrophotometrically, as described above. Then, 3 mL of distilled water at 4°C was added to each sample to lyse all RBCs, and these were vortexed and centrifuged again to remove bacteria. The RBC membranes were isolated by flotation on a sucrose density gradient, as described elsewhere [9]. The material at the 44%/25% sucrose interface was collected, diluted in Tris-saline, and concentrated by centrifugation in a TLA-100.3 rotor (Beckman) at 450,000 g for 20 min at 4°C. The pellets were resuspended in Laemmli sample buffer and analyzed by Western blotting.
Immunoblotting analysis.
Immunoblotting analysis was performed with primary PAbs against YopB (gift from . Forsberg, Ume University), PopB, PopD, and PcrV [9]; monoclonal antibodies (MAbs) against YopD [20] and LcrV [11]; and a secondary antibody conjugated to horseradish peroxidase (Sigma). Membranes were developed by use of an enhanced chemiluminescence kit (Amersham Biosciences).
RESULTS AND DISCUSSION
Since PcrV has been found to be necessary for assembly of PopB/D pores in RBC membranes [9], we asked whether antibodies against PcrV would interfere with the PopB/D-dependent hemolysis induced by cytotoxic P. aeruginosa strain CHA. Affinity-purified PAbs against translocator proteins PopB and PopD did not inhibit hemolysis. Concentrations of both antibodies up to 50 ng/L had no protective effect. In contrast, affinity-purified anti-PcrV PAbs [9] mixed with either bacteria or RBCs before infection inhibited hemolysis in a dose-dependent manner (figure 1A). Concentrations of antibodies as low as 0.25 ng/L were able to reduce P. aeruginosainduced lysis by 50%. Increasing the concentration of antibodies up to 10 ng/L in the infection assay resulted in complete protection of RBCs from lysis. MAb166 is an anti-PcrV MAb that has been shown to protect mice from lethal P. aeruginosa infection [27]. In the hemolysis assay, MAb166 prevented hemolysis, but an 18-fold greater concentration of MAb166 than of PAbs was required to obtain 50% inhibition. The higher neutralization activity of the PAbs could be due to the binding of several PcrV epitopes or to a higher affinity of some antibody component, compared with MAb166. The former hypothesis is favored because, in a competition ELISA, a 100-fold excess of PAb did not hinder the binding of MAb166 to recombinant PcrV (rPcrV) (data not shown). This could have practical importance, because efficient neutralization of PcrV by MAbs would then require a combination of several MAbs, as seems to be the case for botulinum toxin type A [34].
LcrV from Yersinia species and PcrV from P. aeruginosa share 41% amino acid identity [16], and they most likely share the same function in the assembly of the translocon. We raised mouse PAbs against recombinant LcrV (rLcrV) from Y. pestis. Similar to anti-PcrV PAbs and MAb166, anti-LcrV PAbs inhibited P. aeruginosainduced hemolysis (i.e., 50% inhibition was obtained with 10 ng/L anti-LcrV PAbs). In summary, these data show that the PAbs against PcrV and LcrV, as well as the protective MAb166, hinder cytotoxic P. aeruginosa from lysing RBCs.
LcrV and PcrV are surface-exposed antigens ([29, 35] and I.A., unpublished data) and, therefore, are probably accessible to antibodies. To further confirm that the protection against hemolysis was due to the direct interaction between antibodies and the antigen during the infection process, we set up competition experiments in which the antibodies necessary to achieve 90%100% protection were kept constant and increasing amounts of the recombinant antigen were added to the infection mixture. With rPcrV, as few as 34 molecules/10 molecules of antibodies were sufficient to restore 50% hemolysis, showing that the protection is due to direct PcrV/antibody interaction (figure 1B).
RBC lysis occurs by osmotic shock following the formation of PopB/D pores within erythrocyte membranes. Since PcrV is required for formation of functional Pop pores, we addressed the question of whether PopB/D inserts into membranes in the presence of anti-V antibodies. The hemolysis assay was scaled up, and antibodies were added at levels necessary to achieve 90%100% protection. After 1 h of incubation, the RBCs were lysed by the addition of water, and the membranes were purified by sucrose density gradient, as described elsewhere [9]. The purified membranes were examined for the presence of Pop translocators by Western blotting. P. aeruginosainfected RBC membranes contained both translocators, PopB and PopD, as reported elsewhere (figure 2) [9]. Notably, in the presence of anti-PcrV PAbs, PopD protein was absent from infected RBC membranes (figure 2), corroborating the phenotype of the PcrV knockout bacteria that were unable to assemble the functional translocon (figure 2) [9]. It should be noted that systematically less PopB could be detected in membranes purified from these immunoprotection experiments. Antibodies against PopB were not able to disturb PopB/D pores inserted into host membranes (figure 2), which is in agreement with the incapacity of these antibodies to immunoprotect infected RBCs. These results show that anti-PcrV antibodies either block the proper insertion of pore-forming proteins or destabilize them within membranes. That PopB was still found in immunoprotected membranes, although it was unable to form functional pores, suggests that protective antibodies do not prevent contact between the host cell and the bacterial injectisome.
Since the serum raised against the Y. pestis rLcrV prevented, to some degree, P. aeruginosa T3Sinduced hemolysis, we applied the hemolysis test to several Y. enterocolitica strains. As was already reported by several authors [5, 20], the wt Yersinia strains had very low hemolytic activity, even when the bacteriahost cell contact was forced with centrifugation. Indeed, no detectable hemolysis could be observed at 1 h after infection (figure 3A). Neyt et al. [20] and Marenne et al. [11] reported that, in contrast to the wt strain, the mutant strain HOPEMN, which lacks T3S effectors (YopH, -O, -P, -E, and -M) as well as the control protein YopN [36], provokes macrophage cell lysis through the formation of pores presumably constituted by the translocators YopB and YopD. Notably, in the standard RBC assay, HOPEMN bacteria lysed 30% of the erythrocytes within 1 h of coincubation (MOI, 1), a value lower than that observed with cytotoxic P. aeruginosa but still very significant (figure 3A). HOPEMN-induced hemolysis was dependent on the presence of LcrV, since the mutant HOPEMNV showed only a basal level of hemolysis. In addition, LcrV was not directly hemolytic to RBCs, since the mutants HOPEMNB and HOPEMND, which secrete the same amounts of LcrV as wt bacteria, were not hemolytic. This result is in agreement with data showing that P. aeruginosa strains that secrete PcrV are nonhemolytic [9]. lcrV mutants are known to produce less YopB and YopD, compared with wt bacteria [3739], and this effect can be attenuated by also mutating yopQ (figure 3C) [11]. We thus also tested a HOPEMNVQ mutant in the hemolysis assay, and it was also found to be nonhemolytic (figure 3A), showing that the lack of hemolysis observed with HOPEMNV was not due to insufficient secretion of YopB and YopD but was directly due to the absence of LcrV (figure 3C).
Since PcrV is required for correct formation of PopB/D pores, we addressed the function of LcrV in Y. enterocolitica. RBC membrane fractions were examined for the presence of the translocators after infection with the same strains of Y. enterocolitica (figure 3D). Only YopD was readily detectable in membranes of RBCs infected with wt bacteria. In contrast, YopB and YopD were readily detectable in membranes of RBCs infected with HOPEMN and N bacteria. This difference reflects the fact that wt bacteria produce and secrete less Yops on contact with RBCs than do yopN mutant bacteria, which are deregulated for Yop secretion (figure 3B and 3C) [36]. This shows that centrifugation-achieved contact with RBCs does not trigger optimal Yop secretion of wt bacteria. Both YopB and YopD were detected in membranes of RBCs infected with HOPEMNVQ, but the amount of both translocators, especially YopB, was reduced, compared with that in HOPEMN or N bacteria. This may simply reflect the fact that HOPEMNVQ bacteria secrete less YopB/D than do HOPEMN bacteria, but it should be noted that they secrete at least as much YopB as do wt bacteria (figure 3C). Nevertheless, although both YopB and YopD were detected in the membrane, no pore was formed. This suggests that, although LcrV is necessary for the proper assembly of the YopB/D pore in the host cell membrane, it is not necessary for the insertion of the translocators within host membranes.
Next, we addressed the question of whether YopB/D pores are formed in the presence of anti-LcrV antibodies. HOPEMN bacteria were preincubated for 30 min with antibodies at levels necessary to achieve 90%100% protection. Immunoprotected membranes were purified and examined for the presence of the translocators by Western blotting. The amount of YopB was severely reduced in the presence of protective antibodies. It should be noted that less YopD could be detected in immunoprotected membranes (figure 4B). This suggests that anti-LcrV antibodies, similar to anti-PcrV antibodies, prevent the assembly of the functional translocation pore.
PcrV/LcrV protective antigens would thus act as extrabacterial chaperones or scaffolds in the sense that they would bind unfolded PopB/YopB and PopD/YopD emerging from the bacterium and ensure their correct folding and assembly into a functional pore. This hypothesis, which is supported by the fact that the T3S apparatus exports unfolded proteins [40], is compatible with the known localization of LcrV at the bacterial surface [29, 35]. It implies that the V antigen interacts, at least transiently, with the B and D antigens, which is in agreement with the observation of Sarker et al. [39]. However, it does not imply that the V antigen remains bound to the assembled pore and, thus, is not contradictory to results showing the absence of direct interactions between PcrV and Pop proteins folded in vitro [9, 23]. Finally, although it is inconsistent with the idea that the purified LcrV forms channels on its own, the chaperone or scaffold hypothesis is compatible with the observation that LcrV determines the size of the pore [10, 18].
In conclusion, we have demonstrated that the protective antibodies against the V antigens (PcrV and LcrV) of P. aeruginosa and Yersinia species act by preventing the assembly of a functional type III translocon in host cell membranes. In addition, our work has shown that the hemolysis assay is well suited for study of the function of the type III translocon and could be adapted for screening of anti-infectious molecules that specifically target bacterial toxin translocons.
Acknowledgments
We thank Prof. Dara W. Frank for providing MAb166, Dr. ke Forsberg for providing anti-YopB antibody, Dr. Elisabeth Carniel for providing Yersinia pestis EV76 genomic DNA, Sylvie Elsen for helpful discussions, and Valerie Gros for excellent technical assistance.
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