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A structural overview of the Helicobacter cytotoxin
J M
InU Med. Microbial. 290,375-379 (2000) © Urban &Fischer Verlag http://www.urbanfischer.de/journals/ijmm
A structural overview of the Helicobacter cytotoxin J.-M. Reyrat 1, 2, R. Rappuoli1, 1. L. Telford 1 1 2
IRIS, Chiron S. p. A., via Fiorentina 1,1-53100 Siena, Italy Present address: Unite de Genetique Mycobacterienne, Institut Pasteur, F-75724 Paris, France
Abstract VacA, the major exotoxin produced by Helicobacter pylori, is composed of identical 87-kDa monomers that assemble into flower-shaped oligomers. The monomers can be proteolytically cleaved into two moieties of 37 and 58 kDa, or P37 and P58. The most studied property of VacA is the alteration of intracellular vesicular trafficking in eukaryotic cells leading to the formation of large vacuoles containing markers of late endosomes and lysosomes. However, VacA also causes a reduction in trans-epithelial electrical resistance in polarized mono layers and forms ion channels in lipid bilayers. The ability to induce vacuoles is localized mostly but not entirely in P37, while P58 is involved in cell targeting. Here, we review the structural aspects of VacA biology. Key words: Helicobacter pylori - cytotoxin - 3D structure
Introduction Helicobacter pylori was identified as the etiologic agent of gastritis and peptic ulcer more than 15 years ago (Warren and Marshall, 1983) (for a review (Blaser, 1993)). Contrary to acute infectious diseases, H. pylori, a microaerophilic gram-negative bacterium, causes a long lasting infection with a time scale in decades. Furthermore, H. pylori favours the development of gastroduodenal carcinoma and gastric lymphoma (Parsonnet et al., 1994). The mode of transmission of H. pylori is currently unknown but the most widely held hypothesis is that the bacterium is transmitted directly from person to person by human feces and gastric contents. The family is the core unit of H. pylori transmission. Frequently children are infected by a strain with a genetic fingerprint identical to that of one of the parents (Van der Ende et al., 1996). The children chronically maintain the same strain even after leaving home and establishing their own family. Husbands and wifes do not exchange their strains, and infection is rarely transmitted to an uninfected partner. Soon after the isolation of H. pylori, it was found that broth culture filtrates of the majority of clinical
isolates exhibited a cytotoxic activity. This cytotoxic activity is characterized by the vacuolation of diverse mammalian cell lines and the induction of gastric epithelial erosion in animal models (Leunk et al., 1988) (for a review (Cover, 1996)). Vacuole formation arises from an alteration of intracellular vesicular trafficking leading to the formation of large vacuoles containing markers of late endosomes and lysosomes (Papini et al., 1994; Molinari et al., 1997). Vacuolation requires the activity of the small GTP-binding protein Rab 7 (Satin et al., 1997). Moreover, VacA has been shown to impair the degradative properties of the endocytic pathway in HeLa cells and antigen presentation by Bcells by preventing processing and maturation of antigens (Papini et al., 1997; Molinari et al., 1998). The agent responsible for this toxic activity is a secreted oligomeric protein composed of identical 87-kDa monomers (Cover, 1996; Telford et al., 1994a). The toxin, produced as a 140-kDa precursor, is exported as a 95-kDa polypeptide after cleavage of the C-terminal 45-kDa (Telford et al., 1994a; Loveless and Saier, 1997). After release from the bacterium each monomer can be cleaved at a specific hydrophilic loop into two fragments of 37-kDa and 58-kDa that remain associat-
Corresponding author: John Telford, IRIS, Chiron S. p. A., via Fiorentina 1, 1-53100 Siena, Italy, E-mail: [email protected] 1438-4221100/290/4-5-375 $ 15.00/0
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ed in the oligomeric structure, suggesting that they may represent two distinct cytotoxin subunits (Lupetti et ai., 1996), however the location of the subunits in the holotoxin is not yet described. . Reverse genetics has permitted the characterization of the vacA gene (vacuolating cytotoxin gene) which is about 3.9 kb in size (for a review (Cover, 1996)). The gene is composed of an orthodox N-terminal signal sequence and a C-terminal domain which is cleaved during export across the outer-mebrane (Figure 1). This Cterminal domain, known as autotransporter module, is a recurrent theme among exported virulence factors (Loveless and Saier, 1997). Recently new toxic properties of VacA have been described, including permeability changes in polarized epithelial cell mono layers and channel formation in lipid bilayers (Papini et ai., 1998; Tombola et ai., 1999). It is still unknown whether vacuolization, monolayer TER decrease and gastric atrophy represent different manifestations of the same toxic activity, or whether they correspond to different activities on different targets. Transfection experiments of eukaryotic cells have shown that the entire NHrterminal domain of VacA (P37) plus a short sequence belonging to PS8 were sufficient to achieve vacuolation (de Bernard et ai., 1998; Ye et ai., 1999). It was consequently inferred that the PS8 domain was involded in cell targeting. However, it is currently unknown if the part of the PS8
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Fig. 1. (a) Phase contrast microscopy of HeLa cells vacuolated by SI-tg/ml of purified VacA; (b) schematic representation of the wild-type vacA gene and the obtained product. The level of amino-acid identity between m1 and m2 forms is indicated below. sp = signal peptide; loop = flexible loop between P37 and PS8 containing the proteolytic cleavage site.
subunit required for full toxic activity is part of a functional toxic domain or if it is required to assist folding and oligomerisation of the P37 subunit. A unique feature of the VacA cytotoxin is the allelic variation occurring in the P58 subunit (Atherton et ai., 1995). There are two alleles, m1 and m2 of a 300-amino acids region of the PS8 subunit which have target cell specificity (Pagliaccia et ai., 1998) (for a review (Covacci et ai., 1999)). Only the ml form is toxic to HeLa cells in the standard assay of vacuolization. However, both of them are active on primary gastric cells. The lack of toxicity in the HeLa cell assay was shown to be due to the inability of the m2 form to bind to the surface of HeLa cells. This was a further indication that the PS8 subunit was the binding domain of the cytotoxin. The fact that the m2 cytotoxin interacts with gastric cells but not with He La cells is not compatible with an interaction through non-specific membrane insertion since biological membranes are highly similar. Whether or not this binding process is receptor mediated remains to be determined.
Structural overview Observed by quick-freeze deep etch microscopy, VacA appears as a flower with either 6 or 7 petals, each of them corresponding to a 87-kDa monomer (Figure 2) (Lupetti et ai., 1996; Lanzavecchia et ai., 1998). The oligomeric protein is inactive, and dissociation into the 87-kDa monomer by treatment at low pH is required to reveal its activity (de Bernard et ai., 1995; Cover et ai., 1997). VacA associated with the surface of the bacteria is, however, active in the absence of low pH dissociation, suggesting that the toxin forms oligomers only after release from the bacteria (Pelicic et ai., 1999). The 87-kDa monomer of VacA can be cleaved into 2 subunits of 37 and 58-kDa following proteolysis of a flexible surfaced-exposed loop (Telford et al., 1994b). Interestingly, the connecting loop, i. e. the region where processing occurs, has been shown to play a structural role (Burroni et al., 1998). Deletion and substitution studies have shown that the shortening of the loop favours an organization into hexamers. It is likely that shortening of the connecting loop between the P37 and the PS8 subunit pull these subunits closer together which reduce the flexibility of the interaction and consequently the number of monomers which can enter into the oligomeric structure. However, despite the fact that a petal represents a subunit, the molecular organisation between the P37 and the P58 subunit remained largely obscure. In order to decipher the structural relationship between each building block, the PS8 moiety was subjected to analysis (Reyrat et ai., 1999). Using the sacB counter-selectable marker, we
Structural overview of Helicobacter cytotoxin have engineered a strain of H. pylori to delete the gene sequence coding for the P37 subunit. The remaining PS8 subunit is expressed efficiently and exported as a soluble dimer, suggesting a structural autonomy for this domain. This PS8 dimer is able to bind target cells in a manner similar to the holotoxin, thus confiming previous studies suggesting that the binding activity is localized in the PS8 subunits (Ye et al., 1999; Pagliaccia et al., 1998; Garner and Cover, 1996). However, the bound dimer is not internalized by the target cell, suggesting that the hexameric structure is needed for efficient endocytosis. In addition the PS8 molecule is non-toxic in every model tested (Tombola et al., 1999; Reyrat et al., 1999), thus confirming that the P37 part is largely involved in toxic activity (de Bernard et al., 1998; Ye et al., 1999). However, the nature of the toxic activity and the cytosolic target remain so far uncharacterized. The PS8 molecule has been purified to homogeneity and subjected to structural analysis by quick-freeze deep-etch microscopy. 3D image reconstruction of the PS8 moiety replicas permitted the reconstruction of the global architecture of the P58 molecule and to propose a model for the wild-type holotoxin. The images confirmed the dime ric structure of the PS8 molecule and revealed that each half of the molecule had a curved appearance remarkably similar to the peripheral arms of the wild-type oligomer (Figure 2). The dimeric structure of the PS8 molecule may reflect the interaction, in the intact molecule, involved in holding together the oligomeric structure. On the other hand, the dimerization may be due to interaction between hydrophobic surfaces normally masked in the wild-type molecule. This has led to a model in which the monomers are intercalated with each other to form the ring structure (Fig. 3). In Figure 3B, the structure of half a dimer of the P58 molecule has been arranged in a hexameric ring corresponding to the diameter of the intact oli-
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Fig. 2. 3D top views of the reconstructions of the mold. (A) wild-type molecule, (B) PS8 molecule.
gomer. The two parts match each other perfectly, and the difference in height gives a clear idea of the arrangement of the P37 subunits with respect to the PS8 subunits. However, it is not clear which P37 and PS8 subunits belong to the same 87-kDa monomer. However, we favour a model in which the monomers are intercalated with each other to form the ring structure in such a way that the 37 -kDa subunit of one monomer lies on top of the S8-kDa subunit of the adjacent monomer as shown in Figure 3C. In brief, the oligomeric structure is maintained by interaction of one monomer with the S8-kDa subunit of the adjacent monomer (Reyrat et al., 1999).
Concluding remarks A large amount of data has been accumulated on VacA biology. Genetic studies have shown that the toxic activity is localized mostly in the P37 domain, whereas the binding activity is localized in the P58 subunit. This relates VacA to the AB type family, however caution is suggested by the fact that endocytosis after surface binding requires the putative active domain (P37).
Fig. 3. (A) 3D top view of WT VacA replicas, (B) 3D top view of the hexameric ring of PS8 subunits, assembled in silico by use of reconstructed replicas. (C) model of the interaction of the 87-kDa monomer in the oligomeric structure. In the proposed model, alternate monomers in the oligomeric structure are shown in different shadings in order to represent the interaction of the P37 subunit of one monomer with the PS8 subunit of the adjacent monomer. PS8, PS8 subunit; P37, P37 subunit, dotted line, connecting loop between PS8 and P37 subunit.
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Moreover PS8 alone lacks pore-forming actlVlty a property which again requires the presence of P37. VacA may possess an interesting and perhaps unique molecular structure as it shares both structural and intoxication features of both AB and pore-forming toxins. It is not clear why the allelic variation of the cellbinding subunit has evolved, but it may reflect human genetic polymorphism because the m1 form is predominant in Western, Korean, and Japanese isolates, whereas the m2 form is found in 7S % of Chinese isolates (Pan et al., 1998). VacA is clearly a key player of peptic ulcer and the next step is to characterize both the cellular target and the putative surface receptor. Acknowledgements. We are grateful to J. Triccas for critical reading of the manuscript. G. Corsi is acknowledged for artwork. We thank Bruce di Montechiaro for stimulating discussIOns.
References Atherton, J. c., Cao, P., Peek, R. M., Jr., Tummuru, M. K. R., Blaser, M. J., Cover, T. L.: Mosaicism in vacuolating cytotoxin alleles of Helicobaeter pylori. J. BioI. Chern. 270, 17771-17777 (1995). Blaser, M.J. : Helicobaeter pylori: microbiology of a "slow" bacterial infection, Trends Microbiol. 1,255-260 (1993). Burroni, D., Lupetti, P., Pagliaccia, c., Reyrat, J.-M., Dallai, R., Rappuoli, R., Telford, J. L.: Deletion of the major proteolytic site of Helieobaeter pylori cytotoxin does not influence toxin activity but favors assembly of the toxin into hexameric structures. Infect. Immun. 66, 5547-5550 (1998). Covacci, A., Telford, J. L., del Giudice, G., Parsonnet, J., Rappuoli, R.: Helieobacter pylori virulence and genetic geography. Science 284, 1328-1333 (1999). Cover, T. L.: The vacuolating cytotoxin of Helicobaeter pylori. Mol. Microbiol. 20,241-246 (1996). Cover, T. L., Hanson, P. I., Heuser, J. E.: Acid-induced dissociation of VacA, the Helicobaeter pylori vacuolating cytotoxin, reveals its pattern of assembly. J. Cell BioI. 138, 759-769 (1997). de Bernard, M., Burroni, D., Papini, E., Rappuoli, R., Telford, J., Montecucco, c.: Identification of the Helicobaeter pylori VacA toxin domain active in the cell cytosol. Infect. Immun. 66, 6014-6016 (1998). de Bernard, M., Papini, E., de Filippis, v., Gottardi, E., Telford, J., Manetti, R., Fontana, A., Rappuoli, R., Montecucco, c.: Low pH activates the vacuolating toxin of Helieobacter pylori, which becomes acid and pepsin resistant. J. BioI. Chern. 270, 23937-23940 (1995) . Garner, J. A., Cover, T. L.: Binding and internalization of Helieobaeter pylori vacuolating cytotoxin by epithelial cells. Infect. Immun. 64,4197-4203 (1996). Lanzavecchia, S., Bellon, P. L., Lupetti, P., Dallai, R., Rappuoli, R., Telford, J. L.: Three-dimensional reconstruction
of metal replicas of the Helieobaeter pylori vacuolating cytotoxin. J. Struct. BioI. 121,9-18 (1998). Leunk, R. D., Johnson, P. T., David, B. c., Kraft, W. G., Morgan, D. R.: Cytotoxic activity in broth-culture filtrates of Campylobaeter pylori. J. Med. Microbiol. 26, 93-99 (1988). Loveless, B. J., Saier, M. H., Jr.: A novel family of channelforming, autotransporting, bacterial virulence factors. Mol. Membr. BioI. 14,113-123 (1997) . Lupetti, P., Heuser, J. E., Manetti, R., Massari, P., Lanzavecchia, S., Bellon, P. L., Dallai, R., Rappuoli, R., Telford, ]. L.: Oligomeric and subunit structure of the Helicobaeter pylori vacuolating cytotoxin. ]. Cell. BioI. 133, 801807 (1996). Molinari, M., Galli, c., Norais, N., Telford,]. L., Rappuoli, R., Luzio, J. P., Montecucco, c.: Vacuoles induced by Helicobacter pylori toxin contain both late endosomal and lysosomal markers. J. BioI. Chern. 272, 25339-25344 (1997). Molinari, M., Salio, M., Galli, c., Norais, N., Rappuoli, R., Lanzavecchia, A., Montecucco, c.: Selective inhibition of Li-dependent antigen presentation by Helicobaeter pylori toxin VacA. j. Exp. Med. 187, 135-140 (1998). Pagliaccia, c., de Bernard, M., Lupetti, P., Ji, X., Burroni, D., Cover, T. L., Papini, E., Rappuoli, R., Telford, j. L., Reyrat, J.-M.: The m2 form of the Helicobaeter pylori cytotoxin has bell type-specific vacuolating activity. Proc. Nat\. Acad. Sci. USA 95,10212-10217 (1998). Pan, Z.]., Berg, D. E., van der Hulst, R. W., Su, W. W., Raudoni kiene, A., Xiao, S. D., Dankert,]., Tytgat, G. N., van der Ende, A.: Prevalence of vacuolating cytotoxin production and distribution of distinct vaeA alleles in Helicobaeter pylori from China. J. Infect. Dis. 178, 220-226 (1998). Papini, E., de Bernard, M., Milia, E., Bugnoli, M., Zerial, M., Rappuoli, R., Montecucco, c.: Cellular vacuoles induced by Helieobaeter pylori originate from late endosomal compartments. Proc. Nat!' Acad. Sci. USA 91, 97209724 (1994). Papini, E., Satin, B., Bucci, c., de Bernard, M., Telford, j. L., Manetti, R., Rappuoli, R., Zerial, M., Montecucco, c.: The small GTP binding protein Rab7 is essential for cellular vacuolation induced by Helieobaeter pylori cytotoxin. EMBO j. 16, 15-24 (1997) . Papini, E., Satin, B., Norais, N., de Bernard, M., Telford,j. L., Rappuoli, R., Montecucco, c.: Helicobaeter pylori vacuolating toxin increases the permeability of polarized epithelial cell monolayers. J. Clin. Invest. 102, 813-820 (1998). Parsonnet, J., Hansen, S., Rodriguez, L., Gelb, A., Warnke, R., Jellum, E., Orentreich, N., Vogelman, J. H., Friedman, G. G.: Helieobaeter pylori infection and gastric lymphoma. N. Engl. J. Med. 330, 1267-1271 (1994). Pelicic, v., Reyrat, J.-M., Sartori, L., Pagliaccia, c., RappuoIi, R., Telford, j., Montecucco, c., Papini, E.: Helieobaeter pylori VacA cytotoxin associated with the bacteria increases epithelial permeability independently of its vacuolating activity. Microbiology 145,2043-2050 (1999). Reyrat, J.-M., Lanzavecchia, S., Lupetti, S., de Bernard, M., Pagliaccia, c., Pelicic, V., Charrel, M., Ulivieri, c., Norais, N., Ji, X., Cabiaux, V., Papini, E., Rappuoli, R., Telford, j. L.: 3D structure and location in the holotoxin
Structural overview of Helicobacter cytotoxin oligomer of the 58-kDa cell binding subunit of the Helicobacter cytotoxin.]. Mol. BioI. 290, 459-470 (1999). Satin, B., Norais, N., Telford, ]. L., Rappuoli, R., Murgia, M., Montecucco, c., Papini, E.: Effect of Helicobacter pylori vacuolating toxin on maturation and extracellular release of procathepsin D and epidermal growth factor degradation.]. BioI. Chern. 272, 25022-25028 (1997). Telford, ]. L., Covacci, A., Ghiara, P., Montecucco, c., Rappuoli, R.: Unravelling the pathogenic role of Helicobacter pylori in peptic ulcer: potential new therapies and vaccines. Trends Biotechnol. 12,420-426 (1994a). Telford, ]., Ghiara, P., Dell'Orco, M., Comanducci, M., Burroni, D., Bugnoli, M., Tecce, M. E, Censini, S., Covacci, A., Xiang, Z., Papini, E., Montecucco, c., Parente, L., Rappuoli, R.: Gene structure of the Helicobacter pylori cytotoxin and evidence of its key role in gastric disease. ]. Exp. Med. 179, 1653-1658 (1994b).
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Tombola, E, Carlesso, c., Szabo, I., de Bernard, M., Reyrat, ].-M., Telford,]. L., Rappuoli, R., Montecucco, c., Papini, E., Zoratti, M.: Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: possible implications for the mechanism of cellular vacuolation. Biophys.]. 76, 1401-1409 (1999). Van der Ende, A., Rauws, E. A., Feller, M., Mulder, C. ]., Tytgat, G. N., Dankert, ].: Heterogeneous Helicobacter pylori isolates from members of a family with a history of peptic ulcer disease. Gastroenterology 111,638-717 (1996). Warren, ]. R., Marshall, B.: Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1, 1273-1275 (1983). Ye, D., Willhite, D. c., Blanke, S. R.: Identification of the minimal intracellular vacuolating domain of the Helicobacter pylori vacuolating toxin.]' BioI. Chern. 14,92779282 (1999).
InU Med. Microbial. 290,375-379 (2000) © Urban &Fischer Verlag http://www.urbanfischer.de/journals/ijmm
A structural overview of the Helicobacter cytotoxin J.-M. Reyrat 1, 2, R. Rappuoli1, 1. L. Telford 1 1 2
IRIS, Chiron S. p. A., via Fiorentina 1,1-53100 Siena, Italy Present address: Unite de Genetique Mycobacterienne, Institut Pasteur, F-75724 Paris, France
Abstract VacA, the major exotoxin produced by Helicobacter pylori, is composed of identical 87-kDa monomers that assemble into flower-shaped oligomers. The monomers can be proteolytically cleaved into two moieties of 37 and 58 kDa, or P37 and P58. The most studied property of VacA is the alteration of intracellular vesicular trafficking in eukaryotic cells leading to the formation of large vacuoles containing markers of late endosomes and lysosomes. However, VacA also causes a reduction in trans-epithelial electrical resistance in polarized mono layers and forms ion channels in lipid bilayers. The ability to induce vacuoles is localized mostly but not entirely in P37, while P58 is involved in cell targeting. Here, we review the structural aspects of VacA biology. Key words: Helicobacter pylori - cytotoxin - 3D structure
Introduction Helicobacter pylori was identified as the etiologic agent of gastritis and peptic ulcer more than 15 years ago (Warren and Marshall, 1983) (for a review (Blaser, 1993)). Contrary to acute infectious diseases, H. pylori, a microaerophilic gram-negative bacterium, causes a long lasting infection with a time scale in decades. Furthermore, H. pylori favours the development of gastroduodenal carcinoma and gastric lymphoma (Parsonnet et al., 1994). The mode of transmission of H. pylori is currently unknown but the most widely held hypothesis is that the bacterium is transmitted directly from person to person by human feces and gastric contents. The family is the core unit of H. pylori transmission. Frequently children are infected by a strain with a genetic fingerprint identical to that of one of the parents (Van der Ende et al., 1996). The children chronically maintain the same strain even after leaving home and establishing their own family. Husbands and wifes do not exchange their strains, and infection is rarely transmitted to an uninfected partner. Soon after the isolation of H. pylori, it was found that broth culture filtrates of the majority of clinical
isolates exhibited a cytotoxic activity. This cytotoxic activity is characterized by the vacuolation of diverse mammalian cell lines and the induction of gastric epithelial erosion in animal models (Leunk et al., 1988) (for a review (Cover, 1996)). Vacuole formation arises from an alteration of intracellular vesicular trafficking leading to the formation of large vacuoles containing markers of late endosomes and lysosomes (Papini et al., 1994; Molinari et al., 1997). Vacuolation requires the activity of the small GTP-binding protein Rab 7 (Satin et al., 1997). Moreover, VacA has been shown to impair the degradative properties of the endocytic pathway in HeLa cells and antigen presentation by Bcells by preventing processing and maturation of antigens (Papini et al., 1997; Molinari et al., 1998). The agent responsible for this toxic activity is a secreted oligomeric protein composed of identical 87-kDa monomers (Cover, 1996; Telford et al., 1994a). The toxin, produced as a 140-kDa precursor, is exported as a 95-kDa polypeptide after cleavage of the C-terminal 45-kDa (Telford et al., 1994a; Loveless and Saier, 1997). After release from the bacterium each monomer can be cleaved at a specific hydrophilic loop into two fragments of 37-kDa and 58-kDa that remain associat-
Corresponding author: John Telford, IRIS, Chiron S. p. A., via Fiorentina 1, 1-53100 Siena, Italy, E-mail: [email protected] 1438-4221100/290/4-5-375 $ 15.00/0
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ed in the oligomeric structure, suggesting that they may represent two distinct cytotoxin subunits (Lupetti et ai., 1996), however the location of the subunits in the holotoxin is not yet described. . Reverse genetics has permitted the characterization of the vacA gene (vacuolating cytotoxin gene) which is about 3.9 kb in size (for a review (Cover, 1996)). The gene is composed of an orthodox N-terminal signal sequence and a C-terminal domain which is cleaved during export across the outer-mebrane (Figure 1). This Cterminal domain, known as autotransporter module, is a recurrent theme among exported virulence factors (Loveless and Saier, 1997). Recently new toxic properties of VacA have been described, including permeability changes in polarized epithelial cell mono layers and channel formation in lipid bilayers (Papini et ai., 1998; Tombola et ai., 1999). It is still unknown whether vacuolization, monolayer TER decrease and gastric atrophy represent different manifestations of the same toxic activity, or whether they correspond to different activities on different targets. Transfection experiments of eukaryotic cells have shown that the entire NHrterminal domain of VacA (P37) plus a short sequence belonging to PS8 were sufficient to achieve vacuolation (de Bernard et ai., 1998; Ye et ai., 1999). It was consequently inferred that the PS8 domain was involded in cell targeting. However, it is currently unknown if the part of the PS8
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Fig. 1. (a) Phase contrast microscopy of HeLa cells vacuolated by SI-tg/ml of purified VacA; (b) schematic representation of the wild-type vacA gene and the obtained product. The level of amino-acid identity between m1 and m2 forms is indicated below. sp = signal peptide; loop = flexible loop between P37 and PS8 containing the proteolytic cleavage site.
subunit required for full toxic activity is part of a functional toxic domain or if it is required to assist folding and oligomerisation of the P37 subunit. A unique feature of the VacA cytotoxin is the allelic variation occurring in the P58 subunit (Atherton et ai., 1995). There are two alleles, m1 and m2 of a 300-amino acids region of the PS8 subunit which have target cell specificity (Pagliaccia et ai., 1998) (for a review (Covacci et ai., 1999)). Only the ml form is toxic to HeLa cells in the standard assay of vacuolization. However, both of them are active on primary gastric cells. The lack of toxicity in the HeLa cell assay was shown to be due to the inability of the m2 form to bind to the surface of HeLa cells. This was a further indication that the PS8 subunit was the binding domain of the cytotoxin. The fact that the m2 cytotoxin interacts with gastric cells but not with He La cells is not compatible with an interaction through non-specific membrane insertion since biological membranes are highly similar. Whether or not this binding process is receptor mediated remains to be determined.
Structural overview Observed by quick-freeze deep etch microscopy, VacA appears as a flower with either 6 or 7 petals, each of them corresponding to a 87-kDa monomer (Figure 2) (Lupetti et ai., 1996; Lanzavecchia et ai., 1998). The oligomeric protein is inactive, and dissociation into the 87-kDa monomer by treatment at low pH is required to reveal its activity (de Bernard et ai., 1995; Cover et ai., 1997). VacA associated with the surface of the bacteria is, however, active in the absence of low pH dissociation, suggesting that the toxin forms oligomers only after release from the bacteria (Pelicic et ai., 1999). The 87-kDa monomer of VacA can be cleaved into 2 subunits of 37 and 58-kDa following proteolysis of a flexible surfaced-exposed loop (Telford et al., 1994b). Interestingly, the connecting loop, i. e. the region where processing occurs, has been shown to play a structural role (Burroni et al., 1998). Deletion and substitution studies have shown that the shortening of the loop favours an organization into hexamers. It is likely that shortening of the connecting loop between the P37 and the PS8 subunit pull these subunits closer together which reduce the flexibility of the interaction and consequently the number of monomers which can enter into the oligomeric structure. However, despite the fact that a petal represents a subunit, the molecular organisation between the P37 and the P58 subunit remained largely obscure. In order to decipher the structural relationship between each building block, the PS8 moiety was subjected to analysis (Reyrat et ai., 1999). Using the sacB counter-selectable marker, we
Structural overview of Helicobacter cytotoxin have engineered a strain of H. pylori to delete the gene sequence coding for the P37 subunit. The remaining PS8 subunit is expressed efficiently and exported as a soluble dimer, suggesting a structural autonomy for this domain. This PS8 dimer is able to bind target cells in a manner similar to the holotoxin, thus confiming previous studies suggesting that the binding activity is localized in the PS8 subunits (Ye et al., 1999; Pagliaccia et al., 1998; Garner and Cover, 1996). However, the bound dimer is not internalized by the target cell, suggesting that the hexameric structure is needed for efficient endocytosis. In addition the PS8 molecule is non-toxic in every model tested (Tombola et al., 1999; Reyrat et al., 1999), thus confirming that the P37 part is largely involved in toxic activity (de Bernard et al., 1998; Ye et al., 1999). However, the nature of the toxic activity and the cytosolic target remain so far uncharacterized. The PS8 molecule has been purified to homogeneity and subjected to structural analysis by quick-freeze deep-etch microscopy. 3D image reconstruction of the PS8 moiety replicas permitted the reconstruction of the global architecture of the P58 molecule and to propose a model for the wild-type holotoxin. The images confirmed the dime ric structure of the PS8 molecule and revealed that each half of the molecule had a curved appearance remarkably similar to the peripheral arms of the wild-type oligomer (Figure 2). The dimeric structure of the PS8 molecule may reflect the interaction, in the intact molecule, involved in holding together the oligomeric structure. On the other hand, the dimerization may be due to interaction between hydrophobic surfaces normally masked in the wild-type molecule. This has led to a model in which the monomers are intercalated with each other to form the ring structure (Fig. 3). In Figure 3B, the structure of half a dimer of the P58 molecule has been arranged in a hexameric ring corresponding to the diameter of the intact oli-
377
I
Fig. 2. 3D top views of the reconstructions of the mold. (A) wild-type molecule, (B) PS8 molecule.
gomer. The two parts match each other perfectly, and the difference in height gives a clear idea of the arrangement of the P37 subunits with respect to the PS8 subunits. However, it is not clear which P37 and PS8 subunits belong to the same 87-kDa monomer. However, we favour a model in which the monomers are intercalated with each other to form the ring structure in such a way that the 37 -kDa subunit of one monomer lies on top of the S8-kDa subunit of the adjacent monomer as shown in Figure 3C. In brief, the oligomeric structure is maintained by interaction of one monomer with the S8-kDa subunit of the adjacent monomer (Reyrat et al., 1999).
Concluding remarks A large amount of data has been accumulated on VacA biology. Genetic studies have shown that the toxic activity is localized mostly in the P37 domain, whereas the binding activity is localized in the P58 subunit. This relates VacA to the AB type family, however caution is suggested by the fact that endocytosis after surface binding requires the putative active domain (P37).
Fig. 3. (A) 3D top view of WT VacA replicas, (B) 3D top view of the hexameric ring of PS8 subunits, assembled in silico by use of reconstructed replicas. (C) model of the interaction of the 87-kDa monomer in the oligomeric structure. In the proposed model, alternate monomers in the oligomeric structure are shown in different shadings in order to represent the interaction of the P37 subunit of one monomer with the PS8 subunit of the adjacent monomer. PS8, PS8 subunit; P37, P37 subunit, dotted line, connecting loop between PS8 and P37 subunit.
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Moreover PS8 alone lacks pore-forming actlVlty a property which again requires the presence of P37. VacA may possess an interesting and perhaps unique molecular structure as it shares both structural and intoxication features of both AB and pore-forming toxins. It is not clear why the allelic variation of the cellbinding subunit has evolved, but it may reflect human genetic polymorphism because the m1 form is predominant in Western, Korean, and Japanese isolates, whereas the m2 form is found in 7S % of Chinese isolates (Pan et al., 1998). VacA is clearly a key player of peptic ulcer and the next step is to characterize both the cellular target and the putative surface receptor. Acknowledgements. We are grateful to J. Triccas for critical reading of the manuscript. G. Corsi is acknowledged for artwork. We thank Bruce di Montechiaro for stimulating discussIOns.
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