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1.
Biophys J ; 81(6): 3204-15, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11720986

RESUMO

VacA is a pore-forming cytotoxin produced by Helicobacter pylori in several strain-specific isoforms, which have been classified in two main families, m1 and m2, according to the sequence of a variable "midregion." Both forms are associated with gastric pathologies and can induce vacuolation of cultured cells. The comparison of two representative toxins, m1 17874 and m2 9554, has indicated that the m2 form is less powerful in vacuolation assays and that its effects are more strongly cell type dependent. To rationalize these differences and to investigate structure-function relationships in this toxin, we have compared the properties of the channels formed by these two variants and by a construct derived from 17874 by deleting a loop that connects the two toxin domains, which is shorter in 9554 than in 17874. Although the channels formed by all three proteins are similar, m2 9554 channels have, on average, a lower conductance and are less anion-selective and more voltage-dependent than the m1 pores. Furthermore, the rate of incorporation of 9554 VacA into planar bilayers depends on lipid composition much more strongly than that of 17874. The comparison with the behavior of the loop deletion mutant indicates that this latter property, as well as a portion of the conductance decrease, may be attributed to the reduction in loop length. The differences in pore properties are proposed to account in part for the different cytotoxicity exhibited by the two toxin isoforms. We furthermore present evidence suggesting that the conformation of the membrane-embedded toxin may be influenced by the lipid composition of the membrane itself.


Assuntos
Proteínas de Bactérias/química , Helicobacter pylori/química , Sequência de Aminoácidos , Aminoácidos/química , Condutividade Elétrica , Eletrofisiologia , Células HeLa , Humanos , Cinética , Lipídeos/química , Dados de Sequência Molecular , Isoformas de Proteínas , Estrutura Terciária de Proteína , Sais/química , Homologia de Sequência de Aminoácidos
2.
Infect Immun ; 68(6): 3754-7, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10816542

RESUMO

There are two alleles of the vacuolating cytotoxin gene from Helicobacter pylori, which code for toxins with different cell specificities. By analyzing the phenotypes of natural and artificial chimeras between the two forms of the protein, we have delimited a short stretch of amino acids which determine the cell specificity.


Assuntos
Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Citotoxinas/toxicidade , Helicobacter pylori/patogenicidade , Polimorfismo Genético , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Citotoxinas/genética , Células Epiteliais , Células HeLa , Helicobacter pylori/genética , Humanos , Rim/citologia , Dados de Sequência Molecular , Coelhos , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Vacúolos
3.
Eur J Biochem ; 267(1): 104-9, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10601856

RESUMO

In its mature form, the VacA toxin of Helicobacter pylori is a 95-kDa protein which is released from the bacteria as a low-activity complex. This complex can be activated by low-pH treatment that parallels the activity of the toxin on target cells. VacA has been previously shown to insert itself into lipid membranes and to induce anion-selective channels in planar lipid bilayers. Binding of VacA to lipid vesicles and its ability to induce calcein release from these vesicles were systematically compared as a function of pH. These two phenomena show a different pH-dependence, suggesting that the association with the lipid membrane may be a two-step mechanism. The secondary and tertiary structure of VacA as a function of pH and the presence of lipid vesicles were investigated by Fourier-transform infrared spectroscopy. The secondary structure of VacA is identical whatever the pH and the presence of a lipid membrane, but the tertiary structure in the presence of a lipid membrane is dependent on pH, as evidenced by H/D exchange.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Helicobacter pylori/química , Bicamadas Lipídicas/metabolismo , Deutério/metabolismo , Fluoresceínas/metabolismo , Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Bicamadas Lipídicas/química , Lipossomos/química , Lipossomos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolipídeos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier
4.
J Mol Biol ; 290(2): 459-70, 1999 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-10390344

RESUMO

Pathogenic strains of Helicobacter pylori produce a potent exotoxin, VacA, which intoxicates gastric epithelial cells and leads to peptic ulcer. The toxin is released from the bacteria as a high molecular mass homo-oligomer of a 95 kDa polypeptide which undergoes specific proteolytic cleavage to 37 kDa and 58 kDa subunits. We have engineered a strain of H. pylori to delete the gene sequence coding for the 37 kDa subunit. The remaining 58 kDa subunit is expressed efficiently and exported as a soluble dimer that is non-toxic but binds target cells in a manner similar to the holotoxin. A 3D reconstruction of the molecule from electron micrographs of quick-freeze, deep-etched preparations reveals the contribution of each building block to the structure and permits the reconstruction of the oligomeric holotoxin starting from individual subunits. In this model P58 subunits are assembled in a ring structure with P37 subunits laying on the top. The data indicate that the 58 kDa subunit is capable of folding autonomously into a discrete structure recognizable within the holotoxin and containing the cell binding domain.


Assuntos
Proteínas de Bactérias/ultraestrutura , Citotoxinas/química , Helicobacter pylori , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Sobrevivência Celular , Citotoxinas/metabolismo , Citotoxinas/toxicidade , Dimerização , Endocitose , Escherichia coli/genética , Técnica de Congelamento e Réplica , Células HeLa , Humanos , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Solubilidade , Vacúolos/ultraestrutura
5.
Infect Immun ; 66(11): 5547-50, 1998 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9784571

RESUMO

The Helicobacter pylori cytotoxin is proteolytically cleaved at a flexible hydrophilic loop into two subunits. Deletion of the loop sequences had no effect on biological activity of the toxin in the HeLa cell vacuolation assay but favored the organization of the protein into hexameric rather than heptameric structures.


Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Citotoxinas/genética , Helicobacter pylori/imunologia , Deleção de Sequência/genética , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Citotoxinas/química , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Deleção de Sequência/imunologia
6.
Proc Natl Acad Sci U S A ; 95(17): 10212-7, 1998 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-9707626

RESUMO

The Helicobacter pylori toxin VacA causes vacuolar degeneration in mammalian cell lines in vitro and plays a key role in peptic ulcer disease. Two alleles, m1 and m2, of the mid-region of the vacA gene have been described, and the m2 cytotoxin always has been described as inactive in the in vitro HeLa cell assay. However, the m2 allele is associated with peptic ulcer and is prevalent in populations in which peptic ulcer and gastric cancer have high incidence. In this paper, we show that, despite the absence of toxicity on HeLa cells, the m2 cytotoxin is able to induce vacuolization in primary gastric cells and in other cell lines such as RK-13. The absence of Hela cell activity is due to an inability to interact with the cell surface, suggesting a receptor-mediated interaction. This result is consistent with the observation that the m2 allele is found in a population that has a high prevalence of peptic ulcer disease and gastric cancer. VacA is the first bacterial toxin described for which the same active subunit can be delivered by different receptor binding domains.


Assuntos
Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Citotoxinas/toxicidade , Helicobacter pylori/patogenicidade , Alelos , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Sequência de Bases , Citotoxinas/genética , Citotoxinas/fisiologia , Primers do DNA/genética , DNA Bacteriano/genética , Genes Bacterianos , Células HeLa , Helicobacter pylori/genética , Helicobacter pylori/fisiologia , Humanos , Úlcera Péptica/etiologia , Úlcera Péptica/microbiologia , Reação em Cadeia da Polimerase , Neoplasias Gástricas/etiologia , Neoplasias Gástricas/microbiologia , Transfecção , Vacúolos/efeitos dos fármacos , Virulência
7.
FEMS Microbiol Lett ; 165(1): 79-84, 1998 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-9711842

RESUMO

The vacuolating cytotoxin (VacA) is a major virulence factor of Helicobacter pylori which is not yet well characterised and is difficult to obtain in large quantities. Here we describe the production of a monoclonal antibody that recognises the native but not the denatured form of VacA. The antibody can be efficiently used in affinity chromatography for one-step purification of large quantities of VacA from culture supernatants. Elution at acidic pH dissociates the oligomeric molecule into monomers that reanneal in a time-dependent fashion. The purified cytotoxin is able to bind, and to intoxicate HeLa cells.


Assuntos
Anticorpos Monoclonais , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Helicobacter pylori/química , Animais , Anticorpos Antibacterianos/biossíntese , Anticorpos Antibacterianos/isolamento & purificação , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Células HeLa/metabolismo , Helicobacter pylori/imunologia , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Microscopia de Fluorescência
8.
Vaccine ; 16(1): 33-7, 1998 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9607006

RESUMO

We have previously shown that infection of mice with H. pylori can be prevented by oral immunization with H. pylori antigens given together with E. coli heat-labile enterotoxin (LT) as adjuvant. Since LT cannot be used in humans because of its unacceptable toxicity, we investigated whether protection of mice could be achieved by co-administration of antigens with non-toxic LT mutants. Here we show that CD1/SPF mice are protected against infection after oral vaccination with either purified H. pylori antigens (native and recombinant VacA, urease and CagA), or whole-cell vaccine formulations, given together with the non-toxic mutant LTK63 as a mucosal adjuvant. Furthermore we show that such protection is antigen-specific since immunization with recombinant or native VacA plus LTK63 conferred protection against infection by an H. pylori Type I strain, which expresses VacA, but not against challenge with a Type II strain which is not able to express this antigen. These results show that: (1) protection against H. pylori can be achieved in the mouse model of infection using subunit recombinant constructs plus non-toxic mucosal adjuvants; and (2) this mouse model is an useful tool in testing H. pylori vaccine formulations for eventual use in humans.


Assuntos
Adjuvantes Imunológicos , Antígenos de Bactérias/imunologia , Toxinas Bacterianas , Vacinas Bacterianas/uso terapêutico , Enterotoxinas , Proteínas de Escherichia coli , Infecções por Helicobacter/prevenção & controle , Helicobacter pylori/imunologia , Animais , Vacinas Bacterianas/imunologia , Masculino , Camundongos , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/uso terapêutico
9.
Arch Microbiol ; 168(5): 437-40, 1997 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9325433

RESUMO

Pertactin, which is a membrane-associated antigen of Bordetella pertussis and which is present in many acellular vaccines against whooping cough, has been reported to be similar to the homologous protein in Bordetella bronchiseptica. By running parallel experiments using proteins derived from the two species, we show that the isoelectric point of pertactin from B. pertussis is lower than reported and clearly distinguishable from the homologous protein of B. bronchiseptica.


Assuntos
Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/química , Bordetella bronchiseptica/imunologia , Bordetella pertussis/imunologia , Fatores de Virulência de Bordetella , Antígenos de Bactérias/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Ponto Isoelétrico , Especificidade da Espécie
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