RESUMEN

The three parts (Stx17B, Stx27B and StxB) of Shiga toxin B subunit have been fused into a cell surface exposed loop of the LamB protein at a BamH I site between residues 153 and 154. Western blotting revealed that the three parts of Shiga toxin B subunit could be expressed as the LamB fusion proteins in E. coli. Indirect immunofluorescence and immunoelectron microscopy analyses showed fusion proteins LamB/Stx17B and LamB/Stx27B could be expressed at cell surface in E. coli, but fusion protein LamB/StxB could not be expressed at cell surface; it was aggregated in cytoplasm and was toxic to host. This expression system provided a new way to construct an oral live vaccine against Shigella dysenteriae 1.

Asunto(s)

Toxinas Bacterianas/genética , Escherichia coli/genética , Receptores Virales/genética , Shigella dysenteriae , Proteínas de la Membrana Bacteriana Externa , Toxinas Bacterianas/biosíntesis , Bacteriófago lambda/metabolismo , Escherichia coli/metabolismo , Expresión Génica , Porinas , Receptores Virales/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Toxinas Shiga

RESUMEN

Lipopolysaccharide (LPS) and outer membrane protein (OMP) preparations of Bordetella pertussis were incorporated into multilamellar liposomes composed of soya bean-derived phospholipids which were then used for oral and intranasal immunization of mice. Specific antibody responses of animals immunized by either route were measured in lung washes. A specific IgA response to LPS was detected after immunization with the OMP-containing liposomes but not with the LPS-containing liposomes, indicating adjuvant activity of the proteins. The OMP-containing liposomes were significantly more effective in inducing immune responses than the OMP preparation alone. Responses were highest when mice were given a booster 30 days after primary immunization. Maximum responses occurred 20 days after the booster but specific antibody was still detected 75 days after the secondary immunization. These results suggest that this liposome antigen delivery system has potential in stimulating secretory antibody responses which may be helpful in protecting against infection from B. pertussis.

Asunto(s)

Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Antígenos de Superficie/inmunología , Bordetella pertussis/inmunología , Pulmón/inmunología , Administración Intranasal , Administración Oral , Animales , Antígenos Bacterianos/administración & dosificación , Antígenos de Superficie/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/inmunología , Western Blotting , Femenino , Humanos , Lipopolisacáridos , Liposomas , Ratones , Ratones Endogámicos BALB C

RESUMEN

In this study, the Shiga toxin B subunit has been fused to haemolysin A C-terminus. The fusion protein StxB/HlyA(CT) can be exported to the external medium not only from E. coli K-12 but also from S. typhimurium aroA strain SL3261. When the plasmid pUC18 was used as vector, StxB/HlyA(CT) was toxic to hosts. But the fusion protein was stable and safe to hosts when the pUC18 was replaced with pBR322. The fusion protein can be expressed and exported to the external medium under either the aerobactin promoter or beta-lactomase promoter. Oral and i.p. immunization of mice with StxB/HlyA(CT) carrying S. typhimurium aroA strain SL3261 resulted in significant B subunit specific mucosal and serum antibody responses. This the first report demonstrating that foreign polypeptides fused to the 23kD C-terminus of E. coli haemolysin A can be exported from attenuated Salmonella Vaccine strains and that such exported polypeptides can result in antigen specific immune responses.

Asunto(s)

Toxinas Bacterianas/biosíntesis , Proteínas Hemolisinas/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Shigella dysenteriae/patogenicidad , Animales , Anticuerpos Antibacterianos/biosíntesis , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Western Blotting , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Toxinas Shiga

RESUMEN

The Shiga toxin B-subunit has been fused to the 23-kD C-terminus of Escherichia coli haemolysin A (HlyA) and exported from attenuated antigen carrier strain of Salmonella typhimurium aroA (SL3261). The expression of the gene fusion under the control of a synthetic modified beta-lactamase promoter (constitutive expression) and under the iron-regulated aerobactin promoter showed that the fusion protein could be stably expressed and exported out of the bacterial cell in significant amounts so long as high copy number plasmids were not used. Oral and i.p. immunization of mice with the hybrid salmonellae resulted in significant B-subunit specific mucosal and serum antibody responses. A comparative analysis of the location of hybrid proteins in the antigen carrier bacterial cell (i.e. cytoplasmic expression and extracellular export) has shown that both modes of expression result in antigen-specific immune responses. This is the first report demonstrating that foreign polypeptides fused to the 23-kD C-terminus of E. coli haemolysin A can be exported from attenuated Salmonella vaccine strains and that such exported polypeptides can result in antigen-specific immune responses.

Asunto(s)

Transferasas Alquil y Aril , Anticuerpos Antibacterianos/sangre , Toxinas Bacterianas/inmunología , Proteínas de Escherichia coli , Shigella/inmunología , Vacunas Sintéticas/inmunología , 3-Fosfoshikimato 1-Carboxiviniltransferasa , Administración Oral , Animales , Formación de Anticuerpos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/genética , Western Blotting , Escherichia coli/genética , Femenino , Proteínas Hemolisinas/biosíntesis , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/inmunología , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos BALB C , Plásmidos/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Salmonella typhimurium/genética , Toxinas Shiga

RESUMEN

The complete Shiga toxin B subunit and two N-terminal segments of the B subunit have been inserted into a cell surface exposed loop of the LamB protein, and expression of the hybrid proteins from three different promoter systems, i.e., (i) an in vitro-inducible tac promoter that provides high-level expression, (ii) the iron-regulated aerobactin promoter presumably induced in vivo under the iron-limiting conditions of the intestinal mucosal environment, and (iii) a synthetic, modified beta-lactamase promoter providing moderate level constitutive expression, has been analyzed in Escherichia coli, Salmonella typhimurium, and attenuated antigen carrier strains of S. typhimurium (aroA mutants). The hybrid vaccine strains were used to immunize mice by the oral and intraperitoneal routes. S. typhimurium aroA mutants apparently have a membrane export defect which prevents the transport of LamB and its derivatives across the cytoplasmic membrane. High-level expression of hybrid proteins through use of the tac promoter proved deleterious to the vaccine strains and prevented the production of viable cells at reasonable cell densities. The lower levels of gene expression observed with the beta-lactamase and aerobactin promoters did not have this effect. Immunization of mice with S. typhimurium aroA strains carrying the hybrid genes expressed from these two promoters resulted in significant B subunit-specific mucosal and serum antibody responses. This suggests that such expression systems may be useful when incorporated into candidate antidysentery live oral vaccines for inducing protection against the effect of Shiga toxin in infections caused by Shigella dysenteriae 1 and other Shiga toxin-or Shiga-like toxin-producing pathogens.

Asunto(s)

Anticuerpos Antibacterianos/análisis , Proteínas de la Membrana Bacteriana Externa/inmunología , Toxinas Bacterianas/inmunología , Vacunas Bacterianas/inmunología , Receptores Virales/inmunología , Proteínas Recombinantes de Fusión/inmunología , Salmonella typhimurium/genética , Shigella dysenteriae/inmunología , Vacunas Sintéticas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Toxinas Bacterianas/análisis , Toxinas Bacterianas/genética , Secuencia de Bases , Western Blotting , Femenino , Operón Lac , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Plásmidos , Porinas , Regiones Promotoras Genéticas , Receptores Virales/análisis , Receptores Virales/genética , Proteínas Recombinantes de Fusión/análisis , Toxinas Shiga

Asunto(s)

Endotoxinas/metabolismo , Lipopolisacáridos/metabolismo , Shigella/química , Animales , Vacunas Bacterianas/inmunología , Bacteriófagos/genética , Secuencia de Carbohidratos , Endotoxinas/química , Herencia Extracromosómica , Genes Bacterianos , Humanos , Lipopolisacáridos/química , Datos de Secuencia Molecular , Antígenos O , Plásmidos , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/inmunología , Polisacáridos Bacterianos/metabolismo , Shigella/genética , Shigella/inmunología , Shigella/patogenicidad , Especificidad de la Especie , Virulencia

RESUMEN

Biosynthesis of the Salmonella typhimurium LT2 O antigen is encoded by genes which map in the rfb cluster. The cloning and restriction enzyme analysis of part of this cluster have been described previously (H. N. Brahmbhatt, N. B. Quigley, and P. R. Reeves, Mol. Gen. Genet. 203:172-176, 1986). The entire rfb gene cluster has now been cloned, and a detailed restriction enzyme map has been constructed which has enabled us to map the approximate positions of individual rfb genes.

Asunto(s)

Antígenos Bacterianos/genética , Genes Bacterianos , Salmonella typhimurium/genética , Mapeo Cromosómico , Clonación Molecular , Enzimas de Restricción del ADN , ADN Bacteriano/genética , Hibridación de Ácido Nucleico , Antígenos O , Plásmidos , Salmonella typhimurium/enzimología , Salmonella typhimurium/inmunología

RESUMEN

A low copy number cosmid was constructed by subcloning the pair of cos sites and the kanamycin resistance gene of pcos2EMBL into pGB2. The resulting cosmid, pPR691, has the pSC101 replicon and specifies resistance to kanamycin, spectinomycin, and streptomycin. pPR691 also carries restriction sites suitable for cloning partial Sau3A digests using the strategy of Bates and Swift (P. F. Bates and R. A. Swift, 1983, Gene 26, 137-146). A library of Salmonella typhimurium chromosomal DNA was made using this cosmid and the rfb gene cluster (map position 42) was isolated from this library.

Asunto(s)

Clonación Molecular , Cósmidos , Escherichia coli/genética , Enzimas de Restricción del ADN , Farmacorresistencia Microbiana , Kanamicina/farmacología , Factores R

RESUMEN

The rfb gene cluster of Salmonella typhimurium encodes the enzymes required for the biosynthesis of the O-Antigen. A part of it has been cloned in plasmid vectors pBR322 and pUC9 using an adjacent, previously cloned, part of the his operon (Barnes 1981) as a molecular probe for the first clone. A detailed restriction enzyme map of 7.57 kb of rfb DNA is presented and the approximate locations of two of the genes, rfbK and rfbM have been defined.

Asunto(s)

Antígenos Bacterianos/genética , Antígenos de Superficie/genética , Clonación Molecular , Genes Bacterianos , Genes , Lipopolisacáridos/genética , Salmonella typhimurium/genética , Enzimas de Restricción del ADN , Vectores Genéticos , Antígenos O , Plásmidos , Salmonella typhimurium/inmunología