RESUMEN
Bacterial ATP-binding cassette (ABC) transporters play a crucial role in the physiology and pathogenicity of different bacterial species. Components of ABC transporters have also been tested as target antigens for the development of vaccines against different bacterial species, such as those belonging to the Streptococcus genus. Streptococcus mutans is the etiological agent of dental caries, and previous studies have demonstrated that deletion of the gene encoding PstS, the substrate-binding component of the phosphate uptake system (Pst), reduced the adherence of the bacteria to abiotic surfaces. In the current study, we generated a recombinant form of the S. mutans PstS protein (rPstS) with preserved structural features, and we evaluated the induction of antibody responses in mice after sublingual mucosal immunization with a formulation containing the recombinant protein and an adjuvant derived from the heat-labile toxin from enterotoxigenic Escherichia coli strains. Mice immunized with rPstS exhibited systemic and secreted antibody responses, measured by the number of immunoglobulin A-secreting cells in draining lymph nodes. Serum antibodies raised in mice immunized with rPstS interfered with the adhesion of bacteria to the oral cavity of naive mice challenged with S. mutans. Similarly, mice actively immunized with rPstS were partially protected from oral colonization after challenge with the S. mutans NG8 strain. Therefore, our results indicate that S. mutans PstS is a potential target antigen capable of inducing specific and protective antibody responses after sublingual administration. Overall, these observations raise interesting perspectives for the development of vaccines to prevent dental caries.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Caries Dental/prevención & control , Inmunización/métodos , Boca/microbiología , Proteínas de Unión a Fosfato/inmunología , Streptococcus mutans/crecimiento & desarrollo , Streptococcus mutans/inmunología , Adyuvantes Inmunológicos , Administración Sublingual , Animales , Antígenos Bacterianos/inmunología , Adhesión Bacteriana , Vacunas Bacterianas/inmunología , Caries Dental/microbiología , Escherichia coli Enterotoxigénica/química , Femenino , Inmunidad Mucosa , Inmunoglobulina A/análisis , Ratones , Proteínas de Unión a Fosfato/administración & dosificación , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Saliva/inmunologíaRESUMEN
Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigA(C)) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigA(C), either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigA(C) or LigA(C) coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Flagelina/administración & dosificación , Leptospira interrogans/inmunología , Leptospirosis/prevención & control , Hidróxido de Aluminio/administración & dosificación , Animales , Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/genética , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Inmunoglobulina G/sangre , Riñón/microbiología , Leptospira interrogans/genética , Leptospirosis/inmunología , Masculino , Mesocricetus , Análisis de Supervivencia , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
The cause of Alzheimer's disease is still unknown, but the disease is distinctively characterized by the accumulation of ß-amyloid plaques and neurofibrillary tangles in the brain. These features have become the primary focus of much of the research looking for new treatments for the disease, including immunotherapy and vaccines targeting ß-amyloid in the brain. Adverse effects observed in a clinical trial based on the ß-amyloid protein were attributed to the presence of the target antigen and emphasized the relevance of finding safer antigen candidates for active immunization. For this kind of approach, different vaccine formulations using DNA, peptide, and heterologous prime-boost immunization regimens have been proposed. Promising results are expected from different vaccine candidates encompassing B-cell epitopes of the ß-amyloid protein. In addition, recent results indicate that targeting another protein involved in the etiology of the disease has opened new perspectives for the effective prevention of the illness. Collectively, the evidence indicates that the idea of finding an effective vaccine for the control of Alzheimer's disease, although not without challenges, is a possibility.
Asunto(s)
Enfermedad de Alzheimer/prevención & control , Vacunas contra el Alzheimer/clasificación , Péptidos beta-Amiloides/metabolismo , Placa Amiloide/inmunología , Enfermedad de Alzheimer/patología , Anticuerpos Monoclonales/uso terapéutico , Encéfalo/patología , Ensayos Clínicos como Asunto/tendencias , Humanos , Ovillos Neurofibrilares , Vacunación/métodosRESUMEN
Flagellin is the structural protein and most abundant component of bacterial flagella. The flagellum filament contains around 20,000 100,000 subunits of 50 kDa flagellin that can have diversebiotechnological applications such as vaccine adjuvant and cellular protector during chemo- and radiotherapy.The main aim of this work was to study a production process of purified native FliC flagellin of Salmonella Typhimurium. The culture conditions in shakers were established with medium devoid of animal-derived components. In bioreactors, culture conditions were established in order to obtain flagellin from the culture supernatant by tangential ultrafiltration (TUF). The concentrated 750 kDa cut-off TUF fraction had a purification factor of 1.5 and a recovery yield of 52.2% for flagellin. The volumetric production of flagellin using the described procedure achieved around 307 mg/L of culture, which represented a significant improvement over previously reported methods. These results permit the development of production and purification processes that can be easily scaled up.
Asunto(s)
Flagelina/aislamiento & purificación , Reactores Biológicos/microbiología , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/aislamiento & purificación , Fermentación , Ultrafiltración/métodosRESUMEN
Anti-cancer DNA vaccines have attracted growing interest as a simple and non-invasive method for both the treatment and prevention of tumors induced by human papillomaviruses. Nonetheless, the low immunogenicity of parenterally administered vaccines, particularly regarding the activation of cytotoxic CD8+ T cell responses, suggests that further improvements in both vaccine composition and administration routes are still required. In the present study, we report the immune responses and anti-tumor effects of a DNA vaccine (pgD-E7E6E5) expressing three proteins (E7, E6, and E5) of the human papillomavirus type 16 genetically fused to the glycoprotein D of the human herpes simplex virus type 1, which was administered to mice by the intradermal (id) route using a gene gun. A single id dose of pgD-E7E6E5 (2 µg/dose) induced a strong activation of E7-specific interferon-γ (INF-γ)-producing CD8+ T cells and full prophylactic anti-tumor effects in the vaccinated mice. Three vaccine doses inhibited tumor growth in 70 percent of the mice with established tumors. In addition, a single vaccine dose consisting of the co-administration of pgD-E7E6E5 and the vector encoding interleukin-12 or granulocyte-macrophage colony-stimulating factor further enhanced the therapeutic anti-tumor effects and conferred protection to 60 and 50 percent of the vaccinated mice, respectively. In conclusion, id administration of pgD-E7E6E5 significantly enhanced the immunogenicity and anti-tumor effects of the DNA vaccine, representing a promising administration route for future clinical trials.
Asunto(s)
Animales , Femenino , Ratones , Vacunas contra el Cáncer/administración & dosificación , /inmunología , Proteínas Oncogénicas Virales/inmunología , Simplexvirus/inmunología , Vacunas de ADN/administración & dosificación , Proteínas del Envoltorio Viral/inmunología , /inmunología , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , /genética , Inyecciones Intradérmicas , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/prevención & control , Proteínas Oncogénicas Virales/genética , Simplexvirus/genética , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
Anti-cancer DNA vaccines have attracted growing interest as a simple and non-invasive method for both the treatment and prevention of tumors induced by human papillomaviruses. Nonetheless, the low immunogenicity of parenterally administered vaccines, particularly regarding the activation of cytotoxic CD8+ T cell responses, suggests that further improvements in both vaccine composition and administration routes are still required. In the present study, we report the immune responses and anti-tumor effects of a DNA vaccine (pgD-E7E6E5) expressing three proteins (E7, E6, and E5) of the human papillomavirus type 16 genetically fused to the glycoprotein D of the human herpes simplex virus type 1, which was administered to mice by the intradermal (id) route using a gene gun. A single id dose of pgD-E7E6E5 (2 µg/dose) induced a strong activation of E7-specific interferon-γ (INF-γ)-producing CD8+ T cells and full prophylactic anti-tumor effects in the vaccinated mice. Three vaccine doses inhibited tumor growth in 70% of the mice with established tumors. In addition, a single vaccine dose consisting of the co-administration of pgD-E7E6E5 and the vector encoding interleukin-12 or granulocyte-macrophage colony-stimulating factor further enhanced the therapeutic anti-tumor effects and conferred protection to 60 and 50% of the vaccinated mice, respectively. In conclusion, id administration of pgD-E7E6E5 significantly enhanced the immunogenicity and anti-tumor effects of the DNA vaccine, representing a promising administration route for future clinical trials.
Asunto(s)
Vacunas contra el Cáncer/administración & dosificación , Papillomavirus Humano 16/inmunología , Proteínas Oncogénicas Virales/inmunología , Simplexvirus/inmunología , Vacunas de ADN/administración & dosificación , Proteínas del Envoltorio Viral/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Femenino , Papillomavirus Humano 16/genética , Inyecciones Intradérmicas , Ratones , Ratones Endogámicos C57BL , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/prevención & control , Proteínas Oncogénicas Virales/genética , Simplexvirus/genética , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) producedbyenterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains.
Até o presente o momento, não há vacina ou imunoterapia disponível para pacientes com Síndrome Hemolítica Urêmica (SHU) induzida pela toxina Shiga-like (Stx) produzida por linhagens de Escherichia coli entero-hemorragica (EHEC), tais como as pertencentes ao sorotipo O157:H7. Neste trabalho, avaliamos a performance de Bacillus subtilis, uma espécie bacteriana gram-positiva não-patogênica formadora de esporos, como veículo vacinal para a expressão da subunidade B da Stx2B (Stx2B). Uma linhagem vacinal recombinante de B. subtilis expressando Stx2B, sob o controle de um promoter induzível por estresse, foi administrada a camundongos BALB/c por via oral, nasal ou subcutânea usando células vegetativas e esporos. Camundongos imunizados com células vegetativas e esporos pela via oral desenvolveram títulos anti-Stx2B baixos, mas específicos, de IgG sérico e IgA fecal, enquanto camundongos imunizados com esporos recombinates desenvolveram resposta anti-Stx2B apenas após a administração pela via parenteral. No entanto, anticorpos produzidos em camundongos imunizados com a linhagem recombinante de B. subtilis não inibiram os efeitos tóxicos da toxina nativa em condições in vitro e in vivo, sugerindo que a quantidade e/ou a qualidade da resposta imune gerada não suportam uma neutralização efetiva da Stx2 produzidas por linhagens de EHEC.
Asunto(s)
Animales , Ratones , Escherichia coli Enterohemorrágica , Anticuerpos Antibacterianos/análisis , Bacillus subtilis/aislamiento & purificación , Técnicas In Vitro , Vacunas Bacterianas , Ratones , Esporas Bacterianas , Métodos , Serotipificación , MétodosRESUMEN
Maltose-binding protein is the periplasmic component of the ABC transporter responsible for the uptake of maltose/maltodextrins. The Xanthomonas axonopodis pv. citri maltose-binding protein MalE has been crystallized at 293 K using the hanging-drop vapour-diffusion method. The crystal belonged to the primitive hexagonal space group P6(1)22, with unit-cell parameters a = 123.59, b = 123.59, c = 304.20 A, and contained two molecules in the asymetric unit. It diffracted to 2.24 A resolution.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Xanthomonas axonopodis/química , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Secuencia Conservada , Cristalización , Recolección de Datos/métodos , Maltosa/química , Maltosa/metabolismo , Proteínas de Unión a Maltosa , Polisacáridos/química , Polisacáridos/metabolismo , Relación Estructura-Actividad , Xanthomonas axonopodis/patogenicidadRESUMEN
No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) produced by enterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains.
RESUMEN
No effective vaccine or immunotherapy is presently available for patients with the hemolytic uremic syndrome (HUS) induced by Shiga-like toxin (Stx) producedbyenterohaemorragic Escherichia coli (EHEC) strains, such as those belonging to the O157:H7 serotype. In this work we evaluated the performance of Bacillus subtilis strains, a harmless spore former gram-positive bacterium species, as a vaccine vehicle for the expression of Stx2B subunit (Stx2B). A recombinant B. subtilis vaccine strain expressing Stx2B under the control of a stress inducible promoter was delivered to BALB/c mice via oral, nasal or subcutaneous routes using both vegetative cells and spores. Mice immunized with vegetative cells by the oral route developed low but specific anti-Stx2B serum IgG and fecal IgA responses while mice immunized with recombinant spores developed anti-Stx2B responses only after administration via the parenteral route. Nonetheless, serum anti-Stx2B antibodies raised in mice immunized with the recombinant B. subtilis strain did not inhibit the toxic effects of the native toxin, both under in vitro and in vivo conditions, suggesting that either the quantity or the quality of the induced immune response did not support an effective neutralization of Stx2 produced by EHEC strains.
Até o presente o momento, não há vacina ou imunoterapia disponível para pacientes com Síndrome Hemolítica Urêmica (SHU) induzida pela toxina Shiga-like (Stx) produzida por linhagens de Escherichia coli entero-hemorragica (EHEC), tais como as pertencentes ao sorotipo O157:H7. Neste trabalho, avaliamos a performance de Bacillus subtilis, uma espécie bacteriana gram-positiva não-patogênica formadora de esporos, como veículo vacinal para a expressão da subunidade B da Stx2B (Stx2B). Uma linhagem vacinal recombinante de B. subtilis expressando Stx2B, sob o controle de um promoter induzível por estresse, foi administrada a camundongos BALB/c por via oral, nasal ou subcutânea usando células vegetativas e esporos. Camundongos imunizados com células vegetativas e esporos pela via oral desenvolveram títulos anti-Stx2B baixos, mas específicos, de IgG sérico e IgA fecal, enquanto camundongos imunizados com esporos recombinates desenvolveram resposta anti-Stx2B apenas após a administração pela via parenteral. No entanto, anticorpos produzidos em camundongos imunizados com a linhagem recombinante de B. subtilis não inibiram os efeitos tóxicos da toxina nativa em condições in vitro e in vivo, sugerindo que a quantidade e/ou a qualidade da resposta imune gerada não suportam uma neutralização efetiva da Stx2 produzidas por linhagens de EHEC.
RESUMEN
In the present study we investigated the flagellin-specific serum (IgG) and fecal (IgA) antibody responses elicited in BALB/c mice immunized with isogenic mutant derivatives of the attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) SL3261 strain expressing phase 1 (FliCi), phase 2 (FljB), or no endogenous flagellin. The data reported here indicate that mice orally immunized with recombinant S. Typhimurium strains do not mount significant systemic or secreted antibody responses to FliCi, FljB or heterologous B-cell epitopes genetically fused to FliCi. These findings are particularly relevant for those interested in the use of flagellins as molecular carriers of heterologous antigens vectored by attenuated S. Typhimurium strains.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Flagelina/inmunología , Vacunas contra la Salmonella/inmunología , Salmonella typhimurium/inmunología , Administración Oral , Animales , Vacunas Bacterianas/inmunología , Ratones , Ratones Endogámicos BALB C , Vacunas contra la Salmonella/administración & dosificación , Vacunas Atenuadas/inmunologíaRESUMEN
In the present study we investigated the flagellin-specific serum (IgG) and fecal (IgA) antibody responses elicited in BALB/c mice immunized with isogenic mutant derivatives of the attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) SL3261 strain expressing phase 1 (FliCi), phase 2 (FljB), or no endogenous flagellin. The data reported here indicate that mice orally immunized with recombinant S. Typhimurium strains do not mount significant systemic or secreted antibody responses to FliCi, FljB or heterologous B-cell epitopes genetically fused to FliCi. These findings are particularly relevant for those interested in the use of flagellins as molecular carriers of heterologous antigens vectored by attenuated S. Typhimurium strains.
Asunto(s)
Animales , Ratones , Anticuerpos Antibacterianos/sangre , Flagelina/inmunología , Vacunas contra la Salmonella/inmunología , Salmonella typhimurium/inmunología , Administración Oral , Vacunas Bacterianas/inmunología , Ratones Endogámicos BALB C , Vacunas contra la Salmonella/administración & dosificación , Vacunas Atenuadas/inmunologíaRESUMEN
The oligopeptide-binding protein, OppA, binds and ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides expressed by several bacterial species. In the present study, we report the cloning, purification, refolding and conformational analysis of a recombinant OppA protein derived from Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The oppA gene was expressed in Escherichia coli BL21 (DE3) strain under optimized inducing conditions and the recombinant protein remained largely insoluble. Solubilization was achieved following refolding of the denatured protein. Circular dichroism analysis indicated that the recombinant OppA protein preserved conformational features of orthologs expressed by other bacterial species. The refolded recombinant OppA represents a useful tool for structural and functional analyses of the X. citri protein.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Membrana/aislamiento & purificación , Proteínas de Transporte de Membrana/metabolismo , Pliegue de Proteína , Xanthomonas axonopodis/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Dicroismo Circular , Clonación Molecular , Biología Computacional/métodos , Escherichia coli/genética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Datos de Secuencia Molecular , Operón , Plásmidos , Conformación Proteica , Desnaturalización Proteica , Renaturación de Proteína , Señales de Clasificación de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Solubilidad , Especificidad por Sustrato , Xanthomonas axonopodis/metabolismoRESUMEN
The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT(+) (25 strains) only or LT(+)/ST(+) (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.
Asunto(s)
Escherichia coli Enterotoxigénica/genética , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Variación Genética , Animales , Línea Celular , Enzimas de Restricción del ADN/metabolismo , Escherichia coli Enterotoxigénica/clasificación , Escherichia coli Enterotoxigénica/aislamiento & purificación , Enterotoxinas/química , Enterotoxinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Calor , Humanos , Íleon/microbiología , Ratones , Datos de Secuencia Molecular , Fenotipo , Filogenia , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo de Nucleótido Simple , Estructura Secundaria de Proteína , Conejos , Análisis de Secuencia de ADN , SerotipificaciónRESUMEN
The oligopeptide-binding protein, OppA, binds and ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides expressed by several bacterial species. In the present study, we report the cloning, purification, refolding and conformational analysis of a recombinant OppA protein derived from Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The oppA gene was expressed in Escherichia coli BL21 (DE3) strain under optimized inducing conditions and the recombinant protein remained largely insoluble. Solubilization was achieved following refolding of the denatured protein. Circular dichroism analysis indicated that the recombinant OppA protein preserved conformational features of orthologs expressed by other bacterial species. The refolded recombinant OppA represents a useful tool for structural and functional analyses of the X. citri protein.
Asunto(s)
Pliegue de Proteína , Proteínas Bacterianas/aislamiento & purificación , Proteínas de Transporte de Membrana/aislamiento & purificación , Proteínas Portadoras/metabolismo , Xanthomonas axonopodis/genética , Secuencia de Aminoácidos , Secuencia de Bases , Biología Computacional/métodos , Dicroismo Circular , Clonación Molecular , Escherichia coli/genética , Datos de Secuencia Molecular , Operón , Plásmidos , Conformación Proteica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Xanthomonas axonopodis/metabolismoRESUMEN
The natural diversity of the elt operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT+ (25 strains) only or LT+/ST+ (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the elt operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.
Asunto(s)
Humanos , Escherichia coli Enterotoxigénica/genética , Toxinas BacterianasRESUMEN
INTRODUCTION: The Opp system is an ATP-binding cassette-type transporter formed by membrane-associated proteins required for the uptake of oligopeptides in bacteria. In gram-positive bacteria, the Opp system, and particularly the oligopeptide-binding protein (OppA), has been shown to be involved in different aspects of cell physiology, including intercellular communication and binding to host proteins. METHODS: In the present study we began to investigate the Opp system of Streptococcus mutans, the main etiological agent of dental caries. RESULTS: Five opp genes (oppABCDF) organized in a single operon were identified in the genome of the S. mutans UA159 strain. Amino acid sequence analyses showed that the S. mutans OppA is closely related to an ortholog found in Streptococcus agalactiae. Incubation of S. mutans UA159 cells with an anti-OppA-specific serum did not inhibit biofilm formation on polystyrene plates. Moreover, S. mutans UA159 derivatives carrying deletions on the oppA or oppB genes did not show significant growth impairment, increased sensitivity to aminopterin, or defective capacity to form biofilms on polystyrene wells in the presence or not of saliva. Remarkably, only two out of three laboratory strains and one out of seven clinical strains recovered from tooth decay processes harbored a copy of the oppA gene and expressed the OppA protein. CONCLUSION: Collectively, these results indicate that, in contrast to other Streptococcus species, the S. mutans Opp system, and particularly the OppA protein, does not represent an important trait required for growth and colonization.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/genética , Genes Bacterianos , Oligopéptidos/metabolismo , Streptococcus mutans/genética , Transportadoras de Casetes de Unión a ATP/fisiología , Secuencia de Aminoácidos , Adhesión Bacteriana/genética , Proteínas Bacterianas/fisiología , Biopelículas , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Caries Dental/microbiología , Humanos , Lipoproteínas/genética , Lipoproteínas/fisiología , Familia de Multigenes , Operón , Proteínas RecombinantesRESUMEN
The oligopeptide-binding protein, OppA, ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides by several bacterial species. In the present study, we report a structural model and an oligopeptide docking analysis of the OppA protein expressed by Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The X. citri OppA structural model showed a conserved three-dimensional structure, irrespective of the low amino acid identities with previously defined structures of Bacillus subtilis and Salmonella typhimurium orthologs. Oligopeptide docking analysis carried out with the proposed model indicated that the X. citri OppA preferentially binds tri- and tetrapeptides. The present study represents the first structural analysis of an OppA ortholog expressed by a phytopathogen and contributes to the understanding of the physiology and nutritional strategies of X. citri.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Lipoproteínas/química , Lipoproteínas/metabolismo , Oligopéptidos/metabolismo , Xanthomonas axonopodis/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Proteínas Portadoras/genética , Ligandos , Lipoproteínas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/química , Enfermedades de las Plantas/microbiología , Unión Proteica , Conformación Proteica , Xanthomonas axonopodis/genética , Xanthomonas axonopodis/patogenicidadRESUMEN
The oligopeptide-binding protein, OppA, ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides by several bacterial species. In the present study, we report a structural model and an oligopeptide docking analysis of the OppA protein expressed by Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The X. citri OppA structural model showed a conserved three-dimensional structure, irrespective of the low amino acid identities with previously defined structures of Bacillus subtilis and Salmonella typhimurium orthologs. Oligopeptide docking analysis carried out with the proposed model indicated that the X. citri OppA preferentially binds tri- and tetrapeptides. The present study represents the first structural analysis of an OppA ortholog expressed by a phytopathogen and contributes to the understanding of the physiology and nutritional strategies of X. citri.