RESUMEN

Science teaching in most Brazilian Universities tends to focus mainly on lectures and provides few opportunities for the development of modern teaching skills. Our group developed an online tool called Adopt a Bacterium, which consists on a Facebook group where teacher assistants (TAs) can interact with students and have a first contact with student-focused learning approaches. This work shows the TAs' own assessment of how the tool could be further explored to help them develop skills and become better teachers.

Asunto(s)

Educación a Distancia/métodos , Ciencia/educación , Formación del Profesorado/métodos , Enseñanza , Brasil , Curriculum , Humanos , Universidades

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/inmunología

RESUMEN

The Pst system is a high-affinity inorganic phosphate transporter found in many bacterial species. Streptococcus mutans, the etiological agent of tooth decay, carries a single copy of the pst operon composed of six cistrons (pstS, pstC1, pstC, pstB, smu.1134 and phoU). Here, we show that deletion of pstS, encoding the phosphate-binding protein, reduces phosphate uptake and impairs cell growth, which can be restored upon enrichment of the medium with high concentrations of inorganic phosphate. The relevance of Pst for growth was also demonstrated in the wild-type strain treated with an anti-PstS antibody. Nevertheless, a reduced ability to bind to saliva-coated surfaces was observed, along with the reduction of extracellular polysaccharide production, although no difference on pH acidification was observed between mutant and wild-type strains. Taken together, the present data indicate that the S. mutans Pst system participates in phosphate uptake, cell growth and expression of virulence-associated traits.

Asunto(s)

Adhesión Bacteriana/fisiología , Proteínas de Transporte de Fosfato/fisiología , Streptococcus mutans/fisiología , Transportadoras de Casetes de Unión a ATP/análisis , Adenosina Trifosfatasas/análisis , Proteínas Bacterianas/análisis , Película Dental/metabolismo , Técnicas de Inactivación de Genes , Silenciador del Gen , Genes Bacterianos/genética , Humanos , Concentración de Iones de Hidrógeno , Proteínas de Transporte de Membrana/análisis , Mutación/genética , Operón/genética , Proteínas de Transporte de Fosfato/genética , Proteínas de Unión a Fosfato/análisis , Fosfatos/análisis , Polisacáridos Bacterianos/metabolismo , Análisis de Secuencia de ADN , Streptococcus mutans/genética , Streptococcus mutans/metabolismo , Factores de Transcripción/análisis , Virulencia/genética

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.

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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 , Métodos

RESUMEN

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

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/metabolismo

RESUMEN

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/metabolismo

RESUMEN

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 Recombinantes

RESUMEN

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/patogenicidad

RESUMEN

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)

Lipoproteínas/química , Oligopéptidos/metabolismo , Proteínas Bacterianas/química , Proteínas Portadoras/química , Xanthomonas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Enfermedades de las Plantas/microbiología , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica

RESUMEN

Environmental and genetic factors affecting the in vitro spontaneous mutation frequencies to aminoglycoside resistance in Escherichia coli K12 were investigated. Spontaneous mutation frequencies to kanamycin resistance were at least 100 fold higher on modified Luria agar (L2) plates, when compared to results obtained in experiments carried out with Nutrient agar (NA) plates. In contrast to rifampincin, the increased mutability to kanamycin resistance could not be attributed to a mutator phenotype expressed by DNA repair defective strains. Kanamycin mutant selection windows and mutant preventive concentrations on L2 plates were at least fourfold higher than on NA plates, further demonstrating the role of growth medium composition on the mutability to aminoglycosides. Mutability to kanamycin resistance was increased following addition of sorbitol, suggesting that osmolarity is involved on the spontaneous mutability of E. coli K12 strains to aminoglycosides. The spontaneous mutation rates to kanamycin resistance on both L2 and NA plates were strictly associated with the selective antibiotic concentrations. Moreover, mutants selected at different antibiotic concentrations expressed heterogeneous resistance levels to kanamycin and most of them expressing multiple resistance to all tested aminoglycoside antibiotics (gentamicin, neomycin, amykacin and tobramycin). These results will contribute to a better understanding of the complex nature of aminoglycoside resistance and the emergence of spontaneous resistant mutants among E. coli K12 strains

Asunto(s)

Aminoglicósidos , Escherichia coli , Mutación/genética , Farmacorresistencia Microbiana , Ambiente

RESUMEN

The genus Xanthomonas is a diverse and economically important group of bacterial phytopathogens, belonging to the gamma-subdivision of the Proteobacteria. Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, which affects most commercial citrus cultivars, resulting in significant losses worldwide. Symptoms include canker lesions, leading to abscission of fruit and leaves and general tree decline. Xanthomonas campestris pv. campestris (Xcc) causes black rot, which affects crucifers such as Brassica and Arabidopsis. Symptoms include marginal leaf chlorosis and darkening of vascular tissue, accompanied by extensive wilting and necrosis. Xanthomonas campestris pv. campestris is grown commercially to produce the exopolysaccharide xanthan gum, which is used as a viscosifying and stabilizing agent in many industries. Here we report and compare the complete genome sequences of Xac and Xcc. Their distinct disease phenotypes and host ranges belie a high degree of similarity at the genomic level. More than 80% of genes are shared, and gene order is conserved along most of their respective chromosomes. We identified several groups of strain-specific genes, and on the basis of these groups we propose mechanisms that may explain the differing host specificities and pathogenic processes.

Asunto(s)

Genoma Bacteriano , Plantas/microbiología , Xanthomonas/genética , Xanthomonas/fisiología , Orden Génico/genética , Interacciones Huésped-Parásitos , Datos de Secuencia Molecular , Filogenia , Regulón/genética , Origen de Réplica/genética , Especificidad de la Especie , Virulencia/genética , Xanthomonas/clasificación , Xanthomonas/patogenicidad , Xanthomonas campestris/genética , Xanthomonas campestris/patogenicidad , Xanthomonas campestris/fisiología

RESUMEN

1. The mutagenicity of serum and urine from fuinea pigs treated with a single oral dose (500 mg/Kg) of benznidazole and nifurtimox was assayed using the Salmonella/plate incorporation test with strain TA100 and a nitroreductase-deficient derivative, TA100NR. 2. The urine and blood of animals treated with nifurtimox were not mutagenic for either tester strain. 3. The urine and blood of animals receiving benznidazole were mutagenic to the TA100 but not to the TA100NR strain. Similar results were obtained with nitrofurantoin-treated animals. Maximum mutagenicity values were obtained in serum and urine of treated animals 90 min and 24 h after administration, respectively. 4. Mutagenicity induced by benznidazole in the serum and urine of treated animals was not altered when assayed in anaerobic environments. 5. These results indicate that benznidazole and nifurtimox are not metabolized by the mammalian host into stable mutagenic derivatives detectable by the Ames test. Based on these data, we suggest that the potential cancer risk to patients treated with these drugs is small but should be further evaluated

Asunto(s)

Cobayas , Animales , Masculino , Femenino , Pruebas de Mutagenicidad , Mutación , Nifurtimox/metabolismo , Nitroimidazoles/metabolismo

RESUMEN

O derivado nitroimizadole-tiadizol CL 64.855 (2-amino-5-(1-metil-5-nitro-2-imidazoli)-1, 3, 4-tiadiazol), um potente agente tripanomicida, foi submetido a um ensaio mutagênico bacteriano com as linhagens indicadoras de Salmonella typhimurium TA 98, TA 100 e TA 102. Os resultados indicaram que o CL 64.855 é um potente mutagênico tipo troca de referencial detectado pelas linhagens TA 98 e TA 102. O CL 64.855 foi capaz de reverter as linhagens indicadoras em concentraçöes täo baixas quatro 0,1microng/placa. Ativaçäo metabólica com fraçöes microssomais de fígado de rato foram incapazes de aumentar a açäo mutagênica do CL 64.855

Asunto(s)

Mutación/efectos de los fármacos , Salmonella typhimurium/genética , Tripanocidas/farmacología , Trypanosoma cruzi/genética , Pruebas de Mutagenicidad

Asunto(s)

Mutación/efectos de los fármacos , Nifurtimox/toxicidad , Nitroimidazoles/toxicidad , Salmonella typhimurium/efectos de los fármacos , Pruebas de Mutagenicidad/métodos , Salmonella typhimurium/genética