RESUMEN

Chagas disease, caused by the protozoan Trypanosoma cruzi, remains a major public health challenge affecting millions in Latin America and worldwide. Although significant progress has been made in vector control, no vaccine exists to prevent infection or mitigate disease pathogenesis. We developed a rationally designed chimeric protein vaccine, N-Tc52/TSkb20, incorporating immunodominant epitopes from two T. cruzi antigens, the amino-terminal portion of Tc52 and the TSkb20 epitope derived from trans-sialidase. The objectives of this study were to construct and characterize the antigen and evaluate its protective potential in an immunoprophylactic murine model of T. cruzi infection. The N-Tc52/TSkb20 protein was recombinantly expressed in E. coli and its identity was confirmed using mass spectrometry and Western blotting. Immunization with the chimeric protein significantly controlled parasitemia and reduced the heart, colon, and skeletal muscle parasite burdens compared to non-vaccinated mice. Protection was superior to vaccination with the individual parental antigen components. Mechanistically, the vaccine induced potent CD8+ T-cell and IFNγ responses against the incorporated epitopes and a protective IgG antibody profile. A relatively low IL-10 response favored early parasite control. These results validate the promising multi-epitope approach and support the continued development of this type of rational vaccine design strategy against Chagas disease.

RESUMEN

: Aggregated forms of the synaptic protein α-synuclein (αS) have been proposed to operate as a molecular trigger for microglial inflammatory processes and neurodegeneration in Parkinson´s disease. Here, we used brain microglial cell cultures activated by fibrillary forms of recombinant human αS to assess the anti-inflammatory and neuroprotective activities of the antibiotic rifampicin (Rif) and its autoxidation product rifampicin quinone (RifQ). Pretreatments with Rif and RifQ reduced the secretion of prototypical inflammatory cytokines (TNF-, IL-6) and the burst of oxidative stress in microglial cells activated with αS fibrillary aggregates. Note, however, that RifQ was constantly more efficacious than its parent compound in reducing microglial activation. We also established that the suppressive effects of Rif and RifQ on cytokine release was probably due to inhibition of both PI3K- and non-PI3K-dependent signaling events. The control of oxidative stress appeared, however, essentially dependent on PI3K inhibition. Of interest, we also showed that RifQ was more efficient than Rif in protecting neuronal cells from toxic factors secreted by microglia activated by αS fibrils. Overall, data with RifQ are promising enough to justify further studies to confirm the potential of this compound as an anti-parkinsionian drug.

Asunto(s)

Microglía/efectos de los fármacos , Microglía/metabolismo , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/metabolismo , Rifampin/análogos & derivados , Rifampin/farmacología , alfa-Sinucleína/metabolismo , Citocinas/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Modelos Biológicos , Estructura Molecular , Enfermedades Neurodegenerativas/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 2/metabolismo

RESUMEN

Microorganisms produce siderophores to facilitate iron uptake and even though this trait has been extensively studied, there is growing evidence suggesting that siderophores may have other physiological roles aside from iron acquisition. In support of this notion, we previously linked the archetypal siderophore enterobactin with oxidative stress alleviation. To further characterize this association, we studied the sensitivity of Escherichia coli strains lacking different components of the enterobactin system to the classical oxidative stressors hydrogen peroxide and paraquat. We observed that strains impaired in enterobactin production, uptake and hydrolysis were more susceptible to the oxidative damage caused by both compounds than the wild-type strain. In addition, meanwhile iron supplementation had little impact on the sensitivity, the reducing agent ascorbic acid alleviated the oxidative stress and therefore significantly decreased the sensitivity to the stressors. This indicated that the enterobactin-mediated protection is independent of its ability to scavenge iron. Furthermore, enterobactin supplementation conferred resistance to the entE mutant but did not have any protective effect on the fepG and fes mutants. Thus, we inferred that only after enterobactin is hydrolysed by Fes in the cell cytoplasm and iron is released, the free hydroxyl groups are available for radical stabilization. This hypothesis was validated testing the ability of enterobactin to scavenge radicals in vitro. Given the strong connection between enterobactin and oxidative stress, we studied the transcription of the entE gene and the concomitant production of the siderophore in response to such kind of stress. Interestingly, we observed that meanwhile iron represses the expression and production of the siderophore, hydrogen peroxide and paraquat favour these events even if iron is present. Our results support the involvement of enterobactin as part of the oxidative stress response and highlight the existence of a novel regulation mechanism for enterobactin biosynthesis.

Asunto(s)

Enterobactina/biosíntesis , Escherichia coli/genética , Regulación de la Expresión Génica , Sideróforos/biosíntesis , Estrés Fisiológico/genética , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Cloruros/farmacología , Enterobactina/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Compuestos Férricos/farmacología , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/farmacología , Hidrólisis , Hierro/metabolismo , Ligasas/genética , Ligasas/metabolismo , Mutación , Oxidantes/antagonistas & inhibidores , Oxidantes/farmacología , Oxidación-Reducción , Estrés Oxidativo , Paraquat/antagonistas & inhibidores , Paraquat/farmacología , Sideróforos/genética , Transcripción Genética

RESUMEN

Numerous bacteria have evolved different iron uptake systems with the ability to make use of their own and heterologous siderophores. However, there is growing evidence attributing alternative roles for siderophores that might explain the potential adaptive advantages of microorganisms having multiple siderophore systems. In this work, we show the requirement of the siderophore enterobactin for Escherichia coli colony development in minimal media. We observed that a strain impaired in enterobactin production (entE mutant) was unable to form colonies on M9 agar medium meanwhile its growth was normal on LB agar medium. Given that, neither iron nor citrate supplementation restored colony growth, the role of enterobactin as an iron uptake-facilitator would not explain its requirement for colony development. The absence of colony development was reverted either by addition of enterobactin, the reducing agent ascorbic acid or by incubating in anaerobic culture conditions with no additives. Then, we associated the enterobactin requirement for colony development with its ability to reduce oxidative stress, which we found to be higher in media where the colony development was impaired (M9) compared with media where the strain was able to form colonies (LB). Since oxyR and soxS mutants (two major stress response regulators) formed colonies in M9 agar medium, we hypothesize that enterobactin could be an important piece in the oxidative stress response repertoire, particularly required in the context of colony formation. In addition, we show that enterobactin has to be hydrolyzed after reaching the cell cytoplasm in order to enable colony development. By favoring iron release, hydrolysis of the enterobactin-iron complex, not only would assure covering iron needs, but would also provide the cell with a molecule with exposed hydroxyl groups (hydrolyzed enterobactin). This molecule would be able to scavenge radicals and therefore reduce oxidative stress.

Asunto(s)

Enterobactina/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Estrés Oxidativo , Carga Bacteriana , Medios de Cultivo , Enterobactina/genética , Escherichia coli/genética , Hidrólisis , Mutación , Especies Reactivas de Oxígeno

RESUMEN

BACKGROUND: Microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide produced by Escherichia coli (E. coli). MccJ25 enters into the sensitive E. coli strains by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD and SbmA. The resistance of Salmonella enterica serovar Typhimurium (S. Typhimurium) to MccJ25 is attributed to the inability of its FhuA protein to incorporate the antibiotic into the cell. RESULTS: In this work we demonstrate that S. Typhimurium becomes notably susceptible to MccJ25 when replicating within macrophages. In order to determine the possible cause of this phenomenon, we studied the sensitivity of S. Typhimurium to MccJ25 at conditions resembling those of the internal macrophage environment, such as low pH, low magnesium and iron deprivation. We observed that the strain was only sensitive to the antibiotic at low pH, leading us to attribute the bacterial sensitization to this condition. A MccJ25-resistant E. coli strain in which fhuA is deleted was also inhibited by the antibiotic at low pH. Then, we could assume that the MccJ25 sensitivity change observed in both E. coli fhuA and S. Typhimurium is mediated by a MccJ25 uptake independent of the FhuA receptor. Moreover, low pH incubation also sensitized S. Typhimurium to the hydrophobic antibiotic novobiocin, which does not affect enteric bacteria viability because it is unable to penetrate the bacterial outer membrane. This observation supports our hypothesis about low pH producing a modification in the bacterial membrane permeability that allows an unspecific MccJ25 uptake. On the other hand, MccJ25 inhibited S. Typhimurium when cells were preincubated in acidic pH medium and then treated at neutral pH with the antibiotic. CONCLUSIONS: Our results suggest that acidic condition does not alter MccJ25 hydrophobicity but irreversibly modifies bacterial membrane permeability. This would allow an unspecific antibiotic uptake into the cell.From our data it is possible to infer that intracellular pathogenic strains, which are in vitro resistant to MccJ25, could become susceptible ones in vivo. Therefore, the MccJ25 action spectrum would be broader than what in vitro experiments indicate.

Asunto(s)

Antibacterianos/farmacología , Bacteriocinas/farmacología , Macrófagos/microbiología , Salmonella typhimurium/efectos de los fármacos , Animales , Línea Celular , Permeabilidad de la Membrana Celular , Medios de Cultivo/química , Citosol/microbiología , Escherichia coli/efectos de los fármacos , Concentración de Iones de Hidrógeno , Ratones , Pruebas de Sensibilidad Microbiana , Modelos Teóricos , Novobiocina/farmacología

RESUMEN

BACKGROUND: Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition. METHODS AND PRINCIPAL FINDINGS: Here we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity. CONCLUSIONS: We have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.

Asunto(s)

Antibacterianos/farmacología , Enterobactina/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fenoles/farmacología , Tiazoles/farmacología , Enterobactina/fisiología , Escherichia coli/efectos de los fármacos , Escherichia coli/fisiología , Proteínas de Escherichia coli/fisiología , Hierro/farmacología , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Sideróforos/metabolismo , Sideróforos/fisiología

RESUMEN

Microcin J25 (MccJ25) is a plasmid-encoded, 21-amino-acid, antibacterial peptide produced by Escherichia coli. MccJ25 inhibits RNA polymerase and the membrane respiratory chain. MccJ25 uptake into E. coli-sensitive strains is mediated by the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbB, ExbD, and SbmA. This peptide is active on some E. coli, Salmonella, and Shigella species strains, while other Gram-negative bacteria, such as clinical isolates of Enterobacter cloacae, Citrobacter freundii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Moraxella catarrhalis, and Salmonella enterica serovar Typhimurium, are completely resistant. In the present work, we demonstrated that the membrane-permeabilizing peptide (KFF)3K made some resistant strains sensitive to MccJ25, among them S. Typhimurium, where the antibiotic inhibits in vitro cell growth and bacterial replication within macrophages. The results demonstrate that the membrane permeabilization induced by (KFF)3K allows MccJ25 penetration in an FhuA and SbmA-independent manner and suggest that the combination of both peptides could be considered as a therapeutic agent against pathogenic Salmonella strains.

Asunto(s)

Antibacterianos/metabolismo , Antibacterianos/farmacología , Bacteriocinas/metabolismo , Bacteriocinas/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Péptidos/farmacología , Recuento de Colonia Microbiana , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Gramnegativas/metabolismo , Bacterias Gramnegativas/fisiología , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Oxígeno/metabolismo

RESUMEN

The SbmA protein is involved in the transport of MccB17-, MccJ25-, bleomycin- and proline-rich peptides into the Escherichia coli cytoplasm. sbmA gene homologues were found in a variety of bacteria. However, the physiological role of this protein still remains unknown. Previously, we found that a combination of sbmA and tolC mutations in Tn10-carrying E. coli K-12 strains results in hypersusceptibility to tetracycline. In this work, we studied sbmA expression in a tolC mutant background and observed an increased expression throughout growth. We ruled out the global transcriptional regulator RpoS and the small RNA micF as intermediates in this regulation. The tolC mutation induced the expression of other well-characterized strong σ(E) -dependent promoters in E. coli. We observed that the increase in σ(E) activity led to a greater sbmA expression, conversely eliminating σ(E) prevented expression of sbmA. We also observed that the sbmA upregulation in a tolC mutant context was abolished in an rpoE-null strain. These results suggest a σ(E) -dependent positive regulation on sbmA by the tolC mutation. We hypothesize that this mechanism might be part of a compensatory cell envelope stress response.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Factor sigma/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Escherichia coli/genética , Factor sigma/genética