RESUMO
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.
Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Macrófagos/microbiologia , Salmonella typhimurium/efeitos dos fármacos , Animais , Linhagem Celular , Permeabilidade da Membrana Celular , Meios de Cultura/química , Citosol/microbiologia , Escherichia coli/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Camundongos , Testes de Sensibilidade Microbiana , Modelos Teóricos , Novobiocina/farmacologiaRESUMO
Copper is both an essential nutrient and a toxic element able to catalyze free radicals formation which damage lipids and proteins. Although the available copper redox species in aerobic environment is Cu(II), proteins that participate in metal homeostasis use Cu(I). With isolated Escherichia coli membranes, we have previously shown that electron flow through the respiratory chain promotes cupric ions reduction by NADH dehydrogenase-2 and quinones. Here, we determined Cu(II)-reductase activity by whole cells using strains deficient in these respiratory chain components. Measurements were done by the appearance of Cu(I) in the supernatants of cells exposed to sub-lethal Cu(II) concentrations. In the absence of quinones, the Cu(II)-reduction rate decreased ~70% in respect to the wild-type strain, while this diminution was about 85% in a strain lacking both NDH-2 and quinones. The decrease was ~10% in the absence of only NDH-2. In addition, we observed that quinone deficient strains failed to grow in media containing either excess or deficiency of copper, as we have described for NDH-2 deficient mutants. Thus, the Cu(II)-reduction by E. coli intact cells is mainly due to quinones and to a lesser extent to NDH-2, in a quinone-independent way. To our knowledge, this is the first in vivo demonstration of the involvement of E. coli respiratory components in the Cu(II)-reductase activity which contributes to the metal homeostasis.
Assuntos
Cobre/metabolismo , Transporte de Elétrons , Escherichia coli/citologia , Escherichia coli/metabolismo , NADH Desidrogenase/metabolismo , Membrana Celular/metabolismo , Escherichia coli/química , Ferro/metabolismo , NADH Desidrogenase/deficiência , NADH Desidrogenase/genética , Oxirredução , Oxirredutases/metabolismo , Quinonas/metabolismoRESUMO
Microcin J25 (MccJ25) is a 21 amino acid lasso-peptide antibiotic produced by Escherichia coli and composed of an 8-residues ring and a terminal 'tail' passing through the ring. We have previously reported two cellular targets for this antibiotic, bacterial RNA polymerase and the membrane respiratory chain, and shown that Tyr9 is essential for the effect on the membrane respiratory chain which leads to superoxide overproduction. In the present paper we investigated the redox behavior of MccJ25 and the mutant MccJ25 (Y9F). Cyclic voltammetry measurements showed irreversible oxidation of both Tyr9 and Tyr20 in MccJ25, but infrared spectroscopy studies demonstrated that only Tyr9 could be deprotonated upon chemical oxidation in solution. Formation of a long-lived tyrosyl radical in the native MccJ25 oxidized by H2O2 was demonstrated by Electron Paramagnetic Resonance Spectroscopy; this radical was not detected when the reaction was carried out with the MccJ25 (Y9F) mutant. These results show that the essential Tyr9, but not Tyr20, can be easily oxidized and form a tyrosyl radical.
Assuntos
Antibacterianos/química , Bacteriocinas/química , Tirosina/química , Espectroscopia de Ressonância de Spin Eletrônica , Ferricianetos , Peróxido de Hidrogênio/química , Oxirredução , Espectrofotometria Infravermelho , VibraçãoRESUMO
Thyroid hormones (THs) exert a broad range of actions on development, growth, and cell differentiation by both genomic and nongenomic mechanisms. THs regulate lymphocyte function, but the participation of nongenomic actions is still unknown. Here the contribution of both genomic and nongenomic effects on TH-induced division of T cells was studied by using free and noncell permeable THs coupled to agarose (TH-ag). THs-ag led to cell division, but to a lesser extent than free hormones. THs induced nongenomically the rapid translocation of protein kinase C (PKC) ζ isoform to cell membranes, extracellular-signal-regulated kinases (ERK1/2) phosphorylation and nuclear factor-κB (NF-κB) activation. The signaling cascade include sphingomyelinases acting up-stream the activation of PKCζ isoform, while ERK and NF-κB are activated downstream this PKC isoenzyme. Both free and THs-ag increased the protein and mRNA levels of TH nuclear receptor TRα1, while only free hormones incremented the inducible NOS gene and protein levels as well as a calcium independent NOS activity. Both effects were blunted by PKCζ inhibition. These results indicate that THs, by triggering a nongenomic signaling cascade that involves Smases-mediated activation of PKCζ, lead to ERK 1/2 and NF-κB activation and to the genomic increase of TRs and the inducible nitric oxide synthase protein and mRNA levels, improving T lymphocyte proliferation. These finding not only contribute to the understanding of the mechanisms involved in TH modulation of lymphocyte physiology, but would also point out for the first time the interplay between genomic and nongenomic TH actions in T cells.
Assuntos
Proliferação de Células , Óxido Nítrico Sintase Tipo II/metabolismo , Linfócitos T/enzimologia , Receptores alfa dos Hormônios Tireóideos/metabolismo , Tiroxina/metabolismo , Tri-Iodotironina/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico , RNA Mensageiro/metabolismo , Transdução de Sinais , Esfingomielina Fosfodiesterase/metabolismo , Linfócitos T/efeitos dos fármacos , Receptores alfa dos Hormônios Tireóideos/genética , Fatores de Tempo , Regulação para CimaRESUMO
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.
Assuntos
Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bacteriocinas/metabolismo , Bacteriocinas/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Peptídeos/farmacologia , Contagem de Colônia Microbiana , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Negativas/fisiologia , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Oxigênio/metabolismoRESUMO
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme related with Huntington's, Parkinson's and Alzheimer's diseases. The ability of negatively charged membranes to induce a rapid formation of GAPDH amyloid fibrils has been demonstrated, but the mechanisms by which GAPDH reaches the fibrillar state remains unclear. In this report, we describe the structural changes undergone by GAPDH at physiological pH and temperature conditions right from its interaction with acidic membranes until the amyloid fibril is formed. According to our results, the GAPDH-membrane binding induces a beta-structuring process along with a loss of quaternary structure in the enzyme. In this way, experimental evidences on the initial steps of GAPDH amyloid fibrils formation pathway are provided.
Assuntos
Amiloide/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Membranas Artificiais , Amiloide/ultraestrutura , Gliceraldeído-3-Fosfato Desidrogenases/ultraestrutura , Humanos , Microscopia Eletrônica de Transmissão , Multimerização Proteica , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Escherichia coli microcin J25 (MccJ25) is a lasso-peptide antibiotic comprising 21 L-amino acid residues (G(1)-G-A-G-H(5)-V-P-E-Y-F(10)-V-G-I-G-T(15)-P-I-S-F-Y(20)-G). MccJ25 has two independent substrates: RNA-polymerase (RNAP) and the membrane respiratory chain. The latter is mediated by oxygen consumption inhibition together with an increase of superoxide production. In the present paper, the antibiotic MccJ25 was engineered by substituting Tyr(9) or Tyr(20) with phenylalanine. Both mutants were well transported into the cells and remained active on RNAP. Only the Y9F mutant lost the ability to overproduce superoxide and inhibit oxygen consumption. The last results confirm that the Tyr(9), and not Tyr(20), is involved in the MccJ25 action on the respiratory chain target.
Assuntos
Antibacterianos/química , Bacteriocinas/química , Escherichia coli/metabolismo , Superóxidos/metabolismo , Tirosina/química , Antibacterianos/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Escherichia coli/química , Escherichia coli/genética , Tirosina/genética , Tirosina/metabolismoRESUMO
We found that Escherichia coli grown in media with >37 mM phosphate maintained a high polyphosphate level in late stationary phase, which could account for changes in gene expression and enzyme activities that enhance stationary-phase fitness.
Assuntos
Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Polifosfatos/metabolismo , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Fosfatos/farmacologiaRESUMO
Inhibition or reversion of protein self-aggregation has been suggested as a possible preventive mechanism against amyloid diseases, and many efforts are underway to found out molecules capable to restrain the protein aggregation process. In this paper, the inhibitory effects of thyroid hormone analogues on heat-induced fibrillation process of serum albumin are reported. Among the analogues tested, 3,5,3',5'-tetraiodothyroacetic and 3,5,3'-triiodothyroacetic acid showed the most important inhibitory effects on amyloid formation. Thyroxine exhibits a lesser protective effect, while 3,5,3'-triiodothyronine showed no significant inhibition. The gaining of a negative charge together with a size reduction of the hormone molecule could play an essential role in the inhibition of fibrils formation. According to infrared spectroscopy results, the thyroid hormones analogues protective effects proceed via the stabilization of the protein native structure. The current work demonstrates the effectiveness of naturally occurring molecules in the inhibition of albumin fibril formation.
Assuntos
Albumina Sérica/metabolismo , Tiroxina/análogos & derivados , Tri-Iodotironina/análogos & derivados , Amiloide/metabolismo , Animais , Bovinos , Relação Dose-Resposta a Droga , Desenho de Fármacos , Temperatura Alta , Cinética , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Albumina Sérica/química , Espectroscopia de Infravermelho com Transformada de Fourier , Tiroxina/farmacologia , Tri-Iodotironina/farmacologiaRESUMO
Escherichia coli gradually decline the capacity to resist oxidative stress during stationary phase. Besides the aerobic electron transport chain components are down-regulated in response to growth arrest. However, we have previously reported that E. coli cells grown in media containing at least 37mM phosphate maintained ndh expression in stationary phase, having high viability and low NADH/NAD(+) ratio. Here we demonstrated that, in the former condition, other aerobic respiratory genes (nuoAB, sdhC, cydA, and ubiC) expression was maintained. In addition, reactive oxygen species production was minimal and consequently the levels of thiobarbituric acid-reactive substances and protein carbonylation were lower than the expected for stationary cells. Interestingly, defense genes (katG and ahpC) expression was also maintained during this phase. Our results indicate that cells grown in high phosphate media exhibit advantages to resist endogenous and exogenous oxidative stress in stationary phase.
Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Fosfatos/metabolismo , Meios de Cultura , Transporte de Elétrons/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Peróxido de Hidrogênio/farmacologia , Cinética , Estresse Oxidativo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Escherichia coli NADH dehydrogenase-2 (NDH-2) is a primary dehydrogenase in aerobic respiration that shows cupric-reductase activity. The enzyme is encoded by ndh, which is highly regulated by global transcription factors. It was described that the gene is expressed in the exponential growth phase and repressed in late stationary phase. We report the maintenance of NDH-2 activity and ndh expression in the stationary phase when cells were grown in media containing at least 37 mM phosphate. Gene regulation was independent of RpoS and other transcription factors described to interact with the ndh promoter. At this critical phosphate concentration, cell viability, oxygen consumption rate, and NADH/NAD+ ratio were maintained in the stationary phase. These physiological parameters gradually changed, but NDH-2 activity remained high for up to 94 h. Phosphate seems to trigger an internal signal in the stationary phase mediated by systems not yet described.
Assuntos
Transporte de Elétrons , Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , NADH Desidrogenase/biossíntese , Fosfatos/metabolismo , Aerobiose , Fusão Gênica Artificial , Proteínas de Bactérias/metabolismo , Escherichia coli/química , Expressão Gênica , Genes Reporter , Viabilidade Microbiana , NAD/metabolismo , Oxigênio/metabolismo , Piridinas/análise , Fator sigma/metabolismo , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
As Escherichia coli strains enter the stationary phase of growth they become more resistant to the peptide antibiotic microcin J25. It is known that starvation for nutrients such as amino acids or glucose leads to increases in guanosine 3',5'-bispyrophosphate (ppGpp) levels and that the intracellular concentration of this nucleotide increases as cells enter the stationary phase of growth. Therefore, we examined the effects of artificially manipulating the ppGpp levels on sensitivity to microcin J25. A direct correlation was found between ppGpp accumulation and microcin resistance. Our results indicate that the nucleotide is required to induce production of YojI, a chromosomally encoded efflux pump which, in turn, expels microcin from cells. This would maintain the intracellular level of the antibiotic below a toxic level.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteriocinas/farmacologia , Escherichia coli/efeitos dos fármacos , Guanosina Tetrafosfato/metabolismo , Proteínas de Bactérias/genética , Bacteriocinas/metabolismo , Transporte Biológico/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ligases/genética , Ligases/metabolismo , Viabilidade Microbiana/efeitos dos fármacosRESUMO
Microcin J25 (MccJ25) uptake by Escherichia coli requires the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbD, ExbB, and SbmA. MccJ25 appears to have two intracellular targets: (i) RNA polymerase (RNAP), which has been described in E. coli and Salmonella enterica serovars, and (ii) the respiratory chain, reported only in S. enterica serovars. In the current study, it is shown that the observed difference between the actions of microcin on the respiratory chain in E. coli and S. enterica is due to the relatively low microcin uptake via the chromosomally encoded FhuA. Higher expression by a plasmid-encoded FhuA allowed greater uptake of MccJ25 by E. coli strains and the consequent inhibition of oxygen consumption. The two mechanisms, inhibition of RNAP and oxygen consumption, are independent of each other. Further analysis revealed for the first time that MccJ25 stimulates the production of reactive oxygen species (O(2)(*-)) in bacterial cells, which could be the main reason for the damage produced on the membrane respiratory chain.
Assuntos
Bacteriocinas/farmacologia , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Superóxidos/metabolismo , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteriocinas/farmacocinética , Catalase/genética , Catalase/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/genética , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Genótipo , Consumo de Oxigênio/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: The pyridine nucleotide disulfide reductase (PNDR) is a large and heterogeneous protein family divided into two classes (I and II), which reflect the divergent evolution of its characteristic disulfide redox active site. However, not all the PNDR members fit into these categories and this suggests the need of further studies to achieve a more comprehensive classification of this complex family. RESULTS: A workflow to improve the clusterization of protein families based on the array of linear conserved motifs is designed. The method is applied to the PNDR large family finding two main groups, which correspond to PNDR classes I and II. However, two other separate protein clusters, previously classified as class I in most databases, are outgrouped: the peroxide reductases (NAOX, NAPE) and the type II NADH dehydrogenases (NDH-2). In this way, two novel PNDR classes III and IV for NAOX/NAPE and NDH-2 respectively are proposed. By knowledge-driven biochemical and functional data analyses done on the new class IV, a linear array of motifs putatively related to Cu(II)-reductase activity is detected in a specific subset of NDH-2. CONCLUSION: The results presented are a novel contribution to the classification of the complex and large PNDR protein family, supporting its reclusterization into four classes. The linear array of motifs detected within the class IV PNDR subfamily could be useful as a signature for a particular subgroup of NDH-2.
Assuntos
Oxirredutases/química , Piridinas/química , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Motivos de Aminoácidos , Sequência de Aminoácidos , Simulação por Computador , Sequência Conservada , Análise Discriminante , Dissulfetos/química , Modelos Lineares , Modelos Químicos , Dados de Sequência Molecular , Nucleotídeos/química , Oxirredutases/classificação , Homologia de Sequência de AminoácidosRESUMO
OBJECTIVES: To study the possible therapeutic utility of microcin J25 (MccJ25), a peptide RNA polymerase inhibitor. METHODS: We subjected the antibiotic to two types of assays. First, with an ex vivo assay, we evaluated the stability and efficacy of MccJ25 in complex fluid biomatrices such as human whole blood, plasma and serum, compared with that in conventional laboratory media. Antimicrobial efficacy of MccJ25 was assessed by quantitative culture 2 h after inoculation of the biomatrices with a Salmonella Newport target organism and compared with that of MccJ25-free controls. Second, the antibiotic was tested in a mouse model of Salmonella infection. The latter was induced by intraperitoneal inoculation of 10(6) cfu of Salmonella Newport and the treatment with MccJ25 was initiated at 2 h post-infection. RESULTS: MccJ25 retained full activity after 24 h of incubation in whole blood, plasma or serum. In addition, it did not show any haemolytic activity. In whole blood, homologous plasma and serum, introduction of MccJ25 was associated with a significant reduction in cfu versus the respective peptide-free controls. The counts of viable bacteria in the spleen and liver of mice treated with MccJ25 at a total dosage of 3 mg/mouse during either 24 h (0.5 mg/mouse every 4 h) or 6 days (0.5 mg/mouse every 24 h) significantly decreased by two or three orders of magnitude (P Assuntos
Antibacterianos/uso terapêutico
, Bacteriocinas/uso terapêutico
, Infecções por Salmonella/tratamento farmacológico
, Ampicilina/uso terapêutico
, Animais
, Antibacterianos/farmacocinética
, Bacteriocinas/farmacocinética
, Contagem de Colônia Microbiana
, Meia-Vida
, Hemólise/efeitos dos fármacos
, Humanos
, Técnicas In Vitro
, Fígado/microbiologia
, Camundongos
, Infecções por Salmonella/microbiologia
, Baço/microbiologia
RESUMO
NADH dehydrogenase-2 (NDH-2) from Escherichia coli respiratory chain is a membrane-bound cupric-reductase encoded by ndh gene. Here, we report that the respiratory system of a ndh deficient strain suffered a faster inactivation than that of the parental strain in the presence of tert-butyl hydroperoxide due to endogenous copper. The inactivation was similar for both strains when copper concentration increased in the culture media. Furthermore, several ndh deficient mutants grew less well than the corresponding parental strains in media containing either high or low copper concentrations. A mutant strain complemented with ndh gene almost recovered the parental phenotype for growing in copper limitation or excess. Then, NDH-2 gives the bacteria advantages to diminish the susceptibility of the respiratory chain to damaging effects produced by copper and hydroperoxides and to survive in extreme copper conditions. These results suggest that NDH-2 contributes in the bacterial oxidative protection and in the copper homeostasis.
Assuntos
Cobre/toxicidade , Escherichia coli/efeitos dos fármacos , Homeostase/efeitos dos fármacos , NADH Desidrogenase/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Oxirredutases/fisiologia , terc-Butil Hidroperóxido/toxicidade , Cobre/metabolismo , Meios de Cultura , Relação Dose-Resposta a Droga , Transporte de Elétrons/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Homeostase/fisiologia , Membranas/enzimologia , Mutação , NADH Desidrogenase/metabolismo , Estresse Oxidativo/fisiologia , Oxirredutases/metabolismo , Fenótipo , Fatores de Tempo , terc-Butil Hidroperóxido/metabolismoRESUMO
Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded antibiotic peptide consisting of 21 L-amino acid residues (G1-G-A-G-H5-V-P-E-Y-F10-V-G-I-G-T15-P-I-S-F-Y20-G). E. coli RNA polymerase (RNAP) is the intracellular target of MccJ25. MccJ25 enters cells after binding to specific membrane transporters: FhuA in the outer membrane and SbmA in the inner membrane. Here, we studied MccJ25 mutants carrying a substitution of His5 by Lys, Arg, or Ala. The inhibitory effects on cellular growth and in vitro RNAP activity were determined for each mutant microcin. The results show that all mutants inhibited RNAP in vitro. However, the mutants were defective in their ability to inhibit cellular growth. Experiments in which the FhuA protein was bypassed showed that substitutions of MccJ25 His5 affected the SbmA-dependent transport. Our results thus suggest that MccJ25 His5 located in the lariat ring is involved, directly or indirectly, in specific interaction with SbmA and is not required for MccJ25 inhibition of RNAP.
Assuntos
Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Histidina/fisiologia , Proteínas de Membrana/metabolismo , Substituição de Aminoácidos , Antibacterianos/farmacologia , Bacteriocinas/genética , Bacteriocinas/farmacologia , Transporte Biológico , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Histidina/genética , Mutação , RNA Bacteriano/biossínteseRESUMO
In the present study, we showed that yojI, an Escherichia coli open reading frame with an unknown function, mediates resistance to the peptide antibiotic microcin J25 when it is expressed from a multicopy vector. Disruption of the single chromosomal copy of yojI increased sensitivity of cells to microcin J25. The YojI protein was previously assumed to be an ATP-binding-cassette-type exporter on the basis of sequence similarities. We demonstrate that YojI is capable of pumping out microcin molecules. Thus, one obvious explanation for the protective effect against microcin J25 is that YojI action keeps the intracellular concentration of the peptide below a toxic level. The outer membrane protein TolC in addition to YojI is required for export of microcin J25 out of the cell. Microcin J25 is thus the first known substrate for YojI.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Peptídeos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Cromossomos Bacterianos , Clonagem Molecular , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Proteínas de Membrana Transportadoras , Fases de Leitura Aberta/genéticaRESUMO
Microcin J25 appears to have two intracellular targets: (1) RNA polymerase, which was described in Escherichia coli and Salmonella enterica serovars, and (2) cell respiration in Salmonella enterica serovars. C-terminal glycine amidation of the threaded segment localized in the MccJ25 lariat ring region specifically blocked the RNA-polymerase inhibition, but not the cell respiration inhibition and peptide uptake. These results suggest that different regions of the molecule are responsible for each cellular effect, they are localized far away from the beta-hairpin region and the C-terminal region is an important determinant for RNAP inhibition.
Assuntos
Bacteriocinas/química , Bacteriocinas/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Amidas/metabolismo , Bacteriocinas/genética , Respiração Celular/fisiologia , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/classificação , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Consumo de Oxigênio , RNA/biossíntese , Salmonella/classificação , Salmonella/genética , Salmonella/crescimento & desenvolvimento , Salmonella/metabolismo , Transcrição GênicaRESUMO
The antibiotic microcin J25 (MccJ25) was cleaved by hydrolysis with thermolysin giving a two-chain peptide (MccJ25-Th19) of 10 and 9 amino acid residues. MccJ25-Th19 with deep modifications in beta-hairpin region had no effect on Escherichia coli growth, but still inhibited RNA polymerase in vitro and oxygen consumption in Salmonella strains. MccJ25-Th19 showed antibiotic activity on E. coli transformed with plasmids containing either fhuA or sbmA genes, which code for proteins involved in MccJ25 transport. These results suggest that an intact beta-hairpin region is crucial for MccJ25 import but not for inhibition of E. coli RNA polymerase or oxygen consumption in Salmonella strains.