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1.
J Phys Chem Lett ; 11(5): 1934-1939, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32067463

RESUMO

Slow polypeptide conformational changes on time scales of >1 s are generally assumed to be highly cooperative two-state transitions, reflecting the high energy barrier. However, few experimental characterizations have tested the validity of this assumption. We performed residue-specific NMR thermodynamic analysis of the 27-residue lantibiotic peptide, nukacin ISK-1, to characterize the isomerization between two topological states on the second time scale. Unexpectedly, the thermal transition behaviors were distinct among peptide regions, indicating that the topological isomerization process is a mosaic of different degrees of cooperativity. The conformational change path between the two NMR structures was deduced by a targeted molecular dynamics simulation. The unique side-chain threading motions through the monosulfide rings are the structural basis of the high energy barrier, and the nonlocal interactions in the hydrophobic core are the structural basis of the cooperativity. Taken together, we provide an energetic description of the topological isomerization of nukacin ISK-1.


Assuntos
Bacteriocinas/química , Ressonância Magnética Nuclear Biomolecular , Bacteriocinas/metabolismo , Dicroísmo Circular , Isomerismo , Simulação de Dinâmica Molecular , Staphylococcus/metabolismo , Termodinâmica
2.
World J Microbiol Biotechnol ; 35(9): 133, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432254

RESUMO

There is a significant increase in the discovery of new antimicrobial compounds in recent past to combat drug resistant pathogens. Members of the genus Bacillus and related genera have been screened extensively due to their ability to produce wide range of antimicrobial compounds. In this study, we have isolated and characterized a new antimicrobial peptide from a marine bacterium identified as Virgibacillus species. The low molecular mass and stability of the antimicrobial substance pointed towards the bacteriocinogenic nature of the compound. The RAST analysis of genome sequence showed presence of a putative bacteriocin biosynthetic cluster containing genes necessary for synthesis of a lanthipeptide. Translated amino acid sequence of mature C-terminal propeptide showed identity with salivaricin A (52.2%) and lacticin A (33.3%). Accordingly, the mass (2417 Da) obtained by MALDI analysis was in agreement with posttranslational modifications of the leader peptide to yield three methyl lanthionine rings and a disulfide bond between two free cysteine residues. The lanthipeptide was named as virgicin, which selectively inhibited the growth of Gram-positive bacteria and biofilm formation by Enterococcus faecalis. Inhibition of biofilm formation by E. faecalis was also observed in in vitro model experiments using hydroxyapatite discs. Thus, virgicin appears to be a promising new bacteriocin to control oral biofilm formation by selective pathogens.


Assuntos
Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Virgibacillus/metabolismo , Bacteriocinas/química , Bacteriocinas/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Vias Biossintéticas/genética , Genoma Bacteriano , Peso Molecular , Família Multigênica , Peptídeos/química , Peptídeos/genética , Água do Mar/microbiologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Virgibacillus/classificação , Virgibacillus/isolamento & purificação
3.
J Med Microbiol ; 68(9): 1359-1366, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31364964

RESUMO

Methodology. Biochemical and molecular methods were used to identify 100 lactobacilli isolated from rectal swabs. Among these, L. paracasei ssp. paracasei LP5 and L. brevis LP9 showed significant antibacterial activity against S. agalactiae and L. monocytogenes. Accordingly, characterization of their bacteriocins, BacLP5 and BacLP9, was conducted to obtain information on their kinetic production, sensitivity to chemico-physical parameters and molecular weight. To investigate the possible use of the two Lactobacillus strains as probiotics, their gastrointestinal resistance, cellular adhesiveness and sensitivity to antibiotics were also studied.Results. The obtained data show that BacLP5 and BacLP9 most likely belong to class II bacteriocins and both have a molecular weight of approximately 3 kDa. The production of BacLP5 and BacLP9 started after 4 h (40 and 80 AU ml-1), respectively. Both of the Lactobacillus strains survived gastric and intestinal juices well and showed adhesive capability on HEp-2 cells.Conclusion. Due to their peculiar antimicrobial characteristics, L. paracasei ssp. paracasei LP5 and L. brevis LP9 are suitable for use in the treatment of vaginal disorders, through both oral and transvaginal administration.


Assuntos
Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Lactobacillus brevis/metabolismo , Lactobacillus paracasei/metabolismo , Antibacterianos/química , Antibacterianos/isolamento & purificação , Aderência Bacteriana , Bacteriocinas/química , Bacteriocinas/isolamento & purificação , Ácidos e Sais Biliares/farmacologia , Linhagem Celular , Fenômenos Químicos , Suco Gástrico/metabolismo , Humanos , Lactobacillus brevis/classificação , Lactobacillus brevis/isolamento & purificação , Lactobacillus paracasei/classificação , Lactobacillus paracasei/isolamento & purificação , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Peso Molecular , Probióticos , Reto/microbiologia , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/crescimento & desenvolvimento
4.
Molecules ; 24(13)2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31324069

RESUMO

The production of a bacteriocin-like substance with antimicrobial activity, named peocin, by the probiotic Paenibacillus ehimensis NPUST1 was previously reported by our laboratory. The present study aimed to identify peocin and increase the peocin yield by heterologous expression in Escherichia coli BL21(DE3). Peocin was identified as a DNA starvation/stationary phase protection protein, also called DNA-binding protein from starved cells (Dps), by gel overlay and LC-MS/MS analysis. For mass production of peocin, fed-batch cultivation of E. coli was performed using a pH-stat control system. Purification by simple nickel affinity chromatography and dialysis yielded 45.3 mg of purified peocin from a 20-mL fed-batch culture (49.3% recovery). The biological activity of the purified peocin was confirmed by determination of the MIC and MBC against diverse pathogens. Purified peocin exhibited antimicrobial activity against aquatic, food spoilage, clinical and antibiotic-resistant pathogens. In an in vivo challenge test, zebrafish treated with purified peocin exhibited significantly increased survival rates after A. hydrophila challenge. The present study is the first to show the antimicrobial activity of Dps and provides an efficient strategy for production of bioactive peocin, which will aid the development of peocin as a novel antimicrobial agent with potential applications in diverse industries.


Assuntos
Bacteriocinas/biossíntese , Fermentação , Paenibacillus/metabolismo , Sequência de Aminoácidos , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Bacteriocinas/química , Bacteriocinas/genética , Bacteriocinas/isolamento & purificação , Expressão Gênica , Paenibacillus/genética , Plasmídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
5.
Eur Biophys J ; 48(7): 621-633, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31324942

RESUMO

Antimicrobial peptides are a large group of natural compounds which present promising properties for the pharmaceutical and food industries, such as broad-spectrum activity, potential for use as natural preservatives, and reduced propensity for development of bacterial resistance. Plantaricin 149 (Pln149), isolated from Lactobacillus plantarum NRIC 149, is an intrinsically disordered peptide with the ability to inhibit bacteria from the Listeria and Staphylococcus genera, and which is capable of promoting inhibition and disruption of yeast cells. In this study, the interactions of Pln149 with model membranes composed of zwitterionic and/or anionic phospholipids were investigated using a range of biophysical techniques, including isothermal titration calorimetry, surface tension measurements, synchrotron radiation circular dichroism spectroscopy, oriented circular dichroism spectroscopy, and optical microscopy, to elucidate these peptides' mode of interactions and provide insight into their functional roles. In anionic model membranes, the binding of Pln149 to lipid bilayers is an endothermic process and induces a helical secondary structure in the peptide. The helices bind parallel to the surfaces of lipid bilayers and can promote vesicle disruption, depending on peptide concentration. Although Pln149 has relatively low affinity for zwitterionic liposomes, it is able to adsorb at their lipid interfaces, disturbing the lipid packing, assuming a similar parallel helix structure with a surface-bound orientation, and promoting an increase in the membrane surface area. Such findings can explain the intriguing inhibitory action of Pln149 in yeast cells whose cell membranes have a significant zwitterionic lipid composition.


Assuntos
Bacteriocinas/química , Bacteriocinas/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Adsorção , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Ligação Proteica , Tensão Superficial , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo
6.
Carbohydr Polym ; 222: 115021, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31320086

RESUMO

We reported the preparation of antibacterial corn starch film (57% reduction in bacterial count) with enhanced tensile strength (69%) by incorporating immobilized bacteriocin. Whisker shaped crystalline nanocellulose (CNC, length 71.2 ± 20.7 nm and width 27.8 ± 11.2 nm) was prepared from cotton linters by bio-mechanical process, having the degree of polymerization 250. Bacteriocins extracted from broth cultures of P. acidilactici and E. faecium were immobilized on the surface of CNC and used to reinforce the starch film. The biodegradability of reinforced films was affected due to the use of bacteriocin in fillers. Surface morphology and roughness of reinforced films were studied by SEM and AFM. In an ambient environment, the films incorporated with bacteriocin immobilized CNC stayed fresh for 28 days while that of bacteriocin alone had fungal degradation in 14 days. This supports the requirement of CNC immobilization for better stability of bacteriocin on storage.


Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Plásticos Biodegradáveis/química , Celulose/química , Nanopartículas/química , Amido/química , Antibacterianos/química , Bacteriocinas/química , Escherichia coli/efeitos dos fármacos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/farmacologia , Permeabilidade , Solubilidade , Staphylococcus aureus/efeitos dos fármacos , Propriedades de Superfície , Resistência à Tração , Água/química , Zea mays/química
7.
Org Biomol Chem ; 17(27): 6519-6527, 2019 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-31232404

RESUMO

Fmoc-based solid-phase synthesis provides efficient access to both linear and macrocyclic peptides. To synthesize complex macrocyclic polyamides using Fmoc chemistry, multiple protective groups with orthogonal reactivities are generally employed because the free amines and carboxylic acids of specific residues must be selectively exposed prior to amide formation. This review focuses on four-dimensionally orthogonal protective group strategies for the full solid-phase synthesis of macrocyclic peptides with branched chains (polymyxin E2 and daptomycin) and a tricyclic natural peptide (lacticin 481).


Assuntos
Antibacterianos/síntese química , Produtos Biológicos/síntese química , Peptídeos Cíclicos/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Antibacterianos/química , Bacteriocinas/síntese química , Bacteriocinas/química , Produtos Biológicos/química , Colistina/síntese química , Colistina/química , Daptomicina/síntese química , Daptomicina/química
8.
J Food Sci ; 84(7): 1864-1870, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31237974

RESUMO

In this study, an active antibacterial packaging film was developed by coating a polyethylene terephthalate/polyvinylidene chloride/retort casting polypropylene (PPR) plastic multilayer film with plantaricin BM-1 and chitosan. The characteristics of the active packaging film and its antibacterial effect for chilled meat preservation were evaluated. Our results indicated that the barrier properties against oxygen were improved significantly and the tensile strength and the elongation at break were changed slightly. The active plantaricin film significantly (P < 0.05) decreased the viable counts of Listeria monocytogenes by 3.6 log10 CFU/mL in liquid medium and approximately 1.4 log10 CFU/g in meat stored at 4 °C for 8 days compared with the control. Moreover, the viable counts of aerobes and anaerobes in the meat packaged with the active plantaricin film were significantly (P < 0.05) decreased by approximately 0.6 log10 CFU/g and 1.1 log10 CFU/g when compared with that packaged with PPR film stored at 4 °C for 12 days. The total volatile base (TVB-N) in the meat packaged with the active plantaricin film was significantly (P < 0.05) lower than that in the control during the entire storage period. Our results indicated that the active film could extend the meat shelf life by inhibiting the L. monocytogenes and the background spoilage bacteria in chilled meat stored at 4 °C. This outcome suggests that plastic multilayer film incorporating plantaricin BM-1 can be potentially used for fresh meat packaging. PRACTICAL APPLICATION: Fresh meat is highly perishable product. This study developed a plantaricin BM-1 active plastic multilayer film that can inhibit the growth of microorganisms in chilled meat during storage at 4 °C.


Assuntos
Antibacterianos/química , Bacteriocinas/química , Embalagem de Alimentos/instrumentação , Carne/microbiologia , Plásticos/química , Animais , Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Contagem de Colônia Microbiana , Microbiologia de Alimentos , Embalagem de Alimentos/métodos , Conservação de Alimentos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Carne/análise , Suínos
9.
ACS Chem Biol ; 14(7): 1583-1592, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31243957

RESUMO

Lanthipeptides, which belong to the superfamily of ribosomally synthesized and posttranslationally modified peptides (RiPPs), are associated with various interesting biological activities. Lanthipeptides can be subdivided into four classes that are defined by the characteristics of the corresponding posttranslational modification enzymes. Class IV lanthipeptide synthetases consist of an N-terminal lyase, a central kinase, and a C-terminal cyclase domain. Here, we present the first in-depth characterization of such a kinase domain from the globisporin maturation enzyme SgbL that originates from Streptomyces globisporus sp. NRRL B-2293. Catalytic residues were identified by alignments with homologues and structural modeling. Their roles were confirmed by employing proteins with Ala substitutions in in vitro modification and fluorescence polarization binding assays. Furthermore, the protein region that is binding the leader peptide was identified by hydrogen-deuterium exchange-mass spectrometry experiments. By fusion of this protein region to the maltose binding protein, a protein was generated that can specifically bind the SgbA leader peptide, albeit with reduced binding affinity compared to that of full length SgbL. Combined, the results of this study provide a firmer grasp of how lanthipeptide biosynthesis is accomplished by class IV synthetases and suggest by homology analysis that biosynthetic mechanisms are similar in class III lanthipeptide processing enzymes.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriocinas/metabolismo , Ligases/metabolismo , Streptomyces/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Bacteriocinas/química , Domínio Catalítico , Ligases/química , Domínios Proteicos , Sinais Direcionadores de Proteínas , Streptomyces/química , Especificidade por Substrato
10.
Talanta ; 203: 322-327, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31202346

RESUMO

Fast detection of bacteria in samples presumed to be un-contaminated, such as blood, is of great importance. Indeed, rapid diagnosis allows the set-up of appropriate antibiotic treatment. Besides clinical issues, there are many other domains, such as food processing or drug manufacturing, where the strict absence of any bacteria has to be assessed. Because the bacterial load found in most contaminated samples is often below the limit of detection for currently validated assays, a preliminary enrichment step is required to allow bacterial multiplication before proceeding to the analysis step, whatever it might be - cultural, immunological or molecular methods. In this study, we describe the use of a biosensor for single-step bacteria detection. The whole analysis is performed in less than 20 h, during the growth phase of the micro-organisms, using an array of antimicrobial peptides (AMPs) coupled with a surface plasmon resonance imager (SPRI). A wide range of bacterial strains are assayed, showing differentiated affinity patterns with the immobilized peptides, which are confirmed by multivariate analysis. This work establishes the evidence that antimicrobial peptides, mostly used so far in the antibiotic drug industry, are suited for the wide-spectrum detection of unknown bacteria in samples, even at very low initial loads. Moreover, the small set of AMPs that were assayed provided a specific affinity profile for each pathogen, as confirmed by multivariate analyses. Furthermore, this work opens up the possibility of applying this method in more complex and relevant samples such as foodstuff, urine or blood.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Bactérias/isolamento & purificação , Bacteriocinas/metabolismo , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Bactérias/metabolismo , Bacteriocinas/química , Técnicas Biossensoriais/métodos , Limite de Detecção , Análise Multivariada , Análise de Componente Principal , Ligação Proteica , Ressonância de Plasmônio de Superfície/métodos
11.
J Microbiol Biotechnol ; 29(7): 1033-1042, 2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31216789

RESUMO

Bacillus velezensis BS2 was isolated from meongge (common sea squirt) jeotgal, a Korean fermented seafood, and produces a bacteriocin, BacBS2, which strongly inhibits Listeria monocytogenes and Bacillus cereus. BacBS2 was partially purified by Q-Sepharose column chromatography after ammonium sulfate precipitation of the culture supernatant, then further purified by Sephadex G-50 column chromatography. Partially purified BacBS2 was estimated to be 6.5 kDa in size by Tricine-SDS PAGE and activity detection by gel-overlay. Enzyme treatment and FT-IR spectrum of partially purified BacBS2 confirmed its proteinaceous nature. BacBS2 was fully stable at pH 4-9, and half of activity was retained at pH 1-3. Full activity was retained after exposure to 80°C for 15 min, but half of the activity was retained upon exposure to 90°C for 15 min or 100°C for 10 min. BacBS2 inhibited L. monocytogenes by bactericidal mode of action. B. velezensis BS2 and its BacBS2 seem useful as biopreservatives for fermented foods such as jeotgal.


Assuntos
Antibacterianos/metabolismo , Bacillus/metabolismo , Bacteriocinas/isolamento & purificação , Bacteriocinas/metabolismo , Conservantes de Alimentos/metabolismo , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Antibiose , Bacillus/crescimento & desenvolvimento , Bacillus/fisiologia , Bacillus cereus/efeitos dos fármacos , Bacteriocinas/química , Bacteriocinas/farmacologia , Meios de Cultura , Conservantes de Alimentos/química , Conservantes de Alimentos/isolamento & purificação , Conservantes de Alimentos/farmacologia , Temperatura Alta , Concentração de Íons de Hidrogênio , Listeria monocytogenes/efeitos dos fármacos , Peso Molecular , Estabilidade Proteica
12.
Phys Chem Chem Phys ; 21(23): 12530-12539, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31147666

RESUMO

The emergence of antibiotic-resistance is a major concern to global human health and identification of novel antibiotics is critical to mitigate the threat. Mutacin 1140 (MU1140) is a promising antimicrobial lanthipeptide and is effective against Gram-positive bacteria. Like nisin, MU1140 targets and sequesters lipid II and interferes with its function, which results in the inhibition of bacterial cell wall synthesis, and leads to bacteria cell lysis. MU1140 contains a structurally similar thioether cage for binding the lipid II pyrophosphate as for nisin. In addition to lipid II binding, nisin is known to form membrane pores. Membrane pore formation and membrane disruption is a common mode of action for many antimicrobial peptides, including gallidermin, a lantibiotic peptide with similar structural features as MU1140. However, whether and how MU1140 and its variants can form permeable membrane pores remains to be demonstrated. In this work, we explored the potential mechanisms of membrane pore formation by performing molecular simulations of the MU1140-lipid II complex in the bacterial membrane. Our results suggest that MU1140-lipid II complexes are able to form water permeating membrane pores. We find that a single chain of MU1140 complexed with lipid II in the transmembrane region can permeate water molecules across the membrane via a single-file water transport mechanism. The ordering of the water molecules in the single-file chain region as well as the diffusion behavior is similar to those observed in other biological water channels. Multiple complexes of MU1140-lipid II in the membrane showed enhanced permeability for the water molecules, as well as a noticeable membrane distortion and lipid relocation, suggesting that a higher concentration of MU1140 assembly in the membrane can cause significant disruption of the bacterial membrane. These investigations provide an atomistic level insight into a novel mode of action for MU1140 that can be exploited to develop optimized peptide variants with improved antimicrobial properties.


Assuntos
Bacteriocinas/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Simulação de Dinâmica Molecular , Peptídeos/farmacologia , Bacteriocinas/química , Membrana Celular/efeitos dos fármacos , Bactérias Gram-Positivas/citologia , Lipídeos/química , Lipídeos/farmacologia , Testes de Sensibilidade Microbiana , Peptídeos/química , Água/química
13.
Int J Biol Macromol ; 134: 1132-1144, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31136751

RESUMO

The study sought to purify and characterize a novel bacteriocin from oral L. salivarius and studying the effect of L. salivarius and its bacteriocin against multidrug-resistant (MDR) P. aeruginosa in vivo and in vitro. Saliva Lactobacillus salivarius bacteriocin was prepared and purified. The molecular weight of purified L. salivarius bacteriocin was 13,500Da protein. The antibacterial activity of purified salivaricin LHM was higher than crude (P<0.05) and was active at a wide range of pH values, thermostable and has no lipid or carbohydrate moiety. The antibiofilm activity of salivaricin LHM was observed. In vivo, Lactobacillus salivarius and salivaricin LHM significantly decrease the effect of bacteria in the kidney and bladder, while there is an improvement of P. aeruginosa infection in ureter salivaricin LHM-treated groups (P<0.05). Analysis of serum IL-10 and IL-4 levels revealed salivaricin LHM has prophylaxis effect. In conclusion, salivaricin LHM is protein in nature, without lipid or carbohydrate moieties, heat-stable and active at a wide range of pH values and can be classified as type II bacteriocin. Lactobacillus salivarius and salivaricin LHM has anti-pseudomonas activity, immunomodulatory by increasing pro-inflammatory cytokines and antibiofilm against P. aeruginosa urinary tract infection model in vivo and in vitro.


Assuntos
Bacteriocinas/farmacologia , Biofilmes/efeitos dos fármacos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia , Adolescente , Adulto , Animais , Bacteriocinas/química , Modelos Animais de Doenças , Estabilidade de Medicamentos , Humanos , Concentração de Íons de Hidrogênio , Imunomodulação/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/patologia , Termodinâmica , Infecções Urinárias/imunologia , Infecções Urinárias/patologia , Adulto Jovem
14.
Int J Antimicrob Agents ; 53(6): 838-843, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30928682

RESUMO

The alarming burden of antibiotic resistance in nosocomial pathogens warrants the discovery and development of new and effective antimicrobial compounds. Small cationic antimicrobial peptides seem to be a promising therapeutic alternative to fight multi-drug resistance. This study investigated the in-vitro potential of a previously reported lantibiotic, paenibacillin, from the clinical perspective. An antimicrobial peptide, M152-P4, was isolated, purified and characterized from a mud isolate, and its susceptibility was determined in clinical isolates of Staphylococcus aureus and Enterococcus spp. Time-kill kinetics, resistance, probable mode of action, haemolytic activity and mammalian cytotoxicity were investigated. M152-P4 was identified as paenibacillin based on mass spectroscopy data, amino acid analysis and biosynthetic gene cluster analysis. It had potent antibacterial activity against the Gram-positive pathogens tested, with minimum inhibitory concentrations from 0.1 to 1.56 µM. It appeared very challenging for S. aureus to develop resistance to this compound. Also, paenibacillin penetrated the outer layer of bacteria, and depolarized the membrane completely by creating pores in the plasma membrane with better potential than nisin. Paenibacillin showed no haemolysis up to 60 µM, and the half maximal inhibitory concentration on mammalian cell lines was >100 µM. These results highlight the excellent antibacterial properties of paenibacillin in clinically relevant pathogens. It is stable in the presence of serum, and non-haemolytic and non-cytotoxic even above the therapeutic concentration. Further research efforts regarding toxicity and in-vivo efficacy are necessary to develop paenibacillin as a next-generation therapeutic drug to overcome multi-drug resistance in Gram-positive pathogens.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacteriocinas/farmacologia , Enterococcus/efeitos dos fármacos , Paenibacillus/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/toxicidade , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/toxicidade , Bacteriocinas/química , Bacteriocinas/isolamento & purificação , Bacteriocinas/toxicidade , Vias Biossintéticas/genética , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Farmacorresistência Bacteriana , Humanos , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Família Multigênica , Paenibacillus/classificação , Paenibacillus/isolamento & purificação , Análise de Sequência de Proteína , Esgotos/microbiologia
15.
Dokl Biochem Biophys ; 484(1): 42-44, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31012010

RESUMO

Avicin A is a bacteriocin from the gram-positive bacterium Enterococcus avium. It exhibits a high microbicidal activity against bacteria of the genus Listeria, a causative agent of the severe human infection listeriosis. We developed a biotechnological method for obtaining avicin A and characterized its structure and biological activity. We also proposed a possible mechanism of the antimicrobial action of avicin A.


Assuntos
Antibacterianos , Bacteriocinas , Enterococcus/química , Listeria/crescimento & desenvolvimento , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Bacteriocinas/química , Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia
16.
J Sci Food Agric ; 99(10): 4731-4738, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30924936

RESUMO

BACKGROUND: Sakacin-A due to its specific antimicrobial activity may represent a good candidate to develop active packaging solutions for food items supporting Listeria growth. In the present study a protein extract containing the bacteriocin sakacin-A, produced by Lactobacillus sakei Lb 706 in a low-cost culture medium containing deproteinized cheese whey, was adsorbed onto cellulose nanofibers (CNFs) to obtain an active material to be used as a mat (or a separator) in direct contact with foods. RESULTS: The applied fermentation conditions allowed 4.51 g L-1 of freeze-dried protein extract to be obtained, characterized by an antimicrobial activity of near 16 700 AU g-1 , that was used for the preparation of the active material by casting. The active material was then characterized by infrared spectra and thermogravimetric analyses. Antimicrobial trials were carried out in vitro using Listeria innocua as indicator strain; results were also confirmed in vivo, employing smoked salmon fillets intentionally inoculated with Listeria innocua: its final population was reduced to about 2.5-3 Log cycles after 28 days of storage at 6 °C in presence of sakacin-A, compared with negative control mats produced without the bacteriocin extract. CONCLUSION: This study demonstrates the possibility of producing an antimicrobial active material containing sakacin-A absorbed onto CNFs to decrease Listeria population in smoked salmon, a ready-to eat-food product. © 2019 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.


Assuntos
Antibacterianos/química , Bacteriocinas/química , Produtos Pesqueiros/análise , Conservação de Alimentos/métodos , Conservantes de Alimentos/química , Nanofibras/química , Animais , Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Celulose/química , Fast Foods/análise , Fast Foods/microbiologia , Produtos Pesqueiros/microbiologia , Conservação de Alimentos/instrumentação , Conservantes de Alimentos/farmacologia , Listeria/crescimento & desenvolvimento , Salmão/microbiologia
17.
Carbohydr Polym ; 209: 172-180, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30732796

RESUMO

Enterococcus faecium (E. faecium) isolated from Vigna mungo (Black gram) produced bacteriocin that inhibits both Gram positive and Gram negative bacteria and better heat stability (100 °C for 30 min). The bacteriocin was sensitive to protease treatment and most active in acidic pH. Bacteriocin produced by Pediococcus acidilactici was used for comparison. To enhance stability for diversified applications, the bacteriocin was immobilized by physical adsorption onto cellulose nanocrystals (CNC) extracted from cotton linters. The bacteriocin immobilization yield was 64.91% for P. acidilactici and 53.63% for E. faecium. The bacteriocin immobilized CNC was characterized by DLS particle sizing, FTIR and AFM to evaluate size distribution, chemical nature and surface morphology. The bacteriocins immobilized on CNC showed 50% increase in stability in terms of antibacterial activity. The enzymatic synthesis of CNC in combination with physical adsorption immobilization method for bacteriocin makes it an efficient system of producing antibacterial nanofillers for food packaging and bio-composites applications.


Assuntos
Antibacterianos/química , Bacteriocinas/química , Celulose/química , Enterococcus faecium/química , Proteínas Imobilizadas/química , Nanopartículas/química , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bacteriocinas/isolamento & purificação , Bacteriocinas/metabolismo , Bacteriocinas/farmacologia , Concentração de Íons de Hidrogênio , Proteínas Imobilizadas/isolamento & purificação , Proteínas Imobilizadas/metabolismo , Proteínas Imobilizadas/farmacologia , Testes de Sensibilidade Microbiana , Tamanho da Partícula , Estabilidade Proteica , Proteólise , Cloreto de Sódio/química , Temperatura Ambiente
18.
Proc Natl Acad Sci U S A ; 116(4): 1273-1278, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30626643

RESUMO

We report crystal structures of the antibacterial lasso peptides microcin J25 (MccJ25) and capistruin (Cap) bound to their natural enzymatic target, the bacterial RNA polymerase (RNAP). Both peptides bind within the RNAP secondary channel, through which NTP substrates enter the RNAP active site, and sterically block trigger-loop folding, which is essential for efficient catalysis by the RNAP. MccJ25 binds deep within the secondary channel in a manner expected to interfere with NTP substrate binding, explaining the partial competitive mechanism of inhibition with respect to NTPs found previously [Mukhopadhyay J, Sineva E, Knight J, Levy RM, Ebright RH (2004) Mol Cell 14:739-751]. The Cap binding determinant on RNAP overlaps, but is not identical to, that of MccJ25. Cap binds further from the RNAP active site and does not sterically interfere with NTP binding, and we show that Cap inhibition is partially noncompetitive with respect to NTPs. This work lays the groundwork for structure determination of other lasso peptides that target the bacterial RNAP and provides a structural foundation to guide lasso peptide antimicrobial engineering approaches.


Assuntos
Bacteriocinas/química , Peptídeos/química , Transcrição Genética/efeitos dos fármacos , Antibacterianos/química , Bactérias/efeitos dos fármacos , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/química , Conformação Proteica
19.
Folia Microbiol (Praha) ; 64(4): 535-545, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30627971

RESUMO

Seventy-eight isolates of lactic acid bacteria from Ukraine and Thailand were screened for bacteriocinogenic activity against indicator strain Lactobacillus sakei subsp. sakei JCM 1157. One isolate showed an antagonistic activity of cell-free supernatant eliminated after the treatment with Proteinase K. Based on 16S rRNA gene sequence, this isolate was identified as Enterococcus italicus. Bacteriocin produced by this strain showed antimicrobial activity against L. sakei subsp. sakei JCM 1157, Brochothrix thermosphacta DSMZ 20171, and Listeria ivanovii subsp. ivanovii DSMZ 20750 in agar well diffusion assay. This bacteriocin was cationic and hydrophobic. The partially purified bacteriocin was thermostable, while heating of cell-free supernatant increased its activity more than twofold. Molecular mass of the partially purified bacteriocin as determined by SDS-PAGE differed from enterocin A and B previously known for E. italicus. Concentrated bacteriocin decreased the level of biofilm formation in L. sakei subsp. sakei JCM 1157 and Pseudomonas aeruginosa PAO1 in 52.5 and 48.0%, respectively (p < 0.05). We suggest that the studied bacteriocin could be a perspective antibiofilm agent in food conservation and medicine.


Assuntos
Antibacterianos/química , Antibacterianos/metabolismo , Bacteriocinas/química , Bacteriocinas/metabolismo , Brassica/microbiologia , Enterococcus/metabolismo , Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Biofilmes/efeitos dos fármacos , Estabilidade de Medicamentos , Enterococcus/química , Enterococcus/genética , Enterococcus/isolamento & purificação , Fermentação , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Tailândia
20.
Mol Cell ; 73(4): 749-762.e5, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30661981

RESUMO

The introduction of azole heterocycles into a peptide backbone is the principal step in the biosynthesis of numerous compounds with therapeutic potential. One of them is microcin B17, a bacterial topoisomerase inhibitor whose activity depends on the conversion of selected serine and cysteine residues of the precursor peptide to oxazoles and thiazoles by the McbBCD synthetase complex. Crystal structures of McbBCD reveal an octameric B4C2D2 complex with two bound substrate peptides. Each McbB dimer clamps the N-terminal recognition sequence, while the C-terminal heterocycle of the modified peptide is trapped in the active site of McbC. The McbD and McbC active sites are distant from each other, which necessitates alternate shuttling of the peptide substrate between them, while remaining tethered to the McbB dimer. An atomic-level view of the azole synthetase is a starting point for deeper understanding and control of biosynthesis of a large group of ribosomally synthesized natural products.


Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Bacteriocinas/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Complexos Multienzimáticos/metabolismo , Ribossomos/enzimologia , Inibidores da Topoisomerase II/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacteriocinas/química , Bacteriocinas/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Difração de Raios X
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