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1.
Metab Eng ; 68: 34-45, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34492380

RESUMO

Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms. Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACDCgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l-asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family.


Assuntos
Bacteriocinas , Corynebacterium glutamicum , Listeria , Bacteriocinas/genética , Corynebacterium glutamicum/genética , Pediocinas/genética
2.
PLoS One ; 16(9): e0251951, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34473709

RESUMO

The purpose of this study was to explore potential mechanisms of cytotoxicity towards HeLa and HT29 cells displayed by Pediocin PA-1. We did this by carrying out sequence alignments and 3D modelling of related bacteriocins which have been studied in greater detail: Microcin E492, Enterocin AB heterodimer and Divercin V41. Microcin E492 interacts with Toll-Like Receptor 4 in order to activate an apoptosis reaction, sequence alignment showed a high homology between Pediocin PA-1 and Microcin E492 whereas 3D modelling showed Pediocin PA-1 interacting with TLR-4 in a way reminiscent of Microcin E492. Furthermore, Pediocin PA-1 had the highest homology with the Enterocin heterodimer, particularly chain A; Enterocin has also shown to cause an apoptotic response in cancer cells. Based on this we are led to strongly believe Pediocin PA-1 interacts with TLRs in order to cause cell death. If this is the case, it would explain the difference in cytotoxicity towards HeLa over HT29 cells, due to difference in expression of particular TLRs. Overall, we believe Pediocin PA-1 exhibits a dual effect which is dose dependant, like that of Microcin. Unfortunately, due to the COVID-19 pandemic, we were unable to carry out experiments in the lab, and the unavailability of important data meant we were unable to provide and validate out solid conclusions, but rather suggestions. However, bioinformatic analysis is still able to provide information regarding structure and sequence analysis to draw plausible and evidence based conclusions. We have been able to highlight interesting findings and how these could be translated into future research and therapeutics in order to improve the quality of treatment and life of cancer patients.


Assuntos
Bacteriocinas/química , Bacteriocinas/farmacologia , Pediocinas/química , Pediocinas/farmacologia , Conformação Proteica , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Apoptose/efeitos dos fármacos , Bacteriocinas/genética , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/farmacologia , COVID-19/epidemiologia , COVID-19/prevenção & controle , COVID-19/virologia , Sobrevivência Celular/efeitos dos fármacos , Células HT29 , Células HeLa , Humanos , Modelos Moleculares , Pandemias , Pediocinas/genética , SARS-CoV-2/fisiologia , Homologia de Sequência de Aminoácidos , Receptor 4 Toll-Like/metabolismo
3.
Bioresour Technol ; 338: 125565, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34315131

RESUMO

Listeria monocytogenes is one of the foodborne pathogens of most concern for food safety. To limit its presence in foods, bacteriocins have been proposed as natural bio-preservatives. Herein, a bacteriocin was produced on hemicellulose hydrolysate of sugarcane bagasse by Pediococcus pentosaceous ET34, whose genome sequencing revealed an operon with 100% similarity to that of pediocin PA-1. ET34 grown on hydrolysate-containing medium led to an increase in the expression of PA-1 genes and a non-optimized purification step sequence resulted in a yield of 0.8 mg·L-1 of pure pediocin (purity > 95%). Culture conditions were optimized according to a central composite design using temperature and hydrolysate % as independent variables and validated in 3-L Erlenmeyers. Finally, a process for scaled-up implementation by sugar-ethanol industry was proposed, considering green chemistry and biorefinery concepts. This work stands up as an approach addressing a future proper sugarcane bagasse valorisation for pediocin production.


Assuntos
Bacteriocinas , Saccharum , Celulose , Pediocinas , Pediococcus , Pediococcus pentosaceus , Polissacarídeos
4.
Folia Microbiol (Praha) ; 65(6): 955-961, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32578013

RESUMO

Pediocin PA-1 is a bacteriocin that shows strongly anti-microbial activity against some Gram-positive pathogens such as Listeria monocytogenes, Staphylococcus aureus, and Enterococcus faecalis. With the broad inhibitory spectrum as well as high-temperature stability, pediocin has a potential application in the food preservation and pharmaceutical industry. Pediocin has been studied to express in many heterologous expression systems such as Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris as a free peptide. Here we showed in this study a new strategy by using yeast surface display system to produce the anchored pediocin PA-1 on the cell surface of Saccharomyces cerevisiae, which could be used directly as a pediocin resource. We had successfully constructed a recombinant S. cerevisiae W303 strain that could express pediocin PA-1 on the cell surface. The pediocin-expressing yeast could inhibit the growth of Shigella boydii and Shigella flexneri, which have never been reported before for pediocin activity. Besides, the pediocin expression level of the recombinant S. cerevisiae strain was also evaluated in three different media: synthetic defined (SD), basic medium (BM), and fermentation medium (FM). BM medium was shown to give the highest production yield of the recombinant yeast (4.75 ± 0.75 g dry cell weight per 1 L of culture) with the ratio number of the pediocin-expressing cells of 93.46 ± 2.45%. Taken together, the results clearly showed that pediocin can be displayed on yeast cell surface as anchored protein. The application of yeast cell surface system enables a new door of pediocin application on either food or feed industries. Graphical abstract.


Assuntos
Pediocinas/genética , Pediocinas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bacteriocinas , Fermentação , Vetores Genéticos , Testes de Sensibilidade Microbiana , Pediocinas/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
5.
Biochim Biophys Acta Biomembr ; 1862(10): 183346, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32428447

RESUMO

Pediocin PA-1 (PA-1) is a membrane-targeting bacteriocin from lactic acid bacteria, which shows antimicrobial activity against a wide range of Gram-positive pathogens. However, the outer membrane of Gram-negative bacteria does not allow pediocin access to its target. In this work, the synergistic inhibitory mechanism of PA-1 with L-lactic acid against Gram-negative aquaculture and food pathogen Aeromonas hydrophila (A. hydrophila) was analyzed. The combined treatment of 3.5 mmol/L L-lactic acid and 50 µmol/L (or 30 µmol/L) PA-1 had strong bacteriostatic and bactericidal activity against A. hydrophila. Full wavelength scanning and ELISA assay revealed the release of lipopolysaccharide (LPS) from the outer membrane of A. hydrophila caused by L-lactic acid treatment. Laser confocal microscopic imaging of A. hydrophila with FITC-labeled pediocin PA-1 proved the accumulation of PA-1 on lactic acid-treated bacterial cells. PA-1 then caused a rapid dissipation of membrane potential (Δψ) and a proton gradient difference (ΔpH) in lactic acid-treated A. hydrophila. Pediocin PA-1 also caused an increase in the extracellular ATP level. Morphology revealed by SEM and TEM showed that combined treating with lactic acid and PA-1 induced vesicles on the cell surface, the outer and inner membrane disruption, and even cytoplasm leakage and cell lysis. The results proved a potential mechanism of the synergistic inhibition of lactic acid and PA-1 against A. hydrophila, by which L-lactic acid released the outer membrane LPS, making it possible for PA-1 to contact the plasma membrane of A. hydrophila, resulting in the dissipation of proton-motive force in the inner membrane and cell death.


Assuntos
Aeromonas hydrophila/efeitos dos fármacos , Antibacterianos/farmacologia , Ácido Láctico/farmacologia , Pediocinas/farmacologia , Aeromonas hydrophila/metabolismo , Sequência de Aminoácidos , Antibacterianos/química , Sinergismo Farmacológico , Lipopolissacarídeos/metabolismo , Testes de Sensibilidade Microbiana , Pediocinas/química
6.
World J Microbiol Biotechnol ; 36(5): 72, 2020 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-32363424

RESUMO

The bacteriocinogenic lactic acid bacterium Pediococcus pentosaceus LJR1 isolated from rumen liquor of goat had strong anti-bacterial activity toward Listeria monocytogenes in vitro. This antibacterial activity was lost on treatment with protease indicating that the bacteriocin is proteinaceous in nature. The bacteriocin LJR1 produced by P. pentosaceus was purified following a three step procedure consisting of ammonium sulphate precipitation, gel filtration chromatography and reverse phase-high performance liquid chromatography. The molecular weight of purified bacteriocin was determined to be 4.6 kDa using Tricine SDS-PAGE. Further, we found that the proteinaceous bacteriocin was stable at 100 °C as well as 121 °C for 30 min and 15 min respectively and also at different pH ranging from 4 to 10 when stored for 15 min at 37 °C. Its minimum inhibitory concentration for S. typhi MTCC134 and L. monocytogenes MTCC 1143 was 7.81 µg/ml and 15.63 µg/ml respectively. Scanning electron microscopy analysis of the surface of S. typhi treated with the bacteriocin showed the presence of craters; while in the case of treated L. monocytogenes blebs were observed. The addition of the bacteriocin to shrimp (white leg shrimp) has led to reduction of about 1 log units of L. monocytogenes on day 1 and maintained for 7 days on storage at 4 °C. It is clear that the purified bacteriocin has good potential as a bio preservative for application in food industry.


Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Conservação de Alimentos/métodos , Pediococcus pentosaceus/metabolismo , Penaeidae/microbiologia , Animais , Antibacterianos/química , Bacteriocinas/química , Bacteriocinas/genética , Microbiologia de Alimentos , Listeria monocytogenes/citologia , Listeria monocytogenes/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Peso Molecular , Pediocinas/genética , Pediococcus pentosaceus/genética , Salmonella typhi/citologia , Salmonella typhi/efeitos dos fármacos , Alimentos Marinhos/microbiologia
7.
J Food Prot ; 83(10): 1693-1700, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32421790

RESUMO

ABSTRACT: The quorum-sensing regulation of class II bacteriocin (AcH) synthesis in Lactobacillus plantarum subsp. plantarum Zhang-LL was studied. No detectable inhibition zone was formed by the supernatant of L. plantarum subsp. plantarum Zhang-LL culture in skim milk (SM) with an inoculum size of 7 × 102 CFU/mL after incubation for 36 h. Hence, this culture system was used to investigate the induced regulation mechanism of bacteriocin production in L. plantarum subsp. plantarum Zhang-LL. Bacteriocin production by this bacterium in SM medium was induced by treatment with inactivated culture supernatant from de Man Rogosa Sharpe (MRS) medium (supernatant-MRS). Pediocin AcH encoded by the papA gene in a plasmid in strain Zhang-LL was the inducer present in supernatant-MRS. This is the first report of the role of pediocin AcH in the quorum-sensing regulation of class II bacteriocin synthesis. The mRNA of the papA, papB, papC, and papD genes involved in bacteriocin synthesis by strain Zhang-LL in SM medium was upregulated significantly after being induced by pediocin AcH. This study offers the first evidence that the ABT40_05745, ABT40_05750, and ABT40_11975 components of two-component systems in L. plantarum subsp. plantarum Zhang-LL are involved in the induced regulation of AcH bacteriocin production.


Assuntos
Bacteriocinas , Lactobacillus plantarum , Humanos , Lactobacillus , Lactobacillus plantarum/genética , Pediocinas
8.
Res Microbiol ; 171(3-4): 115-121, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32119904

RESUMO

Listeria ivanovii is one of the two pathogenic species within the genus Listeria, the other being Listeria monocytogenes. In this study, we generated a stable pediocin resistant mutant Liv-r1 of a L. ivanovii strain, compared phenotypic differences between the wild-type and the mutant, localised the pediocin-induced mutations in the chromosome, and analysed the mechanisms behind the bacteriocin resistance. In addition to pediocin resistance, Liv-r1 was also less sensitive to nisin. The growth of Liv-r1 was significantly reduced with glucose and mannose, but less with cellobiose. The cells of Liv-r1 adsorbed less pediocin than the wild-type cells. Consequently, with less pediocin on the cell surface, the mutant was also less leaky, as shown as the release of intracellular lactate dehydrogenase to the supernatant. The surface of the mutant cells was more hydrophobic than that of the wild-type. Whole genome sequencing revealed numerous changes in the Liv-r1 chromosome. The mutations were found e.g., in genes encoding sigma-54-dependent transcription regulator and internalin B, as well as in genes involved in metabolism of carbohydrates such as glucose and cellobiose. Genetic differences observed in the mutant may be responsible for resistance to pediocin but no direct evidence is provided.


Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Farmacorresistência Bacteriana , Listeria/efeitos dos fármacos , Listeria/genética , Listeriose/microbiologia , Pediocinas/genética , Pediocinas/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Metabolismo dos Carboidratos , Genoma Bacteriano , Genômica/métodos , Listeria/metabolismo , Listeriose/tratamento farmacológico , Testes de Sensibilidade Microbiana , Pediocinas/química , Sequenciamento Completo do Genoma
9.
PLoS One ; 15(3): e0230345, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32160253

RESUMO

With regards to the frequently reported findings of spoilage bacteria and pathogens in various foods there is a need to explore new ways to control hazards in food production and to improve consumer safety. Fermented sausages from traditional and industrial production in Germany were screened for lactic acid bacteria with antibacterial effects towards important foodborne pathogens (Escherichia coli DSM 1103, Listeria innocua DSM 20649, Listeria monocytogenes DSM 19094, Pseudomonas aeruginosa DSM 939, Staphylococcus aureus DSM 799 and Salmonella Typhimurium DSM 19587). The obtained isolates and their cell-free supernatants were tested for their antibacterial activity by agar well diffusion assay. Isolates with an inhibitory effect were examined for the underlying antibacterial mechanism. Among the 169 collected isolates, 12.4% showed antibacterial effects only against Listeria innocua DSM 20649 and Listeria monocytogenes DSM 19094. In 6.5% of the isolates, bacteriocins were responsible for the effect. On the remaining test strains, the lactic bacteria isolates exerted no antibacterial effect. Two isolates were selected based on their antibacterial potential against Listeria spp. and the thermostability of the deriving cell free supernatants, traditional product: Pediococcus pentosaceus LMQS 331.3, industrial product: Pediococcus acidilactici LMQS 154.1, were investigated further and confirmed for the presence of bacteriocin structural genes by real-time PCR. Enriched crude bacteriocin preparations were obtained by ammonium sulfate precipitation and were found to remain stable under different pH milieus and to be active towards an extended set of Listeria spp. strains. Fermented meat products from German production are a promising source for bacteriocin-producing lactic acid bacteria. Two bacteriocin-producing isolates were identified which have the potential to contribute to product and consumer safety.


Assuntos
Antibacterianos/metabolismo , Pediocinas/metabolismo , Pediococcus acidilactici/isolamento & purificação , Pediococcus pentosaceus/isolamento & purificação , Fermentação , Indústria Alimentícia , Microbiologia de Alimentos , Inocuidade dos Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Doenças Transmitidas por Alimentos/prevenção & controle , Alemanha , Produtos da Carne/microbiologia , Testes de Sensibilidade Microbiana , Pediococcus acidilactici/metabolismo , Pediococcus pentosaceus/metabolismo
10.
Int J Biol Macromol ; 143: 555-572, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31785295

RESUMO

Human-milk-based probiotics play a major role in the early colonization and protection of infants against gastrointestinal infection. We investigated potential probiotics in human milk. Among 41 Lactic acid bacteria (LAB) strains, four strains showed high antimicrobial activity against Escherichia coli 0157:H7, Listeria monocytogenes ATCC 15313, Bacillus cereus ATCC 14576, Staphylococcus aureus ATCC 19095, and Helicobacter pylori. The selected LAB strains were tested in simulated gastrointestinal conditions for their survival. Four LAB strains showed high resistance to pepsin (82%-99%), bile with pancreatine stability (96%-100%), and low pH (80%-94%). They showed moderate cell surface hydrophobicity (22%-46%), auto-aggregation abilities (12%-34%), and 70%-80% co-aggregation abilities against L. monocytogenes ATCC 15313, S. aureus ATCC 19095, B. cereus ATCC 14576, and E. coli 0157:H7. All four selected isolates were resistant to gentamicin, imipenem, novobiocin, tetracycline, clindamycin, meropenem, ampicillin, and penicillin. The results show that Pediococcus acidilatici is likely an efficient probiotic strain to produce < 3 Kda pediocin-based antimicrobial peptides, confirmed by applying amino acid sequences), using liquid chromatography mass spectrometry and HPLC with the corresponding sequences from class 2 bacteriocin, and based on the molecular docking, the mode of action of pediocin was determined on LipoX complex, further the 13C nuclear magnetic resonance structural analysis, which confirmed the antimicrobial peptide as pediocin.


Assuntos
Antibacterianos , Infecções Bacterianas , Caenorhabditis elegans/microbiologia , Pediocinas , Pediococcus acidilactici/química , Probióticos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Pediocinas/química , Pediocinas/farmacologia , Probióticos/química , Probióticos/farmacologia
11.
Food Microbiol ; 86: 103315, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31703881

RESUMO

Non-thermal food processing and replacement of chemical additives by natural antimicrobials are promising trends in the food industry. The objective of the present work was to evaluate the effect of a process which combines mild high hydrostatic pressure - HHP (200 and 300 MPa, 5 min, 10 °C), phage Listex™ P100 and the bacteriocin pediocin PA-1 as a new non-thermal process for destruction of Listeria monocytogenes (104 CFU mL-1 or 107 CFU mL-1) in milk. For inoculum levels of 104 CFU mL-1, HHP combined with phage P100 eliminated L. monocytogenes immediately after pressurization. When L. monocytogenes was inoculated at levels of 107 CFU mL-1, a synergistic effect between phage P100, pediocin PA-1 and HHP (300 MPa) on the inactivation of L. monocytogenes was observed during storage of milk at 4 °C. For non-pressure treated samples inoculated with phage or pediocin or both, L. monocytogenes counts decreased immediately after biocontrol application, but regrowth was observed in a few samples during storage. Phage particles were stable during refrigerated storage for seven days while pediocin PA-1 remained stable only during three days. Further studies will have to be performed to validate the findings of this work in specific applications (e.g. production of raw milk cheese).


Assuntos
Bacteriófagos/fisiologia , Conservação de Alimentos/métodos , Conservantes de Alimentos/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/virologia , Leite/microbiologia , Pediocinas/farmacologia , Animais , Bovinos , Contagem de Colônia Microbiana , Conservação de Alimentos/instrumentação , Pressão Hidrostática , Listeria monocytogenes/química , Listeria monocytogenes/crescimento & desenvolvimento
12.
Food Microbiol ; 85: 103282, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31500713

RESUMO

Two biopreservation approaches for fresh lettuce, rocket salad, parsley and spinach were studied. The potential of Pediococcus pentosaceus DT016, as a protective culture, to suppress Listeria monocytogenes in vegetables during storage was evaluated. The pathogen numbers in the vegetables inoculated with P. pentosaceus DT016 were significantly (p < 0.01) lower throughout the storage period and, at the last storage day, a minimum difference of 1.4 log CFU/g was reported when compared with the vegetables without the protective culture. Moreover, by using two levels of L. monocytogenes (about 6 and 4 log CFU/g), it was observed that the antagonist effect of P. pentosaceus was higher for the lower pathogen numbers. The second approach evaluated a pediocin DT016 solution to inactivate and control L. monocytogenes proliferation. The pathogen load was studied after washing with: water, chlorine and the pediocin solution and along storage at 4  °C. Comparing the various washing solutions, the vegetables washed with pediocin presented significantly (p < 0.01) lower pathogen numbers throughout storage, by a minimum of 3.2 and 2.7 log CFU/g, than in vegetables washed with water and chlorine, respectively. The proposed methodologies are promising alternatives to maintain the safety of fresh vegetables during extended storage at refrigeration temperature.


Assuntos
Antibiose , Microbiologia de Alimentos/métodos , Conservação de Alimentos/métodos , Listeria monocytogenes/fisiologia , Verduras/microbiologia , Carga Bacteriana , Cloro/farmacologia , Temperatura Baixa , Qualidade de Produtos para o Consumidor , Manipulação de Alimentos/métodos , Alface/microbiologia , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Pediocinas/farmacologia , Pediococcus pentosaceus/fisiologia , Petroselinum/microbiologia , Refrigeração , Spinacia oleracea/microbiologia , Água
13.
Biochimie ; 165: 183-195, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31381962

RESUMO

Class II bacteriocins are unmodified membrane-active peptides that act over a narrow spectrum of target bacteria. They bind a specific receptor protein on the membrane to form a pore, leading to membrane permeabilization and cell death. However, little is known about the molecular events triggering the pore formation after the bacteriocin recognizes the receptor. It is not clear yet if the pore is the same receptor forced into an open conformation or if the pore results from the bacteriocin insertion and oligomeric assembly in the lipid bilayer. In order to reveal which model is more suitable to explain the toxicity mechanism, in this work we use chimeric peptides, resulting from the fusion of the bitopic membrane protein EtpM with different class II bacteriocins: enterocin CRL35, pediocin PA-1 and microcin V. E. coli strains lacking the specific receptors for these bacteriocins were chosen as expression hosts. As these constructs display a lethal effect when they are heterologously expressed, they are called "suicide probes". The results suggest that, indeed, the specific receptor would act as a docking molecule more than as a structural piece of the pore, as long as the bacteriocin is somehow anchored to the membrane. These set of chimeric peptides also represent an in vivo system that allows to study the interaction of the bacteriocins with real bacterial membranes, instead of model membranes. Hence, the effects of these suicide probes in membrane fluidity and transmembrane potential were also assessed, using fluorescence spectroscopy. The data show that the different suicide probes are able to increase phospholipid order and depolarize the membranes of receptor-free bacterial cells.


Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Escherichia coli/fisiologia , Pediocinas/farmacologia , Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos
14.
Biochemistry (Mosc) ; 84(5): 464-478, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31234762

RESUMO

Bacteriocins are bacterial antimicrobial peptides that, unlike classical peptide antibiotics, are products of ribosomal synthesis and usually have a narrow spectrum of antibacterial activity against species closely related to the producers. Pediocin-like bacteriocins (PLBs) belong to the class IIa of the bacteriocins of Gram-positive bacteria. PLBs possess high activity against pathogenic bacteria from Listeria and Enterococcus genera. Molecular target for PLBs is a membrane protein complex - bacterial mannose-phosphotransferase. PLBs can be synthesized by components of symbiotic microflora and participate in the maintenance of homeostasis in various compartments of the digestive tract and on the surface of epithelial tissues contacting the external environment. PLBs could give a rise to a new group of antibiotics of narrow spectrum of activity.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Bactérias Gram-Positivas/metabolismo , Pediocinas/metabolismo , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Enterococcus/efeitos dos fármacos , Bactérias Gram-Positivas/imunologia , Listeria/efeitos dos fármacos , Pediocinas/química , Pediocinas/farmacologia , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Estrutura Secundária de Proteína , Alinhamento de Sequência
15.
Folia Microbiol (Praha) ; 64(6): 765-778, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30796707

RESUMO

Pediococcus pentosaceus GS4 (MTCC 12683), a probiotic lactic acid bacterium (LAB), was found to produce bacteriocin in spent culture. Antibacterial and antagonistic potential of this bacteriocin against reference strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 25619), and Listeria monocytogenes (ATCC 15313) was proven by double-layer and well diffusion methods wherein nisin and ampicillin were used as positive controls. Bacteriocin in supernatant was purified and analyzed by SDS-PAGE, RP-HPLC, and circular dichroism (CD). The physico-chemical properties of purified bacteriocin were characterized being treated at different temperatures (30 to 110 °C), pH (3.0 to 12.0), with different enzymes (α-amylase, pepsin, and lysozyme), and organic solvents (hexane, ethanol, methanol, and acetone) respectively. The molar mass of bacteriocin (named pediocin GS4) was determined as 9.57 kDa. The single peak appears at the retention time of 2.403 with area amounting to 25.02% with nisin as positive control in RP-HPLC. CD analysis reveals that the compound appears to have the helix ratio of 40.2% with no beta sheet. The antibacterial activity of pediocin GS4 was optimum at 50 °C and at pH 5.0 and 7.0. The pediocin GS4 was not denatured by the treatment of amylase and lysozyme but was not active in the presence of organic solvents. This novel bacteriocin thus m ay be useful in food and health care industry.


Assuntos
Antibacterianos/química , Antibacterianos/isolamento & purificação , Pediocinas/química , Pediocinas/isolamento & purificação , Pediococcus pentosaceus/química , Probióticos , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Hidrolases/metabolismo , Peso Molecular , Nisina/química , Pediocinas/farmacologia , Pediococcus pentosaceus/metabolismo , Estabilidade Proteica , Estrutura Secundária de Proteína , Solventes , Temperatura
16.
São Paulo; s.n; s.n; 2019. 81 p. ilus, graf, tab.
Tese em Português | LILACS | ID: biblio-1015240

RESUMO

Um dos grandes desafios nas indústrias alimentícia, farmacêutica e agropecuária é a busca por novos compostos para substituir os antibióticos. Como possíveis candidatos estão as bacteriocinas para serem utilizados paralelamente aos antibióticos ou até substituí-los. As bactérias ácido-láticas podem produzir substâncias inibitórias semelhantes às bacteriocinas (BLIS - bacteriocin-like inhibitory substances) que possuem efeito bacteriostático ou bactericida contra diferentes grupos de bactéria sendo largamente utilizadas como bioconservantes alimentares. Neste contexto, o objetivo deste trabalho foi o emprego de um resíduo agroindustrial, o hidrolisado de bagaço de cana-de-açúcar, como fonte de carbono em cultivos fermentativos para produção do BLIS pela cepa Pediococcus pentosaceus ET 34. Resultados revelaram que as células de ET34 foram capazes de crescer utilizando este resíduo agroindustrial como fonte de carbono e ensaios utilizando planejamento fatorial demonstraram que a agitação não influencia na produção de BLIS. Ao escalonar o cultivo em biorreatores, foi verificado que tanto o crescimento como a atividade antimicrobiana foram semelhantes aos obtidos em bancada com exceção da condição de 25% (v/v) de HBC (hidrolisado de bagaço de cana) que devido a maior viscosidade do meio, resultou em uma diminuição no crescimento e de produção de BLIS. O BLIS produzido por células de ET34 utilizando o HBC como fonte de carbono foi parcialmente purificado por sulfato de amônio e demonstrou atividade contra Listeria monocytogenes ATCC 934 e Salmonella enterica CECT 724. Desta maneira, pode-se concluir que Pediococcus pentosaceus ET34 foi capaz de crescer em meio contendo HBC como fonte de carbono produzindo BLIS em seu sobrenadante com ação frente a diferentes bactérias patogênicas. A possibilidade de utilizar uma fonte alternativa de carbono pode diminuir o custo de processo consideravelmente. Além disso, ensaios de planejamento fatorial, superfície de resposta e escalonamento em biorreator de bancada indicaram que concentrações baixas de HBC (5-15%, v/v) a 35 °C resultaram na maior produção de BLIS


The great challenge in the food, pharmaceutical and agricultural industries is the search for new compounds to replace antibiotics. Bacteriocins are possible candidates that can be used in parallel with the antibiotics or even to replace them. Lactic-acid bacteria can produce bacteriocin inhibitory substances (BLIS - bacteriocin-like inhibitory substances) that have a bacteriostatic or bactericidal effect against different bacterial species and are widely used as food bioconservatives. In this context, the aim of this work was to use of an agroindustrial waste, hydrolyzed sugarcane bagasse, as a carbon source in fermentative cultures for the production of BLIS by Pediococcus pentosaceus ET 34 strain. Results revealed that ET34 cells were able to grow using this agroindustrial residue as a carbon source, and trials using factorial design showed that agitation did not influence on the production of BLIS. When it was perform cultivation scale up in bioreactors, it was verified that both the growth and the antimicrobial activity were similar to those obtained in the workbench with the exception of the condition of 25% (v/v) of HBC (sugarcane bagasse hydrolyzate) that due to its higher viscosity, resulted in a decrease in growth and BLIS production. BLIS produced by ET34 cells using HBC as a carbon source that was partially purified by ammonium sulfate showed activity against Listeria monocytogenes ATCC 934 and Salmonella enterica CECT 724. Thus, it can be concluded that Pediococcus pentosaceus ET34 was able to grow in medium containing HBC as carbon source producing BLIS in its supernatant with action against different pathogenic bacteria. The possibility of using an alternative carbon source can greatly reduce the process cost. In addition, factorial design, response surface and scale up trials in bench bioreactors indicated that low concentrations of HBC (5-15% v/v) at 35 ºC resulted in higher BLIS production


Assuntos
Resíduos/classificação , Pediococcus pentosaceus/classificação , Pediocinas/análise , Saccharum
17.
São Paulo; s.n; s.n; 2019. 65 p. tab, graf.
Tese em Português | LILACS | ID: biblio-1007563

RESUMO

Bacteriocinas produzidas por bactérias láticas (BAL) apresentam um importante potencial de aplicação na bioconservação de alimentos, por sua ação antimicrobiana contra algumas espécies de microrganismos patogênicos de relevância, como Listeria monocytogenes. Este estudo analisou o efeito da interação entre cepas selecionadas de BAL produtoras de bacteriocinas com outras BAL viáveis ou não viáveis (bacteriocinogênicas ou não) na indução da produção de bacteriocinas. O efeito dos metabólitos produzidos por estas cepas na indução da bacteriocinogênese também foi avaliado. As cepas produtoras de bacteriocinas selecionadas para o estudo foram Lactobacillus sakei MBSa1, produtora de sakacina A e Pediococcus acidilactici ET34, produtora de pediocina, isoladas de salame e salmão defumado, respectivamente. A produção de pediocina por P. acidilactici ET34 foi avaliada também em leite em pó desnatado reconstituído, além de meio de cultura (caldo MRS). Os resultados indicaram que, quando em co-cultura com Enterococcus faecalis ATCC12755, Lactobacillus sakei ATCC15521 ou Listeria monocytogenes (cepas 104, 711 e 637), ou na presença do sobrenadante livre de células (SLC) dessas culturas, nenhuma das duas cepas testadas produziu maior quantidade de bacteriocina do que a produzida quando em monocultura ou na ausência do SLC. A bacteriocina produzida por P. acidilactici ET34 apresentou um efeito bacteriostático contra L. monocytogenes 104 no leite em pó desnatado reconstituído nas 12 h analisadas, com extensão da fase lag, de forma dose-dependente. Os resultados indicaram, também, que P. acidilactici ET34 não foi capaz de produzir pediocina no leite em pó desnatado reconstituído quando em monocultura ou em co-cultura, ao contrário do observado para o caldo MRS. Mais investigação é necessária para esclarecer os efeitos de possíveis interações entre as BAL presentes em um alimento, bem como o efeito dos componentes dos alimentos na produção das bacteriocinas pelas BAL bacteriocinogênicas


Bacteriocins produced by lactic acid bacteria (LAB) present an important application potential in food biopreservation, by their antimicrobial activity against some species of pathogenic microorganisms of relevance, such as Listeria monocytogenes. This study analyzed the effect of the interaction between selected strains of bacteriocin-producing LAB with other viable or non-viable LAB (bacteriocinogenic or not) in the induction of bacteriocin production. The effect of the metabolites produced by these strains on the induction of bacteriocinogenesis was also evaluated. The bacteriocin-producing strains selected for the study were Lactobacillus sakei MBSa1, producer of sakacin A and Pediococcus acidilactici ET34, producer of pediocin, isolated from salami and smoked salmon, respectively. The production of pediocin by P. acidilactici ET34 was also evaluated in reconstituted skimmed milk powder as well as culture medium (MRS broth). The results indicated that when co-cultivated with Enterococcus faecalis ATCC12755, Lactobacillus sakei ATCC15521 or Listeria monocytogenes (strains 104, 711 and 637), or in the presence of the cell free supernatant (SLC) of these cultures, neither of the two strains tested produced greater amount of bacteriocin than that produced in monoculture or in the absence of SLC. The bacteriocin produced by P. acidilactici ET34 presented a bacteriostatic effect against L. monocytogenes 104 in skimmed milk powder reconstituted in 12h, with extension of lag phase, in a dose-dependent manner. The results also indicated that P. acidilactici ET34 was not able to produce pediocin in the reconstituted skimmed milk powder when in monoculture or in co-culture, unlike that observed for the MRS broth. More research is needed to clarify the effects of possible interactions between BAL present in a food and the effect of food components on bacteriocin production by bacteriocinogenic BAL


Assuntos
Bacteriocinas/análise , Ácido Láctico , Alimentos/toxicidade , Pediocinas/efeitos adversos , Listeria monocytogenes/isolamento & purificação
18.
Appl Microbiol Biotechnol ; 102(24): 10393-10408, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30338356

RESUMO

Antimicrobial peptides (AMPs) from prokaryotic source also known as bacteriocins are ribosomally synthesized by bacteria belonging to different eubacterial taxonomic branches. Most of these AMPs are low molecular weight cationic membrane active peptides that disrupt membrane by forming pores in target cell membranes resulting in cell death. While these peptides known to exhibit broad-spectrum antimicrobial activity, including antibacterial and antifungal, they displayed minimal cytotoxicity to the host cells. Their antimicrobial efficacy has been demonstrated in vivo using diverse animal infection models. Therefore, we have discussed some of the promising peptides for their ability towards potential therapeutic applications. Further, some of these bacteriocins have also been reported to exhibit significant biological activity against various types of cancer cells in different experimental studies. In fact, differential cytotoxicity towards cancer cells as compared to normal cells by certain bacteriocins directs for a much focused research to utilize these compounds as novel therapeutic agents. In this review, bacteriocins that demonstrated antitumor activity against diverse cancer cell lines have been discussed emphasizing their biochemical features, selectivity against extra targets and molecular mechanisms of action.


Assuntos
Antineoplásicos/farmacologia , Bacteriocinas/genética , Bacteriocinas/farmacologia , Azurina/farmacologia , Bacteriocinas/química , Cátions , Membrana Celular/efeitos dos fármacos , Humanos , Nisina/farmacologia , Pediocinas/farmacologia , Engenharia de Proteínas/métodos , Piocinas/química , Piocinas/farmacologia
19.
Sci Rep ; 8(1): 9029, 2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29899567

RESUMO

The antimicrobial peptide pediocin PA-1 is a class IIa bacteriocin that inhibits several clinically relevant pathogens including Listeria spp. Here we report the synthesis and characterization of whole pediocin PA-1 and novel analogs thereof using a combination of solid- and solution-phase strategies to overcome difficulties due to instability and undesired reactions. Pediocin PA-1 thus synthesized was a potent inhibitor of Listeria monocytogenes (MIC = 6.8 nM), similar to the bacteriocin produced naturally by Pediococcus acidilactici. Of particular interest is that linear analogs lacking both of the disulfide bridges characterizing pediocin PA-1 were as potent. One linear analog was also a strong inhibitor of Clostridium perfringens, another important food-borne pathogen. These results are discussed in light of conformational information derived from circular dichroism, solution NMR spectroscopy and structure-activity relationship studies.


Assuntos
Anti-Infecciosos/farmacologia , Bacteriocinas/farmacologia , Pediocinas/farmacologia , Relação Estrutura-Atividade , Sequência de Aminoácidos , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Bacteriocinas/química , Clostridium perfringens/efeitos dos fármacos , Listeria monocytogenes/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana/métodos , Modelos Químicos , Modelos Moleculares , Pediocinas/síntese química , Pediocinas/química , Conformação Proteica
20.
Sci Rep ; 8(1): 5878, 2018 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-29650991

RESUMO

One of the most challenging aspects of probiotics as a replacement for antibiotics is to enhance their antimicrobial activity against pathogens. Given that prebiotics stimulate the growth and/or activity of probiotics, we developed phthalyl inulin nanoparticles (PINs) as prebiotics and observed their effects on the cellular and antimicrobial activities of Pediococcus acidilactici (PA). First, we assessed the internalization of PINs into PA. The internalization of PINs was largely regulated by glucose transporters in PA, and the process was energy-dependent. Once internalized, PINs induced PA to produce substantial amounts of antimicrobial peptide (pediocin), which is effective against both Gram-positive (Salmonella Gallinarum) and Gram-negative (Listeria monocytogenes) pathogens. When treated with small-sized PINs, PA witnessed a nine-fold increase in antimicrobial activity. The rise in pediocin activity in PA treated with PINs was accompanied by enhanced expression of stress response genes (groEL, groES, dnaK) and pediocin biosynthesis genes (pedA, pedD). Although the mechanism is not clear, it appears that the internalization of PINs by PA causes mild stress to activate the PA defense system, leading to increased production of pediocin. Overall, we identified a prebiotic in nanoparticle form for intracellular stimulation of probiotics, demonstrating a new avenue for the biological production of antimicrobial peptides.


Assuntos
Anti-Infecciosos/administração & dosagem , Inulina/química , Nanopartículas/química , Pediocinas/farmacologia , Anti-Infecciosos/química , Citoplasma/efeitos dos fármacos , Citoplasma/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inulina/farmacologia , Listeria monocytogenes , Pediocinas/biossíntese , Pediocinas/genética , Pediococcus acidilactici/química , Probióticos/química , Probióticos/farmacologia
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