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1.
Int J Food Microbiol ; 339: 109007, 2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33341684

RESUMO

Cast films obtained from polyvinyl alcohol (PVOH) blended with casein hydrolysates (HCas) in a weight ratio of 1:1 were employed to carry nisin-producing L. lactis and phytic acid in order to broaden the antimicrobial spectrum of L. lactis to Gram-positive and Gram-negative spoilage and pathogen bacteria. For this purpose, the effect of the antimicrobial activity of various film formulations and combinations of films on the growth of E. coli at 37 °C for 24 h was studied. The film system that showed antimicrobial activity against Gram-negative bacteria consisted of phytic acid and L. lactis incorporated in separate films. When the active agents were in the same film the viability of L. lactis decreased considerably and it did not exert antimicrobial activity against the bacterium. Therefore, the combination of L. lactis and phytic acid in separate films was chosen as the reliable system, and the effect of its activity on the growth of Gram-negative bacteria (E. coli, Salmonella enterica, and Pseudomonas fluorescens) and Gram-positive bacteria (Listeria monocytogenes) in liquid culture medium was tested at refrigeration temperature (4 °C), and with simulated breaks in the cold chain (14 °C and 24 °C). The survival of L. lactis in coexistence with these bacteria was also studied. The film system exerted an antimicrobial effect against the Gram-negative bacteria tested, and the activity depended on the bacteria and the temperature assayed. With regard to the antimicrobial activity against L. monocytogenes, phytic acid improved the antimicrobial capacity of L.lactis. The survival of L. lactis was maintained at 7-8 log (CFU/mL) culture in liquid medium throughout the storage period. The films developed were intended to be used as coatings in the design of a double-sided active bag for a non-fermented dairy product. The bags were filled with homemade preservative-free pastry cream, and the microbiological shelf life and evolution of pH of the packaged ready-to-eat food stored at 4 °C was studied for 20 days. The results showed a reduction in the growth of spoilage bacteria and therefore an increase in the shelf life of the packaged product. The films developed could be applied in the design of packages for perishable dairy foods in order to increase their microbiological shelf life.


Assuntos
Microbiologia de Alimentos/métodos , Embalagem de Alimentos/métodos , Bactérias Gram-Negativas/efeitos dos fármacos , Lactococcus lactis/metabolismo , Nisina/farmacologia , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Contagem de Colônia Microbiana , Bactérias Gram-Positivas/efeitos dos fármacos , Lactococcus lactis/crescimento & desenvolvimento , Nisina/metabolismo , Álcool de Polivinil/química , Refrigeração
2.
Virology ; 552: 107-111, 2021 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-33130382

RESUMO

Nisin, a food-grade antimicrobial peptide produced by lactic acid bacteria has been examined for its probable interaction with the human ACE2 (hACE2) receptor, the site where spike protein of SARS-CoV-2 binds. Among the eight nisin variants examined, nisin H, nisin Z, nisin U and nisin A showed a significant binding affinity towards hACE2, higher than that of the RBD (receptor binding domain) of the SARS-CoV-2 spike protein. The molecular interaction of nisin with hACE2 was investigated by homology modeling and docking studies. Further, binding efficiency of the most potent nisin H was evaluated through the interaction of hACE2:nisin H complex with RBD (receptor-binding domain) of SARS-CoV-2 and that of hACE2:RBD complex with nisin H. Here, nisin H acted as a potential competitor of RBD to access the hACE2 receptor. The study unravels for the first time that a globally used food preservative, nisin has the potential to bind to hACE2.


Assuntos
/metabolismo , Nisina/metabolismo , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Nisina/química , Ligação Proteica , Domínios Proteicos , Receptores Virais/química , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química
3.
Methods Mol Biol ; 2220: 243-257, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975780

RESUMO

This chapter describes methods used to isolate, identify, and partially characterize lactic acid bacteria (LAB) which exhibit inhibitory activity against Listeria monocytogenes from foods. Vegetal (plant based) sources are rich in naturally occurring LAB and therefore provide an easily accessible source of strains with potential antimicrobial activity for use in food-processing applications. From our previous work, the majority of LAB with inhibitory activity against L. monocytogenes were identified as generally recognized as safe (GRAS) Lactococcus lactis. Although these bacteria are most commonly known for their role in industrial dairy fermentations, they are believed to have originally derived from natural plant-based habitats. These isolates with anti-Listeria activity were all found to carry the genes involved in the production of nisin, which is an approved food-grade preservative (E234). These isolates may find various applications for in situ production of nisin allowing control of L. monocytogenes in various fermented and non-fermented foods and other environments.


Assuntos
Microbiologia de Alimentos , Lactococcus lactis/isolamento & purificação , Lactococcus lactis/fisiologia , Listeria monocytogenes/fisiologia , Interações Microbianas , Antibacterianos/metabolismo , Conservantes de Alimentos/metabolismo , Humanos , Listeriose/microbiologia , Listeriose/prevenção & controle , Nisina/metabolismo
4.
Appl Environ Microbiol ; 86(19)2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32709730

RESUMO

NisI confers immunity against nisin, with high substrate specificity to prevent a suicidal effect in nisin-producing Lactococcus lactis strains. However, the NisI maturation process as well as its influence on nisin resistance has not been characterized. Here, we report the roles of lipoprotein signal peptidase II (Lsp) and prolipoprotein diacylglyceryl transferase (Lgt) in NisI maturation and nisin resistance of L. lactis F44. We found that the resistance of nisin of an Lsp-deficient mutant remarkably decreased, while no significant differences in growth were observed. We demonstrated that Lsp could cleave signal peptide of NisI precursor in vitro Moreover, diacylglyceryl modification of NisI catalyzed by Lgt played a decisive role in attachment of NisI on the cell envelope, while it exhibited no effects on cleavage of the signal peptides of NisI precursor. The dissociation constant (KD ) for the interaction between nisin and NisI exhibited a 2.8-fold increase compared with that between nisin and pre-NisI with signal peptide by surface plasmon resonance (SPR) analysis, providing evidence that Lsp-catalyzed signal peptide cleavage was critical for the immune activity of NisI. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production.IMPORTANCE Nisin, a safe and natural antimicrobial peptide, has a long and impressive history as a food preservative and is also considered a novel candidate to alleviate the increasingly serious threat of antibiotic resistance. Nisin is produced by certain L. lactis strains. The nisin immunity protein NisI, a membrane-bound lipoprotein, is expressed by nisin producers to avoid suicidal action. Here, we report the roles of Lsp and Lgt in NisI maturation and nisin resistance of L. lactis F44. The results verified the importance of Lsp to NisI-conferred immunity and Lgt to localization. Our study revealed the process of NisI maturation in L. lactis and presented a potential strategy to enhance industrial nisin production.


Assuntos
Proteínas de Bactérias/genética , Lactococcus lactis/genética , Lipoproteínas/genética , Proteínas de Membrana/genética , Nisina/genética , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Proteínas de Bactérias/metabolismo , Lactococcus lactis/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Nisina/metabolismo , Transferases/genética , Transferases/metabolismo
5.
J Med Microbiol ; 69(4): 605-616, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32125268

RESUMO

Introduction. Against the backdrop of increasing resistance to conventional antibiotics, bacteriocins represent an attractive alternative, given their potent activity, novel modes of action and perceived lack of issues with resistance.Aim. In this study, the nature of the antibacterial activity of a clinical isolate of Streptococcus gallolyticus was investigated.Methods. Optimization of the production of an inhibitor from strain AB39 was performed using different broth media and supplements. Purification was carried out using size exclusion, ion exchange and HPLC. Gel diffusion agar overlay, MS/MS, de novo peptide sequencing and genome mining were used in a proteogenomics approach to facilitate identification of the genetic basis for production of the inhibitor.Results. Strain AB39 was identified as representing Streptococcus gallolyticus subsp. pasteurianus and the successful production and purification of the AB39 peptide, named nisin P, with a mass of 3133.78 Da, was achieved using BHI broth with 10 % serum. Nisin P showed antibacterial activity towards clinical isolates of drug-resistant bacteria, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and penicillin-resistant Streptococcus pneumoniae. In addition, the peptide exhibited significant stability towards high temperature, wide pH and certain proteolytic enzymes and displayed very low toxicity towards sheep red blood cells and Vero cells.Conclusion. To the best of our knowledge, this study represents the first production, purification and characterization of nisin P. Further study of nisin P may reveal its potential for treating or preventing infections caused by antibiotic-resistant Gram-positive bacteria, or those evading vaccination regimens.


Assuntos
Nisina/isolamento & purificação , Nisina/farmacologia , Streptococcus gallolyticus/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia , Cromatografia Líquida de Alta Pressão , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Estrutura Molecular , Nisina/química , Nisina/metabolismo , Ovinos , Streptococcus gallolyticus/química , Streptococcus gallolyticus/classificação , Streptococcus gallolyticus/genética , Espectrometria de Massas em Tandem
6.
Appl Environ Microbiol ; 86(8)2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32086306

RESUMO

Nisin A is a bacteriocin produced by Lactococcus lactis and is widely used as a food preservative. Staphylococcus aureus has the BraRS-VraDE system that provides resistance against low concentrations of nisin A. BraRS is a two-component system that induces the expression of the ABC transporter VraDE. Previously, we isolated a highly nisin A-resistant strain with increased VraDE expression due to a mutation in braRS In this study, we isolated S. aureus MW2 mutants with BraRS-VraDE-independent nisin A resistance. These mutants, designated SAN2 ( S. aureus nisin resistant) and SAN469, had a mutation in pmtR, which encodes a transcriptional regulator responsible for the expression of the pmtABCD operon. As a result, these mutants exhibited increased expression of PmtABCD, a transporter responsible for the export of phenol-soluble modulin (PSM). Characterization of the mutants revealed that they have decreased susceptibility to human ß-defensin-3 (hBD3) and LL37, which are innate immune factors. Additionally, these mutants showed higher hemolytic activity than the original MW2 strain. Furthermore, in a mouse bacteremia model, the SAN2 strain exhibited a lower survival rate than the original MW2 strain. These results indicate that the increased expression of pmtABCD due to a pmtR mutation is an alternative nisin A resistance mechanism that also affects virulence in S. aureus IMPORTANCE Recently, the emergence of antibiotic-resistant bacteria has resulted in serious problems for chemotherapy. In addition, many antibacterial agents, such as disinfectants and food additives, are widely used. Therefore, there is a possibility that bacteria are becoming resistant to some antibacterial agents. In this study, we investigated whether Staphylococcus aureus can become resistant to nisin A, one of the bacteriocins applied as a food additive. We isolated a highly nisin A-resistant strain designated SAN2 that displayed increased expression of Pmt proteins, which are involved in the secretion of virulence factors called phenol-soluble modulins (PSMs). This strain also showed decreased susceptibility to human antimicrobial peptides and increased hemolytic activity. In addition, SAN2 showed increased lethal activity in a mouse bacteremia model. Our study provides new insights into the possibility that the acquisition of resistance against food preservatives may modulate virulence in S. aureus, suggesting that we need to pay more attention to the use of food preservatives together with antibiotics.


Assuntos
Bacteriocinas/genética , Farmacorresistência Bacteriana/genética , Lactococcus lactis/fisiologia , Nisina/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Antibacterianos/farmacologia , Bacteriocinas/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Nisina/metabolismo , Staphylococcus aureus/genética , Virulência/fisiologia
7.
Int J Food Microbiol ; 322: 108545, 2020 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-32109681

RESUMO

PVOH-based polymer matrices in the form of films were evaluated as carriers of living Lactococcus lactis subsp. Lactis. These lactic acid bacteria are capable of producing nisin, which is an effective antilisterial peptide. A low percentage (1:0.125 w/w) of yeast extract, gelatin, sodium caseinate, gelatin, or casein hydrolysates was incorporated in PVOH matrices with the aim of increasing the viability of bacteria in the film. The films were obtained by casting after incorporating L. lactis. Then they were evaluated for antilisterial activity in liquid medium at 37 °C for 24 h, and also at 4 °C for 21 days in order to simulate the storage of liquid foods in refrigeration conditions. The survival of the lactic acid bacteria was also evaluated at both temperatures during the experiment. L. lactis remained viable in all the films tested at 37 and 4 °C. The antimicrobial activity of the films was greater at 4 °C than at 37 °C. With regard to the effect of the film composition, the activity of the films was higher when protein hydrolysates and sodium caseinate were incorporated in the formulation. Films supplemented with protein hydrolysates or sodium caseinate inhibited growth of the pathogen during the 21 days of storage at 4 °C. At 37 °C, after 24 h the films had slowed the growth of the inoculated pathogen by between 2 and 4 log CFU/mL. Finally, as the films developed are intended to be used in the design of active packaging of foods, they were tested in pasteurized milk inoculated with 4 log CFU/mL of Listeria monocytogenes and stored at 4 °C for 21 days. The pathogen began to grow after the second day of storage with or without film, but when the films were added to the medium the growth of the pathogen was slowed down, without reaching >6 log CFU, whereas the control reached a maximum growth of 8.5 log CFU. The pH of the milk was monitored throughout the experiment, and it decreased with time. This was due to the generation of organic acids by the lactic bacteria. Buffering the food stabilized the pH without modifying the activity of the films. Thus, the current study shows that PVOH films supplemented with nutrients can act as carriers of L. lactis, and they can help to increase the safety of refrigerated dairy beverages and sauces.


Assuntos
Conservação de Alimentos/métodos , Lactobacillales/fisiologia , Listeria monocytogenes/crescimento & desenvolvimento , Leite/microbiologia , Álcool de Polivinil , Animais , Antibacterianos/metabolismo , Contagem de Colônia Microbiana , Microbiologia de Alimentos , Conservantes de Alimentos/metabolismo , Lactobacillales/química , Lactobacillales/metabolismo , Lactococcus lactis/química , Lactococcus lactis/metabolismo , Lactococcus lactis/fisiologia , Nisina/metabolismo , Proteínas/química , Refrigeração
8.
Antonie Van Leeuwenhoek ; 113(5): 651-662, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31838601

RESUMO

Lactococcus lactis subsp. lactis bv. diacetylactis is a relevant microorganism for the dairy industry because of its role in the production of aromatic compounds. Despite this technological property, the identification of bacteriocinogenic potential of obtained strains can offer the additional positive aspect of biosafety. A panel of 15 L. lactis subsp. lactis bv. diacetylactis strains was characterised for the presence and expression of bacteriocin related genes, and further investigated regarding the nisin operon. Eight strains were positive only for nisA, and one strain (SBR4) presented a full nisin operon, with sequencing that was shown to be similar to nisin Z. Only SBR4 presented inhibitory activity against 16 microbial target strains. The growth curves of selected targets strains confirmed the inhibitory activity of SBR4 and consequently the nisin production. This research has demonstrated the inhibitory potential of L. lactis subsp. lactis bv. diacetylactis strain, SBR4, due to its ability to produce nisin Z. This biopreservative potential, associated to previously characterised technological properties, allow the indication of this strain as a promising candidate to be used by the dairy industry as a starter or adjunct culture.


Assuntos
Indústria de Laticínios/métodos , Lactococcus lactis/metabolismo , Nisina/metabolismo , Antibacterianos/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Fermentação , Genes Bacterianos , Lactococcus lactis/genética , Nisina/análogos & derivados , Nisina/genética
9.
J Biosci Bioeng ; 129(4): 435-440, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31757606

RESUMO

Detection of bioactive peptides in complex ecosystems like intestinal environment is a difficult task. In this study, we developed two new bioreporters for nisin based on Lactococcus lactis NZ9000 transformed with the vector pNZ:Nis-aFP or pNZ:Nis-mCherry, that encoded for the anaerobic fluorescent protein evoglow-Pp1 (aFP) or the fluorescent protein mCherry, respectively. The biosensors were used to study nisin A production by L. lactis INIA 650 in milk and in a colonic model. The use of L. lactis NZ9000 pNZ:Nis-aFP as a biosensor allowed the detection of nisin produced by L. lactis INIA 650 in milk, but not in the in vitro colonic model. In milk, this reporter was induced by direct addition of 10 ng/ml nisin while, in the colonic model, nisin concentrations of 50 ng/ml were necessary. However, the reporter system based on pNZ:Nis-mCherry showed a higher sensibility, detecting nisin concentrations of 1 ng/ml produced by L. lactis INIA 650 in colonic media using agar diffusion or cross streak bioassays.


Assuntos
Técnicas Biossensoriais/métodos , Lactococcus lactis/fisiologia , Proteínas Luminescentes , Leite/microbiologia , Nisina/análise , Animais , Técnicas de Cultura Celular por Lotes , Bioensaio/métodos , Fermentação , Lactococcus lactis/genética , Lactococcus lactis/crescimento & desenvolvimento , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Nisina/metabolismo , Organismos Geneticamente Modificados , Células-Tronco
10.
J Bacteriol ; 202(3)2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31740495

RESUMO

The skin microbiota is thought to play a key role in host protection from infection. Nisin J is a novel nisin variant produced by Staphylococcus capitis APC 2923, a strain isolated from the toe web space area in a screening study performed on the human skin microbiota. Whole-genome sequencing and mass spectrometry of the purified peptide confirmed that S. capitis APC 2923 produces a 3,458-Da bacteriocin, designated nisin J, which exhibited antimicrobial activity against a range of Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and Cutibacterium acnes The gene order in the nisin J gene cluster (nsjFEGBTCJP) differs from that of other nisin variants in that it is lacking the nisin regulatory genes, nisRK, as well as the nisin immunity gene nisI Nisin J has 9 amino acid changes compared to prototypical nisin A, with 8 amino acid substitutions, 6 of which are not present in other nisin variants (Ile4Lys, Met17Gln, Gly18Thr, Asn20Phe, Met21Ala, Ile30Gly, Val33His, and Lys34Thr), and an extra amino acid close to the C terminus, rendering nisin J the only nisin variant to contain 35 amino acids. This is the first report of a nisin variant produced by a Staphylococcus species and the first nisin producer isolated from human skin.IMPORTANCE This study describes the characterization of nisin J, the first example of a natural nisin variant, produced by a human skin isolate of staphylococcal origin. Nisin J displays inhibitory activity against a wide range of bacterial targets, including MRSA. This work demonstrates the potential of human commensals as a source for novel antimicrobials that could form part of the solution to antibiotic resistance across a broad range of bacterial pathogens.


Assuntos
Nisina/genética , Nisina/metabolismo , Pele/microbiologia , Staphylococcus capitis/metabolismo , Anti-Infecciosos/farmacologia , Humanos , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Família Multigênica/genética , Nisina/efeitos dos fármacos , Propionibacteriaceae/efeitos dos fármacos , Propionibacteriaceae/genética , Propionibacteriaceae/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus capitis/efeitos dos fármacos , Staphylococcus capitis/genética , Sequenciamento Completo do Genoma
11.
J Dairy Sci ; 103(1): 161-165, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31733872

RESUMO

Lactococcus lactis, one of the most important probiotic lactic acid bacteria (LAB), is widely used in the dairy industry as a cell factory for recombinant protein production. Currently, a nisin-controlled inducible expression system is used for this purpose and represents the only commercial expression system in LAB. However, the available genetic modification methods are rather limited for modulating gene expression in L. lactis. Here, we developed a 2-plasmid system for gene transcription repression in L. lactis NZ9000 that uses inducible clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9. An inducible promoter Pnisin was used to drive the expression of dCas9 from Streptococcus pyogenes, whereas a strong constitutive promoter P44 drove single guide RNA expression for single or multiple target genes. dCas9 enabled CRISPR interference-mediated silencing of single or multiple target genes with significant reduction of gene expression, up to 99%. In addition, LLNZ_07335, a putative penicillin acylase, was identified as bile salt hydrolase for bile salt resistance in NZ9000 using this system. To our knowledge, this report is the first for a functional gene for bile salt tolerance in L. lactis. Overall, our work introduces a new gene repression tool for various applications in L. lactis or other LAB.


Assuntos
Lactobacillales/genética , Lactococcus lactis/genética , RNA Guia/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Marcação de Genes , Lactobacillales/enzimologia , Lactococcus lactis/enzimologia , Nisina/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas/genética
12.
Int J Mol Sci ; 20(23)2019 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-31771178

RESUMO

Breast cancer is the second most common cause of cancer-related mortality among women around the world. Conventional treatments in the fight against breast cancer, such as chemotherapy, are being challenged regarding their effectiveness. Thus, strategies for the treatment of breast cancer need to be continuously refined to achieve a better patient outcome. We know that a number of bacteria are pathogenic and some are even associated with tumor development, however, recent studies have demonstrated interesting results suggesting some bacteria may have potential for cancer therapy. Therefore, the therapeutic role of bacteria has aroused attention in medical and pharmaceutical studies. Furthermore, genetic engineering has been used in bacterial therapy and may led to greater efficacy with few side effects. Some genetically modified non-pathogenic bacterial species are more successful due to their selectivity for cancer cells but with low toxicity for normal cells. Some live, attenuated, or genetically modified bacterias are capable to multiply in tumors and inhibit their growth. This article aims to review the role of bacteria and their products including bacterial peptides, bacteriocins, and toxins for the treatment of breast cancer.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Animais , Bactérias/metabolismo , Bacteriocinas/metabolismo , Colicinas/metabolismo , Humanos , Nisina/metabolismo , Peptídeos Cíclicos/metabolismo
13.
World J Microbiol Biotechnol ; 35(12): 185, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31728760

RESUMO

Glutathione (GSH) and S-adenosyl methionine (SAM) have been applied as liver-protective factors to prevent and treat many different liver damages and diseases. Due to their low stability and short half-life, oral administration of GSH or SAM might be replaced by continuous supplying through living lactic bacteria in yogurt. In this study, Lactococcus lactis was engineered via synthetic biology strategies to produce these two important molecules. The bi-functional GSH synthase gene (gshF) and SAM synthase gene (metK) were transformed into food-grade L. lactis together with an adhesion factor gene (cwaA). The highest accumulation of SAM (9.0 mg/L) and GSH (17.3 mg/L) was achieved after 17 h cultivation of the recombinant L. lactis. Meanwhile, the autoaggregation and hydrophobicity were also improved significantly, which suggested that this engineered L. lactis might have an increased colonization-prone ability in human GI. Our studies demonstrated one potential route to self-produce and deliver the liver-healthy factors within living probiotic bacteria.


Assuntos
Glutationa/metabolismo , Lactococcus lactis/metabolismo , Engenharia Metabólica/métodos , S-Adenosilmetionina/metabolismo , Adesinas Bacterianas/genética , Vias Biossintéticas , Fermentação , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Lactococcus lactis/enzimologia , Lactococcus lactis/genética , Lactococcus lactis/crescimento & desenvolvimento , Metionina Adenosiltransferase/genética , Nisina/metabolismo , Probióticos
14.
Chemistry ; 25(64): 14572-14582, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31599485

RESUMO

Natural products that target lipid II, such as the lantibiotic nisin, are strategically important in the development of new antibacterial agents to combat the rise of antimicrobial resistance. Understanding the structural factors that govern the highly selective molecular recognition of lipid II by the N-terminal region of nisin, nisin(1-12), is a crucial step in exploiting the potential of such compounds. In order to elucidate the relationships between amino acid sequence and conformation of this bicyclic peptide fragment, we have used solid-phase peptide synthesis to prepare two novel analogues of nisin(1-12) in which the dehydro residues have been replaced. We have carried out an NMR ensemble analysis of one of these analogues and of the wild-type nisin(1-12) peptide in order to compare the conformations of these two bicyclic peptides. Our analysis has shown the effects of residue mutation on ring conformation. We have also demonstrated that the individual rings of nisin(1-12) are pre-organised to an extent for binding to the pyrophosphate group of lipid II, with a high degree of flexibility exhibited in the central amide bond joining the two rings.


Assuntos
Nisina/análogos & derivados , Peptídeos/síntese química , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Sequência de Aminoácidos , Ligação de Hidrogênio , Nisina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Uridina Difosfato Ácido N-Acetilmurâmico/química , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismo
15.
World J Microbiol Biotechnol ; 35(11): 169, 2019 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-31654140

RESUMO

In the two-component system of NisRK from Lactococcus lactis, the production of nisin is affected by transmembrane NisK and activation of intracellular NisR. The transcription of nisin structural genes can be induced by derivatives of nisin. NisR activation leads to the activation of nisA/Z transcription, which encodes the nisin maturation machinery, nisin regulation and activation of the nisFEG operon to confer immunity. The aim of this study was to express the Lactococcus lactis histidine phosphokinase NisK and response regulator NisR in E. coli, and to perform activity assays and in silico analysis. In silico methods were applied to study the properties and structures of the NisK and NisR proteins, including prediction of physicochemical characteristics, secondary and tertiary structure, stability and ligand-receptor interactions.pET32a and pET28a vectors containing synthetic nisK and nisR genes were transformed into E. coli followed by IPTG induction. SDS-PAGE and western blotting methods were applied to confirm the presence and identity of the amplified proteins. Following purification, the proteins were dialyzed and then prepared for activity assay. The CAI index showed that the genes was compatible with the E. coli host and that the proteins have effective expression. Also, the mRNA prediction results suggest that there is enough mRNA stability for efficient translation in the new host. NisK and NisR recombinant proteins were expressed in E. coli with half - lives of around 10 h and were confirmed with molecular weights of 27 kDa and 69 kDa, respectively, by SDS-PAGE and western blotting. The secondary structure of the recombinant proteins as predicted by circular dichroism spectroscopy was similar to the in silico protein structures. Activity assay of recombinant NisK was performed by measuring the amount of consumed ATP according to the light produced by luciferase. Because NisK and NisR have a direct impact on each other, they have an essential role in increasing the production of nisin and they can be used in different research fields. Our results demonstrated that recombinant proteins NisK and NisR preserved their structure and function after expression.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Histidina Quinase/genética , Lactococcus lactis/genética , Proteínas Recombinantes/genética , Fatores de Transcrição/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Simulação por Computador , Ensaios Enzimáticos , Escherichia coli/genética , Instabilidade Genômica , Histidina Quinase/química , Histidina Quinase/isolamento & purificação , Histidina Quinase/metabolismo , Lactococcus lactis/enzimologia , Simulação de Acoplamento Molecular , Peso Molecular , Nisina/metabolismo , Conformação de Ácido Nucleico , Óperon , Conformação Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Análise de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Transformação Genética
16.
Colloids Surf B Biointerfaces ; 183: 110491, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31518956

RESUMO

Recently, molybdenum disulfide functionalized with poly-ethylene glycol (PEGylated MoS2) has been widely used as a new drug delivery vehicle in biomedical field. However, the weak antibacterial activity of PEGylated MoS2 limits its application as an antibacterial agent. In this work, a novel silkworm-like conjugate of nisin loaded PEGylated MoS2 (nisin@PEGylated MoS2) was developed for antibacterial application. The morphology and structure of PEGylated MoS2 were strongly dependent on the Mo/S molar ratio of precursors during the solvothermal process. The silkworm-like skeleton was well kept after loading with nisin. A high level of reactive oxygen species (ROS) induced by the conjugate was an important cause of bacteria death. Due to the different structure of cell membranes, the sharp edges could more easily puncture into Escherichia coli (E. coli) as compared with Staphylococcus aureus (S. aureus) and produced more intracellular ROS, which improved the antibacterial activity of nisin against E. coli. As a result, nisin@PEGylated MoS2 displayed the antibacterial activity against both gram-positive and gram-negative bacteria. Furthermore, the toxicity of the conjugate was very low. Therefore, the target conjugate of nisin@PEGylated MoS2 may have great potential application as an antibacterial agent.


Assuntos
Antibacterianos/farmacologia , Dissulfetos/química , Proteínas de Insetos/química , Molibdênio/química , Nisina/química , Polietilenoglicóis/química , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Bombyx/metabolismo , Dissulfetos/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Insetos/metabolismo , Molibdênio/metabolismo , Nisina/metabolismo , Polietilenoglicóis/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo
17.
Carbohydr Polym ; 223: 115094, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31426998

RESUMO

This research attempted to inspect the contribution of lactic acid bacteria (LAB) with nanoparticle application in antimicrobial enhancement. Seven lactic acid cultures-free supernatants (CFSs) in both free and nanoparticles-loaded states were examined against seven foodborne microorganisms. Lactobacillus helveticus followed by Lactobacillus Plantarum possessed considerable antimicrobial activity. Headspace GC-MS characterization of Lactobacillus helveticus CFS identified a mixture of antimicrobial and health-promoting compounds. Minimal inhibitory concentration (MIC) values for tested Gram-positive bacteria represented 50% of that for Gram-negative bacteria, 20% and 7.35% of those for fungus and yeast respectively. Nanoparticles were prepared through chitosan-tripolyphosphate nanoparticle formation giving nanospheres from in the range from 5 to 10 nm, and narrow size distribution. CFS-loaded chitosan nanoparticles (CS-NPs) significantly enhanced the overall inhibition zone diameter, as well as, the decline in MIC values for Salmonella enterica (50%) and Penicillium chrysogenum (12.5%) was observed. Lactobacillus helveticus CFS, however, displayed lower antimicrobial activity vs. nisin and natamycin, it has both antibacterial and antifungal promising activities.


Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Queijo/microbiologia , Quitosana/análogos & derivados , Contaminação de Alimentos/prevenção & controle , Nanopartículas/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antifúngicos/química , Antifúngicos/metabolismo , Quitosana/química , Quitosana/metabolismo , Quitosana/farmacologia , Relação Dose-Resposta a Droga , Egito , Fermentação , Lactobacillus helveticus/efeitos dos fármacos , Lactobacillus helveticus/metabolismo , Lactobacillus plantarum/efeitos dos fármacos , Lactobacillus plantarum/metabolismo , Testes de Sensibilidade Microbiana , Nanopartículas/química , Natamicina/química , Natamicina/metabolismo , Natamicina/farmacologia , Nisina/química , Nisina/metabolismo , Nisina/farmacologia
18.
Pol J Microbiol ; 68(2): 269-280, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31257793

RESUMO

Two glucose-limited realkalized fed-batch cultures of Lactococcus lactis CECT 539 were carried out in a diluted whey medium (DW) using two different feeding media. The cultures were fed a mixture of a 400 g/l concentrated lactose and a concentrated mussel processing waste (CMPW, 101.72 g glucose/l) medium (fermentation I) or a CMPW medium supplemented with glucose and KH2PO4 up to concentrations of 400 g glucose/l and 3.21 g total phosphorus/l, respectively (fermentation II). For an accurate description and a better understanding of the kinetics of both cultures, the growth and product formation by L. lactis CECT 539 were both modelled, for the first time, as a function of the amounts of glucose (G) added and the pH gradient (VpH) generated in every realkalization and feeding cycle, by using an empirical polynomial model. With this modeling procedure, the kinetics of biomass, viable cell counts, nisin, lactic acid, acetic acid and butane-2,3-diol production in both cultures were successfully described (R 2 values > 0.970) and interpreted for the first time. In addition, the optimum VpH and G values for each product were accurately calculated in the two realkalized fed-batch cultures. This approach appears to be useful for designing feeding strategies to enhance the productions of biomass, bacteriocin, and metabolites by the nisin-producing strain in wastes from the food industry.Two glucose-limited realkalized fed-batch cultures of Lactococcus lactis CECT 539 were carried out in a diluted whey medium (DW) using two different feeding media. The cultures were fed a mixture of a 400 g/l concentrated lactose and a concentrated mussel processing waste (CMPW, 101.72 g glucose/l) medium (fermentation I) or a CMPW medium supplemented with glucose and KH2PO4 up to concentrations of 400 g glucose/l and 3.21 g total phosphorus/l, respectively (fermentation II). For an accurate description and a better understanding of the kinetics of both cultures, the growth and product formation by L. lactis CECT 539 were both modelled, for the first time, as a function of the amounts of glucose (G) added and the pH gradient (VpH) generated in every realkalization and feeding cycle, by using an empirical polynomial model. With this modeling procedure, the kinetics of biomass, viable cell counts, nisin, lactic acid, acetic acid and butane-2,3-diol production in both cultures were successfully described (R 2 values > 0.970) and interpreted for the first time. In addition, the optimum VpH and G values for each product were accurately calculated in the two realkalized fed-batch cultures. This approach appears to be useful for designing feeding strategies to enhance the productions of biomass, bacteriocin, and metabolites by the nisin-producing strain in wastes from the food industry.


Assuntos
Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Conservantes de Alimentos/metabolismo , Glucose/metabolismo , Lactococcus lactis/crescimento & desenvolvimento , Nisina/metabolismo , Probióticos/metabolismo , Ácido Acético/metabolismo , Biomassa , Fermentação/fisiologia , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo
19.
Probiotics Antimicrob Proteins ; 11(4): 1391-1402, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31124051

RESUMO

The purpose of this study was to select the promising biopreservation bacteriocin producer strain from goat milk and characterize the expressed bacteriocin, related to its physiological and biochemical properties and specificity of operon encoding production and expression of antimicrobial peptide. Brazilian goat milk was used as the source for the selection of bacteriocin-producing lactic acid bacteria. One strain (DF105Mi) stood out for its strong activity against several Listeria monocytogenes strains. Selected strain was identified based on the biochemical and physiological characteristics and 16s rRNA analysis. The bacteriocin production and inhibitory spectrum of strain DF105Mi were studied, together with the evaluation of the effect of temperature, pH, and chemicals on bacteriocin stability and production, activity, and adsorption to target cells as well as to the cell surface of bacteriocin producers. Physiological and bio-molecular analyses based on targeting of different genes, parts of nisin operon were performed in order to investigate the hypothesis that the studied strain can produce and express nisin. Based on biochemical, physiological, and 16s rRNA analysis, the strain DF105Mi was classified as Enterococcus hirae. The selected strain produces a bacteriocin which is stable in a wide range of pH (2.0-12.0), temperature (up to 120 °C), presence of selected chemicals and presents adsorption affinity to different test organisms, process influenced by environmental conditions. Higher bacteriocin production by Ent. hirae DF105Mi was recorded during stationary growth phase, but only when the strain was cultured at 37 °C. The strain's genetic analysis indicated presence of the genes coding for the production of the bacteriocin nisin. This result was confirmed by cross-checking the sensitivity of the produced strain to commercial nisin A. The strong anti-Listeria activity, bacteriocin adsorption, and stability of produced bacteriocin indicate that Ent. hirae DF105Mi presents a differentiated potential application for biopreservation of fermented dairy products.


Assuntos
Streptococcus faecium ATCC 9790/isolamento & purificação , Streptococcus faecium ATCC 9790/metabolismo , Leite/microbiologia , Nisina/metabolismo , Animais , Brasil , Streptococcus faecium ATCC 9790/classificação , Streptococcus faecium ATCC 9790/genética , Cabras , Concentração de Íons de Hidrogênio , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Nisina/química , Nisina/farmacologia
20.
Appl Environ Microbiol ; 85(11)2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30952662

RESUMO

Lantibiotics subtilin and nisin are produced by Bacillus subtilis and Lactococcus lactis, respectively. To prevent toxicity of their own lantibiotic, both bacteria express specific immunity proteins, called SpaI and NisI. In addition, ABC transporters SpaFEG and NisFEG prevent lantibiotic toxicity by transporting the respective peptides to the extracellular space. Although the three-dimensional structures of SpaI and NisI have been solved, very little is known about the molecular function of either lipoprotein. Using laser-induced liquid bead ion desorption (LILBID)-mass spectrometry, we show here that subtilin interacts with SpaI monomers. The expression of either SpaI or NisI in a subtilin-nonproducing B. subtilis strain resulted in the respective strain being more resistant against either subtilin or nisin. Furthermore, pore formation provided by subtilin and nisin was prevented specifically upon the expression of either SpaI or NisI. As shown with a nisin-subtilin hybrid molecule, the C-terminal part of subtilin but not any particular lanthionine ring was needed for SpaI-mediated immunity. With respect to growth, SpaI provided less immunity against subtilin than is provided by the ABC transporter SpaFEG. However, SpaI prevented pore formation much more efficiently than SpaFEG. Taken together, our data show the physiological function of SpaI as a fast immune response to protect the cellular membrane.IMPORTANCE The two lantibiotics nisin and subtilin are produced by Lactococcus lactis and Bacillus subtilis, respectively. Both peptides have strong antimicrobial activity against Gram-positive bacteria, and therefore, appropriate protection mechanisms are required for the producing strains. To prevent toxicity of their own lantibiotic, both bacteria express immunity proteins, called SpaI and NisI, and in addition, ABC transporters SpaFEG and NisFEG. Whereas it has been shown that the ABC transporters protect the producing strains by transporting the toxic peptides to the extracellular space, the exact mode of action and the physiological function of the lipoproteins during immunity are still unknown. Understanding the exact role of lantibiotic immunity proteins is of major importance for improving production rates and for the design of newly engineered peptide antibiotics. Here, we show (i) the specificity of each lipoprotein for its own lantibiotic, (ii) the specific physical interaction of subtilin with its lipoprotein SpaI, (iii) the physiological function of SpaI in protecting the cellular membrane, and (iv) the importance of the C-terminal part of subtilin for its interaction with SpaI.


Assuntos
Bacillus subtilis/imunologia , Bacillus subtilis/metabolismo , Bacteriocinas/metabolismo , Imunidade , Nisina/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/farmacologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Bacteriocinas/genética , Farmacorresistência Bacteriana , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Lactococcus lactis , Lipoproteínas/genética , Lipoproteínas/imunologia , Lipoproteínas/isolamento & purificação , Lipoproteínas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo
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