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1.
PLoS One ; 15(7): e0223395, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645024

RESUMO

Development of the cerebral cortex may be influenced by the composition of the maternal gut microbiota. To test this possibility, we administered probiotic Lactococcus lactis in drinking water to mouse dams from day 10.5 of gestation until pups reached postnatal day 1 (P1). Pups were assessed in a battery of behavioral tests starting at 10 weeks old. We found that females, but not males, exposed to probiotic during prenatal development spent more time in the center of the open field and displayed decreased freezing time in cue associated learning, compared to controls. Furthermore, we found that probiotic exposure changed the density of cortical neurons and increased the density of blood vessels in the cortical plate of P1 pups. Sex-specific differences were observed in the number of mitotic neural progenitor cells, which were increased in probiotic exposed female pups. In addition, we found that probiotic treatment in the latter half of pregnancy significantly increased plasma oxytocin levels in mouse dams, but not in the offspring. These results suggest that exposure of naïve, unstressed dams to probiotic may exert sex-specific long-term effects on cortical development and anxiety related behavior in the offspring.


Assuntos
Ansiedade/prevenção & controle , Córtex Cerebral/efeitos dos fármacos , Lactococcus lactis , Efeitos Tardios da Exposição Pré-Natal/psicologia , Probióticos/farmacologia , Animais , Animais Recém-Nascidos , Contagem de Células , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/citologia , Córtex Cerebral/crescimento & desenvolvimento , Medo , Feminino , Aprendizagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Ocitocina/metabolismo , Gravidez , Caracteres Sexuais
2.
PLoS One ; 15(7): e0227395, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628681

RESUMO

The FluidFM enables the immobilization of single cells on a hollow cantilever using relative underpressure. In this study, we systematically optimize versatile measurement parameters (setpoint, z-speed, z-length, pause time, and relative underpressure) to improve the quality of force-distance curves recorded with a FluidFM. Using single bacterial cells (here the gram negative seawater bacterium Paracoccus seriniphilus and the gram positive bacterium Lactococcus lactis), we show that Single Cell Force Spectroscopy experiments with the FluidFM lead to comparable results to a conventional Single Cell Force Spectroscopy approach using polydopamine for chemical fixation of a bacterial cell on a tipless cantilever. Even for the bacterium Lactococcus lactis, which is difficult to immobilze chemically (like seen in an earlier study), immobilization and the measurement of force-distance curves are possible by using the FluidFM technology.


Assuntos
Aderência Bacteriana , Lactococcus lactis/fisiologia , Microscopia de Força Atômica/métodos , Paracoccus/fisiologia , Células Imobilizadas/fisiologia , Vidro/química , Indóis/química , Polímeros/química , Água do Mar/microbiologia , Análise de Célula Única , Propriedades de Superfície , Titânio/química
3.
Int J Food Microbiol ; 329: 108686, 2020 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-32516659

RESUMO

Clostridium tyrobutyricum has been identified as a major species associated with the late blowing defect (LBD) of semi-hard and hard cheeses, due to undesirable butyric acid fermentation. To find new strategies to control this spoilage bacterium, we investigated the delivery of a bacteriophage endolysin by a cheese starter culture. The nisin producer Lactococcus lactis subsp. lactis INIA 415 was engineered to produce the CTP1L endolysin, encoded by the virulent bacteriophage ΦCTP1 of C. tyrobutyricum and with a demonstrated lytic activity in vitro, to the cheese matrix. The presence of the nisRK two-component regulatory system in the host strain allowed constitutive expression of the endolysin under the control of the nisA promoter (PnisA), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. Engineered lysins with a second cell wall binding domain were also tested and shown to have improved lytic activity. Transformation of L. lactis subsp. lactis INIA 415 with endolysin delivery plasmids had a detrimental effect on its ability to produce nisin in milk, but did not affect its acidifying capacity. Transformed L. lactis subsp. lactis INIA 415 were evaluated as starters in cheeses contaminated with spores of C. tyrobutyricum. Evolution of microbiological parameters, pH and dry matter of cheeses were studied, and Clostridium metabolism and LBD in cheeses were monitored by sensory and instrumental analyses during ripening. Cheese made with the parental strain L. lactis subsp. lactis INIA 415 delayed LBD by one month, attributable to the activity of the nisin, but it was not sufficient to arrest the growth of C. tyrobutyricum during ripening completely. The use of the endolysin-producing strains in cheese manufacture as single cultures also delayed the appearance of LBD by one month, attributable to the activity of the endolysin produced in situ during ripening, because nisin activity in these cheeses was very low at day 1 and undetectable from 15 days onwards. Endolysin was more effective than nisin in inhibiting Clostridium growth, since cheeses made with the CTP1L or the chimeric derivative producers only as starters showed lower LBD symptoms, higher lactic acid levels and lower concentrations of propionic and butyric acids (associated with off-flavours) than cheese made with the parental strain. Investigation of different promoters to maximise endolysin production may help to implement CTP1L as a tool to control C. tyrobutyricum by L. lactis cheese starter and reduce LBD even further.


Assuntos
Bacteriófagos , Queijo/microbiologia , Clostridium tyrobutyricum/efeitos dos fármacos , Endopeptidases/genética , Endopeptidases/farmacologia , Microbiologia de Alimentos/métodos , Lactococcus lactis/genética , Bacteriófagos/enzimologia , Bacteriófagos/genética , Lactococcus lactis/enzimologia , Nisina/farmacologia , Organismos Geneticamente Modificados
4.
Nat Commun ; 11(1): 2610, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451391

RESUMO

Lactic acid bacteria (LAB) are fundamental in the production of fermented foods and several strains are regarded as probiotics. Large quantities of live LAB are consumed within fermented foods, but it is not yet known to what extent the LAB we ingest become members of the gut microbiome. By analysis of 9445 metagenomes from human samples, we demonstrate that the prevalence and abundance of LAB species in stool samples is generally low and linked to age, lifestyle, and geography, with Streptococcus thermophilus and Lactococcus lactis being most prevalent. Moreover, we identify genome-based differences between food and gut microbes by considering 666 metagenome-assembled genomes (MAGs) newly reconstructed from fermented food microbiomes along with 154,723 human MAGs and 193,078 reference genomes. Our large-scale genome-wide analysis demonstrates that closely related LAB strains occur in both food and gut environments and provides unprecedented evidence that fermented foods can be indeed regarded as a possible source of LAB for the gut microbiome.


Assuntos
Microbiologia de Alimentos , Microbioma Gastrointestinal/genética , Lactobacillales/genética , Lactobacillales/isolamento & purificação , Animais , Bases de Dados Genéticas , Alimentos e Bebidas Fermentados/microbiologia , Humanos , Lactococcus lactis/genética , Lactococcus lactis/isolamento & purificação , Estilo de Vida , Metagenoma , Primatas/microbiologia , Probióticos , Streptococcus thermophilus/genética , Streptococcus thermophilus/isolamento & purificação
5.
Nucleic Acids Res ; 48(11): 6198-6209, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32379323

RESUMO

Group II introns are self-splicing ribozymes and mobile genetic elements. Splicing is required for both expression of the interrupted host gene and intron retromobility. For the pRS01 plasmid-encoded Lactococcus lactis group II intron, Ll.LtrB, splicing enables expression of the intron's host relaxase protein. Relaxase, in turn, initiates horizontal transfer of the conjugative pRS01 plasmid and stimulates retrotransposition of the intron. Little is known about how splicing of bacterial group II introns is influenced by environmental conditions. Here, we show that low temperatures can inhibit Ll.LtrB intron splicing. Whereas autocatalysis is abolished in the cold, splicing is partially restored by the intron-encoded protein (IEP). Structure profiling reveals cold-induced disruptions of key tertiary interactions, suggesting that a kinetic trap prevents the intron RNA from assuming its native state. Interestingly, while reduced levels of transcription and splicing lead to a paucity of excised intron in the cold, levels of relaxase mRNA are maintained, partially due to diminished intron-mediated mRNA targeting, allowing intron spread by conjugal transfer. Taken together, this study demonstrates not only the intrinsic cold sensitivity of group II intron splicing and the role of the IEP for cold-stress adaptation, but also maintenance of horizontal plasmid and intron transfer under cold-shock.


Assuntos
Temperatura Baixa , Conjugação Genética , Íntrons/genética , Lactococcus lactis/genética , Processamento de RNA , Sequência de Bases , Resposta ao Choque Frio , DNA Nucleotidiltransferases/metabolismo , Evolução Molecular , Transferência Genética Horizontal , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Catalítico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retroelementos
6.
Nat Commun ; 11(1): 1203, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32139702

RESUMO

Auxotrophy, the inability to produce an organic compound essential for growth, is widespread among bacteria. Auxotrophic bacteria rely on transporters to acquire these compounds from their environment. Here, we study the expression of both low- and high-affinity transporters of the costly amino acid methionine in an auxotrophic lactic acid bacterium, Lactococcus lactis. We show that the high-affinity transporter (Met-transporter) is heterogeneously expressed at low methionine concentrations, resulting in two isogenic subpopulations that sequester methionine in different ways: one subpopulation primarily relies on the high-affinity transporter (high expression of the Met-transporter) and the other subpopulation primarily relies on the low-affinity transporter (low expression of the Met-transporter). The phenotypic heterogeneity is remarkably stable, inherited for tens of generations, and apparent at the colony level. This heterogeneity results from a T-box riboswitch in the promoter region of the met operon encoding the high-affinity Met-transporter. We hypothesize that T-box riboswitches, which are commonly found in the Lactobacillales, may play as-yet unexplored roles in the predominantly auxotrophic lifestyle of these bacteria.


Assuntos
Lactococcus lactis/genética , Riboswitch/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Lactococcus lactis/citologia , Proteínas de Membrana Transportadoras/metabolismo , Modelos Biológicos , Óperon/genética , Fenótipo , Análise de Célula Única , Transcrição Genética
7.
J Food Prot ; 83(3): 542-551, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32084256

RESUMO

ABSTRACT: Mixed thermophilic and mesophilic commercial starter cultures (CSCs), particularly those including Streptococcus thermophilus as a primary milk acidifier, have been found to reduce growth and counteract in situ nisin A (NisA+) antilisterial effects by the novel, indigenous Lactococcus lactis subsp. cremoris M78 costarter in traditional Graviera thermized milk cheese curds. Therefore, this model challenge study evaluated growth and in situ NisA+ activity of strain M78 in coculture with S. thermophilus ST1 singly in sterilized raw milk (SRM). Strain ST1, derived from a CSC for cheese, was challenged at two inoculation levels (5 and 7 log CFU/mL) in SRM against 6 and 3 log CFU/mL of strain M78 and Listeria monocytogenes, respectively. Pure cultures of each strain and cocultures of strain ST1 with the CSC L. lactis LL2, in replacement of strain M78, served as controls. At the high (7-log) inoculation level, the rapid, competitive growth (>9.3 log CFU/mL) of S. thermophilus ST1 reduced growth of both L. lactis by at least 10-fold; the industrial strain LL2 retained slightly higher relative population densities (7.4 to 9.1%) than the wild NisA+ strain M78 (3.8 to 5.6%) after 6 h at 37°C, followed by an additional 66 h of incubation at 22°C. In full contrast, at the low (5-log) inoculation level, S. thermophilus ST1 failed to predominate in SRM at 6 h; thus, the starter lactic acid bacteria populations were reversed in favor of L. lactis. Notably, strain M78 retained higher relative population densities (83.0 to 90.1%) than the CSC strain LL2 (80.3 to 85.2%) at 22°C. Moreover, at the 5-log ST1 level, the direct and deferred in situ NisA+ activities of strain M78 were at similar levels with its pure culture with L. monocytogenes in SRM, whereas at the 7-log ST1 level, the respective NisA+ effects were counteracted. Hence, 10- to 100-fold lowered inoculation levels of CSC S. thermophilus are required to enhance the performance of the M78 costarter in traditional Greek cheese technologies.


Assuntos
Queijo/microbiologia , Lactococcus lactis , Lactococcus , Leite/microbiologia , Nisina , Animais , Grécia , Lactococcus/efeitos dos fármacos , Lactococcus/crescimento & desenvolvimento , Nisina/análise , Streptococcus thermophilus
8.
J Dairy Sci ; 103(4): 3038-3044, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32037169

RESUMO

Nisin, a natural peptide produced by Lactococcus lactis cultivation in milk whey, is widely used as a preservative in industrial production. However, nisin can be degraded by endogenous enzymes in foods. In this study, we investigated the antibacterial activity of nisin-soybean protein and nisin-egg white protein and compared them with that of free nisin in cantaloupe juice, which was used as a model of endogenous protease environment. Results showed that endogenous proteases in the model resulted in a loss of nisin activity, but combining nisin with protein (soybean or egg white) resulted in greater protection of its antimicrobial activity by inhibiting endogenous proteases. The microbial addition experiment (Staphylococcus aureus and Micrococcus luteus) and preservation experiment in the food model showed that the antibacterial activity of nisin combined with either of the 2 proteins was higher than that of nisin alone in an endogenous protease environment. In summary, soybean protein and egg white protein improved the protease tolerance of nisin, expanding the application scope of nisin in food.


Assuntos
Antibacterianos/farmacologia , Nisina/farmacologia , Peptídeo Hidrolases/metabolismo , Proteínas de Soja/farmacologia , Animais , Cucurbitaceae , Endopeptidases/metabolismo , Microbiologia de Alimentos , Lactococcus lactis/metabolismo , Leite/metabolismo , Peptídeo Hidrolases/farmacologia , Inibidores de Proteases/farmacologia , Staphylococcus aureus/metabolismo , Proteínas do Soro do Leite/metabolismo
9.
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
10.
Nat Commun ; 11(1): 309, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949154

RESUMO

Cellular interactions are a major driver for the assembly and functioning of microbial communities. Their strengths are shown to be highly variable in nature; however, it is unclear how such variations regulate community behaviors. Here we construct synthetic Lactococcus lactis consortia and mathematical models to elucidate the role of interaction variability in ecosystem succession and to further determine if casting variability into modeling empowers bottom-up predictions. For a consortium of bacteriocin-mediated cooperation and competition, we find increasing the variations of cooperation, from either altered labor partition or random sampling, drives the community into distinct structures. When the cooperation and competition are additionally modulated by pH, ecosystem succession becomes jointly controlled by the variations of both interactions and yields more diversified dynamics. Mathematical models incorporating variability successfully capture all of these experimental observations. Our study demonstrates interaction variability as a key regulator of community dynamics, providing insights into bottom-up predictions of microbial ecosystems.


Assuntos
Ecossistema , Consórcios Microbianos/fisiologia , Interações Microbianas/fisiologia , Biologia Sintética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Cocultura , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Concentração de Íons de Hidrogênio , Lactococcus lactis/genética , Lactococcus lactis/fisiologia , Consórcios Microbianos/genética , Interações Microbianas/genética , Modelos Teóricos
11.
Food Microbiol ; 87: 103392, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31948633

RESUMO

Genetic diversity and metabolic properties of Lactococcus lactis subsp. lactis were explored using phylogenetic, pan-genomic and metatranscriptomic analysis. The genomes, used in the current study, were available and downloaded from the GenBank which were primarily related with microorganisms isolated from dairy products and secondarily from other foodstuffs. To study the genetic diversity of the microorganism, various bioinformatics tools were employed such as average nucleotide identity, digital DNA-DNA hybridization, phylogenetic analysis, clusters of orthologous groups analysis, KEGG orthology analysis and pan-genomic analysis. The results showed that Lc. lactis subsp. lactis strains cannot be sufficiently separated into phylogenetic lineages based on the 16S rRNA gene sequences and core genome-based phylogenetic analysis was more appropriate. Pan-genomic analysis of the strains indicated that the core, accessory and unique genome comprised of 1036, 3146 and 1296 genes, respectively. Considering the results of pan-genomic and KEGG orthology analyses, the metabolic network of Lc. lactis subsp. lactis was rebuild regarding its carbohydrates' metabolic capabilities. Based on the metatranscriptomic data during the ripening of the Swiss-type Maasdam cheese at 20 °C and 5 °C, it was shown that the microorganism performed mixed acid fermentation producing lactate, formate, acetate, ethanol and 2,3-butanediol. Mixed acid fermentation was more pronounced at higher ripening temperatures. At lower ripening temperatures, the genes involved in mixed acid fermentation were repressed while lactate production remained unaffected resembling to a homolactic fermentation. Comparative genomics and metatranscriptomic analysis are powerful tools to gain knowledge on the genomic diversity of the lactic acid bacteria used as starter cultures as well as on the metabolic activities occurring in fermented dairy products.


Assuntos
Metabolismo dos Carboidratos , Queijo/microbiologia , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Carboidratos/química , Fermentação , Microbiologia de Alimentos , Variação Genética , Genômica , Lactococcus lactis/classificação , Lactococcus lactis/isolamento & purificação , Filogenia
12.
PLoS Negl Trop Dis ; 14(1): e0007939, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31899767

RESUMO

Cutaneous leishmaniasisis a vector-borne disease transmitted by Leishmania infected sand flies. PpSP15 is an immunogenic salivary protein from the sand fly Phlebotomus papatasi. Immunization with PpSP15 was shown to protect against Leishmania major infection. Lactococcus lactis is a safe non-pathogenic delivery system that can be used to express antigens in situ. Here, the codon-optimized Ppsp15-egfp gene was cloned in pNZ8121 vector downstream of the PrtP signal peptide that is responsible for expression and secretion of the protein on the cell wall. Expression of PpSP15-EGFP recombinant protein was monitored by immunofluorescence, flow cytometry and Western blot. Also, expression of protein in cell wall compartment was verified using whole cell ELISA, Western blot and TEM microscopy. BALB/c mice were immunized three times with recombinant L. lactis-PpSP15-EGFPcwa, and the immune responses were followed up, at short-term (ST, 2 weeks) and long-term (LT, 6 months) periods. BALB/c mice were challenged with L. major plus P. papatasi Salivary Gland Homogenate. Evaluation of footpad thickness and parasite burden showed a delay in the development of the disease and significantly decreased parasite numbers in PpSP15 vaccinated animals as compared to control group. In addition, immunized mice showed Th1 type immune responses. Importantly, immunization with L. lactis-PpSP15-EGFPcwa stimulated the long-term memory in mice which lasted for at least 6 months.


Assuntos
Lactococcus lactis/metabolismo , Leishmania major , Proteínas e Peptídeos Salivares/metabolismo , Animais , Feminino , Proteínas de Insetos/imunologia , Lactococcus lactis/genética , Leishmaniose Cutânea/transmissão , Camundongos Endogâmicos BALB C , Phlebotomus/genética , Proteínas Recombinantes/imunologia , Proteínas e Peptídeos Salivares/genética
13.
J Biosci Bioeng ; 129(1): 47-51, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31371162

RESUMO

Lactic acid bacteria (LAB) grow by producing lactate from sugar. However, the accumulation of lactate inhibits their growth. Here, the lactate productivity per cell in a semi-solid medium prepared with a chlorella powder in several LAB strains was much lower than that in the conventional MRS medium. Furthermore, the lactate production was suppressed not only in semi-solid medium, but also in chlorella liquid medium. The lactate productivity by Lactococcus lactis subsp. lactis NBRC 12007 in the chlorella liquid medium and MRS medium was 3.0 and 6.9 g-lactate·g-cell-1, respectively. The productivity of lactate in the chlorella liquid medium decreased to 44% of that in MRS medium. Gas chromatography/mass spectrometry (GC/MS) analysis of the culture supernatants revealed that the utilization of sucrose in the chlorella powder led to the suppression of lactate production. Comparison of the metabolites extracted from the cells indicated that the two ATP generating pathways, the arginine deiminase pathway and the decarboxylation reaction of glutamate and GABA, which are usually repressed by glucose, are activated in chlorella medium. It was considered that these pathways which do not require NAD+ for generation of ATP are not repressed when sucrose is used as a carbon source. Thus, the utilization of these pathways results in the suppression of the lactate production.


Assuntos
Ácido Láctico/metabolismo , Lactococcus lactis/metabolismo , Sacarose/metabolismo , Trifosfato de Adenosina/metabolismo , Chlorella/metabolismo , Meios de Cultura/metabolismo , Glucose/metabolismo , Lactococcus lactis/crescimento & desenvolvimento , NAD/metabolismo
14.
J Appl Microbiol ; 128(3): 862-874, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31758869

RESUMO

AIM: To study the prophylactic effect of recombinant Lactococcus lactis (rLl) harbouring Ara h 2.02 peanut allergen, in sensitized and challenged mice. METHODS AND RESULTS: Ara h 2.02 cDNA was cloned into pNZ8048 for heterologous expression in L. lactis. The purified recombinant allergen showed IgE binding comparable with native Ara h 2. Balb/c mice were fed with either recombinant (rLl), nonrecombinant L. lactis (Ll) or NaHCO3 (Sham) prior to sensitization and challenged with rAra h 2.02, whereas the baseline group was only fed with Ll. Allergen-specific immunoglobulin and splenocyte cytokines responses were determined for each mouse. Mice fed with either Ll or rLl showed significant alleviation of IgE and IgG1 compared to the Sham group. Despite no significant decrease in Th2 (IL-4, IL-13, IL-6) or increase in Th1 (IFN-γ) cytokines, both groups showed lower IL-10 level, while the IL-4 : IFN-γ ratio was significantly lower for rLl compared to Ll group. CONCLUSIONS: Oral administration of rLl harbouring Ara h 2.02 demonstrated alleviation of Th2-associated responses in allergen-challenged mice and a possible added allergen-specific prophylactic effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Ara h 2.02 coupled with the intrinsic properties of probiotic L. lactis as a delivery vehicle can be explored for the development of a commercially scalable vaccine.


Assuntos
Albuminas 2S de Plantas/imunologia , Antígenos de Plantas/imunologia , Lactococcus lactis/genética , Lactococcus lactis/imunologia , Hipersensibilidade a Amendoim/prevenção & controle , Albuminas 2S de Plantas/genética , Administração Oral , Animais , Antígenos de Plantas/genética , Citocinas/imunologia , Feminino , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Hipersensibilidade a Amendoim/imunologia , Probióticos/administração & dosagem
15.
Can J Microbiol ; 66(2): 161-168, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31743042

RESUMO

Nisin is a class I polycyclic bacteriocin produced by the bacterium Lactococcus lactis, which is used extensively as a food additive to inhibit the growth of foodborne Gram-positive bacteria. Nisin also inhibits growth of Gram-negative bacteria when combined with membrane-disrupting chelators such as citric acid. To gain insight into nisin's mode of action, we analyzed chemical-genetic interactions and identified nisin-sensitive Escherichia coli strains in the Keio library of knockout mutants. The most sensitive mutants fell into two main groups. The first group accords with the previously proposed mode of action based on studies with Gram-positive bacteria, whereby nisin interacts with factors involved in cell wall, membrane, envelope biogenesis. We identified an additional, novel mode of action for nisin based on the second group of sensitive mutants that involves cell cycle and DNA replication, recombination, and repair. Further analyses supported these two distinct modes of action.


Assuntos
Antibacterianos/farmacologia , Conservantes de Alimentos/farmacologia , Lactococcus lactis/química , Nisina/farmacologia , Bactérias/metabolismo , Parede Celular/metabolismo , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Escherichia coli/efeitos dos fármacos , Técnicas de Inativação de Genes , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos
16.
Nucleic Acids Res ; 48(1): 432-444, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31713614

RESUMO

SP_0782 from Streptococcus pneumoniae is a dimeric protein that potentially binds with single-stranded DNA (ssDNA) in a manner similar to human PC4, the prototype of PC4-like proteins, which plays roles in transcription and maintenance of genome stability. In a previous NMR study, SP_0782 exhibited an ssDNA-binding property different from YdbC, a prokaryotic PC4-like protein from Lactococcus lactis, but the underlying mechanism remains unclear. Here, we show that although SP_0782 adopts an overall fold similar to those of PC4 and YdbC, the ssDNA length occupied by SP_0782 is shorter than those occupied by PC4 and YdbC. SP_0782 exhibits varied binding patterns for different lengths of ssDNA, and tends to form large complexes with ssDNA in a potential high-density binding manner. The structures of SP_0782 complexed with different ssDNAs reveal that the varied binding patterns are associated with distinct capture of nucleotides in two major DNA-binding regions of SP_0782. Moreover, a comparison of known structures of PC4-like proteins complexed with ssDNA reveals a divergence in the binding interface between prokaryotic and eukaryotic PC4-like proteins. This study provides insights into the ssDNA-binding mechanism of PC4-like proteins, and benefits further study regarding the biological function of SP_0782, probably in DNA protection and natural transformation.


Assuntos
Proteínas de Bactérias/química , DNA Bacteriano/química , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Streptococcus pneumoniae/genética , Fatores de Transcrição/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Cinética , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Streptococcus pneumoniae/metabolismo , Termodinâmica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Can J Microbiol ; 66(1): 39-45, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31574230

RESUMO

The use of the food-grade bacterium Lactococcus lactis as a new cell factory is a promising alternative expression system for producing a desired protein. The Omp16-IL2 fusion protein antigen was cloned, expressed, and purified in this study. The Omp16-IL2 fusion gene was designed and cloned in pGH plasmid with appropriate restriction sites and subcloned in pAMJ2008 expression vector digested with the same enzymes. The purified recombinant constructed pAMJ-rOmp-IL2 was introduced into L. lactis subsp. cremoris MG1363 by electrotransformation. Finally, the expression and purification of Omp16-IL2 fusion protein was investigated. This study reports the construction of a recombinant L. lactis expressing the Omp16-IL2 fusion protein as an oral Lactococcus-based vaccine, as compared with commonly used live attenuated vaccines, for future studies against brucellosis.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Vacina contra Brucelose/genética , Vacina contra Brucelose/imunologia , Brucella melitensis/imunologia , Interleucina-2/genética , Lactococcus lactis/genética , Brucella melitensis/genética , Brucelose/prevenção & controle , Clonagem Molecular , Humanos , Lactococcus lactis/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/metabolismo
18.
J Dairy Sci ; 103(1): 141-149, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31629528

RESUMO

The objective of this study was to develop a novel immobilized system using kefir lactic acid bacteria and sugar cane pieces for the production of fermented milk. Lactobacillus kefiranofaciens HL1, Lactobacillus kefiri HL2, Leuconostoc mesenteroides HL3, and Lactococcus lactis HL4 were isolated from Taiwanese kefir grains and immobilized on pieces of sugar cane using adsorption. Scanning electron micrographs of the cell-immobilized sugar cane pieces (CISCP) showed that the microorganisms were embedded within the porous structures of the sugar cane pieces. During 28 cycles of repeated batch fermentation, viable cells on both sugar cane pieces and fermented products were maintained at 10 log cfu/g (cfu/mL). Random amplified polymorphic DNA PCR analysis revealed that Leu. mesenteroides HL3 (29-43%) and Lc. lactis HL4 (31-49%) were predominant on the CISCP, and the fermented samples had 79% Lc. lactis HL4. When tracking fermentation parameters, the data on the microbial, chemical, and physical properties of the fermented milk suggested that the CISCP had stable fermentative ability over the course of successive fermentations. We found an enhancement of the acid-producing ability of CISCP as the number of fermentations increased, with a significant growth in titratable acidity from 0.65 to 0.81% by the end.


Assuntos
Produtos Fermentados do Leite/microbiologia , Kefir/microbiologia , Lactobacillales/metabolismo , Lactobacillus/isolamento & purificação , Leite/metabolismo , Saccharum , Animais , Células Imobilizadas , Fermentação , Lactobacillales/isolamento & purificação , Lactococcus lactis/isolamento & purificação , Lactococcus lactis/metabolismo , Leuconostoc/isolamento & purificação , Leite/química
19.
J Dairy Sci ; 103(1): 150-160, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31668441

RESUMO

This study aimed to evaluate the possible inhibitory effect of natural lactic acid bacteria on the growth of 2 Bacillus cereus strains. First, we evaluated the behavior of spores of B. cereus GPe2 and D43 when inoculated before cheesemaking using pasteurized or raw milk; no statistical differences were observed between cheese produced with the 2 types of milk. Then, lactic acid bacteria (LAB) were isolated from cheese at the last sampling time, identified, and tested in vitro for their antagonistic activity and organic acid production by using an HPLC method, showing antimicrobial potential. The LAB that produced larger inhibition halos (>9 mm) against B. cereus strains (LAB 3, 6, 9, 10: Lactococcus lactis ssp. lactis; LAB 7: Lactococcus lactis ssp. cremoris) were selected to produce a LAB mixture for subsequent tests. Spores of B. cereus GPe2 and D43 were inoculated in pasteurized milk before cheesemaking with or without addition of the LAB mixture at a high dosage. Bacillus cereus grew more slowly when LAB were added to the dairy matrix (with differences from 2.36 to 2.66 log cfu/g in B. cereus GPe2 and D43 growth).


Assuntos
Bacillus cereus/crescimento & desenvolvimento , Queijo/microbiologia , Microbiologia de Alimentos , Lactobacillales/fisiologia , Lactococcus lactis/fisiologia , Leite/microbiologia , Animais , Ácidos Carboxílicos
20.
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
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