BACKGROUND Although an association between gut microbiota and cholestatic liver disease(CLD)has been reported,the precise functional roles of these microbes in CLD pathogenesis remain largely unknown.AIM To explore th...BACKGROUND Although an association between gut microbiota and cholestatic liver disease(CLD)has been reported,the precise functional roles of these microbes in CLD pathogenesis remain largely unknown.AIM To explore the function of gut microbes in CLD pathogenesis and the effects of gut microbiota on intestinal barrier and bile acid(BA)metabolism in CLD.METHODS Male C57BL/6J mice were fed a 0.05%3,5-diethoxycarbonyl-1,4-dihydrocollidine diet for 2 weeks to induce CLD.The sterile liver tissues of mice were then meticulously harvested,and bacteria in homogenates were identified through culture methods.Furthermore,16S ribosomal DNA sequencing was employed to analyze sterile liver samples collected from eight patients with primary biliary cholangitis(PBC)and three control individuals with hepatic cysts.The functional roles of the identified bacteria in CLD pathogenesis were assessed through microbiota transfer experiments,involving the evaluation of changes in intestinal permeability and BA dynamics.RESULTS Ligilactobacillus murinus(L.murinus)and Lactococcus garvieae(L.garvieae)were isolated from the bacterial culture of livers from CLD mice.L.murinus was prevalently detected in PBC patients and controls,whereas L.garvieae was detected only in patients with PBC but not in controls.Mice inoculated with L.garvieae exhibited increased susceptibility to experimental CLD,with both in vitro and in vivo indicating that L.garvieae disrupted the intestinal barrier function by down-regulating the expression of occludin and zonula occludens-1.Moreover,L.garvieae administration significantly upregulated the expression of the apical sodium-dependent BA transporter in the terminal ileum and increased serum BA levels.CONCLUSION L.garvieae contributes to excessive BA-induced hepatobiliary injury and liver fibrosis by increasing intestinal permeability and enhancing BA reabsorption.展开更多
Salmonella grows better under aerobic conditions as a facultative anaerobic foodborne pathogenic bacteria.The oxygen-scavenging activity of Lactococcus lactis in the intestinal tract is a promising strategy for preven...Salmonella grows better under aerobic conditions as a facultative anaerobic foodborne pathogenic bacteria.The oxygen-scavenging activity of Lactococcus lactis in the intestinal tract is a promising strategy for preventing Salmonella infection.In this study,the aerobic respiration requirement and preventive mechanism of L.lactis subsp.lactis KLDS 4.0325 in murine models infected by Salmonella enterica subsp.enterica serovar Typhimurium(S.Typhimurium)SL1344 were investigated.Results indicate that L.lactis KLDS 4.0325 is capable of aerobic respiratory metabolism in the host intestine when exogenous heme exists,and decrease intestinal oxygen concentration,which in turn trigger autophagy of intestinal cells to reduce S.Typhimurium load,improve gut microbiota composition,alleviate intestinal barrier injury and inflammation response.These results suggest that aerobic respiration L.lactis KLDS 4.0325 can prevent S.Typhimurium infection in a new way in which by restoring intestinal cell hypoxia,maintaining immune balance and regulating intestinal flora.展开更多
Parkinson’s disease is characterized by synucleinopathy-associated neurodegeneration.Previous studies have shown that glucagon-like peptide-1(GLP-1)has beneficial effects in a mouse model of Parkinson’s disease indu...Parkinson’s disease is characterized by synucleinopathy-associated neurodegeneration.Previous studies have shown that glucagon-like peptide-1(GLP-1)has beneficial effects in a mouse model of Parkinson’s disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.However,the effect of GLP-1 on intrinsic synuclein malfunction remains unclear.In this study,we investigated the effect of Lactococcus lactis MG1363-pMG36e-GLP-1 on parkinsonism in SncaA53T transgenic mice and explored the underlying mechanisms.Our data showed that Lactococcus lactis MG1363-pMG36e-GLP-1 inhibited dopaminergic neuronal death,reduced pathological aggregation ofα-synuclein,and decreased movement disorders in SncaA53T transgenic mice.Furthermore,Lactococcus lactis MG1363-pMG36e-GLP-1 downregulated lipopolysaccharide-related inflammation,reduced cerebral activation of microglia and astrocytes,and promoted cell survival via the GLP-1 receptor/PI3K/Akt pathway in the substantia nigra.Additionally,Lactococcus lactis MG1363-pMG36e-GLP-1 decreased serum levels of pro-inflammatory molecules including lipopolysaccharide,lipopolysaccharide binding protein,interleukin-1β,and interleukin-6.Gut histopathology and western blotting further revealed that Lactococcus lactis MG1363-pMG36e-GLP-1 increased the expression of gut integrity-related proteins and reduced lipopolysaccharide-related inflammation by reversing gut dysbiosis in SncaA53T transgenic mice.Our findings showed that the beneficial effect of Lactococcus lactis MG1363-pMG36e-GLP-1 on parkinsonism traits in SncaA53T transgenic mice is mediated by microglial polarization and the reversal of dysbiosis.Collectively,our findings suggest that Lactococcus lactis MG1363-pMG36e-GLP-1 is a promising therapeutic agent for the treatment of Parkinson’s disease.展开更多
[Objective]The aim was to clone TapⅡchalcone isomerases（CHI1 A） from soybean specially and construct expression vector of PNZ8149-CHI1 A,and then transform it into Lactococcus Lactis NICE systers.[Method]Chalcone i...[Objective]The aim was to clone TapⅡchalcone isomerases（CHI1 A） from soybean specially and construct expression vector of PNZ8149-CHI1 A,and then transform it into Lactococcus Lactis NICE systers.[Method]Chalcone isomerases（CHI1 A） was cloned by RTPCR method,and it was sequenced after cloning into pMD18-T vectors,and recombined to expression vector PNZ8149-CHI1 A,then it was transformed into Lactococcus Lactis NZ3900[Result]The sequencing results indicated that the cloned fragment of CHI1 A contained 670 nucleotides,and shared a sequence homology of 92% with that from Genbank accession number AF595413（CHI1 A）.CHI1 A was transformed into NICE expression system successfully by identification of PCR and digestion.[Conclusion]The foundation of using the microorganism fermentation method to produce flavonoids was laid by construction of efficient induction expression vector with chalcone isomerases CHI1 A.展开更多
【目的】寻找精氨酸代谢途径中与酸胁迫相关的关键作用因素。【方法】通过在Lactococcus lactis NZ9000中分别过量表达来源于Lactobacillus casei Zhang的精氨酰琥珀酸合成酶(ASS)和精氨酰琥珀酸裂解酶(ASL)改变精氨酸代谢提高酸胁迫抗...【目的】寻找精氨酸代谢途径中与酸胁迫相关的关键作用因素。【方法】通过在Lactococcus lactis NZ9000中分别过量表达来源于Lactobacillus casei Zhang的精氨酰琥珀酸合成酶(ASS)和精氨酰琥珀酸裂解酶(ASL)改变精氨酸代谢提高酸胁迫抗性。【结果】与对照菌株对比,重组菌株在环境胁迫下表现了较高的生长性能、存活率和发酵性能。生理学分析发现,酸胁迫环境下,重组菌株细胞有较高的胞内NH4+、ATP含量和H+-ATPase活性,并显著提高了精氨酸脱亚胺酶(ADI)途径中的氨基酸浓度。进一步的转录分析发现,天冬氨酸合成、精氨酸代谢相关的基因转录水平上调。【结论】在L.lactis NZ9000中过量表达ASS或ASL可以引发精氨酸代谢流量的上调,进而提高了细胞的多种胁迫抗性。精氨酸合成途径广泛存在于多种微生物中,为微生物,尤其是工业微生物提高胁迫抗性提供了新思路。展开更多
Objective To construct four recombinant Lactococcus lactis strains exhibiting high β-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion....Objective To construct four recombinant Lactococcus lactis strains exhibiting high β-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion. Methods The gene fragments encoding β-galactosidase from two strains of Loctobacillus bulgaricus, wch9901 isolated from yogurt and 1.1480 purchased from the Chinese Academy of Sciences, were amplified and inserted into lactococcal expression vector pMG36e. For fusion expression, the open reading frame of the β-galactosidase gene was amplified, while for non-fusion expression, the open reading frame of the β-galactosidase gene was amplified with its native Shine-Dalgarno sequence upstream. The start codon of the β-galactosidase gene partially overlapped with the stop codon of vector origin open reading frame. Then, the recombinant plasmids were transformed into Escherichia coli DH5α and Lactococcus lactis subsp, lactis MG1363 and confirmed by determining β-galactosidase activities. Results The non-fusion expression plasmids showed a significantly higher β-galactosidase activity in transformed strains than the fusion expression plasmids. The highest enzyme activity was observed in Lactococcus lactis transformed with the non-fusion expression plasmids which were inserted into the β-galactosidase gene from Lactobacillus bulgaricus wch9901. The β-galactosidase activity was 2.75 times as high as that of the native counterpart. In addition, β-galactosidase expressed by recombinant plasmids in Lactococcus lactis could be secreted into the culture medium. The highest secretion rate （27.1%） was observed when the culture medium contained 20 g/L of lactose. Conclusion Different properties of the native bacteria may have some effects on the protein expression of recombinant plasmids. Non-fusion expression shows a higher enzyme activity in host bacteria. There may be a host-related weak secretion signal peptide gene within the structure gene of Lb. bulgaricus β-galactosidase, and its translation product may introduce the enzyme secretion out of cells in special hosts.展开更多
Objective This study is to examine the secretion effects of β-galactosidase in Lactococcus lactis.Methods The usp45 and β-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant pl...Objective This study is to examine the secretion effects of β-galactosidase in Lactococcus lactis.Methods The usp45 and β-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant plasmid pMG36e-usp-lacZ.This recombinant plasmid was transformed into both Escherichia coli DH5α and L.lactis MG1363.The enzyme activity,gene sequencing,SDS-PAGE and hereditary stability were assessed and studied.Results The lacZ gene inserted into plasmids pMG36e-usp-lacZ was 99.37% similar to the GenBank sequence,and SDS-PAGE revealed an evident idio-strap at 116 KDa between L.lactis MG1363/pMG36eusp-lacZ in both supernatant and cell samples.β-Galactosidase activity measured 0.225 U/mL in L.lactis pMG36e-usp-lacZ transformants,and its secretion rate was 10%.The plasmid pMG36e-usp-lacZ appeared more stable in MG1363.Conclusion The authors concluded that these new recombinant bacteria well expressed and secreted β-galactosidase,indicating that the β-galactosidase expression system was successfully constructed,and this might provide a new solution for management of lactose intolerance specifically and promote the use of gene-modified organisms as part of the food-grade plasmid in general.展开更多
AIM： To construct the recombinant Lactococcus/actis as oral delivery vaccination against malaria. METHODS： The C-terminal 19-ku fragments of MSP1 （MSP-119） of Plasmodium yoelii265-BY was expressed in L. lactis and...AIM： To construct the recombinant Lactococcus/actis as oral delivery vaccination against malaria. METHODS： The C-terminal 19-ku fragments of MSP1 （MSP-119） of Plasmodium yoelii265-BY was expressed in L. lactis and the recombinant L. lact/s was administered orally to BALB/c and C57BL/6 mice. After seven interval vaccinations within 4 wk, the mice were challenged with P. yoelii 265-BY parasites of erythroo/tic stage. The protective efficacy of recombinant L. lactiswas evaluated. RESULTS： The peak parasitemias in average for the experiment groups of BALB/c and C57BL/6 mice were 0.8± 0.4% and 20.8±26.5%, respectively, and those of their control groups were 12.0±0.8% and 60.8±9.6%, respectively. None of the BALB/c mice in both experimental group and control group died during the experiment. However, all the C57BL/6 mice in the control group died within 23 d and all the vaccinated mice survived well. CONCLUSION： The results imply the potential of recombinant L. lactis as oral delivery vaccination against malaria.展开更多
This study evaluated efficacy of maternal and larval immunisation against Lactococcus garviae infection and on the lysozyme and immunoglobulin (IgM) levels in rainbow trout Oncorhynchus mykiss (Walaum). Forty-eight-da...This study evaluated efficacy of maternal and larval immunisation against Lactococcus garviae infection and on the lysozyme and immunoglobulin (IgM) levels in rainbow trout Oncorhynchus mykiss (Walaum). Forty-eight-day-old larvae (mean weight 96 mg) originating from injected weekly with letrozole and immunised, only immunised and non-immunised parents were experimentally infected with the L. garvieae, and the mortality rate was recorded daily. Larvae were vaccinated by immersion at 58 days post hatch with live L. garvieae (109 cells/mL) for 15 min. Every third day post larvae vaccination, two larvae from each group were collected for analysis lysozyme (by a method based on the ability of lysozyme to lyse the bacterium Micrococcus lysodeikticus) and IgM (by enzyme-linked immunosorbent assay (ELISA)) parameters. Vaccinated and control larvae were tested for protection against L. garvieae 30 days post larvae immunization when the larvae were 88 days old. Larvae were challenged by bath exposure with live L. garvieae (109 cells/mL) for 2 min and monitored for mortality for at least 10 days following challenge. The challenge experiment with L. garvieae showed a significant reduction in larvae from immunised (54.44% ± 0.64%) and injected weekly with letrozole and immunised fish (52.96% ± 0.97%) compared to larvae from control fish (62.96% ± 2.22%). Vaccinated larvae originated from injected weekly with letrozole and immunised parents showed significantly higher lysozyme activity compared to other fish groups. Vaccinated larvae showed significantly less mortality compared to controls. The relative percent survival (RPS) values of larvae from only immunised, injected weekly with letrozole and immunised and non-immunised parents vaccinated with L. garvieae were 67.36% ± 0.9%, 68.05% ± 0.66% and 48.27% ± 2.79% respectively. The results indicate that the effect of maternal immunization rainbow trout against L. garvieae infection by eliciting the immune responses as indicated by an increase in the IgM level and lysozyme activity.展开更多
To evaluate the specific immune responses induced by recombinant Lactococcus lactis（L.lactis） which expresses porcine epidemic diarrhea virus（PEDV） S1 protein through oral administration,the spike gene fragment of...To evaluate the specific immune responses induced by recombinant Lactococcus lactis（L.lactis） which expresses porcine epidemic diarrhea virus（PEDV） S1 protein through oral administration,the spike gene fragment of PEDV was amplified from PEDV SDLY strain to construct p MG36 e-S1 recombinant plasmid.The recombinant plasmid was then electro-transferred into competent cells of L.lactis MG1363,to prepare the recombinant L.lactis expressing S1 protein of PEDV.The expression of target protein was identified by SDS-PAGE and Western-blot.New Zealand white rabbits were orally administered with the recombinant strain;the antibody titer in intestinal mucosa and serum was detected by neutralizing test;and the specific Ig G in serum was evaluated by indirect ELISA.The results showed that the recombinant L.lactis could effectively induce high level of Ig G in serum and high level of mucosal immune antibody.The recombinant L.lactis is qualified to be a potential oral vaccine because it could successfully stimulate both humoral and mucosal immune responses against PEDV.展开更多
Objective:To determine the inhibition mechanisms of secretome protein extracted from Paenibacillus polymyxa Kp10(Kp10)and Lactococcus lactis Gh1(Gh1)against methicillin-resistant Staphylococcus aureus(MRSA)and vancomy...Objective:To determine the inhibition mechanisms of secretome protein extracted from Paenibacillus polymyxa Kp10(Kp10)and Lactococcus lactis Gh1(Gh1)against methicillin-resistant Staphylococcus aureus(MRSA)and vancomycin-resistant Enterococcus(VRE).Methods:The sensitivity and viability of MRSA and VRE treated with secretome proteins of Kp10 and Gh1 were determined using minimal inhibitory concentration,minimum bactericidal concentration,and time-to-kill assays.The morphological changes were observed using scanning electron microscopy and transmission electron microscopy.To elucidate the antimicrobial mechanism of secretome protein of Kp10 and Gh1 against MRSA and VRE,2D gel proteomic analysis using liquid chromatography-mass spectrometry was run by comparing upregulated and downregulated proteins,and the proton motive force study including the efflux of ATP,pH gradient,and the membrane potential study were conducted.Results:MRSA and VRE were sensitive to Kp10 and Gh1 secretome protein extracts and displayed apparent morphological and internal composition changes.Several proteins associated with cellular component functions were either downregulated or upregulated in treated MRSA and VRE by changing the membrane potential gradient.Conclusions:Kp10 and Gh1 secretome proteins reduce the growth of VRE and MRSA by damaging the cell membrane.Cell division,cell wall biosynthesis,and protein synthesis are involved in the inhibition mechanism.展开更多
In this study, a plasmid was integrated into nisP, creating the first defined mutation in a nisin biosynthetic gene. The mutant strain secreted fully modified nisin with the N-terminal leader still attached.The presen...In this study, a plasmid was integrated into nisP, creating the first defined mutation in a nisin biosynthetic gene. The mutant strain secreted fully modified nisin with the N-terminal leader still attached.The presence of the leader was confirmed by N-terminal sequencing of the purified precursor. The dehydration and lanthionine formation of the precursor were already completed as active nisin could be formed by cleaving the leader from the inactive precursor by a trypsin treatment or by incubation with wild type cells. Nisin immunity of the NisP mutant strain was lowered to about 10% of the wild type immunity. The results show that NisP is needed for precursor processing and for development of high immunity of nisin.展开更多
Bovine mastitis affects the udder health and thus causing significant economic losses. Probiotic products based on the use of lactic acid bacteria (LAB) to limit pathogens multiplication and pre-infection risks can be...Bovine mastitis affects the udder health and thus causing significant economic losses. Probiotic products based on the use of lactic acid bacteria (LAB) to limit pathogens multiplication and pre-infection risks can be an interesting alternative to post infection allopathic treatment with antibiotics. Lactococcus lactis is one of the most important bacteria used in dairy technology. In this work, a total of 21 Lactococcus lactis subsp. Lactis strains, 20 from goat milk whey and one strain from cow milk were used to evaluate their antibacterial activity against four pathogenic germs responsible for mastitis: Escherichia coli, Staphylococcus aureus, Streptococcus uberis and Streptococcus agalactiae. The nisin-producing cow milk strain was active against St. uberis and Str. Agalactiae using the well diffusion method. For the strains isolated from goat milk whey, no antimicrobial effect was observed against these pathogens. However, a different approach based on the growth of pathogenic bacteria interacting with the Lactococcus lactis strains in a minimum medium was used to study the barrier effect of LAB. The Lactococcus lactis strains S1 and S2 from goat milk whey depleted the growth of Sa. aureus, St. uberis and E. coli during 8 h and stopped the development of St. agalactiae.展开更多
Objective: A community-based intervention study was conducted to examine the effect of consumption of JCM 5805 yogurt on influenza incidence rates and the cumulative incidence rates among schoolchildren in Iwate Prefe...Objective: A community-based intervention study was conducted to examine the effect of consumption of JCM 5805 yogurt on influenza incidence rates and the cumulative incidence rates among schoolchildren in Iwate Prefecture, Japan. Methods: Schoolchildren and their parents in Shizukuishi town were told of the purpose, frequency and duration of JCM 5805 yogurt administration. The number of elementary schoolchildren in Shizukuishi town was 780 while that of junior high school students in Shizukuishi town numbered 475. The number of elementary schoolchildren in neighboring town A was 208 and that of junior high school students in town A was 121. JCM 5805 yogurt was delivered three times a week to all elementary schools and junior high schools in Shizukuishi town from January 16 through March 18, 2015. The incidence rate was calculated every week as the maximum case number divided by the number of schoolchildren in each school. The cumulative incidence rate was calculated as the total case number during the period when JCM 5805 yogurt was delivered divided by the number of schoolchildren in each school. Results: JCM 5805 yogurt intake was associated with a two-thirds reduction in influenza incidence rates in Shizukuishi town schoolchildren compared with those of town A. Furthermore, the cumulative incidence rates of the elementary school and combined data from the elementary school and junior high school were significantly lower than those of neighbor town A. Conclusion: JCM 5805 yogurt intake reduced both the incidence rates and cumulative incidence rates of influenza.展开更多
Background/Aims: Administration of a lactic acid bacterial strain, Lactococcus lactis subsp. lactis JCM 5805 (LC-Plasma), is reported to prevent viral infection via activation of plasmacytoid dendritic cells in mouse ...Background/Aims: Administration of a lactic acid bacterial strain, Lactococcus lactis subsp. lactis JCM 5805 (LC-Plasma), is reported to prevent viral infection via activation of plasmacytoid dendritic cells in mouse and human studies. As it is assumed that LC-Plasma is taken in excess when it is commercially provided as a supplement, we conducted a trial using capsules to give 250 mg LC-Plasma (5 times the effective anti-viral dose) every day for four weeks to healthy volunteers to investigate the safety of excessive intake of LC-Plasma. Trial Design: A randomized, double-blind, placebo-controlled, parallel-group trial was conducted. Methods: Forty healthy subjects were randomly assigned to the LC-Plasma group (daily intake of five capsules containing 50 mg heat-killed LC-Plasma cells per capsule) or the placebo group (daily intake of five placebo capsules with no LC-Plasma). Physical, hematological, biochemical and urinary examinations and medical interviews were used to evaluate safety. Results: No abnormal differences were observed after excessive intake of LC-Plasma capsules when compared to the intake of placebo capsules. Conclusions: There are no safety concerns associated with the excessive intake of heat-killed LC-Plasma capsules.展开更多
基金Supported by Tianjin Health Research Project,No.TJWJ2024QN005Beijing iGandan Public Welfare Foundation Artificial Liver Special Fund,No.iGandanF-1082024-RGG122.
文摘BACKGROUND Although an association between gut microbiota and cholestatic liver disease(CLD)has been reported,the precise functional roles of these microbes in CLD pathogenesis remain largely unknown.AIM To explore the function of gut microbes in CLD pathogenesis and the effects of gut microbiota on intestinal barrier and bile acid(BA)metabolism in CLD.METHODS Male C57BL/6J mice were fed a 0.05%3,5-diethoxycarbonyl-1,4-dihydrocollidine diet for 2 weeks to induce CLD.The sterile liver tissues of mice were then meticulously harvested,and bacteria in homogenates were identified through culture methods.Furthermore,16S ribosomal DNA sequencing was employed to analyze sterile liver samples collected from eight patients with primary biliary cholangitis(PBC)and three control individuals with hepatic cysts.The functional roles of the identified bacteria in CLD pathogenesis were assessed through microbiota transfer experiments,involving the evaluation of changes in intestinal permeability and BA dynamics.RESULTS Ligilactobacillus murinus(L.murinus)and Lactococcus garvieae(L.garvieae)were isolated from the bacterial culture of livers from CLD mice.L.murinus was prevalently detected in PBC patients and controls,whereas L.garvieae was detected only in patients with PBC but not in controls.Mice inoculated with L.garvieae exhibited increased susceptibility to experimental CLD,with both in vitro and in vivo indicating that L.garvieae disrupted the intestinal barrier function by down-regulating the expression of occludin and zonula occludens-1.Moreover,L.garvieae administration significantly upregulated the expression of the apical sodium-dependent BA transporter in the terminal ileum and increased serum BA levels.CONCLUSION L.garvieae contributes to excessive BA-induced hepatobiliary injury and liver fibrosis by increasing intestinal permeability and enhancing BA reabsorption.
基金supported by the National Natural Science Foundation of China(32072190 and 32101929)Academic Backbone Plan of Northeast Agricultural University(20XG12)。
文摘Salmonella grows better under aerobic conditions as a facultative anaerobic foodborne pathogenic bacteria.The oxygen-scavenging activity of Lactococcus lactis in the intestinal tract is a promising strategy for preventing Salmonella infection.In this study,the aerobic respiration requirement and preventive mechanism of L.lactis subsp.lactis KLDS 4.0325 in murine models infected by Salmonella enterica subsp.enterica serovar Typhimurium(S.Typhimurium)SL1344 were investigated.Results indicate that L.lactis KLDS 4.0325 is capable of aerobic respiratory metabolism in the host intestine when exogenous heme exists,and decrease intestinal oxygen concentration,which in turn trigger autophagy of intestinal cells to reduce S.Typhimurium load,improve gut microbiota composition,alleviate intestinal barrier injury and inflammation response.These results suggest that aerobic respiration L.lactis KLDS 4.0325 can prevent S.Typhimurium infection in a new way in which by restoring intestinal cell hypoxia,maintaining immune balance and regulating intestinal flora.
基金supported by grants from the Jiangxi Provincial Natural Science Foundation,No.20242BAB26134(to XF)the National Natural Science Foundation of China,Nos.82060638(to TC),82060222(to XF),82460237(to XF)+1 种基金the Major Disciplines of Academic and Technical Leaders Project of Jiangxi Province,Nos.20194BCJ22032(to TC),20213BCJL22049(to XF)Science and Technology Plan of Jiangxi Health Planning Committee,No.202210390(to XF).
文摘Parkinson’s disease is characterized by synucleinopathy-associated neurodegeneration.Previous studies have shown that glucagon-like peptide-1(GLP-1)has beneficial effects in a mouse model of Parkinson’s disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.However,the effect of GLP-1 on intrinsic synuclein malfunction remains unclear.In this study,we investigated the effect of Lactococcus lactis MG1363-pMG36e-GLP-1 on parkinsonism in SncaA53T transgenic mice and explored the underlying mechanisms.Our data showed that Lactococcus lactis MG1363-pMG36e-GLP-1 inhibited dopaminergic neuronal death,reduced pathological aggregation ofα-synuclein,and decreased movement disorders in SncaA53T transgenic mice.Furthermore,Lactococcus lactis MG1363-pMG36e-GLP-1 downregulated lipopolysaccharide-related inflammation,reduced cerebral activation of microglia and astrocytes,and promoted cell survival via the GLP-1 receptor/PI3K/Akt pathway in the substantia nigra.Additionally,Lactococcus lactis MG1363-pMG36e-GLP-1 decreased serum levels of pro-inflammatory molecules including lipopolysaccharide,lipopolysaccharide binding protein,interleukin-1β,and interleukin-6.Gut histopathology and western blotting further revealed that Lactococcus lactis MG1363-pMG36e-GLP-1 increased the expression of gut integrity-related proteins and reduced lipopolysaccharide-related inflammation by reversing gut dysbiosis in SncaA53T transgenic mice.Our findings showed that the beneficial effect of Lactococcus lactis MG1363-pMG36e-GLP-1 on parkinsonism traits in SncaA53T transgenic mice is mediated by microglial polarization and the reversal of dysbiosis.Collectively,our findings suggest that Lactococcus lactis MG1363-pMG36e-GLP-1 is a promising therapeutic agent for the treatment of Parkinson’s disease.
文摘[Objective]The aim was to clone TapⅡchalcone isomerases（CHI1 A） from soybean specially and construct expression vector of PNZ8149-CHI1 A,and then transform it into Lactococcus Lactis NICE systers.[Method]Chalcone isomerases（CHI1 A） was cloned by RTPCR method,and it was sequenced after cloning into pMD18-T vectors,and recombined to expression vector PNZ8149-CHI1 A,then it was transformed into Lactococcus Lactis NZ3900[Result]The sequencing results indicated that the cloned fragment of CHI1 A contained 670 nucleotides,and shared a sequence homology of 92% with that from Genbank accession number AF595413（CHI1 A）.CHI1 A was transformed into NICE expression system successfully by identification of PCR and digestion.[Conclusion]The foundation of using the microorganism fermentation method to produce flavonoids was laid by construction of efficient induction expression vector with chalcone isomerases CHI1 A.
基金a scientific research grant from Health Bureau of Sichuan Province (No. F0201)
文摘Objective To construct four recombinant Lactococcus lactis strains exhibiting high β-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion. Methods The gene fragments encoding β-galactosidase from two strains of Loctobacillus bulgaricus, wch9901 isolated from yogurt and 1.1480 purchased from the Chinese Academy of Sciences, were amplified and inserted into lactococcal expression vector pMG36e. For fusion expression, the open reading frame of the β-galactosidase gene was amplified, while for non-fusion expression, the open reading frame of the β-galactosidase gene was amplified with its native Shine-Dalgarno sequence upstream. The start codon of the β-galactosidase gene partially overlapped with the stop codon of vector origin open reading frame. Then, the recombinant plasmids were transformed into Escherichia coli DH5α and Lactococcus lactis subsp, lactis MG1363 and confirmed by determining β-galactosidase activities. Results The non-fusion expression plasmids showed a significantly higher β-galactosidase activity in transformed strains than the fusion expression plasmids. The highest enzyme activity was observed in Lactococcus lactis transformed with the non-fusion expression plasmids which were inserted into the β-galactosidase gene from Lactobacillus bulgaricus wch9901. The β-galactosidase activity was 2.75 times as high as that of the native counterpart. In addition, β-galactosidase expressed by recombinant plasmids in Lactococcus lactis could be secreted into the culture medium. The highest secretion rate （27.1%） was observed when the culture medium contained 20 g/L of lactose. Conclusion Different properties of the native bacteria may have some effects on the protein expression of recombinant plasmids. Non-fusion expression shows a higher enzyme activity in host bacteria. There may be a host-related weak secretion signal peptide gene within the structure gene of Lb. bulgaricus β-galactosidase, and its translation product may introduce the enzyme secretion out of cells in special hosts.
基金supported by the National Science Foundation of China (NO. 30800910)
文摘Objective This study is to examine the secretion effects of β-galactosidase in Lactococcus lactis.Methods The usp45 and β-galactosidase genes were cloned and inserted into plasmid pMG36e to obtain the recombinant plasmid pMG36e-usp-lacZ.This recombinant plasmid was transformed into both Escherichia coli DH5α and L.lactis MG1363.The enzyme activity,gene sequencing,SDS-PAGE and hereditary stability were assessed and studied.Results The lacZ gene inserted into plasmids pMG36e-usp-lacZ was 99.37% similar to the GenBank sequence,and SDS-PAGE revealed an evident idio-strap at 116 KDa between L.lactis MG1363/pMG36eusp-lacZ in both supernatant and cell samples.β-Galactosidase activity measured 0.225 U/mL in L.lactis pMG36e-usp-lacZ transformants,and its secretion rate was 10%.The plasmid pMG36e-usp-lacZ appeared more stable in MG1363.Conclusion The authors concluded that these new recombinant bacteria well expressed and secreted β-galactosidase,indicating that the β-galactosidase expression system was successfully constructed,and this might provide a new solution for management of lactose intolerance specifically and promote the use of gene-modified organisms as part of the food-grade plasmid in general.
基金Supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), No.980198
文摘AIM： To construct the recombinant Lactococcus/actis as oral delivery vaccination against malaria. METHODS： The C-terminal 19-ku fragments of MSP1 （MSP-119） of Plasmodium yoelii265-BY was expressed in L. lactis and the recombinant L. lact/s was administered orally to BALB/c and C57BL/6 mice. After seven interval vaccinations within 4 wk, the mice were challenged with P. yoelii 265-BY parasites of erythroo/tic stage. The protective efficacy of recombinant L. lactiswas evaluated. RESULTS： The peak parasitemias in average for the experiment groups of BALB/c and C57BL/6 mice were 0.8± 0.4% and 20.8±26.5%, respectively, and those of their control groups were 12.0±0.8% and 60.8±9.6%, respectively. None of the BALB/c mice in both experimental group and control group died during the experiment. However, all the C57BL/6 mice in the control group died within 23 d and all the vaccinated mice survived well. CONCLUSION： The results imply the potential of recombinant L. lactis as oral delivery vaccination against malaria.
文摘This study evaluated efficacy of maternal and larval immunisation against Lactococcus garviae infection and on the lysozyme and immunoglobulin (IgM) levels in rainbow trout Oncorhynchus mykiss (Walaum). Forty-eight-day-old larvae (mean weight 96 mg) originating from injected weekly with letrozole and immunised, only immunised and non-immunised parents were experimentally infected with the L. garvieae, and the mortality rate was recorded daily. Larvae were vaccinated by immersion at 58 days post hatch with live L. garvieae (109 cells/mL) for 15 min. Every third day post larvae vaccination, two larvae from each group were collected for analysis lysozyme (by a method based on the ability of lysozyme to lyse the bacterium Micrococcus lysodeikticus) and IgM (by enzyme-linked immunosorbent assay (ELISA)) parameters. Vaccinated and control larvae were tested for protection against L. garvieae 30 days post larvae immunization when the larvae were 88 days old. Larvae were challenged by bath exposure with live L. garvieae (109 cells/mL) for 2 min and monitored for mortality for at least 10 days following challenge. The challenge experiment with L. garvieae showed a significant reduction in larvae from immunised (54.44% ± 0.64%) and injected weekly with letrozole and immunised fish (52.96% ± 0.97%) compared to larvae from control fish (62.96% ± 2.22%). Vaccinated larvae originated from injected weekly with letrozole and immunised parents showed significantly higher lysozyme activity compared to other fish groups. Vaccinated larvae showed significantly less mortality compared to controls. The relative percent survival (RPS) values of larvae from only immunised, injected weekly with letrozole and immunised and non-immunised parents vaccinated with L. garvieae were 67.36% ± 0.9%, 68.05% ± 0.66% and 48.27% ± 2.79% respectively. The results indicate that the effect of maternal immunization rainbow trout against L. garvieae infection by eliciting the immune responses as indicated by an increase in the IgM level and lysozyme activity.
基金Supported by Priority Academic Talent Team Cultivation Program of Shandong Colleges and Universities and Agricultural Industry Research System of Shandong Province(SDAIT-06-022-08)People’s Livelihood Science and Technology Program of Qingdao City(16-6-2-42-nsh)
文摘To evaluate the specific immune responses induced by recombinant Lactococcus lactis（L.lactis） which expresses porcine epidemic diarrhea virus（PEDV） S1 protein through oral administration,the spike gene fragment of PEDV was amplified from PEDV SDLY strain to construct p MG36 e-S1 recombinant plasmid.The recombinant plasmid was then electro-transferred into competent cells of L.lactis MG1363,to prepare the recombinant L.lactis expressing S1 protein of PEDV.The expression of target protein was identified by SDS-PAGE and Western-blot.New Zealand white rabbits were orally administered with the recombinant strain;the antibody titer in intestinal mucosa and serum was detected by neutralizing test;and the specific Ig G in serum was evaluated by indirect ELISA.The results showed that the recombinant L.lactis could effectively induce high level of Ig G in serum and high level of mucosal immune antibody.The recombinant L.lactis is qualified to be a potential oral vaccine because it could successfully stimulate both humoral and mucosal immune responses against PEDV.
基金supported by the funds of Ministry of Higher Education,Malaysia and Universiti Putra Malaysia through Fundamental Research Grant Scheme (FRGS/1/2017/SKK11/UPM/01/1) and Putra Grant (GP/2017/9571800)
文摘Objective:To determine the inhibition mechanisms of secretome protein extracted from Paenibacillus polymyxa Kp10(Kp10)and Lactococcus lactis Gh1(Gh1)against methicillin-resistant Staphylococcus aureus(MRSA)and vancomycin-resistant Enterococcus(VRE).Methods:The sensitivity and viability of MRSA and VRE treated with secretome proteins of Kp10 and Gh1 were determined using minimal inhibitory concentration,minimum bactericidal concentration,and time-to-kill assays.The morphological changes were observed using scanning electron microscopy and transmission electron microscopy.To elucidate the antimicrobial mechanism of secretome protein of Kp10 and Gh1 against MRSA and VRE,2D gel proteomic analysis using liquid chromatography-mass spectrometry was run by comparing upregulated and downregulated proteins,and the proton motive force study including the efflux of ATP,pH gradient,and the membrane potential study were conducted.Results:MRSA and VRE were sensitive to Kp10 and Gh1 secretome protein extracts and displayed apparent morphological and internal composition changes.Several proteins associated with cellular component functions were either downregulated or upregulated in treated MRSA and VRE by changing the membrane potential gradient.Conclusions:Kp10 and Gh1 secretome proteins reduce the growth of VRE and MRSA by damaging the cell membrane.Cell division,cell wall biosynthesis,and protein synthesis are involved in the inhibition mechanism.
文摘In this study, a plasmid was integrated into nisP, creating the first defined mutation in a nisin biosynthetic gene. The mutant strain secreted fully modified nisin with the N-terminal leader still attached.The presence of the leader was confirmed by N-terminal sequencing of the purified precursor. The dehydration and lanthionine formation of the precursor were already completed as active nisin could be formed by cleaving the leader from the inactive precursor by a trypsin treatment or by incubation with wild type cells. Nisin immunity of the NisP mutant strain was lowered to about 10% of the wild type immunity. The results show that NisP is needed for precursor processing and for development of high immunity of nisin.
文摘Bovine mastitis affects the udder health and thus causing significant economic losses. Probiotic products based on the use of lactic acid bacteria (LAB) to limit pathogens multiplication and pre-infection risks can be an interesting alternative to post infection allopathic treatment with antibiotics. Lactococcus lactis is one of the most important bacteria used in dairy technology. In this work, a total of 21 Lactococcus lactis subsp. Lactis strains, 20 from goat milk whey and one strain from cow milk were used to evaluate their antibacterial activity against four pathogenic germs responsible for mastitis: Escherichia coli, Staphylococcus aureus, Streptococcus uberis and Streptococcus agalactiae. The nisin-producing cow milk strain was active against St. uberis and Str. Agalactiae using the well diffusion method. For the strains isolated from goat milk whey, no antimicrobial effect was observed against these pathogens. However, a different approach based on the growth of pathogenic bacteria interacting with the Lactococcus lactis strains in a minimum medium was used to study the barrier effect of LAB. The Lactococcus lactis strains S1 and S2 from goat milk whey depleted the growth of Sa. aureus, St. uberis and E. coli during 8 h and stopped the development of St. agalactiae.
文摘Objective: A community-based intervention study was conducted to examine the effect of consumption of JCM 5805 yogurt on influenza incidence rates and the cumulative incidence rates among schoolchildren in Iwate Prefecture, Japan. Methods: Schoolchildren and their parents in Shizukuishi town were told of the purpose, frequency and duration of JCM 5805 yogurt administration. The number of elementary schoolchildren in Shizukuishi town was 780 while that of junior high school students in Shizukuishi town numbered 475. The number of elementary schoolchildren in neighboring town A was 208 and that of junior high school students in town A was 121. JCM 5805 yogurt was delivered three times a week to all elementary schools and junior high schools in Shizukuishi town from January 16 through March 18, 2015. The incidence rate was calculated every week as the maximum case number divided by the number of schoolchildren in each school. The cumulative incidence rate was calculated as the total case number during the period when JCM 5805 yogurt was delivered divided by the number of schoolchildren in each school. Results: JCM 5805 yogurt intake was associated with a two-thirds reduction in influenza incidence rates in Shizukuishi town schoolchildren compared with those of town A. Furthermore, the cumulative incidence rates of the elementary school and combined data from the elementary school and junior high school were significantly lower than those of neighbor town A. Conclusion: JCM 5805 yogurt intake reduced both the incidence rates and cumulative incidence rates of influenza.
文摘Background/Aims: Administration of a lactic acid bacterial strain, Lactococcus lactis subsp. lactis JCM 5805 (LC-Plasma), is reported to prevent viral infection via activation of plasmacytoid dendritic cells in mouse and human studies. As it is assumed that LC-Plasma is taken in excess when it is commercially provided as a supplement, we conducted a trial using capsules to give 250 mg LC-Plasma (5 times the effective anti-viral dose) every day for four weeks to healthy volunteers to investigate the safety of excessive intake of LC-Plasma. Trial Design: A randomized, double-blind, placebo-controlled, parallel-group trial was conducted. Methods: Forty healthy subjects were randomly assigned to the LC-Plasma group (daily intake of five capsules containing 50 mg heat-killed LC-Plasma cells per capsule) or the placebo group (daily intake of five placebo capsules with no LC-Plasma). Physical, hematological, biochemical and urinary examinations and medical interviews were used to evaluate safety. Results: No abnormal differences were observed after excessive intake of LC-Plasma capsules when compared to the intake of placebo capsules. Conclusions: There are no safety concerns associated with the excessive intake of heat-killed LC-Plasma capsules.
