Recently,more and more bacteria have been reported to become tolerant to antibiotics.In this study,one tnaA gene involved in indole production,and the effect of exogenous indole on the formation of persister cells spe...Recently,more and more bacteria have been reported to become tolerant to antibiotics.In this study,one tnaA gene involved in indole production,and the effect of exogenous indole on the formation of persister cells specific to tetracycline in Vibrio splendidus were characterized.The tnaA Vs gene was first cloned and conditionally expressed in Escherichia coli Rosetta(DE3).To investigate the regulatory effect of TnaA Vs,the tnaA deletion strain AJ01/ΔtnaA was constructed by in-frame deletion.The undetected extracellular indole in the AJ01/ΔtnaA indicated that TnaA was the solo enzyme to produce indole in V.splendidus.The drop plate method showed that AJ01/ΔtnaA was more tolerant to the higher concentration of tetracycline than that of AJ01,being 340-fold higher in the proportion of survived cells when cell density OD 600≈0.65.Moreover,the synergistic effects of indole and tetracycline on killing of V.splendidus were determined.Results show that addition of 2-mmol/L indole increased the susceptibility of both AJ01 and AJ01/ΔtnaA to 10×minimum inhibitory concentration tetracycline.To explore the genes and pathways regulated by TnaA Vs,the transcriptomic analysis between AJ01 and AJ01/ΔtnaA was performed.Result shows that TCA cycle,arginine biosynthesis,quorum sensing and microbial metabolism in diverse environments were downregulated,while the ribosome pathways,the protein metabolic process,peptide biosynthetic and metabolic process were upregulated in the AJ01/ΔtnaA.This study shows that indole could enhance the bactericidal effect of tetracycline on V.splendidus by decreased ribosome level probably but increased ATP level.展开更多
Sea cucumbers, Apostichopus japonicus Selenka, were fed diets containing non-immunostimulant (basal diet), 0.2% β-glucan and 0.02% glycyrrhizin in a recirculatory water system for 45 days, and subsequently challenged...Sea cucumbers, Apostichopus japonicus Selenka, were fed diets containing non-immunostimulant (basal diet), 0.2% β-glucan and 0.02% glycyrrhizin in a recirculatory water system for 45 days, and subsequently challenged with Vibrio splendidus by injection at 1.0×108 cfu/sea cucumber for 15 days.Phagocytic capacity (PC), intracellular superoxide anion production (ISAP), ly-sozyme (LSZ) activity and superoxide dismutase (SOD) activity in the coelomic fluid were analyzed on the 0th, 5th, 10th and 15th days after injection.Results showed that after the 45-day feeding period, PC, ISAP, LSZ activity and SOD activity in sea cucumbers fed with dietary β-glucan or glycyrrhizin were significantly higher than in those fed with the basal diet.On the 5th day after infection, all the immune parameters examined in the sea cucumbers injected with V.splendidus decreased in value significantly.On the 15th day, PC, ISAP and LSZ activity returned to levels similar to those on the 0th day.For the sea cucumbers injected with saline, there were no significant differences in all the immune parameters examined and in the cumulative morbidity during the 15-day challenging trial.After injecting with V.splendidus, the cumulative morbidity of sea cucumbers fed with the basal diet was significantly higher than those fed with dietary β-glucan or glycyrrhizin when challenged with V.splendidus challenged sea cucumber fed with the basal diet was significantly higher than those fed with dietary β-glucan or glycyrrhizin.There was no significant difference in cumulative morbidity between the dietary β-glucan and glycyrrhizin treatments over time.展开更多
The effects of probiotic Bacillus TC22 (isolated from intestine of infected sea cucumber) and prebiotic fructooligosaccharide (FOS) on growth, immunity and disease resistance in sea cucumber Apostichopus japonicus wer...The effects of probiotic Bacillus TC22 (isolated from intestine of infected sea cucumber) and prebiotic fructooligosaccharide (FOS) on growth, immunity and disease resistance in sea cucumber Apostichopus japonicus were studied. Six experimental diets were formulated with combinations of three levels of TC22 (0, 107 and 109 CFU g-1 diet) and two levels of FOS (0 and 0.5%) in a 3 × 2 factorial experiment. At the end of the 8-week feeding trial,animals were challenged by injecting Vibrio splendidus. The results revealed that the specific growth rates (SGR) of sea cucumbers were not affected by TC22 and FOS, or the interaction between TC22 and FOS (P > 0.05). However,there were significant interactions between TC22 and FOS for immune response and disease resistance in sea cucumbers (P < 0.05). When sea cucumbers were fed with TC22 at 109 CFU g?1 feed and 0.5% FOS alone or in combination, the phagocytosis, respiratory burst and phenoloxidase activity of sea cucumber coelomocytes were significantly enhanced;the disease resistance against V. splendidus infection was also increased significantly (P < 0.05).However, dietary combination of TC22 at 109 CFU g-1 diet and 0.5% FOS did not yield significantly higher efficiency than either TC22 at 109 CFU g-1 diet or 0.5% FOS used alone (P > 0.05). Therefore, further studies should examine the effects of combinations of other levels of FOS(> 0.5% or < 0.5%) and TC22 on the immunity and disease resistance of sea cucumbers.展开更多
Vibrio splendidus is an important opportunistic pathogen ubiquitously present in the marine environment,exhibiting virulence to a variety of cultured animals.The extracellular products secreted by V.splendidus are cru...Vibrio splendidus is an important opportunistic pathogen ubiquitously present in the marine environment,exhibiting virulence to a variety of cultured animals.The extracellular products secreted by V.splendidus are crucial to bacterial survival and virulence.In this study,the secretion of outer membrane vesicles(OMVs)by V.splendidus was determined,purified,and morphologically characterized.The protein composition of OMVs was analyzed by proteomic analysis.The results showed that approximately 120 proteins were contained in these OMVs,including outer membrane proteins,flagellins,ABC transporters,protease,and iron regulation proteins,etc.,which were involved in bacterial motility,formation of biofilms and the cell membrane components,and cellular localization based on their structural molecule activity,passive transmembrane transporter activity,channel activity,neurotransmitter receptor activity,extracellular ligand-gated ion channel activity,glutamate receptor activity,ligand-gated ion channel activity,and transmembrane signaling receptor activity.To explore the biological functions of OMVs in V.splendidus,the effects of OMVs on the bacterial adaption to iron limitation,antibiotic,and the coelomic fluid of the Apostichopus japonicus were confirmed.This study is the first time to show that V.splendidus secretes OMVs,and OMVs carry functional proteins that enhance bacterial survival under various stresses.展开更多
The p21-activated kinase 1(PAK1)is a downstream serine/threonine kinase effector of Rac1/Cdc42 that regulates various biological processes including those associating with pathological inflammation.To investigate the ...The p21-activated kinase 1(PAK1)is a downstream serine/threonine kinase effector of Rac1/Cdc42 that regulates various biological processes including those associating with pathological inflammation.To investigate the function of PAK1 in echinoderms,we isolated a new PAK1 from sea cucumber Apostichopus japonicus(AjPAK1)by transcriptome database mining and with rapid amplification of cDNA ends(RACE).The full-length cDNA AjPAK1 was 2303 bp in length,containing a 1587 bp ORF encoding 528 amino acid residues.The deduced AjPAK1 contained a p21-Rho-binding domain(PBD)and a serine/threonine protein kinase catalytic domain(S_TKc),which was similar to the PAK1 of crown-of-thorns starfish Acanthaster planci and other eukaryotes.AjPAK1 expressed in all tissues of adult A.japonicus analyzed with the highest transcript anumdance detected in coelomocytes.Significant change in AjPAK1 abundance was observed at 4,24,48 and 72 h after Vibrio splendidus infection.Silencing AjPAK1 induced a significant reduction of lysozyme content in coelomic fluid and relative transcript abundances of AjRac1 and AjMKK3/6 in A.japonicus coelomocytes.These results should aid to characterize PAK1 of sea cucumber and decipher its immune function.展开更多
Sea cucumber（Apostichopus japonicus） is one of the economically important farmed echinoderm species in North China. The NF-κB/Rel transcription factors can function as key regulators to modulate the expression of i...Sea cucumber（Apostichopus japonicus） is one of the economically important farmed echinoderm species in North China. The NF-κB/Rel transcription factors can function as key regulators to modulate the expression of immune-related genes in response to immune challenge or environmental stress. And as a crucial enzyme in innate immunity, lysozyme plays a key role in the overall defense against pathogens in A. japonicus. In the present study, the expression of NF-κB/Rel and lysozyme mRNA against bacterial challenge in sea cucumber was investigated. After bacterial challenge, the Aj-rel mRNA transcripts increased significantly and reached the peak at 10 min（3.26-fold, P0.05）; the Aj-p105 gene expression showed a rising trend and reached the peak at 240 min（6.12-fold, P0.05）; and the Aj-p50 mRNA transcripts showed a downward trend and reached the peak at 10 min（17.80-fold, P 0.05）. The lysozyme mRNA expression level showed two peaks at 10 min（1.54-fold, P 0.05） and 240 min（2.48-fold, P 0.05）, respectively. This study will provide certain reference for further understanding on the immune defense mechanism and offer enlightenment to disease control of echinoderm.展开更多
基金Supported by the Zhejiang Provincial Natural Science Foundation for Distinguished Young Scholar(No.LR20C190001)the National Natural Science Foundation of China(No.42376103)+1 种基金the Natural Science Foundation of Ningbo City(No.2021J062)the K.C.Wong Magna Fund in Ningbo University。
文摘Recently,more and more bacteria have been reported to become tolerant to antibiotics.In this study,one tnaA gene involved in indole production,and the effect of exogenous indole on the formation of persister cells specific to tetracycline in Vibrio splendidus were characterized.The tnaA Vs gene was first cloned and conditionally expressed in Escherichia coli Rosetta(DE3).To investigate the regulatory effect of TnaA Vs,the tnaA deletion strain AJ01/ΔtnaA was constructed by in-frame deletion.The undetected extracellular indole in the AJ01/ΔtnaA indicated that TnaA was the solo enzyme to produce indole in V.splendidus.The drop plate method showed that AJ01/ΔtnaA was more tolerant to the higher concentration of tetracycline than that of AJ01,being 340-fold higher in the proportion of survived cells when cell density OD 600≈0.65.Moreover,the synergistic effects of indole and tetracycline on killing of V.splendidus were determined.Results show that addition of 2-mmol/L indole increased the susceptibility of both AJ01 and AJ01/ΔtnaA to 10×minimum inhibitory concentration tetracycline.To explore the genes and pathways regulated by TnaA Vs,the transcriptomic analysis between AJ01 and AJ01/ΔtnaA was performed.Result shows that TCA cycle,arginine biosynthesis,quorum sensing and microbial metabolism in diverse environments were downregulated,while the ribosome pathways,the protein metabolic process,peptide biosynthetic and metabolic process were upregulated in the AJ01/ΔtnaA.This study shows that indole could enhance the bactericidal effect of tetracycline on V.splendidus by decreased ribosome level probably but increased ATP level.
基金supported by grant No.2006AA100313 from the National High Technology Research and Development Project of P.R. China (863 Project)
文摘Sea cucumbers, Apostichopus japonicus Selenka, were fed diets containing non-immunostimulant (basal diet), 0.2% β-glucan and 0.02% glycyrrhizin in a recirculatory water system for 45 days, and subsequently challenged with Vibrio splendidus by injection at 1.0×108 cfu/sea cucumber for 15 days.Phagocytic capacity (PC), intracellular superoxide anion production (ISAP), ly-sozyme (LSZ) activity and superoxide dismutase (SOD) activity in the coelomic fluid were analyzed on the 0th, 5th, 10th and 15th days after injection.Results showed that after the 45-day feeding period, PC, ISAP, LSZ activity and SOD activity in sea cucumbers fed with dietary β-glucan or glycyrrhizin were significantly higher than in those fed with the basal diet.On the 5th day after infection, all the immune parameters examined in the sea cucumbers injected with V.splendidus decreased in value significantly.On the 15th day, PC, ISAP and LSZ activity returned to levels similar to those on the 0th day.For the sea cucumbers injected with saline, there were no significant differences in all the immune parameters examined and in the cumulative morbidity during the 15-day challenging trial.After injecting with V.splendidus, the cumulative morbidity of sea cucumbers fed with the basal diet was significantly higher than those fed with dietary β-glucan or glycyrrhizin when challenged with V.splendidus challenged sea cucumber fed with the basal diet was significantly higher than those fed with dietary β-glucan or glycyrrhizin.There was no significant difference in cumulative morbidity between the dietary β-glucan and glycyrrhizin treatments over time.
基金supported by the National High Technology Research and Development Program of China(863 Program,No.2006AA100313)
文摘The effects of probiotic Bacillus TC22 (isolated from intestine of infected sea cucumber) and prebiotic fructooligosaccharide (FOS) on growth, immunity and disease resistance in sea cucumber Apostichopus japonicus were studied. Six experimental diets were formulated with combinations of three levels of TC22 (0, 107 and 109 CFU g-1 diet) and two levels of FOS (0 and 0.5%) in a 3 × 2 factorial experiment. At the end of the 8-week feeding trial,animals were challenged by injecting Vibrio splendidus. The results revealed that the specific growth rates (SGR) of sea cucumbers were not affected by TC22 and FOS, or the interaction between TC22 and FOS (P > 0.05). However,there were significant interactions between TC22 and FOS for immune response and disease resistance in sea cucumbers (P < 0.05). When sea cucumbers were fed with TC22 at 109 CFU g?1 feed and 0.5% FOS alone or in combination, the phagocytosis, respiratory burst and phenoloxidase activity of sea cucumber coelomocytes were significantly enhanced;the disease resistance against V. splendidus infection was also increased significantly (P < 0.05).However, dietary combination of TC22 at 109 CFU g-1 diet and 0.5% FOS did not yield significantly higher efficiency than either TC22 at 109 CFU g-1 diet or 0.5% FOS used alone (P > 0.05). Therefore, further studies should examine the effects of combinations of other levels of FOS(> 0.5% or < 0.5%) and TC22 on the immunity and disease resistance of sea cucumbers.
基金the Zhejiang Provincial Natural Science Foundation for Distinguished Young Scholars(No.LR20C190001)the National Natural Science Foundation of China(No.31972833)+1 种基金the Fundamental Research Funds for the Provincial Universities of Zhejiang(No.SJ LZ2020001)the K.C.Wong Magna Fund at Ningbo University。
文摘Vibrio splendidus is an important opportunistic pathogen ubiquitously present in the marine environment,exhibiting virulence to a variety of cultured animals.The extracellular products secreted by V.splendidus are crucial to bacterial survival and virulence.In this study,the secretion of outer membrane vesicles(OMVs)by V.splendidus was determined,purified,and morphologically characterized.The protein composition of OMVs was analyzed by proteomic analysis.The results showed that approximately 120 proteins were contained in these OMVs,including outer membrane proteins,flagellins,ABC transporters,protease,and iron regulation proteins,etc.,which were involved in bacterial motility,formation of biofilms and the cell membrane components,and cellular localization based on their structural molecule activity,passive transmembrane transporter activity,channel activity,neurotransmitter receptor activity,extracellular ligand-gated ion channel activity,glutamate receptor activity,ligand-gated ion channel activity,and transmembrane signaling receptor activity.To explore the biological functions of OMVs in V.splendidus,the effects of OMVs on the bacterial adaption to iron limitation,antibiotic,and the coelomic fluid of the Apostichopus japonicus were confirmed.This study is the first time to show that V.splendidus secretes OMVs,and OMVs carry functional proteins that enhance bacterial survival under various stresses.
基金supported by the National Key R&D Program of China (No. 2018YFD0900105)
文摘The p21-activated kinase 1(PAK1)is a downstream serine/threonine kinase effector of Rac1/Cdc42 that regulates various biological processes including those associating with pathological inflammation.To investigate the function of PAK1 in echinoderms,we isolated a new PAK1 from sea cucumber Apostichopus japonicus(AjPAK1)by transcriptome database mining and with rapid amplification of cDNA ends(RACE).The full-length cDNA AjPAK1 was 2303 bp in length,containing a 1587 bp ORF encoding 528 amino acid residues.The deduced AjPAK1 contained a p21-Rho-binding domain(PBD)and a serine/threonine protein kinase catalytic domain(S_TKc),which was similar to the PAK1 of crown-of-thorns starfish Acanthaster planci and other eukaryotes.AjPAK1 expressed in all tissues of adult A.japonicus analyzed with the highest transcript anumdance detected in coelomocytes.Significant change in AjPAK1 abundance was observed at 4,24,48 and 72 h after Vibrio splendidus infection.Silencing AjPAK1 induced a significant reduction of lysozyme content in coelomic fluid and relative transcript abundances of AjRac1 and AjMKK3/6 in A.japonicus coelomocytes.These results should aid to characterize PAK1 of sea cucumber and decipher its immune function.
基金Supported by Key Technologies R&D Program of Shandong Province(2014GHY115024)Presidential Foundation of Marine Biology Institute of Shandong Province(SZJJ201303)~~
文摘Sea cucumber（Apostichopus japonicus） is one of the economically important farmed echinoderm species in North China. The NF-κB/Rel transcription factors can function as key regulators to modulate the expression of immune-related genes in response to immune challenge or environmental stress. And as a crucial enzyme in innate immunity, lysozyme plays a key role in the overall defense against pathogens in A. japonicus. In the present study, the expression of NF-κB/Rel and lysozyme mRNA against bacterial challenge in sea cucumber was investigated. After bacterial challenge, the Aj-rel mRNA transcripts increased significantly and reached the peak at 10 min（3.26-fold, P0.05）; the Aj-p105 gene expression showed a rising trend and reached the peak at 240 min（6.12-fold, P0.05）; and the Aj-p50 mRNA transcripts showed a downward trend and reached the peak at 10 min（17.80-fold, P 0.05）. The lysozyme mRNA expression level showed two peaks at 10 min（1.54-fold, P 0.05） and 240 min（2.48-fold, P 0.05）, respectively. This study will provide certain reference for further understanding on the immune defense mechanism and offer enlightenment to disease control of echinoderm.
