【目的】分析深汕特别合作区某鲈鱼养殖基地大口黑鲈(Micropterus salmoides)突发性死亡原因,探究其病原菌生物学特性,为大口黑鲈养殖中的疾病防控与诊治提供理论参考。【方法】从患病大口黑鲈肝脏、脾、肠等病灶组织中分离得到一株优势...【目的】分析深汕特别合作区某鲈鱼养殖基地大口黑鲈(Micropterus salmoides)突发性死亡原因,探究其病原菌生物学特性,为大口黑鲈养殖中的疾病防控与诊治提供理论参考。【方法】从患病大口黑鲈肝脏、脾、肠等病灶组织中分离得到一株优势菌,命名为SZNH-S20230817;开展形态学特征观察,生理生化特性鉴定,人工感染试验,药物敏感性测试,最小抑菌浓度(MIC)和最小杀菌浓度(MBC)测定,16S rRNA序列、耐药基因及毒力基因的鉴定,分析其耐药性和病理。【结果】菌株SZNH-S20230817的序列与豚鼠气单胞菌(Aeromonas caviae)AP019195具有99.38%同源性;同时携带aer、act、lip和fl a 4个毒力基因;生化鉴定结果显示,该菌的吲哚、氧化酶、苯丙氨酸、甘露醇、木糖、精氨酸双水解酶和葡萄糖产酸等结果为阳性,确定菌株SZNH-S20230817为豚鼠气单胞菌。回归感染试验发现菌株SZNH-S20230817具有较强致病性,对大口黑鲈的半致死浓度(LC_(50))为1.39×10^(7)CFU/mL。药敏试验结果显示,该菌对头孢唑林、卡那霉素、四环素、万古霉素、氨苄西林和克林霉素等12种抗生素耐药,同时携带qnrS、catI、emrB、Sul1、rmtB等13种耐药基因。组织病理学分析表明,菌株SZNH-S20230817感染可引起鱼体脾脏细胞肿胀、游离、肠黏膜断裂破损、绒毛脱落、肾脏细胞坏死、炎症细胞浸润、肝脏细胞呈空泡化等。体外抑菌试验结果显示大黄和五倍子对该菌的MIC和MBC较低,其余7种中草药对该菌的抑菌能力较弱。【结论】豚鼠气单胞菌是引起本次大口黑鲈突发性死亡的病原菌,其同时携带aer、act、lip和fl a 4个毒力基因,对大口黑鲈的LC_(50)为1.39×10^(7)CFU/mL,对头孢唑林、卡那霉素、四环素等抗生素耐药。低剂量的大黄和五倍子能够有效抑制该菌生长,具备良好的预防和抑制效果。展开更多
Understanding aquatic pathogen in sediments or aquacultural water is crucial to protect public health from soilborne and waterborne diseases.Quorum sensing(QS)was increasingly reported in biological wastewater treatme...Understanding aquatic pathogen in sediments or aquacultural water is crucial to protect public health from soilborne and waterborne diseases.Quorum sensing(QS)was increasingly reported in biological wastewater treatment processes because of their inherent roles in biofilm development,bacterial aggregation and so on.The widely QS signals was Antoinducer-2(AI-2),primarily involved to allow the possibility of interspecies communication.However,the cellular components that mediate the response of Streptococcus agalactiae to AI-2 have not been fully characterized.Analysis of the complete genome sequence of S.agalactiae indi-cated that its RbsB protein has similarity to Escherichia coli LsrB and Aggregatibacter actinomycetemcomitans RbsB proteins that bind AI-2.We hypothesized that RbsB protein mediates quorum sensing signal uptake via interaction with AI-2.To evaluate the regulatory effect of RbsB on QS system,the recombinant plasmid pGEX-6p-1-RbsB was constructed and RbsB protein was purified with GST-tag.To further elucidate the role of RbsB protein binding to DPD(AI-2 precursor dihydroxypentanedione),the systemati-cally throughput circular dichroism(CD)spectroscopy,isothermal titration calorimetry200(ITC200)and molecular docking methods were employed.The high expression of soluble RbsB protein with molecular weight of 33 kDa was obtained.The thermodynamics results(ΔH<0,ΔS<0,ΔG<0)with ITC determination indicated that the binding process between DPD and RbsB was exothermic and spontaneous,with hydrogen bonds and van der Waals forces as the main binding forces.Obviously,DPD can be more easily combined with RbsB in a dose-dependent manner,suggesting that RbsB was changed in the microenvironment of DPD when the DPD concentration was between 0.8-1.0mmolL−1 and reaching the maximum binding amount.According to molecular docking,3 hydrophobic residues involved in DPD and RbsB protein stable binding were be found,and also hydrogen bonding plays a key role in the formation of the new complex.RbsB efficiently inhibited V.harveyi bioluminescence induced by both S.agalactiae AI-2 and V.harveyi AI-2 in a dose-dependent manner.However,our results suggest that RbsB may play a role in the response of S.agalactiace to AI-2.展开更多
CD79, a key component of the B cell antigen receptor complex, is composed of CD79a（Igα and CD7913（Igβ） encoded by rnb-1 and B29 respectively, and plays an important role in B cell signaling. In this study, we iso...CD79, a key component of the B cell antigen receptor complex, is composed of CD79a（Igα and CD7913（Igβ） encoded by rnb-1 and B29 respectively, and plays an important role in B cell signaling. In this study, we isolated and characterized rob-1 and B29 from humphead snapper （Lutjanus sanguineus）. Their tissue distribution and expression profiles after stimulations in vitro and in vivo were also investigated. The humphead snapper rob-1 and B29 contain open reading frames of 684 bp and 606 bp, encoding 227 amino acids and 201 amino acids, respectively. Both CD79α and CD79β possess signal peptide, extracellular Ig domain, transmem- brane region and immunoreceptor tyrosine kinase activation motif （ITAM）. Mb-1 is highly expressed in lymphoid organs （thymus, posterior kidney and spleen） and mucosal-associated lymphoid tissues （gill and intestine）, while B29 is mainly detected in posterior kidney, spleen, gill and skin. Furthermore, transcription of rob-1 and B29 in head kidney leucocytes was up-regulated following lipopolysaccharide （LPS）, pokeweed mitogen （PWM）, and polyinosinic-polycytidylic acid （PolyI：C） stimulation, respectively, and their expression level in anterior kidney and spleen was also increased after challenged with formalin-inactived Vibrio harveyi. These results indicated that humphead snapper CD79 molecule might play an important role in immune response to pathogen infection.展开更多
Vibrio alginolyticus, a gram-negative bacterium has been described to be one of the most common and economically important aquatic pathogens of fish and shellfish. Vaccine immunization is an effective approach prevent...Vibrio alginolyticus, a gram-negative bacterium has been described to be one of the most common and economically important aquatic pathogens of fish and shellfish. Vaccine immunization is an effective approach preventing V. alginolyticus infection. Attenuated vaccine stimulates systemic immune response in the host, but few attenuated vaccine against V. alginolyticus is available. The type III secretion system （T3SS）, an important pathogenic factor of V. alginolyticus, is used by bacterial pathogens to inject effector proteins into the cytoplasm of their host cells. The T3SS forms a structure called needle complex with a multi-ring base that spans the bacteria and a needle-like extension that protrudes several nanometers from the bacterial surface, vscO locates at the ＂needle＂ site of T3SS, playing the role of escorting the molecular chaperone and effector proteins into host cells and further inducing the death of host cells. In this paper, an in-frame deletion mu- tant of vscO was constructed using overlap PCR and homologous recombination technology combining with chloramphenicol （Cm） and sucrose screening. The LDs0 changes of ZJO3AvscO mutant strains compared with the strain ZJ03 were e^amined in grouper （ Epinephelus coioides）. The ZJO3AvscO mutant showed about 150 times decrease in virulence in E. coioides compared with wide type ZJ03. After vaccination with ZJO3AvscO in E. coioides through injection and immersion, the spe- cific antibody titers were markedly higher than that in the saline control group （P 〈 0.05 ）. The titers of injection and immersion group on the forth week reached the maximums at 1：2 048 and 1： 128, respectively. The relative percentage survival （RPS） of injection group was 84%, while that in immersion group was 68%. These results indicate that the ZJO3AvscO of V. alginolyticus has a high immunogenicity, and can be used as live attenuated vaccine. In addition, RPS may be affected by vaccination and infection methods. This study can provide technical support for controlling fish diseases caused by V. alginolyticus.展开更多
The two-component regulatory system plays an important role in the growth and virulence of bacteria. In this study, the phosphate transcriptional regulatory protein （KdpE） gene of Vibrio alginolyticus strain HY9901 ...The two-component regulatory system plays an important role in the growth and virulence of bacteria. In this study, the phosphate transcriptional regulatory protein （KdpE） gene of Vibrio alginolyticus strain HY9901 was cloned. Sequence analysis revealed that the length ofkdpE gene is 732 bp and encodes a putative protein of 243 amino acids. The predicted molecular weight （MW） of KdpE was 27.66 kD with an estimated pI of 5.01. Using SignalP 4.0 and TM- HMM Server 2.0 software, it was predicted that the KdpE protein was located in cytoplasm. It did not contain a signal peptide or a transmembranous region. A phylogenetic tree was constructed by MEGA 5.0 software, indicating that the KdpE of V. alginolyticus bad high genetic relationship with Vibrio campbellii and Vibrio parahaemolyticus. Using SWISS-MODEL work-space, the three-dimensional structures of conserved domain REC in the KdpE was determined. These results can provide a basis for further studies on the two-component regulatory systems of V. alginolyticus.展开更多
核糖体蛋白S6(ribosomal protein S6,RPS6)是构成40S小亚基核糖体的关键蛋白之一,在蛋白质合成、调控细胞周期和凋亡、调节肿瘤细胞增殖和转移等方面具有重要作用。为探究RPS6在尼罗罗非鱼(Oreochromis niloticus)中的分子结构、表达特...核糖体蛋白S6(ribosomal protein S6,RPS6)是构成40S小亚基核糖体的关键蛋白之一,在蛋白质合成、调控细胞周期和凋亡、调节肿瘤细胞增殖和转移等方面具有重要作用。为探究RPS6在尼罗罗非鱼(Oreochromis niloticus)中的分子结构、表达特性和生物学功能,本研究从尼罗罗非鱼中克隆获得RPS6基因的编码区序列(GenBank登录号:XM_003454192.4;命名为On-RPS6),并采用qPCR法分析该基因在健康鱼体各组织的分布特征以及无乳链球菌刺激后该基因在鱼体肝脏、脾脏、脑和头肾中的表达模式。结果显示:On-RPS6的开放阅读框长度为750 bp,编码249个氨基酸,理论分子量为28.70 kDa,等电点为10.90。氨基酸序列分析显示,On-RPS6具有一个高度保守的核糖体蛋白S6e结构域,且与其他物种具有较高同源性。系统进化树分析表明,尼罗罗非鱼RPS6与斑马拟丽鱼(Maylandia zebra)RPS6聚为一支,表明二者具有较近的亲缘关系。qPCR结果显示,尼罗罗非鱼各组织均能检测到On-RPS6基因mRNA的表达,且在血液中的表达量最高;经无乳链球菌刺激后,On-RPS6基因在肝脏、脾脏、脑和头肾中的表达模式均呈现先上调后下降的趋势。结果表明,On-RPS6在尼罗罗非鱼抵抗细菌感染的免疫应答过程中发挥了重要作用。展开更多
文摘【目的】分析深汕特别合作区某鲈鱼养殖基地大口黑鲈(Micropterus salmoides)突发性死亡原因,探究其病原菌生物学特性,为大口黑鲈养殖中的疾病防控与诊治提供理论参考。【方法】从患病大口黑鲈肝脏、脾、肠等病灶组织中分离得到一株优势菌,命名为SZNH-S20230817;开展形态学特征观察,生理生化特性鉴定,人工感染试验,药物敏感性测试,最小抑菌浓度(MIC)和最小杀菌浓度(MBC)测定,16S rRNA序列、耐药基因及毒力基因的鉴定,分析其耐药性和病理。【结果】菌株SZNH-S20230817的序列与豚鼠气单胞菌(Aeromonas caviae)AP019195具有99.38%同源性;同时携带aer、act、lip和fl a 4个毒力基因;生化鉴定结果显示,该菌的吲哚、氧化酶、苯丙氨酸、甘露醇、木糖、精氨酸双水解酶和葡萄糖产酸等结果为阳性,确定菌株SZNH-S20230817为豚鼠气单胞菌。回归感染试验发现菌株SZNH-S20230817具有较强致病性,对大口黑鲈的半致死浓度(LC_(50))为1.39×10^(7)CFU/mL。药敏试验结果显示,该菌对头孢唑林、卡那霉素、四环素、万古霉素、氨苄西林和克林霉素等12种抗生素耐药,同时携带qnrS、catI、emrB、Sul1、rmtB等13种耐药基因。组织病理学分析表明,菌株SZNH-S20230817感染可引起鱼体脾脏细胞肿胀、游离、肠黏膜断裂破损、绒毛脱落、肾脏细胞坏死、炎症细胞浸润、肝脏细胞呈空泡化等。体外抑菌试验结果显示大黄和五倍子对该菌的MIC和MBC较低,其余7种中草药对该菌的抑菌能力较弱。【结论】豚鼠气单胞菌是引起本次大口黑鲈突发性死亡的病原菌,其同时携带aer、act、lip和fl a 4个毒力基因,对大口黑鲈的LC_(50)为1.39×10^(7)CFU/mL,对头孢唑林、卡那霉素、四环素等抗生素耐药。低剂量的大黄和五倍子能够有效抑制该菌生长,具备良好的预防和抑制效果。
基金This study was supported by the National Natural Sci-ence Foundation of China(Nos.31702386,31660251,31860245 and 31960203)the International Cooperation Science&Technology Planning Project of Guangdong Province of China(No.2017A050501037)+2 种基金the Natural Science Foundation of Guangxi Province(Nos.2018 GXNSFAA281019,2017GXNSFAA198010)the Central Government Directs Special Funds for Local Science and Technology Development Projects(No.ZY1949015)E.Wangkahart was supported financially by the Ministry of Science and Technology of Thailand and Mahasarakham University.
文摘Understanding aquatic pathogen in sediments or aquacultural water is crucial to protect public health from soilborne and waterborne diseases.Quorum sensing(QS)was increasingly reported in biological wastewater treatment processes because of their inherent roles in biofilm development,bacterial aggregation and so on.The widely QS signals was Antoinducer-2(AI-2),primarily involved to allow the possibility of interspecies communication.However,the cellular components that mediate the response of Streptococcus agalactiae to AI-2 have not been fully characterized.Analysis of the complete genome sequence of S.agalactiae indi-cated that its RbsB protein has similarity to Escherichia coli LsrB and Aggregatibacter actinomycetemcomitans RbsB proteins that bind AI-2.We hypothesized that RbsB protein mediates quorum sensing signal uptake via interaction with AI-2.To evaluate the regulatory effect of RbsB on QS system,the recombinant plasmid pGEX-6p-1-RbsB was constructed and RbsB protein was purified with GST-tag.To further elucidate the role of RbsB protein binding to DPD(AI-2 precursor dihydroxypentanedione),the systemati-cally throughput circular dichroism(CD)spectroscopy,isothermal titration calorimetry200(ITC200)and molecular docking methods were employed.The high expression of soluble RbsB protein with molecular weight of 33 kDa was obtained.The thermodynamics results(ΔH<0,ΔS<0,ΔG<0)with ITC determination indicated that the binding process between DPD and RbsB was exothermic and spontaneous,with hydrogen bonds and van der Waals forces as the main binding forces.Obviously,DPD can be more easily combined with RbsB in a dose-dependent manner,suggesting that RbsB was changed in the microenvironment of DPD when the DPD concentration was between 0.8-1.0mmolL−1 and reaching the maximum binding amount.According to molecular docking,3 hydrophobic residues involved in DPD and RbsB protein stable binding were be found,and also hydrogen bonding plays a key role in the formation of the new complex.RbsB efficiently inhibited V.harveyi bioluminescence induced by both S.agalactiae AI-2 and V.harveyi AI-2 in a dose-dependent manner.However,our results suggest that RbsB may play a role in the response of S.agalactiace to AI-2.
基金supported by the National Science&Technology Pillar Program of China(2012BAD17B02,2012BAD17B03)the National Natural Science Foundation of China(41240041,31402335)
文摘CD79, a key component of the B cell antigen receptor complex, is composed of CD79a（Igα and CD7913（Igβ） encoded by rnb-1 and B29 respectively, and plays an important role in B cell signaling. In this study, we isolated and characterized rob-1 and B29 from humphead snapper （Lutjanus sanguineus）. Their tissue distribution and expression profiles after stimulations in vitro and in vivo were also investigated. The humphead snapper rob-1 and B29 contain open reading frames of 684 bp and 606 bp, encoding 227 amino acids and 201 amino acids, respectively. Both CD79α and CD79β possess signal peptide, extracellular Ig domain, transmem- brane region and immunoreceptor tyrosine kinase activation motif （ITAM）. Mb-1 is highly expressed in lymphoid organs （thymus, posterior kidney and spleen） and mucosal-associated lymphoid tissues （gill and intestine）, while B29 is mainly detected in posterior kidney, spleen, gill and skin. Furthermore, transcription of rob-1 and B29 in head kidney leucocytes was up-regulated following lipopolysaccharide （LPS）, pokeweed mitogen （PWM）, and polyinosinic-polycytidylic acid （PolyI：C） stimulation, respectively, and their expression level in anterior kidney and spleen was also increased after challenged with formalin-inactived Vibrio harveyi. These results indicated that humphead snapper CD79 molecule might play an important role in immune response to pathogen infection.
基金Supported by National Natural Science Foundation of China(31402344)Project of Enhancing School With Innovation of Guangdong Ocean University(GDOU2015050216)
文摘Vibrio alginolyticus, a gram-negative bacterium has been described to be one of the most common and economically important aquatic pathogens of fish and shellfish. Vaccine immunization is an effective approach preventing V. alginolyticus infection. Attenuated vaccine stimulates systemic immune response in the host, but few attenuated vaccine against V. alginolyticus is available. The type III secretion system （T3SS）, an important pathogenic factor of V. alginolyticus, is used by bacterial pathogens to inject effector proteins into the cytoplasm of their host cells. The T3SS forms a structure called needle complex with a multi-ring base that spans the bacteria and a needle-like extension that protrudes several nanometers from the bacterial surface, vscO locates at the ＂needle＂ site of T3SS, playing the role of escorting the molecular chaperone and effector proteins into host cells and further inducing the death of host cells. In this paper, an in-frame deletion mu- tant of vscO was constructed using overlap PCR and homologous recombination technology combining with chloramphenicol （Cm） and sucrose screening. The LDs0 changes of ZJO3AvscO mutant strains compared with the strain ZJ03 were e^amined in grouper （ Epinephelus coioides）. The ZJO3AvscO mutant showed about 150 times decrease in virulence in E. coioides compared with wide type ZJ03. After vaccination with ZJO3AvscO in E. coioides through injection and immersion, the spe- cific antibody titers were markedly higher than that in the saline control group （P 〈 0.05 ）. The titers of injection and immersion group on the forth week reached the maximums at 1：2 048 and 1： 128, respectively. The relative percentage survival （RPS） of injection group was 84%, while that in immersion group was 68%. These results indicate that the ZJO3AvscO of V. alginolyticus has a high immunogenicity, and can be used as live attenuated vaccine. In addition, RPS may be affected by vaccination and infection methods. This study can provide technical support for controlling fish diseases caused by V. alginolyticus.
基金National Natural Science Foundation of China(31402344,31572656)College Student Innovation Training Program(201510566042)+1 种基金Major Program for the Fundamental Research of the Department of Education of Guangdong Province(2014GKXM046)International Cooperation Innovation Platform Project of Universities in Guangdong Province(2013gjhz0008)
文摘The two-component regulatory system plays an important role in the growth and virulence of bacteria. In this study, the phosphate transcriptional regulatory protein （KdpE） gene of Vibrio alginolyticus strain HY9901 was cloned. Sequence analysis revealed that the length ofkdpE gene is 732 bp and encodes a putative protein of 243 amino acids. The predicted molecular weight （MW） of KdpE was 27.66 kD with an estimated pI of 5.01. Using SignalP 4.0 and TM- HMM Server 2.0 software, it was predicted that the KdpE protein was located in cytoplasm. It did not contain a signal peptide or a transmembranous region. A phylogenetic tree was constructed by MEGA 5.0 software, indicating that the KdpE of V. alginolyticus bad high genetic relationship with Vibrio campbellii and Vibrio parahaemolyticus. Using SWISS-MODEL work-space, the three-dimensional structures of conserved domain REC in the KdpE was determined. These results can provide a basis for further studies on the two-component regulatory systems of V. alginolyticus.
