| 136 | 0 | 126 |
| 下载次数 | 被引频次 | 阅读次数 |
溶藻弧菌(Vibrio alginolyticus)引发的弧菌病对水产养殖行业造成了重大威胁，为研究其耐药机制与蛋白翻译后修饰(post-translational modifications, PTMs)的关系，本研究鉴定了溶藻弧菌磷酸丙糖异构酶(triosephosphate isomerase, TPI)的N-ε-赖氨酸乙酰化、琥珀酰化、丙二酰化修饰，发现TPI蛋白PTMs及其表达量在溶藻弧菌野生株和耐药株间的差异。通过实时定量PCR(real-time quantitative PCR, RT-qPCR)和蛋白质免疫印迹法(Western blot, WB)测定溶藻弧菌野生株(HY9901)、氟苯尼考耐药株(FBNK)和恩诺沙星耐药株(ENSX)的tpiA基因相对表达量和蛋白表达量。通过WB鉴定TPI天然蛋白的N-ε-赖氨酸乙酰化、琥珀酰化、丙二酰化修饰并分析其与耐药的关系。RT-qPCR的结果显示，与HY9901相比，在FBNK中tpiA基因的mRNA表达量下调(P<0.01), ENSX中的则上调(P<0.01)。WB结果也证明，与HY9901相比，TPI蛋白在FBNK中的表达量下调，在ENSX中的表达量上调。进一步研究发现，HY9901、 ENSX和FBNK的TPI天然蛋白均具有乙酰化、琥珀酰化、丙二酰化修饰。与HY9901相比，ENSX、 FBNK中的TPI蛋白乙酰化水平降低但琥珀酰化水平升高；ENSX的TPI蛋白丙二酰化修饰水平升高，而FBNK的修饰水平降低。溶藻弧菌野生株和耐药菌株中tpiA的表达量与耐药相关，TPI天然蛋白的N-ε-赖氨酸乙酰化、琥珀酰化、丙二酰化修饰水平存在差异，表明这三种N-ε-赖氨酸修饰与溶藻弧菌的耐药有关。
Abstract：Vibriosis caused by Vibrio alginolyticus poses a major threat to the aquaculture industry. To explore the relationship between drug resistance mechanisms and protein post-translational modifications(PTMs), this study identified the N-ε-lysine acetylation, succinylation, and malonylation modifications of triosephosphate isomerase(TPI) in Vibrio alginolyticus, and revealed differences in TPI protein PTMs and expression levels between wild-type and drug-resistant strains of Vibrio alginolyticus. The relative expression levels of the tpiA gene and protein expression levels in the Vibrio alginolyticus wild-type strain HY9901, enrofloxacin-resistant strain(ENSX), and florfenicol-resistant strain(FBNK) were determined using real-time quantitative PCR(RT-qPCR) and Western blot(WB). WB was used to identify the N-ε-lysine acetylation, succinylation, and malonylation modifications of the TPI protein and their respective relationships with drug resistance. RT-qPCR results showed that compared with that in the Vibrio alginolyticus wild-type strain, the mRNA expression of the tpiA gene was down-regulated in FBNK(P<0.01) and up-regulated in ENSX(P<0.01). WB results also confirmed that the expression level of TPI protein was down-regulated in FBNK and up-regulated in ENSX compared with HY9901. The modifications of TPI protein in FBNK, ENSX, and HY9901 were identified by WB, which showed that TPI was modified by acetylation, succinylation, and malonylation. Compared to HY9901, the acetylation levels of TPI protein were lower, whereas the succinylation levels were higher in both FBNK and ENSX. Notably, compared with HY9901, ENSX exhibited the highest level of malonylation modification, while FBNK displayed the lowest. The mRNA expression level of tpiA and the native protein expression level of TPI were associated with drug resistance in both wild-type and drug-resistant strains of Vibrio alginolyticus. Additionally, variations in the acetylation, succinylation, and malonylation modification levels of TPI native protein were observed between wild-type and drug-resistant strains, which suggests that these three N-ε-lysine modifications are involved in the drug resistance of Vibrio alginolyticus.
黄子威，汪志文，黎源，等，2021.罗非鱼SRA4基因重组表达、 亚细胞定位及组织分布.广东海洋大学学报，41(5):19-27.[HUANG Z W,WANG Z W,LI Y,et al.,2021.Molecular cloning,subcellular localization and expression characterization of SRA4 gene in Nile Tilapia(Oreochromis niloticus).Journal of Guangdong Ocean University,41(5):19-27.]
教郁，高维凡，胡彩光，2013.大肠杆菌耐药性研究进展.现代畜牧兽医，(5):53-58.[JIAO Y,GAO W F,HU C G,2013.The research progress on mechanism of drug resis-tance of Escherichia coli.Modern Journal of Animal Husbandry and Veterinary Medicine,(5):53-58.]
赖福斌，2021.赖氨酸乙酰化修饰对大肠杆菌耐药性的调控机制研究，硕士学位论文.广州：暨南大学.(LAI F B,2021.Lysine acetylation regulates antibiotic resistance in Escherichia coli,Thesis for M.S.Guangzhou:Jinan University.)
李钰芳，杨昆，顾韦维，等，2021.乳源抗菌肽BCp12对金黄色葡萄球菌多靶点抑菌机制.食品科学，42(23):34-41.[LI Y F,YANG K,GU W W,et al.,2021.Multi-target antibacterial mechanism of milk-derived antimicrobial peptide BCp12 against Staphylococcus aureus.Food Science,42(23):34-41.]
吕斌娜，梁文星，2015.蛋白质乙酰化修饰研究进展.生物技术通报，31(4):166-174.[Lü B N,LIANG W X,2015.The research progress of protein acetylation.Biotechnology Bulletin,31(4):166-174.]
卢文卿，车晓芳，郑春雷，等，2019.赖氨酸琥珀酰化修饰的研究进展.现代肿瘤医学，27(20):3720-3724.[LU W Q,CHE X F,ZHENG C L,et al.,2019.Research progress on lysine succinylation modification.Journal of Modern Oncology,27(20):3720-3724.]
沈佳佳，闻浩，2016.蛋白质赖氨酸琥珀酰化修饰研究进展.医学研究生学报，29(3):332-336.[SHEN J J,WEN H,2016.The research progress of protein lysine succinylation.Journal of Medical Postgraduates,29(3):332-336.]
唐禄，董丽平，尹茉莉，等，2021.成纤维细胞生长因子20单克隆抗体的制备及鉴定.生物技术通报，37(10):179-185.[TANG L,DONG L P,YIN M L,et al.,2021.Pre-paration and identification of a novel FGF20 monoclonal antibody.Biotechnology Bulletin,37(10):179-185.]
王旭东，范晨龙，丁燏，2022.溶藻弧菌磷酸甲基嘧啶合酶(ThiC)的原核表达及其乙酰化修饰鉴定.大连海洋大学学报，37(3):435-440.[WANG X D,FAN C L,DING Y,2022.Prokaryotic expression and acetylation identification of phosphomethylpyrimidine synthase (ThiC) in Vibrio alginolyticus.Journal of Dalian Ocean University,37(3):435-440.]
徐洲，范晨龙，丁燏，2020.溶藻弧菌PepA蛋白原核表达载体的构建及其乙酰化鉴定.生物技术通报，36(12):75-81.[XU Z,FAN C L,DING Y,2020.Construction of prokaryotic expression vector of PepA protein of Vibrio alginolyticus and identification of its acetylation.Biotechnology Bulletin,36(12):75-81.]
曾福源，2022.溶藻弧菌PEPCK的赖氨酸琥珀酰化修饰对耐药性的影响研究，硕士学位论文.湛江：广东海洋大学.(ZENG F Y,2022.Effects of lysine succinylation modification of Vibrio alginolyticus PEPCK on drug resistance,Thesis for M.S.Zhanjiang:Guangdong Ocean University.)
曾福源，苏泽辉，周诗慧，等，2021.溶藻弧菌PEPCK蛋白原核表达及其乙酰化、 琥珀酰化修饰的鉴定.生物技术通报，37(5):84-91.[ZENG F Y,SU Z H,ZHOU S H,et al.,2021.Prokaryotic expression of the PEPCK protein of Vibrio alginolyticus and identification of its acetylation and succinylation.Biotechnology Bulletin,37(5):84-91.]
张瑞良，2019.基于蛋白质组学与乙酰化修饰组学对沙门菌耐药机制研究，硕士学位论文.合肥：安徽农业大学.(ZHANG R L,2019.Study on the resistance mechanism of Salmonella based on proteomics and protein acetylation modification,Thesis for M.S.Hefei:Anhui Agricultural University.)
郑玉琦，2011.溶藻弧菌耐四种抗生素的蛋白质组学研究，硕士学位论文.湛江：广东海洋大学.(ZHENG Y Q,2011.Proteomic analysis of Vibrio alginolyticus in response to four types of antibiotics,Thesis for M.S.Zhanjiang:Guangdong Ocean University,2011.)
周诗慧，2021.溶藻弧菌Ⅲ型分泌系统调控蛋白TyeA的功能研究，硕士学位论文.湛江：广东海洋大学.(ZHOU S H,2021.Characterization of the regulatory protein TyeA involved in type Ⅲ secretion system in marine fish pathogen Vibrio alginolyticus,Thesis for M.S.Zhanjiang:Guangdong Ocean University.)
BI J,GUO Q L,ZHOU Z Y,et al.,2022.Malonylome analysis uncovers the association of lysine malonylation with metabolism and acidic stress in pathogenic Mycobacterium tuberculosis.Microbiol.Res.,265:127209.
BULTMANN C A,STEI? J O,LANGNER C,et al.,2016.Complicated sea urchin-induced wound infection caused by Vibrio alginolyticus and Staphylococcus lugdunensis in a 14-year-old boy.JMM Case Rep.,3(6):e005074.
CAI S H,WU Z H,JIAN J C,et al.,2007.Cloning and expression of the gene encoding an extracellular alkaline serine protease from Vibrio alginolyticus strain HY9901,the causative agent of vibriosis in Lutjanus erythopterus (Bloch).J.Fish Dis.,30(8):493-500.
CHEN H L,XU H,POTASH S,et al.,2017.Mild metabolic perturbations alter succinylation of mitochondrial proteins.J.Neurosci.Res.,95(11):2244-2252.
CHENG Z X,YANG M J,PENG B,et al.,2018.The depressed central carbon and energy metabolisms is associated to the acquisition of levofloxacin resistance in Vibrio alginolyticus.J.Proteomics,181:83-91.
DONE H Y,VENKATESAN A K,HALDEN R U,2015.Does the recent growth of aquaculture create antibiotic resistance threats different from those associated with land animal production in agriculture?AAPS J.,17(3):513-524.
DU Z G,LIU X J,CHEN T,et al.,2018.Targeting a Sirt5-positive subpopulation overcomes multidrug resistance in wild-type Kras colorectal carcinomas.Cell Rep.,22(10):2677-2689.
GAVIARD C,COSETTE P,JOUENNE T,et al.,2019.LasB and CbpD virulence factors of Pseudomonas aeruginosa carry multiple post-translational modifications on their lysine residues.J.Proteome Res.,18(3):923-933.
GIBSON G E,XU H,CHEN H L,et al.,2015.Alpha-ketoglu-tarate dehydrogenase complex-dependent succinylation of proteins in neurons and neuronal cell lines.J.Neurochem.,134(1):86-96.
GUAN K L,XIONG Y,2011.Regulation of intermediary meta-bolism by protein acetylation.Trends Biochem.Sci.,36(2):108-116.
KANG C H,SHIN Y,JANG S,et al.,2016.Antimicrobial susceptibility of Vibrio alginolyticus isolated from oyster in Korea.Environ.Sci.Pollut.Res.Int.,23(20):21106-21112.
LI W X,YAO Z J,SUN L N,et al.,2016.Proteomics analysis reveals a potential antibiotic cocktail therapy strategy for Aeromonas hydrophila infection in biofilm.J.Proteome Res.,15(6):1810-1820.
LI X C,XIANG Z Y,XU X M,et al.,2009.Endophthalmitis caused by Vibrio alginolyticus.J.Clin.Microbiol.,47(10):3379-3381.
PANG H Y,CHEN L M,HOARE R,et al.,2016.Identification of DLD,by immunoproteomic analysis and evaluation as a potential vaccine antigen against three Vibrio species in Epinephelus coioides.Vaccine,34(9):1225-1231.
PANG H Y,ZHANG W J,LIN X L,et al.,2024.Vibrio alginolyticus PEPCK mediates florfenicol resistance through lysine succinylation modification.J.Proteome Res.,23(7):2397-2407.
PANG H Y,ZHANG X Z,WU Z H,et al.,2013.Identification of novel immunogenic proteins of Vibrio alginolyticus by immunoproteomic methodologies.Aquac.Res.,44(3):472-484.
PAPANICOLAOU K N,O’ROURKE B,FOSTER D B,2014.Metabolism leaves its mark on the powerhouse:recent progress in post-translational modifications of lysine in mitochondria.Front.Physiol.,5:301.
SGANGA G,COZZA V,SPANU T,et al.,2009.Global climate change and wound care:case study of an off-season Vibrio alginolyticus infection in a healthy man.Ostomy Wound Manage.,55(4):60-62.
XIA Y S,WANG D,PAN X L,et al.,2020.TpiA is a key metabolic enzyme that affects virulence and resistance to aminoglycoside antibiotics through CrcZ in Pseudomonas aeruginosa.mBio,11:e02079-19.
XIE L X,LIU W,LI Q M,et al.,2015a.First succinyl-proteome profiling of extensively drug-resistant Mycobacte-rium tuberculosis revealed involvement of succinylation in cellular physiology.J.Proteome Res.,14(1):107-119.
XIE L X,WANG X B,ZENG J,et al.,2015b.Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis.Int.J.Biochem.Cell Biol.,59:193-202.
XIONG W G,SUN Y X,ZHANG T,et al.,2015.Antibiotics,antibiotic resistance genes,and bacterial community composition in fresh water aquaculture environment in China.Microb.Ecol.,70(2):425-432.
ZENG F Y,PANG H Y,CHEN Y,et al.,2020.First succinylome profiling of Vibrio alginolyticus reveals key role of lysine succinylation in cellular metabolism and virulence.Front.Cell.Infect.Microbiol.,10:626574.
ZHANG Y X,BHARATHI S S,RARDIN M J,et al.,2017.Lysine desuccinylase SIRT5 binds to cardiolipin and regulates the electron transport chain.J.Biol.Chem.,292(24):10239-10249.
ZHANG Z H,TAN M J,XIE Z Y,et al.,2011.Identification of lysine succinylation as a new post-translational modification.Nat.Chem.Biol.,7(1):58-63.
ZUO L,CHEN L C,YANG S,et al.,2022.Cloning and bioinformatics analysis of TpiA gene of Vibrio alginolyticus HY9901.Asian Agricultural Research,14(5):22-26.
基本信息:
DOI：10.13417/j.gab.044.001136
中图分类号:S941.42
引用信息:
[1]张喻嘉,王玉彤,肖星,等.溶藻弧菌TPI蛋白的三种N-ε-赖氨酸修饰与耐药的关联性分析[J].基因组学与应用生物学,2025,44(11):1136-1146.DOI:10.13417/j.gab.044.001136.
基金信息:
广东省自然科学基金项目(2025A1515011061); 广东海洋大学本科生创新团队研究项目(CCTD201802); 广东省研究生教育创新计划项目(粤教研函[2022]1号)共同资助
2025-10-14
2025-10-14
2025-10-14