肠炎沙门菌icdA基因缺失株的构建及重要特性分析
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摘要
为研究肠炎沙门菌异柠檬酸脱氢酶(IDH)的功能,本研究利用λ-Red重组技术构建了肠炎沙门菌IDH编码基因icdA缺失突变株c50336ΔicdA,对其进行生长和生化特性检测,并通过体外模拟应激试验、药敏试验、生物被膜检测、抗蛋清杀伤、胞内存活和动物感染实验,分析icdA基因在肠炎沙门菌致病机制中的重要作用。研究结果显示,icdA基因缺失不影响肠炎沙门菌的生长特性和生化特性,在热应激(42℃)、酸应激(pH3.5)和高渗应激(NaCl 2.4 mol/L)条件下其存活率也未出现显著变化,但icdA基因缺失能够显著降低肠炎沙门菌对碱、低渗透压和过氧化氢应激的抵御能力,降低肠炎沙门菌对多粘菌素B的耐药性和生物被膜的形成能力;同时,该基因缺失能够降低肠炎沙门菌对蛋清杀伤的防御能力、在巨噬细胞内的存活能力和其对小鼠的毒力。研究结果表明icdA基因参与肠炎沙门菌的抗应激能力、耐药性、生物被膜形成和抗蛋清杀伤能力,从而影响细菌的毒力。本研究为肠炎沙门菌疫苗的研究奠定了重要基础。
To investigate the roles of isocitrate dehydrogenase of Salmonella enterica, λ-Red recombination was used for knockout of the icdA gene, and stress test, drug sensitivity test, biofilm detection, anti-killing against egg albumen, intracellular survival, and animal infection experiments were performed to analyze its contribution to pathogenic mechanism of S.enterica. The results indicated that, the icdA-deficient mutant showed significantly weakened defence against alkaline, osmotic, and H_2O_2 oxidative stress; it also displayed reduced tolerance to polymyxin B and biofilm formation. Additionally, deletion of icdA led to impaired defence against egg albumen, intracellular survival ability, and virulence to mouse, compared with the wild type strain.Our results revealed icdA contributed to the anti-stress ability, drug resistance, and bacterial virulence and therefore layed an important foundation for the vaccine development of S.enterica.
To investigate the roles of isocitrate dehydrogenase of Salmonella enterica, λ-Red recombination was used for knockout of the icdA gene, and stress test, drug sensitivity test, biofilm detection, anti-killing against egg albumen, intracellular survival, and animal infection experiments were performed to analyze its contribution to pathogenic mechanism of S.enterica. The results indicated that, the icdA-deficient mutant showed significantly weakened defence against alkaline, osmotic, and H_2O_2 oxidative stress; it also displayed reduced tolerance to polymyxin B and biofilm formation. Additionally, deletion of icdA led to impaired defence against egg albumen, intracellular survival ability, and virulence to mouse, compared with the wild type strain.Our results revealed icdA contributed to the anti-stress ability, drug resistance, and bacterial virulence and therefore layed an important foundation for the vaccine development of S.enterica.
引文
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[4]刘谢,柯碧霞,何冬梅,等.2007~2013年广东省肠炎沙门菌耐药情况分析[J].中华疾病控制杂志,2000,1523(2-3):196-205.
[5]Srikumar S,Kroger C,Hebrard M,et al.RNA-seq brings new insights to the intra-macrophage transcriptome of Salmonella Typhimurium[J].PLo S Pathog,2015,11(11):e1005262.
[6]Tang Xiao-qian,Liu Fu-guo,Sheng Xiu-zhen,et al.Recombinant NADP-dependent isocitrate dehydrogenase of Edwardsiella tarda induces both Th1 and Th2 type immune responses and evokes protective efficacy against edwardsiellosis[J].Vaccine,2018,36(17):2337-2345.
[7]Banerjee S,Nandyala A,Podili R,et al.Mycobacterium tuberculosis(Mtb)isocitrate dehydrogenases show strong B cell response and distinguish vaccinated controls from TB patients[J].Proc Natl Acad Sci USA,2004,101(34):12652-12657.
[8]Hussain M A,Naveed S A,Sechi L A,et al.Isocitrate dehydrogenase of Helicobacter pylori potentially induces humoral immune response in subjects with peptic ulcer disease and gastritis[J].PLo S One,2008,3(1):e1481.
[9]姚丰华,董立伟,张钰,等.肠炎沙门菌hcp基因对生物被膜形成的影响分析[J].中国预防兽医学报,2014,36(07):534-537.
[10]刘松艳,周薇,司微,等.肠炎沙门菌延伸因子EF-Tu缺失株对环境压力的应答变化[J].中国预防兽医学报,2016,38(01):31-34.
[11]Namakchian M,Kassler K,Sticht H,et al.Structure-based functional analysis of effector protein SifA in living cells reveals motifs important for Salmonella intracellular proliferation[J].Int JMed Microbiol,2017,308(1):84-96.
[12]Heimlich D R,Harrison A,Mason K M.Host antimicrobial peptides in bacterial homeostasis and pathogenesis of disease[J].Antibiotics(Basel),2014,3(4):645-676.
[13]Andersson D I,Hughes D,Kubicek-Sutherland J Z.Mechanisms and consequences of bacterial resistance to antimicrobial peptides[J].Drug Resist Updat,2016,26(5):43-57.
[14]Shah D H,Zhou Xiao-hui,Kim H Y,et al.Transposon mutagenesis of Salmonella enterica serovar enteritidis identifies genes that contribute to invasiveness in human and chicken cells and survival in egg albumen[J].Infect Immun,201,80(12):4203-4215.
[15]Crabb H K,Gilkerson J R,Browning G F.Does only the age of the hen matter in Salmonella enterica contamination of eggs?[J].Food Microbiol,2019,77:1-9.
[16]Abudabos A M,Hussein E,Ali M H,et al.The effect of some natural alternative to antibiotics on growth and changes in intestinal histology in broiler exposed to Salmonella challenge[J].Poult Sci,2018,10(4):1-21.