刘思静, 刘婷, 周玉真, 等.ILO与LLO协助李斯特菌黏附、侵袭细胞及胞内增殖的比较研究.四川大学学报(医学版),2019,50(2):152-156
ILO与LLO协助李斯特菌黏附、侵袭细胞及胞内增殖的比较研究
Comparison of the Effects of ILO and LLO in Helping Listeria Adhere, Invade Cell and Intracellularly Multiply
  
中文关键词:  绵羊李斯特菌单增李斯特菌LM溶血素LI溶血素胞内增殖
英文关键词:Listeria ivanoviiListeria monocytogenesListeriolysin OIvanolysin OIntracellular multiplication
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中文摘要:
      目的研究李斯特菌溶血素的主要功能,比较两种李斯特菌——绵羊李斯特菌(Listeria ivanovii, LI)和单增李斯特菌(Listeria monocytogenes, LM)的溶血素对细菌黏附细胞等作用的强弱。方法构建含有LI i-hly基因上、下游同源序列和lacZ基因或hly基因的打靶质粒,利用基因重组技术构建LI溶血素(ILO)缺陷的LI重组菌株LIΔi-hlylacZ和回补表达LM溶血素(LLO)的重组菌株LIΔi-hlyhly;比较2株重组菌与野生LI对人肝癌HepG2细胞的黏附、侵袭能力;比较3株菌在巨噬细胞RAW264.7内的增殖能力。结果重组菌株LIΔi-hlylacZ和LIΔi-hlyhly的基因序列与预期相符;LIΔi-hlyhly、LI和LIΔi-hlylacZ对HepG2细胞的黏附率分别为(3.43±0.82)%、(3.43±1.59)% 和(3.41±1.12)%,侵袭率分别为(1.74±0.46)%、(1.22±0.75)% 和(1.39±0.46)%,差异均无统计学意义;胞内增殖实验结果表明,与野生株相比,ILO缺陷株LIΔi-hlylacZ在巨噬细胞内的增殖量降低,LIΔi-hlyhly的增殖量升高。结论LI在细胞内的增殖水平与ILO有关,ILO缺失抑制了LI在细胞内的增殖能力,LM溶血素LLO协助细菌逃离吞噬泡进入宿主细胞质的能力强于LI溶血素ILO。
英文摘要:
      ObjectiveTo study the primary function of ivanolysin O (ILO) and Listeriolysin O (LLO) and compare the effects of these two hemolysins in helping bacteria adhere, invade cell and intracellularly multiply. MethodsThe targeting plasmids carrying the upstream and downstream sequences of i-hly and lacZ gene sequence or hly gene sequence were constructed. Then two recombinant strains, the ILO deletion strain LIΔi-hlylacZ and LLO compensative expressing strain LIΔi-hly∷hly, were constructed by plasmid targeting recombinant technique. The adhesive and invasive ability of LIΔi-hly∷hly, LI and LIΔi-hly∷lacZ were evaluated in HepG2 cells, and their intracellular multiplication abilities were evaluated in RAW264.7 macrophages. ResultsGenome sequences of the recombinant strains were as expected. The adhesive rate of LIΔi-hly∷hly, LI and LIΔi-hly∷lacZ were (3.43±0.82)%, (3.43±1.59)% and (3.41±1.12)% respectively, and the invasive rate were (1.74±0.46)%, (1.22±0.75)% and (1.39±0.46)% respectively. Difference in adhesive and invasive rates showed no significance. Among three strains, LIΔi-hly∷lacZ showed the lowest intracellular proliferation rate, and LIΔi-hly∷hly possessed the highest intracellular proliferation rate in RAW264.7 macrophages. ConclusionThe intracellular multiplication ability of LI is related to ILO. Deletion of ILO induces a distinct decrease in intracellular multiplication for LI. Compared with ILO, LLO shows a stronger ability in helping the bacteria escape from the phagosome into the host cell cytosol.
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