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

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-hly∷lacZ和回补表达LM溶血素（LLO）的重组菌株LIΔi-hly∷hly；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-hly∷lacZ和LIΔi-hly∷hly的基因序列与预期相符；LIΔi-hly∷hly、LI和LIΔi-hly∷lacZ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-hly∷lacZ在巨噬细胞内的增殖量降低，LIΔi-hly∷hly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。

Comparison of the Effects of ILO and LLO in Helping Listeria Adhere, Invade Cell and Intracellularly Multiply

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-hly∷lacZ 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.