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
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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-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。
Objective To 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. Methods The 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. Results Genome 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. Conclusion The 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.
Objective To 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. Methods The 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. Results Genome 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. Conclusion The 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.
引文
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[8] 周梦莹, 蒲启康, 任晨艳, 等. 致病性李斯特菌毒力因子蛋白组学及基因组学研究进展. 现代预防医学,2015,42(4):694-697.
[9] FABRIZIA S, FREDERIC B, ANNA S, et al. Listeria monocytogenestransiently alters mitochondrial dynamics during infection. Proc Natl Acad Sci U S A,2011,108(9):3612-3617.
[10] FREHEL C, LETY M, AUTRET N, ET AL. Capacity of ivanolysin O to replace listeriolysin O in phagosomal escape and in vivo survival of Listeria monocytogenes. Microbiology,2003,149(3):611-620.
[11] HERNáNDEZFLORES KG, VIVANCOCID H. Biological effects of listeriolysin O: implications for vaccination. Biomed Res Int, 2015, 2015:360741[ 2018-07-22]. https://www.hindawi.com/journals/bmri/2015/360741/. doi: 10.1155/2015/360741.
[2] LE DT, DUBENKSY TW, BROCKSTEDT DG. Clinical development of Listeria monocytogenes-based immunotherapies. Semin Oncol,2012,39(3):311-322.
[3] FABRIZIA S, CRISTEL A, PASCALE C. Cell biology and immunology of Listeria monocytogenes infections: novel insights. Immunol Rev,2011,240(1):160-184.
[4] 刘思静, 蒲启康, 蒋明娟. 李斯特菌溶血素在疫苗研制中的应用进展. 现代预防医学,2016,43(9):699-1701.
[5] LIU D. Molecular approaches to the identification of pathogenic and nonpathogenic Listeriae.Microbiol Insights,2013,6(6):59-69.
[6] LIN Q, ZHOU M, XU Z, et al. Construction of two Listeria ivanoviia ttenuated strains expressing Mycobacterium tuberculosis antigens for TB vaccine purposes. J Biotechnol, 2015, 196-197:20-26[ 2018-07-22]. https://www.sciencedirect.com/science/article/pii/S01681 65615000176 via%3Dihub. doi: 10.1016/j.jbiotec.2015.01.008.
[7] ZHOU M, JIANG M, REN C, et al. Listeria ivanovii infection in mice: restricted to the liver and lung with limited replication in the spleen. Front Microbiol,2016,7:790[2018-07-22].https://www.frontiersin.org/articles/10.3389/fmicb.2016.00790/full. doi: 10.3389/fmicb.2016.00790.
[8] 周梦莹, 蒲启康, 任晨艳, 等. 致病性李斯特菌毒力因子蛋白组学及基因组学研究进展. 现代预防医学,2015,42(4):694-697.
[9] FABRIZIA S, FREDERIC B, ANNA S, et al. Listeria monocytogenestransiently alters mitochondrial dynamics during infection. Proc Natl Acad Sci U S A,2011,108(9):3612-3617.
[10] FREHEL C, LETY M, AUTRET N, ET AL. Capacity of ivanolysin O to replace listeriolysin O in phagosomal escape and in vivo survival of Listeria monocytogenes. Microbiology,2003,149(3):611-620.
[11] HERNáNDEZFLORES KG, VIVANCOCID H. Biological effects of listeriolysin O: implications for vaccination. Biomed Res Int, 2015, 2015:360741[ 2018-07-22]. https://www.hindawi.com/journals/bmri/2015/360741/. doi: 10.1155/2015/360741.