单核细胞增生李斯特菌InlA、InlB和InlJ基因缺失株构建及对MBMEC细胞骨架与细胞紧密连接的影响

Construction of Listeria monocytogenes InlA,InlB and InlJ gene deletion strains and impact on MBMEC cytoskeleton and tight junctions

为探究InlA、InlB和InlJ对单核细胞增生李斯特菌(LM)毒力及在其侵袭MBMEC过程中对细胞骨架与细胞紧密连接的影响,本试验以致绵羊脑炎分离株LM90为亲本株,构建单基因缺失株LM90-△InlA、LM90-△InlB和LM90-△InlJ,通过LD50、溶血试验、MBMEC黏附侵袭、胞内增殖及细胞免疫荧光染色方法,研究LM缺失株和亲本株的差异。结果显示,InlA、InlB和InlJ基因缺失株的LD50分别比LM90高出101.5,101.3,101.33倍;对MBMEC的黏附率、侵袭率及胞内增殖都显著下降,且侵袭率差异显著(P<0.05),溶血性无变化;免疫荧光染色结果表明,InlB缺失株不能使MBMEC细胞骨架发生重排,InlA缺失株细胞骨架重排不明显,InlJ缺失株与亲本株则使细胞骨架完全破坏;LM90与InlA、InlB和InlJ缺失株都使MBMEC细胞间紧密连接蛋白呈不连续分布,而阴性对照则呈连续分布。结果表明,InlA、InlB和InlJ与LM毒力密切相关,且在LM侵袭MBMEC过程中,InlB影响MBMEC细胞骨架重排,InlA对其影响弱于InlB,InlJ则影响微弱或者不影响。LM可引起细胞间紧密连接改变,但InlA、InlB和InlJ在该过程中几乎不发挥作用。

In order to explore the impact of InlA,InlB and InlJ on LM virulence,MBMEC cytoskeleton and tight junction,gene deletion strains LM90-△InlA,LM90-△InlB and LM90-△InlJ were constructed based on sheep encephalitis isolates LM90.The difference of LM mutants and wild strains was studied by LD50 counting,hemolysis test,MBMEC adhesion and invasion,intracellular proliferation and cell immunofluorescence staining.The results showed that LD50 of InlA,InlB and InlJ gene deletion strains were 101.5,101.3 and 101.33 times higher than that of LM90,respectively.The adhesion,invasion to MBMEC and proliferation in MBMEC of deletion strains were declined significantly and the difference of invasion rate was significant（P〈0.05）,but no change of hemolysis activity.Immunofluorescence staining showed that there was no MBMEC cytoskeletal rearrangement in InlB deletion mutant,no obvious cytoskeleton rearrangement in InlA mutant and complete cytoskeleton rearrangement in the InlJ deletion mutant and wild-type strain.There was a discontinuous distribution of MBMEC intercellular tight junction protein induced by LM90 and InlA,InlB and InlJ deletion strains,while a continuous distribution was showed in the negative control.The results showed that InlA,InlB and InlJ were closely related with LM virulence and affectedMBMEC cytoskeleton rearrangement more or less during the process of LM invasion.LM could cause the change of intercellular tight junctions,but InlA,InlB and InlJ did not play any role in this process.