单增李斯特菌内化素家族蛋白Lmo1136生物学特性的研究

Study on the biological characteristics on the internalins lmo1136 of Listeria monocytogenes

单增李斯特菌内化素家族蛋白通常在该菌感染及致病中起重要作用,为探究其内化素家族蛋白Lmo1136在李斯特菌感染致病机制中的作用,本研究以单增李斯特菌EGD-e基因组为模板,经PCR扩增lmo1136基因的上下游基因片段,经重叠延伸PCR将同源臂连接后与温敏型穿梭质粒pKSV7连接构建重组质粒,利用同源重组技术使重组质粒与EGD-e同源片段发生交换,得到缺失株Δlmo1136并经PCR和测序鉴定;以单增李斯特菌EGD-e基因组为模板,PCR扩增lmo1136基因与启动子片段,并与整合型穿梭质粒pIMK2连接后电转化Δlmo1136感受态细胞,得到回补株CΔlmo1136并经PCR和测序鉴定。将单增李斯特菌EGD-e、Δlmo1136和CΔlmo1136分别接种96孔板,37℃培养12h后测定各菌株OD600nm值,并根据OD600nm值绘制各菌株的生长曲线。结果显示各菌株生长速度无显著差异(P>0.05)。将各菌株以感染复数(MOI)10感染人结直肠腺癌细胞(Caco-2细胞)后30min和90min时收集细胞裂解物,接种于BHI培养基进行单菌落计数,结果显示,与EGD-e相比,Δlmo1136的黏附性无显著差异(P>0.05),侵袭力显著下降(P<0.05),而CΔlmo1136的黏附性与侵袭力均无显著差异(P>0.05)。将各菌株均以MOI0.2感染小鼠巨噬细胞(RAW246.7细胞)后2h、5h和8h时收集细胞裂解物接种于BHI培养基,单菌落计数,结果显示,与EGD-e相比,Δlmo1136和CΔlmo1136菌落数均无显著差异(P>0.05)。将各菌株均以1×10^(6) cfu/只经腹腔注射小鼠,48 h后取小鼠脾脏与肝脏研磨,接种于BHI培养基进行单菌落计数,结果显示,与EGD-e相比,Δlmo1136的菌落数显著下降(P<0.05),CΔlmo1136的菌落数无显著差异(P>0.05)。将各菌株均以1×10^(6)cfu/只经腹腔注射小鼠,每隔24h记录小鼠存活情况,结果显示,与EGD-e相比,Δlmo1136组小鼠存活率显著升高(P<0.05),CΔlmo1136组小鼠的存活率无显著差异(P>0.05)。上述结果首次表明lmo1136缺失后,单增李斯特菌的生长能力、细胞黏附性和胞内增殖能力均无显著变化,但对细胞侵袭力、小鼠脏器定植能力显著降低,小鼠存活率显著升高,阐明了内化素家族蛋白Lmo1136在单增李斯特菌细胞侵袭、脏器定植和对小鼠致病机制中发挥重要作用,本研究上述结果为进一步解析Lmo1136介导细菌的感染机制奠定了重要基础。

Listeria monocytogenes internalins usually play important roles in bacterial infections and its pathogenesis.In order to investigate the role of the internalin family protein Lmo1136 in the pathogenesis of Listeria monocytogenes infection,in this study,Listeria monocytogenes EGD-e genome was used as a template,and the upstream and downstream gene fragments of the lmo1136 gene was amplified by PCR.And a recombinant plasmid was constructed by connecting the homologous arm to the temperature-sensitive shuttle plasmid pKSV7 using overlap extension PCR.The resulting recombinant plasmid was exchanged with the homologous fragment of EGD-e through homologous recombination,and the deletion strainΔlmo1136 was obtained.The Listeria monocytogenes EGD-e genome was used as a template,the lmo1136 gene and promoter fragment were amplified with PCR.The obtained amplicons were ligated with the integrative shuttle plasmid pIMK2 before electrotransferring to the competent cells ofΔlmo1136,and then the complementary strain CΔlmo1136 was obtained.Listeria monocytogenes EGD-e,Δlmo1136 and CΔlmo1136 were inoculated into 96-well plates and cultured at 37℃for consecutive 12 hours.The OD600nm values of each strain were determined,and the results showed that there was no significant difference in the growth rates of the strains(P>0.05).Human colorectal adenocarcinoma cells(Caco-2 cells)was infected with each strain for 30 minutes and 90 minutes at the multiplicity of infection(MOI)10,cell lysis was collected and the lysates were plated on BHI medium for single colony counting.The results showed that compared to EGD-e,there was no significant difference for the adhesion ofΔlmo1136(P>0.05),but there was significantly decreased(P<0.05)for the invasion ofΔlmo1136,in contrast for both the adhesion and invasion of CΔlmo1136,there was no significant difference(P>0.05).The mouse macrophages(RAW246.7 cells)were infected with each strain at an MOI of 0.2 for 2 hours,5 hours and 8 hours,cell lysis was collected and the lysates were plated on BHI medium for single colony counting.The results showed there was no significant difference in the intracellular proliferative capacity of bothΔlmo1136 and CΔlmo1136(P>0.05)compared to EGD-e.Mice were injected intraperitoneally with each strain at 1×106 cfu/mouse.After 48 hours,mice were enthanized,and their spleens and livers were harvested,homogenized,and plated on BHI medium.The results showed thatΔlmo1136 had a significantly reduced ability to colonize the organs of mice compared to EGD-e(P<0.05).However,there was no significant difference in the organ colonization ability of CΔlmo1136(P>0.05).Mice were injected with each strain at 1×10^(6) cfu/mouse,and the survival rates of mice were recorded at 24 hours intervals.The results showed that the survival rate of mice in theΔlmo1136 group was significantly higher than that of mice in the EGDe group(P<0.05),whereas there was no significant difference in the CΔlmo1136 group(P>0.05).In summary,the results indicated that after the deletion of lmo1136,there was no significantly change for the growth ability,cell adhesion and intracellular proliferation ability of Listeria monocytogenes,but there was significantly reduced for the invasion,mouse organs colonise ability,in addition,the survival rate of mice was significantly increase.It is elucidated that Lmo1136 was played an important role in cell invasion,organ colonization and survival rate after bacterial infection in mice,providing a crucial foundation for further analysis of Lmo1136-mediated bacterial infection mechanism.