脂磷壁酸合成酶ltaS基因缺失对产单核细胞李氏杆菌致病性的影响

Effect of Deletion of the Lipoteichoic Acid Synthase ltaS Gene on the Pathogenicity of Listeria monocytogenes

本文旨在构建脂磷壁酸合成酶ltaS基因缺失株,探讨ltaS基因对产单核细胞李氏杆菌致病力的影响。本研究利用同源重组技术构建产单核细胞李氏杆菌ltaS基因缺失株,通过生物学特性试验探究ltaS基因缺失株对产单核细胞李氏杆菌生长能力和菌体形态的影响,Western blot试验探究亲本株EGDe-prfA、缺失株ΔltaS对InlA和InlB在细菌表面的锚定情况;使用鸡胚成纤维DF-1细胞进行细胞黏附侵袭试验、小鼠RAW264.7细胞进行吞噬试验与巨噬细胞内增殖试验测定ltaS基因对产单核细胞李氏杆菌侵染能力的影响;小鼠脏器细菌载量和小鼠存活试验评估ltaS基因缺失对产单核细胞李氏杆菌致病性的影响。结果显示:利用同源重组技术成功构建产单核细胞李氏杆菌ltaS基因缺失株,生长曲线试验和光学显微镜观察发现ltaS基因不影响产单核细胞李氏杆菌在BHI中正常生长和细菌形态,通过Western blot试验验证ltaS基因缺失导致细菌表面毒力的InlA和InlB锚定量降低。细胞黏附侵袭试验结果表明,ΔltaS对DF-1的黏附率极显著低于亲本株EGDe-prfA(P<0.01)并且侵袭率也极显著低于亲本株EGDe-prfA(P<0.01);吞噬试验结果显示,小鼠巨噬细胞RAW264.7对ΔltaS的吞噬率与亲本株EGDe-prfA相比无显著性差异(P>0.05);而在增殖试验中,ΔltaS在RAW264.7中增殖量与亲本株EGDe-prfA存在显著差异(P<0.05)。小鼠致病力试验结果显示,经ΔltaS感染的小鼠的肝、脾内细菌载量均显著低于EGDe-prfA(P<0.01)并且EGDe-prfA感染后的小鼠96 h内全部死亡,而缺失株ΔltaS感染的小鼠存活率为80%,测定出亲本株EGDe-prfA半数致死量LD 50为1.7×104 CFU,缺失株ΔltaS半数致死量LD 50为7.49×106 CFU。综上所述,ltaS基因缺失显著减少表面InlA和InlB的锚定量,减弱产单核细胞李氏杆菌对DF-1细胞的黏附侵袭能力与RAW264.7内细菌增殖能力,并且降低对小鼠的致病性。

The aim of this paper was to construct a lipoteichoic acid synthase ltaS gene deletion strain to investigate the effect of the ltaS gene on the pathogenicity of Listeria monocytogenes.In this study,we used homologous recombination to construct a strain of Listeria monocytogenes with a deletion of the ltaS gene.The effect of the ltaS gene on the growth and bacterial morphology of Listeria monocytogenes was explored by growth curves assay and observation of bacterial morphology.The anchoring of InlA and InlB on the bacterial surface of wild strain EGDe-prfA and deletion strainΔltaS were investigated by Western blot assay.The effect of ltaS gene on the ability of Listeria monocytogene s to infest using DF-1 for cell adhesion invasion assay and mice RAW264.7 for phagocytosis assay with multiplication assay was determined.Effect of ltaS gene deletion on pathogenicity of Listeria monocytogenes as assessed by mice organ bacterial load test and survival assay.The results showed:A strain of Listeria monocytogenes with a deletion of the ltaS gene was successfully constructed by homologous recombination technology.Growth curve assay and light microscopic observation revealed that the ltaS gene did not affect normal growth in BHI and bacterial morphology of Listeria monocytogenes.Deletion of the ltaS gene resulted in a significant reduction of the bacterial surface virulence factors InlA and InlB anchor quantification,as confirmed by Western blot analysis.The results of the cell adhesion rate and invasion rate assay showed that the adhesion rate and the invasion rate ofΔltaS to DF-1 was highly significantly lower than that of the parental strain EGDe-prfA(P<0.01).Phagocytosis assay revealed that the phagocytosis ofΔltaS by RAW264.7 was not significantly different from that of the parental strain EGDe-prfA(P>0.05),the amount ofΔltaS multiply in RAW264.7 was significantly different from that of the EGDe-prfA(P<0.05).The results of the mice pathogenicity virulence test showed that the bacterial load in the liver and spleen of mice infected byΔltaS was significantly lower than that of EGDe-prfA(P<0.01).And all mice infected with EGDe-prfA died within 96 h,while the survival rate of mice infected with theΔltaS was 80%.The LD 50 of parental strain EGDe-prfA was 1.7×104 CFU and the LD 50 of deletion strainΔltaS was 7.49×106 CFU.In summary,ltaS gene deletion significantly reduced the anchoring quantities of surface InlA and InlB and diminished the ability of infection ability,pathogenicity of Listeria monocytogenes.