单增李斯特菌srtA基因缺失株的构建及srtA基因对其毒力的影响研究

为了研究srtA 基因对单增李斯特菌LM90SB2毒力的影响,本研究利用同源重组技术构建了LM90SB2 srtA 基因缺失株LM90SB2-ΔsrtA,比较分析了亲本株LM90SB2与缺失株LM90SB2-ΔsrtA对MBMEC、HBMEC、RAW264.7、SIEC 4种细胞系的粘附、侵袭、胞内增值能力及LM90SB2和LM90SB2-ΔsrtA 感染小鼠后,小鼠的LD50及肝脏、脾脏、脑载菌量变化差异。结果显示,本研究成功构建了srtA 基因缺失株;与亲本株LM90SB2比较,缺失株LM90SB2-ΔsrtA 对RAW264.7和SIEC的粘附率、侵袭率及胞内细菌数量均下降,且差异具有显著统计学意义(p <0.05);小鼠 LD50降低了21.38倍,肝脏、脾脏载菌量降低,差异具有显著统计学意义(p <0.05)。本研究结果表明,srtA 基因对单增李斯特菌LM90SB2的毒力具有关键作用,参与粘附、侵袭BMEC,该研究结果为进一步阐明单增李斯特菌毒力因子的致病机制提供理论依据。

CONSTRUCTION AND VIRULENCE EVALUATION OF THE SRTA GENE DELETION STRAIN OF LISTERIA MONOCYTOGENES

The srtA gene of a clinical isolate of Listeria monocytogenes isolated strain LM90SB2 was deleted by homologous recombination technology in order to evaluate its effect on the virulence.Different cells (MBMEC,HBMEC,RAW264.7 and SIEC) were used to examine adhesion,invasion and intracellular growth of the deletion strain LM90SB2-ΔsrtA.Mice were intraperitoneally injected with the parent strain LM90SB2 and deletion strain LM90SB2-ΔsrtA and LD50 and bacteria loads in livers,spleens and brains were measured.Compared with the parental strains LM90SB2,LM90SB2-ΔsrtA strain significant (p <0.05) decreased the adhesion and invasion rate and intracellular bacteria in cells RAW264.7 and SIEC.The LD50 of LM90SB2-ΔsrtA decreased by 21.38 times.The bacterial loads in livers and spleens of LM90SB2-ΔsrtA infected mice were significantly lower than that of the parent LM90SB2 strain (p <0.05).This study suggested that srtA gene played a role in the virulence of Listeria monocytogenes LM90SB2 by involvement in adhesion and invasion to brain microvascular endothelial cells.