单增李斯特菌Lm4b_02325基因缺失株的构建及其生物学特性研究

Construction and biological characteristics of Lm4b_02325 gene deletion strain of Listeria monocytogenes

为研究单增李斯特菌(LM)抗转录抑制因子编码基因Lm4b_02325对LM的生物学特性的影响,本研究采用同源重组技术构建Lm4b_02325基因缺失株LM928ΔLm4b_02325和回补株CLM928ΔLm4b_02325并均经PCR和测序鉴定。同时采用PCR鉴定该菌的遗传稳定性。上述结果表明基因缺失株与回补株均正确构建,且缺失株的遗传稳定性较强。根据培养不同时间的OD600nm值绘制缺失株、回补株和亲本株的生长曲线;对小鼠腹腔注射不同浓度(10^(9) cfu/mL~10^(5) cfu/mL)的3株菌,观察感染后小鼠的临床症状,统计死亡率并计算3株菌对小鼠的半数致死量(LD50)。以MOI 10将上述3种菌分别感染人脑微血管内皮细胞(HBMEC),1 h后裂解各菌,10倍倍比稀释,并涂布于BHI固体培养基,37℃培养1 d后经菌落计数,分析各株菌对HBMEC的粘附力;按照上述方法感染各菌后1 h加入庆大霉素以及感染各菌后不同时间加入庆大霉素杀死未粘附的细菌,裂解各细菌,10倍倍比稀释并涂布于BHI固体培养基,通过菌落计数结果分析各菌株对HBMEC的侵袭力及在该细胞内的增殖能力。生长曲线结果显示3株菌的生长趋势基本相似。小鼠的致病性实验结果显示,接种10^(9) cfu/mL~10^(7) cfu/mL 3株菌的小鼠出现被毛凌乱、厌食或神经症状与昏睡等临床症状,死亡率接近100%。而接种10^(6) cfu/mL~10^(5) cfu/mL 3株菌的小鼠临床症状较轻,死亡率较低。经计算LM928ΔLm4b_02325对小鼠的LD50约10^(5).45 cfu/mL,毒力明显高于LM928株及CLM928ΔLm4b_02325株(P<0.05)。粘附、侵袭力及胞内生存能力结果显示,黏附及侵入HBMEC的LM928ΔLm4b_02325菌株数量极显著(P<0.01)或者显著高于LM928(P<0.01)与CLM928ΔLm4b_02325株(P<0.05);胞内存活试验结果显示,感染后各时间点LM928ΔLm4b_02325较其余两种菌在HBMEC内的增殖速度均显著(P<0.05)或者极显著(P<0.01)加快,且前者在HBMEC中的存活数较后两者均显著(P<0.05)或者极显著(P<0.01)升高;采用荧光定量PCR(qPCR)检测黏附侵袭相关毒力基因inlA、inlB、inlC,胞内感染相关毒力基因plcA、plcB、actA、hly、mpl,内毒素调节基因prfA共9种毒力相关基因的转录水平,结果显示,LM928ΔLm4b_02325株hly、mpl、inlC的转录水平显著降低(P<0.05),actA、plcA的转录水平极显著下调(P<0.01)。其余基因转录水平均无显著变化。本研究结果首次表明Lm4b_02325基因缺失增强了LM对小鼠的致病性、对细胞的粘附性、侵袭力及胞内增殖能力,且其毒力基因hly、actA、plcA、mpl、inlA、inlC转录水平显著下调。本研究为Lm4b_02325基因的功能及在LM致病机致中作用的研究奠定基础。

To investigate the effect of anti-transcription suppressant encoding gene Lm4b_02325 on the biological characteristics of Listeria monocytogenes(LM),the Lm4b_02325 gene deletion strain LM928ΔLm4b_02325 and complementary strain(CLM928ΔLm4b_02325)were constructed by homologous recombination technology and identified by PCR and sequencing,and the genetic stability of the bacteria was identified by PCR.The results showed that the deletion strain and the complementary strain were correctly constructed,and the deletion strain possesses strong genetic stability.The growth curves of the deletion,the complementary and the parental strains were plotted according to the OD600nm value at different culture times.Mice were injected with 3 strains of different concentrations(10^(9) cfu/mL-10^(5) cfu/mL),the clinical symptoms of the mice were observed,the mortality rate was calculated,and the median lethal dose(LD50)of the 3 strains was calculated by Koch's calculation method.Human brain microvascular endothelial cells(HBMEC)were infected with the above bacteria at MOI 10,respectively.The bacteria were lysed after 1 hours,10-fold serially diluted,coated on BHI solid medium,and cultured at 37℃for 1 day,the adhesion of each strain to HBMEC was analyzed by colony counting.According to the above methods,gentamicin was added to the HBMEC at 1 hour after infection with each strain and gentamicin was added at different times after infection with each strain to kill non-adherent bacteria.The bacteria were lysed,10-fold serially diluted,spread on BHI solid medium.The invasion ability of each strain on HBMEC and its proliferation ability in the cell were analyzed by colony counting.The growth curve showed that the growth trends of the three strains were basically similar.The results of pathogenicity test in mice showed that the mice inoculated with 3 strains at a dose of 10^(9)cfu/mL-10^(7)cfu/mL showed clinical symptoms such as rough hair,anorexia or neurological symptoms and lethality,and the mortality rate was close to 100%.The mice inoculated with 3 strains at a dose 10^(6)cfu/mL-10^(5)cfu/mL had mild clinical symptoms and lower mortality.The LD50 of LM928ΔLm4b_02325 to mice was about 10^(5).45cfu/mL,and the virulence of LM928ΔLm4b_02325 was significantly higher than that of LM928 and CLM928ΔLm4b_02325(P<0.05).The results of adhesion,invasion and intracellular viability showed that the survival rate of LM928ΔLm4b_02325 which adhered to and invaded HBMEC cells was significantly higher than that of LM928(P<0.01)and CLM928ΔLm4b_02325(P<0.05).The results of intracellular survival experiment showed that the proliferation rate of LM928ΔLm4b_02325 was significantly(P<0.05)or extremely significantly(P<0.01)faster than that of the other two strains in HBMEC at each time point after infection.The survival rate of the former in HBMEC cells was significantly(P<0.05)or extremely significantly(P<0.01)higher than that of the latter two.Fluorescence quantitative PCR(qPCR)was used to detect the transcription levels of 9 virulence genes including adhesion and invasion related genes(inlA,inlB,and inlC),intracellular infection-related virulence genes(plcA,plcB,actA,hly,and mpl),and endotoxin regulatory gene prfA.The results showed that the transcription levels of hly,mpl and inlC in LM928ΔLm4b_02325 strain were significantly decreased(P<0.05),and the transcription levels of actA and plcA were significantly down-regulated(P<0.01).There were no significant changes in the transcription levels of other tested genes.This work suggested for the first time that the deletion of Lm4b_02325 gene enhanced the pathogenicity,cell adhesion,invasion and intracellular proliferation of LM in mice,and the transcription levels of virulence genes hly,actA,plcA,mpl,inlA and inlC were significantly down-regulated,which laid a foundation for the study of the function of Lm4b_02325 gene and its role in the pathogenesis of LM.