LIPI-4 EⅡC调控单增李斯特菌关键毒力因子转录的研究

Regulation of Transcription of Key Virulence Factors of Listeria monocytogenes by LIPI-4 EⅡC

【目的】探究单增李斯特菌(LM)毒力岛4(LIPI-4)中膜透性酶ⅡC(EⅡC)对LM关键毒力基因表达的影响。【方法】构建分离株LM928 EⅡC基因缺失株LM928ΔEⅡC和回补菌株CLM928ΔEⅡC,测定各菌株的生长曲线和对不同糖的发酵情况,测定侵染人脑微血管内皮细胞(HCMEC/D3)后的黏附、侵袭和胞内增殖能力,利用实时荧光定量PCR方法检测在脑心浸液(BHI)培养条件下和侵染HCMEC/D3后各菌株毒力基因的转录水平。【结果】成功构建LM928 EⅡC基因缺失株LM928ΔEⅡC和回补菌株CLM928ΔEⅡC。EⅡC基因的缺失不影响LM928的生长和对葡萄糖、纤维二糖、果糖、鼠李糖、七叶苷和水杨苷的发酵。EⅡC基因缺失后,LM928对HCMEC/D3的侵袭率极显著下降(P<0.01),黏附能力无显著变化(P>0.05)。LM928ΔEⅡC胞内增殖菌量在2~4 h时显著低于LM928和CLM928ΔEⅡC(P<0.05),但在8~12 h时均显著高于LM928和CLM928ΔEⅡC(P<0.05)。在BHI培养条件下,LM928ΔEⅡC毒力基因hly、prfA、actA、plcB、inlA、inlB、inlC和mpl转录水平极显著下调(P<0.01),毒力基因plcA转录水平极显著上调(P<0.01);在侵染HCMEC/D3后,LM928ΔEⅡC毒力基因hly、prfA、inlB、inlC和mpl转录水平相对于LM928和CLM928ΔEⅡC显著或极显著上调(P<0.05;P<0.01),毒力基因plcA和inlA相比于CLM928ΔEⅡC极显著下调(P<0.01),毒力基因actA和plcB转录水平无显著差异(P>0.05)。【结论】LIPI-4中EⅡC不参与LM928的生长及其对部分糖的发酵,能调控LM中重要的侵袭相关基因和毒力相关基因。本试验结果为进一步探究EⅡC在LM和LIPI-4中的作用奠定基础。

【Objective】This study was aimed to investigate the effect of membrane permeaseⅡC(EⅡC)gene in Listeria monocytogenes(LM)pathogenicity island 4(LIPI-4)on the key virulence factor genes expression.【Method】The EⅡC gene deletion strain LM928ΔEⅡC and complementary strain CLM928ΔEⅡC of isolate LM928 were constructed.The growth curve of each strain and the fermentation of different sugars were determined.The adhesion,invasion and intracellular proliferation of each strain to human brain microvascular endothelial cells(HCMEC/D3)were determined.The transcript levels of virulence genes of each strain were detected using Real-time quantitative PCR under BHI medium and HCMEC/D3.【Result】LM928 EⅡC gene deletion strain LM928ΔEⅡC and complementary strain CLM928ΔEⅡC were successfully constructed.Compared with the LM928,the LM928ΔEⅡC strain showed consistent growth curves and fermentability to glucose,cellobiose,fructose,rhamnose,esculin and salicin.The invasive ability of LM928ΔEⅡC decreased extremely significantly(P<0.01),but the adhesion ability was not change significantly(P>0.05).The amount of intracellular proliferation of LM928ΔEⅡC was significantly lower than that of LM928 and CLM928ΔEⅡC from 2 to 4 h post-infection(P<0.05),but it was significantly higher than that of LM928 and CLM928ΔEⅡC from 8 to 12 h post-infection(P<0.05).Compared with LM928 and CLM928ΔEⅡC,in BHI medium,the transcriptional levels of hly,prfA,actA,plcB,inlA,inlB,inlC and mpl genes of LM928ΔEⅡC were extremely significantly down-regulated(P<0.01),the transcriptional level of plcA gene was extremely significantly up-regulated(P<0.01).While in HCMEC/D3 cell,the transcriptional levels of hly,prfA,inlB,inlC and mpl genes of LM928ΔEⅡC were significant or extremely significantly up-regulated(P<0.05 or P<0.01),the transcriptional levels of plcA and inlA genes were extremely significant down-regulated(P<0.01),and there was no significant difference between the transcriptional levels of actA and plcB genes.【Conclusion】EⅡC in LIPI-4 did not affect the growth and partial sugar fermentation of LM928,could regulate important invasion-related genes and virulence-related genes in LM,which laid a foundation for further exploration of the role of EⅡC in LM and LIPI-4.