LIPI-4 EⅡA基因对单核细胞增生性李斯特菌重要毒力基因转录调控作用的研究

Transcriptional regulation of important virulence genes in Listeria monocytogenes by permease ⅡA of Listeria pathogenicity Island 4

为探究单核细胞增生性李斯特菌(LM)毒力岛4(LIPI-4)的膜透性酶ⅡA(EⅡA)基因在LM中的毒力作用,本研究利用同源重组技术构建了具有遗传稳定性的LM928菌株EⅡA基因缺失株(LM928ΔEⅡA)和回补株(CLM928ΔEⅡA),通过对缺失株、回补株和亲本株生长特性测定分析EⅡA基因对LM体外生长能力的影响。提取各菌株RNA,反转录为c DNA后通过real-time PCR检测各菌株在体外培养条件下LM重要毒力基因的转录水平,分析EⅡA基因缺失对LM毒力基因转录水平的影响。结果显示,缺失EⅡA基因后不影响细菌在体外的生长能力,但细菌plcB和inlA基因的转录水平显著下调(P<0.05),hly、prfA、plcB、inlA、inlB、inlC和mpl基因的转录水平极显著下调(P<0.01),plcA基因的转录水平极显著上调(P<0.01),而actA基因的转录水平无明显变化(P>0.05)。将LM928、LM928ΔEⅡA和CLM928ΔEⅡA菌株分别感染人脑微血管内皮细胞(HCMEC/D3)后,采用CCK8法检测EⅡA基因对HCMEC/D3活性的影响,同时通过胞内增殖试验检测EⅡA基因对LM胞内增殖的影响,进一步采用real-time PCR方法检测EⅡA对细胞毒力基因转录水平的影响,结果显示,与LM928组相比,LM928ΔEⅡA组HCMEC/D3细胞活性无明显改变;LM928ΔEⅡA感染HCMEC/D3后6 h~12 h,胞内LM928ΔEⅡA载菌量极显著高于胞内LM928载菌量(P<0.01),在感染HCMEC/D3后2 h~12 h,胞内LM928ΔEⅡA载菌量也极显著高于胞内CLM928ΔEⅡA的载菌量(P<0.01)。相对于亲本株,LM928ΔEⅡA感染的细胞中毒力基因prf A、plc A、plc B、inl A、inl B、inl C和mpl转录水平均极显著上调(P<0.01),actA基因的转录水平显著上调(P<0.05),hly基因的转录水平极显著下调(P<0.01)。本研究结果首次证实LIPI-4中EⅡA基因对LM体外生长无影响,但其对体内、外LM毒力因子的转录均具有显著的调控作用,提示LM LIPI-4可能通过EⅡA参与调控细菌毒力,该结果为进一步研究LIPI-4在LM致病中的作用奠定了基础。

To investigate the virulence of permease Ⅱ A(E Ⅱ A) gene of Listeria Pathogenicity Island 4(LIPI-4) in Listeria monocytogenes(LM), the deletion mutant(LM928ΔEⅡA) and complementary strain(CLM928ΔEⅡA) with genetic stability were successfully constructed by homologous recombination technology, the growth curves of the mutant, complementary and wildtype strains were determined to evaluate the effect of EIIA gene on LM in vitro growth ability, and the transcriptional profile of important virulence genes of each strain under in vitro culture conditions was examined by real-time PCR to analyze the effect of EIIA deletion on virulence of LM. The results showed that deletion of EⅡA gene did not affect the growth ability of LM in vitro,however, it significantly down-regulated the transcriptional levels of plcB and inlA genes(P<0.05), significantly down-regulated the transcription levels of hly, prfA, plcB, inlA, inlB, inlC and mpl genes(P<0.01), and significantly up-regulated the transcription level of plcA gene(P<0.01), but had no significant effect on the transcription level of actA gene(P>0.05). After infection of the human brain microvascular endothelial cells(HCMEC/D3) with the LM928 and LM928ΔEⅡA strain, respectively, the effect of EIIA on the activity of the cells was examined by CCK8 assay, the effect of EIIA gene on LM intracellular proliferation was detected by intracellular proliferation assay, and the intracellular effect of EIIA on the transcriptional levels of virulence genes was detected using real-time PCR. The results showed that compared with LM928, there was no significant difference in cell activity of HCMEC/D3 infected with LM928ΔEIIA. The amount of intracellular bacteria in LM928ΔEIIA group was significantly higher than that in LM928 group at 6 hours-12 hours post infection(P<0.01). At 2 hours-12 hours post infection, the amount of intracellular LM928ΔEIIA was also significantly higher than that of CLM928ΔEIIA group(P<0.01). The transcriptional levels of virulence genes prfA, plcA, plcB, inlA, inlB, inlC and mpl were significantly up-regulated after deletion of EIIA gene(P<0.01). It was significantly up-regulated for actA gene(P<0.05) and significantly down-regulated for hly gene(P<0.01). This study confirmed for the first time that the EIIA in LIPI-4 has no effect on LM growth in vitro, but it has significant transcriptional regulation on important LM virulence factors in vivo and in vitro, suggesting that LIPI-4 of LM may be involved in regulating bacterial virulence through EIIA, which laid a foundation for further study on the role of LIPI-4 in LM pathogenesis.