[目的]探究单核细胞增生李斯特菌(Listeria monocytogenes,LM)毒力岛3(LIPI-3)和LIPI-4共存对LM毒力的影响,揭示它们对细菌毒力的潜在调节作用,为进一步研究LM的致病机制提供依据.[方法]以LM928(存在LIPI-4)和LM873(同时存在LIPI-3和LIPI-4)2个LM菌株为研究对象,通过侵染HCMEC/D3细胞监测2种菌株对HCMEC/D3细胞的黏附和侵袭情况;通过鸡胚感染试验评估2株菌对鸡胚半数致死量(LD50)的影响;采用溶血试验来测定菌株的溶血活性;通过流式细胞仪分析HCMEC/D3细胞凋亡情况,以评估菌株诱导细胞凋亡的能力;利用实时荧光定量PCR技术比较2株菌在BHI培养条件下及感染HCMEC/D3细胞后毒力基因的转录水平差异.[结果]LM928和LM873株的黏附率无显著差异(P＞0.05),LM928株的侵袭率极显著高于LM873株(P＜0.01).LM873株的LD50是LM928株的约1 000倍;LM928和LM873株的溶血价分别为24和23.LM928株感染24和48 h后诱导的细胞凋亡率显著或极显著高于LM873株(P＜0.05;P＜0.01),而LM873株的凋亡率与对照组间无显著差异(P＞0.05).LM928和LM873株在BHI培养基中培养时,与LM928株相比,LM873株毒力基因mpl、inlB、inlC、inlP、actA、plcA、plcB和sigB的转录水平均显著或极显著上调(P＜0.05;P＜0.01),毒力基因hly、inlA和iap的转录水平均极显著或显著下调(P＜0.01;P＜0.05).LM928和LM873株侵染HCMEC/D3细胞后,与LM928株相比,LM873株毒力基因inlP、actA、plcA、plcB和prfA的转录水平均显著或极显著上调(P＜0.05;P＜0.01);毒力基因hly、mpl、inlA、inlB、inlC和iap的转录水平均极显著下调(P＜0.01).[结论]LIPI-3和LIPI-4共存降低了 LM的毒力,该结果对进一步研究LIPI-3和LIPI-4在细菌毒力调控中的分子机制和复杂的相互作用具有重要意义.
[Objective]This study was aimed to explore the impact of the coexistence of LIPI-3 and LIPI-4 on the virulence of Listeria monocytogenes(LM),reveal their potential regulatory effects on bacterial virulence,and lay a foundation for further research on the pathogenic mechanisms of LM.[Method]LM928(possessing LIPI-4 present)and LM873(possessing both LIPI-3 and LIPI-4)were used as research objects to monitor the adhesion and invasion of two strains by infecting HCMEC/D3 cells.The effect of two strains on the median lethal dose(LD50)of chicken embryos were evaluated by chicken embryo infection test.The hemolytic activity of two strains were determined using hemolysis test.The apoptosis of HCMEC/D3 cells was analyzed using flow cytometry to evaluate the ability of two strain to induce cell apoptosis.The transcriptional differences of virulence genes between two strains under BHI culture conditions and post-infection of HCMEC/D3 cells were compared using Real-time quantitative PCR.[Result]There was no significant difference of adhesion rates between LM928 and LM873 strains(P＞0.05).The invasion rate of LM928 strain was extremely significantly higher than that of LM873 strain(P＜0.01).The LD50 of LM873 strain was approximately 1 000 times that of LM928 strain.The hemolytic values of LM928 and LM873 strains were 24 and 23,respectively.The apoptosis rate induced by LM928 strain at 24 and 48 h post-infection were significantly or extremely significantly higher than that of LM873 strain(P＜0.05 or P＜0.01),while there was no significant difference of the apoptosis rate between LM873 strain and control group(P＞0.05).When cultured in BHI culture,compared with LM928 strain,the transcription levels of virulence genes mpl,inlB,inlC,inlP,actA,plcA,plcB and sigB in LM873 strain were significantly or extremely significantly upregulated(P＜0.05 or P＜0.01),and the transcription levels of hly,inlA and iap genes were extremely significantly or significantly downregulated(P＜0.01 or P＜0.05).After infecting HCMEC/D3 cells,compared with LM928 strain,the transcription levels of virulence genes inlP,actA,plcA,plcB and prfA in LM873 strain were significantly or extremely significantly upregulated(P＜0.05 or P＜0.01),and the transcription levels of hly,mpl,inlA,inlB,inlC and iap genes were extremely significantly downregulated(P＜0.01).[Conclusion]The coexistence of LIPI-3 and LIPI-4 reduced the virulence of LM,which holded significant implications for further research into the molecular mechanisms and complex interactions of LIPI-3 and LIPI-4 in bacterial virulence regulation.
钱瑞宣;殷中科;刘璐;王静;王正荣;蒋建军;李楠;康立超;马勋;刘彩霞;史唯地;寇丽君;任慧杰;祁亚涛
石河子大学动物科技学院,石河子 832000新疆农垦科学院,省部共建绵羊遗传改良与健康养殖国家重点实验室,石河子 832000新疆农垦科学院农业质量标准与检测技术研究所,石河子 832000
畜牧业
单核细胞增生李斯特菌(LM);LIPI-3;LIPI-4;毒力;毒力基因
Listeria monocytogenes(LM);LIPI-3;LIPI-4;virulence;virulence gene
《中国畜牧兽医》 2024 (005)
2027-2036 / 10
国家自然科学基金(32160833、32160834、32260895);省部共建绵羊遗传改良与健康养殖国家重点实验室开放课题(MYSKLKF201905);江苏省人兽共患病学重点实验室(R1901)
10.16431/j.cnki.1671-7236.2024.05.024
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