单增李斯特菌末端氧化酶亚基QoxB的生物学功能探究

Biological functions of terminal oxidase subunit QoxB in Listeria monocytogenes

【目的】本研究旨在构建单增李斯特菌末端细胞色素aa3氧化酶亚基qoxB基因缺失株,并探索其在细菌生长及感染过程中发挥的生物学功能。【方法】利用同源重组方法构建获得缺失株ΔqoxB后,对野生株EGD-e和缺失株ΔqoxB的生长能力、细菌运动能力和细胞内黏附、侵袭、增殖及胞内迁移能力进行比较,同时利用荧光定量PCR方法检测ΔqoxB中鞭毛相关基因转录水平的变化。【结果】缺失qoxB基因后细菌在体外培养过程中生长能力没有差异,细菌的鞭毛运动能力显著降低,在30℃培养24 h和48 h后ΔqoxB运动圈直径分别较EGD-e下降35.86%和34.20%,且22个鞭毛相关基因转录水平显著降低。通过细胞感染试验发现缺失qoxB基因后细胞黏附、侵袭、增殖及胞内迁移能力均显著下降。【结论】本研究首次证实末端氧化酶亚基QoxB能降低单增李斯特菌的运动能力和对细胞的感染能力,此研究为进一步阐明末端细胞色素氧化酶影响单增李斯特菌的致病机制提供重要依据。

[Objective]Listeria monocytogenes is an important zoonotic foodborne intracellular pathogen.Here,we aimed to explore the biological functions of cytochrome aa3terminal oxidase subunit QoxB in L.monocytogenes during bacterial growth and infection.[Methods]We constructed a qoxB deletion mutantΔqoxB based on the wild-type strain EGD-e via homologous recombination.The differences regarding bacterial growth and intracellular infection were analyzed between the wild-type and the mutant strains.In addition,changes in the transcription of flagella-associated genes induced by the deletion of qoxB were determined by real-time quantitative reverse-transcription PCR(qRT-PCR).[Results]The deletion of qoxB did not affect the bacterial growth ability in vitro.However,the motility ofΔqoxB was significantly decreased compared with that of EGD-e.After incubation at 30℃for 24 h and 48 h,the diameter of motility halos ofΔqoxB decreased by 35.86%and 34.2%,respectively,compared with that of EGD-e.The expression abundance of 22 flagella-associated genes reduced significantly inΔqoxB.Moreover,compared with EGD-e,ΔqoxB showed significantly reduced abilities of cell adhesion,invasion,proliferation,and intracellular spread.[Conclusion]We demonstrated for the first time that QoxB played a critical role in the motility and infection of L.monocytogenes.The findings lay an important basis for elucidating the role of cytochrome terminal oxidase in the pathogenesis of bacterial pathogens.