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表皮葡萄球菌vraSR-srrAB突变株的构建及生物学表型
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国家自然科学基金(82060380, 81660346); 云南省中青年学术和技术带头人后备人才项目(202305AC160038);复旦大学教育部/卫健委医学分子病毒学重点实验室开放课题(FDMV-2021002)


Construction and biological phenotype characterization of vraSR-srrAB deletion mutant of Staphylococcus epidermidis
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    摘要:

    【背景】形成生物被膜是表皮葡萄球菌的主要致病方式,双组分系统(two-component regulatory system, TCS) VraSR和SrrAB与细菌的生长、生物被膜形成等多种生物学表型密切相关。敲除表皮葡萄球菌SE1457 vraSR后细胞壁变薄,生物被膜形成量降低,而敲除srrAB后生长进入稳定期的时间延长,生物被膜形成量也降低,且TCS-VraSR和SrrAB均通过ica途径调控表皮葡萄球菌生物被膜的形成。ica操作子是表皮葡萄球菌生物被膜形成的重要调节元件,由icaADBC这4个开放阅读框(open reading frame, ORF)和1个转录方向相反的icaR组成,IcaR是icaADBC的抑制子。【目的】探索TCS-VraSR和SrrAB协同调控表皮葡萄球菌生长及生物被膜形成中的作用,为防控表皮葡萄球菌引起的持续性感染奠定基础。【方法】构建pKOR1-ΔvraSR重组质粒,经大肠杆菌DC10B修饰后转入ΔsrrAB,通过同源重组在ΔsrrAB突变株的基础上进一步敲除vraSR基因,疑似ΔvraSR-srrAB突变株经PCR、RT-PCR和测序验证。检测ΔvraSR-srrAB突变株的生长、生物被膜形成及药物敏感性。【结果】成功构建表皮葡萄球菌ΔvraSR-srrAB突变株。与表皮葡萄球菌SE1457野生株、ΔvraSR和ΔsrrAB突变株相比,ΔvraSR-srrAB突变株生长更为迟缓,药物敏感性增强,几乎不能形成生物被膜;RT-qPCR显示,ΔvraSR和ΔsrrAB突变株中icaA转录水平较SE1457下调约13%-17%,icaR转录水平上调约5-9倍,而ΔvraSR-srrAB突变株中icaA转录水平较SE1457下调约6%,icaR转录水平上调约14倍。【结论】VraSR-SrrAB可能通过ica途径协同调控表皮葡萄球菌生物被膜形成,对压力应激胁迫作用还需进一步研究。

    Abstract:

    [Background] Biofilm formation is the major pathogenic factor of Staphylococcus epidermidis. The two-component regulatory system (TCS) composed of VraSR and SrrAB is involved in the growth, biofilm formation and the other biological phenotypes of bacteria. The deletion of vraSR leads to thinner cell wall and decreased biofilm formation, and the deletion of srrAB results in lagged growth before the stationary phase and decreased biofilm formation of S. epidermidis 1457 (SE1457). Both VraSR and SrrAB modulate biofilm formation in an ica-dependent manner. The ica operon is a key regulatory element of biofilm formation in S. epidermidis, which is composed of four genes (icaADBC) and a transcriptional repressor (icaR). [Objective] To explore the synergistic regulation of TCS-VraSR and SrrAB on the growth and biofilm formation of S. epidermidis, so as to lay a foundation for the prevention and control of persistent infection caused by S. epidermidis. [Methods] The recombinant plasmid pKOR1-ΔvraSR was constructed, modified by E. coli DC10B, and transformed into ΔsrrAB. The vraSR gene was deleted from the genome of ΔsrrAB by homologous recombination. The suspected ΔvraSR-srrAB was verified by PCR, RT-PCR, and sequencing. The growth, biofilm formation, and drug susceptibility of ΔvraSR-srrAB were examined. [Results] ΔvraSR-srrAB was successfully constructed. Compared with SE1457, ΔvraSR, and ΔsrrAB, ΔvraSR-srrAB exhibited retarded growth, increased drug susceptibility, and decreased biofilm formation. The RT-qPCR showed that the deletion of vraSR or srrAB down-regulated the transcriptional level of icaA by 13%–17% and up-regulated that of icaR by 5–9 folds compared with that in SE1457. The deletion of both vraSR and srrAB down-regulated the transcriptional level of icaA by 6% and up-regulated that of icaR by 14 folds compared with that in SE1457. [Conclusion] VraSR and SrrAB may cooperatively regulate the biofilm formation of S. epidermidis through the ica pathway, and the mechanism of VraSR and SrrAB in modulating the responses to stress remains to be studied.

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陈晓婷,乾莲,陈卫国,孙士正,白宗凯,赵纯静,武有聪. 表皮葡萄球菌vraSR-srrAB突变株的构建及生物学表型[J]. 微生物学通报, 2023, 50(8): 3562-3574

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  • 收稿日期:2022-10-14
  • 录用日期:2023-01-01
  • 在线发布日期: 2023-08-08
  • 出版日期: 2023-08-20
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