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III型分泌系统抑制剂对铜绿假单胞菌PAO1毒性因子的影响
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国家自然科学基金面上项目(No. 21272029);The Research Growth Initiative of the University of Wisconsin-Milwaukee;常州市国际科技合作项目(No. CZ20130024)


Regulation of type III secretion system inhibitors on virulence factors of Pseudomonas aeruginosa PAO1
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    摘要:

    【目的】进一步研究III型分泌系统(Type III secretion system, TTSS)抑制剂对条件致病菌Pseudomonas aeruginosa PAO1的TTSS相关蛋白、鞭毛和纤毛等主要毒性因子的影响,评估TTSS抑制剂的防治效果及潜在风险。【方法】构建TTSS效应蛋白合成基因exoY和exoT转录报告质粒pAT-exoY、pAT-exoT,并将其转入菌株PAO1中。菌株PAO1(pAT-exoY)、PAO1(pAT-exoT) 与TTSS抑制剂共同培养后,检测exoY和exoT的表达。通过SDS-PAGE检测TTSS抑制剂对鞭毛结构蛋白FliC的影响。将PAO1单菌落穿刺接种于含有TTSS抑制剂的1%琼脂糖平板,观察细菌纤毛介导的蹭行运动(Twitching motility)。【结果】转录报告实验结果表明4个TTSS抑制剂可显著抑制exoY和exoT的转录;化合物TS52、TS53和TS94虽不影响胞内TTSS针状顶端结构蛋白PcrV的产量,但可抑制PcrV蛋白的胞外运输。化合物TS53可降低鞭毛结构蛋白FliC的产生。另外,化合物TS52、TS53和TS88可降低菌株PAO1的蹭行运动能力,但TS94可提高菌株PAO1的这种运动能力。【结论】TTSS抑制剂除通过抑制TTSS表达外,还可能通过影响其它毒性因子如鞭毛的合成、IV型分泌系统介导的蹭行运动等方式影响菌株PAO1致病性。

    Abstract:

    [Objective] To study the effect of type III secretion system (TTSS) inhibitors on virulence factors, TTSS, bacterial flagella and pilin, of Pseudomonas aeruginosa PAO1. [Methods] The transcriptional reporters of exoT and exoY genes were used to monitor the exoT and exoY expression in the wild type strain PAO1 supplemented with TTSS inhibitors. The protein level of FliC was analyzed by SDS-PAGE. The twitching motility mediated by bacterial pilin was examined on the BM2 medium. [Results] Transcriptional fusion assay indicated that the expression of exoT and exoY was decreased when the TTSS inhibitors were supplemented. Although the production of PcrV was not changed in the cells when compounds TS52, TS53 and TS94 were added in the medium, the translocation of PcrV protein in the medium was decreased. The level of flagella structural protein FliC was decreased when the compound TS53 was supplemented in the medium. In addition, compounds TS52, TS53 and TS88 inhibited the ability of twitching motility, whereas TS94 increased the twitching motility. [Conclusion] Besides the TTSS effector genes, TTSS inhibitors influence other virulence factors, such as flagella and type IV pilus-dependent twitching motility. These data will provide the theoretical basis for further clinical trials.

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顾玲玲,吴小刚,袁小琛,方力为,张成芳,陈新,YANG Ching-Hong. III型分泌系统抑制剂对铜绿假单胞菌PAO1毒性因子的影响[J]. 微生物学通报, 2014, 41(11): 2318-2324

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  • 在线发布日期: 2014-10-30
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