Ⅲ型分泌系统抑制剂对铜绿假单胞菌PAO1毒性因子的影响

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

【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exo Y和exo T转录报告质粒p AT-exo Y、p AT-exo T,并将其转入菌株PAO1中。菌株PAO1(p AT-exo Y)、PAO1(p AT-exo T)与TTSS抑制剂共同培养后,检测exo Y和exo T的表达。通过SDS-PAGE检测TTSS抑制剂对鞭毛结构蛋白Fli C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exo Y和exo T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Pcr V的产量,但可抑制Pcr V蛋白的胞外运输。化合物TS53可降低鞭毛结构蛋白Fli C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。

[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 exo T and exo Y genes were used to monitor the exo T and exo Y expression in the wild type strain PAO1 supplemented with TTSS inhibitors.The protein level of Fli C 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 exo T and exo Y was decreased when the TTSS inhibitors were supplemented.Although the production of Pcr V was not changed in the cells when compounds TS52,TS53 and TS94 were added in the medium,the translocation of Pcr V protein in the medium was decreased.The level of flagella structural protein Fli C 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.