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【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 further investigate the effect of Type Ⅲ secretion system (TTSS) inhibitors on the major TTSS-related proteins, flagella and cilia of pathogenic bacteria Pseudomonas aeruginosa PAO1 and to evaluate the effect of TTSS inhibitors and potential risks. 【Method】 The TTSS effector protein synthesis gene exo Y and exo T transcriptional reporter plasmid p AT-exo Y, p AT-exo T were constructed and transformed into PAO1 strain. Co-cultures of strains PAO1 (p AT-exo Y) and PAO1 (p AT-exo T) with TTSS inhibitors were used to detect exo Y and exo T expression. The effect of TTSS inhibitors on the flagellar structural protein Fli C was examined by SDS-PAGE. PAO1 single colonies were punctured on 1% agarose plates containing TTSS inhibitors to observe bacterial cilia-mediated Twitching motility. 【Results】 The results of transcriptional report showed that four TTSS inhibitors significantly inhibited the transcription of exo Y and exo T; while the compounds TS52, TS53 and TS94 did not affect the intracellular production of Pcr V, but inhibit Pcr V protein extracellular transport. Compound TS53 reduces the production of the flagellar structural protein FliC. In addition, compounds TS52, TS53 and TS88 can reduce the rubbing capacity of PAO1, but TS94 can enhance this kind of exercise capacity of PAO1. 【Conclusion】 TTSS inhibitors can affect the pathogenicity of PAO1 strain by inhibiting the expression of TTSS as well as affecting the pathogenicity of PAO1 by affecting the synthesis of other cytokines such as flagella and type IV secretion system.