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【目的】铜绿假单胞菌是引起医院获得性感染最常见的条件致病菌,而Ⅲ型分泌系统(Type Ⅲ secretion system,TTSS)是其致病的主要因子之一。本文从合成的21个肉桂酸衍生物中筛选影响TTSS效应子(Effector)产生的化合物,并初步研究其作用机制。【方法】将TTSS效应子合成基因exoS的转录报告质粒pAT-exoS转入菌株PAO1中,获得PAO1(pAT-exoS)。待筛选的化合物与PAO1(pAT-exoS)菌株共培养6 h后,检测exoS基因的表达,从中筛选影响exoS基因表达的化合物。【结果】筛选结果表明:21个化合物中,3个化合物抑制exoS基因表达,2个化合物则促进exoS基因表达。此外,化合物TS128、TS143和TS160对菌株生长有明显的抑制作用。Western blot实验进一步证实筛选得到的化合物TS108、TS128和TS165可抑制ExoS的产生;化合物TS139和TS143则促进ExoS的产生。为进一步研究抑制剂的作用机理,过量表达TTSS主要的调控因子exsA基因可部分消除抑制剂TS108和TS165的抑制效果;而rsmZ rsmY双基因突变体PAO6421中添加抑制剂TS108和TS165并不能显著抑制exoS基因的表达,同样,抑制剂TS108和TS165也不影响受Gac/Rsm信号传导系统调控的群体感应信号分子的产生。【结论】抑制剂TS108和TS165的作用机制可能主要是影响esxA基因,从而影响exoS基因表达及蛋白产量。
【Objective】 Pseudomonas aeruginosa is the most common opportunistic pathogen causing hospital-acquired infections, and Type Ⅲ secretion system (TTSS) is one of the major pathogens. In this paper, 21 cinnamic acid derivatives were selected to affect the compounds produced by TTSS effectors (Effector), and preliminary study of its mechanism of action. 【Method】 The transcriptional reporter plasmid pAT-exoS of TTSS effector synthesis gene exoS was transferred into PAO1 to obtain PAO1 (pAT-exoS). The co-cultured PAO1 (pAT-exoS) and the compound to be screened for 6 h, the expression of the exoS gene was detected, and the compounds that affected the expression of the exoS gene were screened out. 【Results】 The results of screening showed that three of the 21 compounds inhibited the expression of exoS gene and the two compounds promoted the expression of exoS gene. In addition, the compounds TS128, TS143 and TS160 significantly inhibited the growth of the strain. The result of Western blot further confirmed that the selected compounds TS108, TS128 and TS165 could inhibit the production of ExoS. Compounds TS139 and TS143 promoted the production of ExoS. To further investigate the mechanism of action of inhibitors, overexpression of exsA, a major regulatory factor of TTSS, partially abolished the inhibitory effect of TS108 and TS165 inhibitors. However, the addition of inhibitors TS108 and TS165 to rsmZ rsmY mutant PAO6421 did not significantly inhibit exoS Similarly, inhibitors TS108 and TS165 also did not affect the generation of population-sensitive signaling molecules regulated by the Gac / Rsm signaling system. 【Conclusion】 The mechanism of action of inhibitors TS108 and TS165 may mainly affect esxA gene, thus affecting exoS gene expression and protein yield.