铜绿假单胞菌pfm基因对三型分泌系统效应蛋白的影响

Pseudomonas aeruginosa gene pfm affects the expression levels of type Ⅲ secretion system effectors

摘要:

【目的】检测铜绿假单胞菌基因pfm对三型分泌系统效应蛋白的影响。【方法】构建pfm基因互补菌株pfmC。提取野生株PAO1、敲除株Δpfm和互补株pfmC的RNA, 利用Real-time PCR从转录水平检测效应蛋白ExoS、ExoT和ExoY转录水平的变化。以ExoS为代表, 检测细胞内和分泌到细胞外效应蛋白的含量。收集铜绿假单胞菌PAO1、Δpfm和pfmC菌体内和分泌到细胞外的总蛋白, 利用ExoS多克隆抗体进行western杂交, 特异检测ExoS的蛋白水平。【结果】与野生型相比, Δpfm中exoS、exoT和exoY转录水平明显降低, 而pfmC中这3个蛋白的转录水平得到回补。Δpfm菌体内和分泌到细胞外的ExoS量均明显低于野生株PAO1, pfmC细胞内和细胞外分泌的ExoS蛋白量均得到恢复。【结论】铜绿假单胞菌基因pfm会影响三型分泌系统效应蛋白的水平。

Abstract:

[Objective] Detect the effect of gene pfm on type Ⅲ secretion system effectors. [Methods] Complementary strain pfmC was constructed. Total RNA was extracted from the wild type strain PAO1, mutant strain Δpfm and complementary strain pfmC respectively and real-time PCR was performed to detect the transcription levels of exoS, exoT and exoY. Further more, both total intracellular proteins and secreted proteins of strains PAO1, Δpfm and pfmC were collected, and detected against the representative effector ExoS by western blot. [Results] The results showed that transcription levels of exoS, exoT and exoY were significantly decreased in Δpfm compared to PAO1, and recovered in pfmC. Western blot showed that both total intracellular and secreted ExoS of Δpfm were significantly lower than that of PAO1 and recovered in pfmC. [Conclusion] In conclusion, gene pfm of Pseudomonas aeruginosa can affect type Ⅲ secretion system effectors.