Title:Novel dual-regulators of Pseudomonas aeruginosa are essential for productive biofilms and

virulence

题目：铜绿假单胞菌的新型双调控因子调控生物被膜和毒力的分子机制研究

Journal：Molecular microbiology(IF=3.97)

Date：2018

Objection:

To present a novel transcriptional regulator named PA1226 could modulate biofilm formation and virulence in P. aeruginosa.

Method:

According to a series experiments such as Transcriptome analysis,ChIP-seq,electrophoretic mobility shift assay,co-immunoprecipitation assay,EMSA and Localted Surface Plasmon Resonance(LSPR) to study the function of PA1226 in P. aeruginosa.

RESULTS:

1. Mutation in the gene encoding this regulator abolished the ability of P. aeruginosa to produce biofilms in vitro, without any effect on the planktonic growth.

2. Transcriptome analysis revealed that PA1226 regulates many essential virulence genes/pathways, including those involved in alginate, pili, and LPS biosynthesis.

3. Genes/operons directly regulated by PA1226 and potential binding sequences were identified via ChIP-seq. Attempts to confirm the binding sequences by electrophoretic mobility shift assay led to the discovery of a co-regulator, PA1413, via co-immunoprecipitation assay.

4. Genes/operons directly regulated by PA1226 and potential binding sequences were identified via ChIP-seq.

本研究为了证明转录调控因子PA1226可调节铜绿假单胞菌的生物被膜形成和毒力。

夏威夷大学微生物学系的研究人员通过PA1226缺失突变体性状研究，转录组分析，结合EMSA，免疫共沉淀和局域表面等离子体共振( LSPR )进行调控因子-共调控因子- DNA的分子互作定性及定量研究。

1. 研究结果表明，将该调控因子编码基因的突变后，铜绿假单胞菌在体外产生生物被膜的能力丧失，对浮游生长没有任何影响。

2. 转录组分析表明，PA1226调控许多重要的毒力基因/途径，包括参与褐藻胶、菌毛和LPS生物合成的基因/途径。

3.除了通过EMSA定性确认调控因子-共调控因子- DNA结合外，还通过局域表面等离子体共振( LSPR )来定量PA1226与DNA的KD值为0.31uM，另外利用LSPR技术检测到两个生物分子之间的结合和解离速率分别为Kon = 1.47 x 104 M-1S-1，Koff = 0.0045 ± 0.0004 S-1。

4. 最后通过ChIP - seq鉴定了PA1226直接调控的基因/操纵子和潜在的结合序列。

本研究发现PA1226和PA1413协同调控生物被膜的形成和多种重要毒力因子的表达。双调控因子PA1226 - PA1413下调参与菌毛/菌毛生物合成的基因表达，同时激活产生荧光铁载体、LPS和褐藻胶的基因。(生物网 www.mamobio.com)

Alm, R.A., Bodero, A.J., Free, P.D., and Mattick, J.S. (1996) Identification of a novel gene, pilZ, essential for type 4 fimbrial biogenesis in Pseudomonas aeruginosa. J Bacteriol 178: 46–53