SadC合成的c-di-GMP信号通过PilZ和FlgZ调节铜绿假单胞菌的泳动能力

Cyclic di-GMP produced by SadC regulates swimming motility of Pseudomonas aeruginosa via PilZ and FlgZ

摘要:

【目的】探究铜绿假单胞菌（Pseudomonas aeruginosa）鸟苷酸环化酶（diguanylate cyclase，DGC） SadC合成的环二鸟苷酸（cyclic di-GMP，c-di-GMP）信号与PilZ结构域受体间的信号传递关系，分析鉴定出特定PilZ结构域受体的调控功能和机制。【方法】SadC突变株和过表达菌株的构建及泳动能力分析；SadC过表达背景下，PilZ结构域受体突变各菌株的泳动表型分析和筛选；基因敲除和过表达解析筛选出的PilZ结构域受体功能；定点突变和遗传互补检测筛选出的PilZ结构域受体是否参与SadC合成c-di-GMP对泳动能力的调控。【结果】SadC通过影响鞭毛功能而非鞭毛形成抑制铜绿假单胞菌的泳动能力；PilZ结构域受体突变菌株筛选发现PilZ、FlgZ这2个受体参与了SadC介导的泳动能力抑制；功能分析发现ΔpilZ或ΔflgZ的泳动能力相比野生型PA14显著增强，而过表达PilZ或FlgZ则抑制了泳动能力；定点突变和回补实验发现PilZ第10位和FlgZ第140位氨基酸R对其介导SadC负调控泳动能力至关重要，多序列比对分析表明这些位点是其保守的c-di-GMP结合位点。【结论】SadC合成的c-di-GMP信号通过PilZ和FlgZ调控铜绿假单胞菌的泳动能力。

关键词:铜绿假单胞菌;SadC;环二鸟苷酸;PilZ结构域蛋白;PilZ;FlgZ;泳动能力

Abstract:

[Objective] To identify the PilZ domain-containing receptor(s) that sense the second messenger cyclic di-GMP (c-di-GMP) produced by the diguanylate cyclase SadC in Pseudomonas aeruginosa and investigate the functions and regulatory mechanisms of the identified receptor(s). [Methods] We constructed the strains in which sadC gene was deleted or overexpressed and tested their ability to swim by using a plate-based approach. We then added sadC in multicopy in each deletion mutant of the eight PilZ domain-containing receptors and screened for the mutants with alleviated swimming repression compared to the wild-type PA14 overexpressing SadC. For the mutations screened out, single gene knockout and overexpression strategies were used to explore the function of the identified receptor(s). Furthermore, site-directed mutagenesis and genetic complementation were employed to test whether the identified receptor's role in SadC-mediated swimming repression requires its c-di-GMP-binding motif. [Results] The SadC-mediated repression of swimming motility was associated with flagellar malfunction rather than flagellum formation. Two PilZ domain-containing receptors, PilZ and FlgZ, were identified to be involved in SadC-mediated swimming repression. The deletion of gene pilZ or flgZ increased the swimming motility, while overexpression of them significantly impaired swimming. A R10A substitution in the conserved c-di-GMP-binding motif of PilZ, or a R140A substitution in FlgZ, resulted in a variant that was no longer able to repress swimming in ΔpilZ or ΔflgZ overexpressing SadC, indicating that the conserved residue required for c-di-GMP binding is critical for PilZ or FlgZ to repress swimming in response to SadC-derived c-di-GMP. [Conclusion] PilZ and FlgZ are the effector relay proteins that respond to SadC c-di-GMP signaling to mediate swimming repression in P. aeruginosa.

Key words:Pseudomonas aeruginosa;SadC;cyclic di-GMP (c-di-GMP);PilZ domain-containing proteins;PilZ;FlgZ;swimming motility