铜绿假单胞菌fur基因缺失突变株的构建及其表型分析

Construction and phenotypic characterization of fur-deleted mutant of Pseudomonas aeruginosa

摘要:

铁摄取调节蛋白(ferric uptake regulator, Fur)是控制铜绿假单胞菌铁代谢和毒力的关键调节因子。许多课题组尝试构建铜绿假单胞菌fur的缺失突变株均失败,因此铜绿假单胞菌的fur一直被认为是必需基因,这导致其生物学功能一直未得到全面的解析。【目的】构建铜绿假单胞菌fur的缺失突变株,并对该突变株的表型进行分析。【方法】以铜绿假单胞菌PAO1为亲本菌株,通过同源重组的方法构建fur缺失突变株,研究该基因对铜绿假单胞菌生长、铁载体生物合成、抗氧胁迫能力、鞭毛形成、生物被膜形成和毒力等的影响。同时,通过遗传分析对fur缺失突变株生长缺陷表型的原因进行探究。【结果】本研究成功构建了铜绿假单胞菌fur基因的缺失突变株,发现缺失突变fur极大地限制了铜绿假单胞菌的生长能力,并降低了该菌对限铁环境的生长适应性,但不影响该菌对高铁环境的生长适应性。铜绿假单胞菌Δfur的这种生长缺陷表型是细胞生长增殖变慢造成的,而不是诱导细胞死亡引起的。然而,其他异源的fur基因能完全互补Δfur的这种生长缺陷表型,暗示铜绿假单胞菌的Fur蛋白在功能上不存在独特性。尽管Fur与毒素-抗毒素系统PacTA存在功能关联性,但是铜绿假单胞菌Δfur的这种生长缺陷表型却与PacT毒素无关。除了影响铜绿假单胞菌的生长表型,缺失突变fur还使铜绿假单胞菌丧失了对铁载体生物合成的抑制作用,导致该菌对H₂O₂更敏感并丧失了鞭毛的形成能力,同时降低了该菌对大蜡螟幼虫的毒力。此外,缺失突变fur还显著提升了铜绿假单胞菌的胞内环二鸟苷酸(cyclic diguanylate, c-di-GMP)水平,从而诱导pelF和pslA基因的表达,进而促进铜绿假单胞菌生物被膜的形成。【结论】fur是可以缺失的非必需基因,在铜绿假单胞菌的正常生长、铁载体生物合成、抗氧胁迫能力、鞭毛形成、生物被膜形成和毒力等方面都发挥着十分重要的作用,这为针对铜绿假单胞菌的疫苗和抗菌药物开发奠定了基础。

Abstract:

Ferric uptake regulator (Fur) is a key regulatory factor of iron metabolism and virulence in Pseudomonas aeruginosa. Many research groups have failed to construct the fur-deleted mutant of P. aeruginosa, so fur has always been considered to be an essential gene in P. aeruginosa, and the knowledge of its biological function is limited. 【Objective】 This study aims to construct a fur-deleted mutant of P. aeruginosa and analyze its phenotypes. 【Methods】 With P. aeruginosa PAO1 as the parental strain, the fur-deleted mutant was constructed by homologous recombination. After that, we studied the effects of fur on the growth, siderophore biosynthesis, resistance to oxygen stress, flagella formation, biofilm formation, and virulence of P. aeruginosa. In addition, we explored the cause of the growth defect phenotype of the fur-deleted mutant by genetic analysis. 【Results】 The fur-deleted mutant of P. aeruginosa was successfully constructed. The deletion of fur greatly limited the growth of P. aeruginosa and reduced the growth adaptability of P. aeruginosa to the iron-limited environment, while it did not affect the growth adaptability of P. aeruginosa to the iron-rich environment. This growth defect phenotype of Δfur was caused by the slow cell growth and proliferation, rather than by cell death. Interestingly, heterologous fur could completely complement the growth defect phenotype of Δfur, suggesting that the Fur of P. aeruginosa was not functionally unique. Although there was a functional relationship between Fur and the toxin-antitoxin system PacTA, the growth defect phenotype of P. aeruginosa Δfur was not associated with PacT toxin. In addition to affecting the growth phenotype of P. aeruginosa, the deletion of fur also made P. aeruginosa lose the inhibitory effect on siderophore biosynthesis and the ability to form flagella and have increased sensitivity to H₂O₂ and reduced virulence to Galleria mellonella larvae. Moreover, the deletion of fur increased the intracellular cyclic diguanylate (c-di-GMP) level of P. aeruginosa to induce the expression of pelF and pslA, thereby promoting the biofilm formation of P. aeruginosa. 【Conclusion】 fur is a non-essential gene that can be deleted and plays a crucial role in the normal growth, siderophore biosynthesis, resistance to oxygen stress, flagellum formation, biofilm formation, and virulence of P. aeruginosa, which lays a foundation for the development of vaccines and agents against P. aeruginosa.