Gac系统中gacS基因的缺失对东湖假单胞菌HYS毒性的影响

Effect of gacS Gene Deletion in Gac System on Toxicity of Pseudomonas donghuensis HYS

细菌致病因子是细菌毒性的重要部分。Gac系统是细菌的一种全局性调控系统,调控着细菌多种毒性因子的产生。在前期研究中,首次从武汉东湖水域分离得到一株具备高产铁载体能力的革兰氏阴性菌--东湖假单胞菌(Pseudomonas donghuensis)HYS菌株。为了探索Gac系统对于HYS毒性的影响,首先通过氨基酸序列Blast P比对以定位HYS中Gac系统Gac S蛋白的位置,经生物信息学分析,确定了HYS中存在Gac系统;再将来源于P.donghuensis HYS和铜绿假单胞菌(P.aeruginosa)PAO1的gac S片段分别通过基因克隆回补至HYS的gac S基因敲除株Δgac S中,并将获得的菌株喂食秀丽隐杆线虫,通过比较线虫的生存数量和生存天数来判断菌株毒性的强弱。结果表明,Δgac S菌株毒性明显减弱,而2株回补菌株对线虫的毒性均有不同程度的恢复,说明HYS中的Gac系统参与了毒性调控途径,且P.aeruginosa PAO1的gac S片段也能在HYS中发挥一定的毒性调控作用。为了进一步探索Δgac S菌株的减毒特性是否与HYS中毒力因子的丧失有关,利用脱脂牛奶平板检测菌株胞外蛋白酶的活性,并利用泳动运动平板、群集运动平板、蹭行运动平板检测菌株的运动能力,同时还对脂多糖、氢氰酸等代谢产物进行了检测。结果表明,相比于HYS,Δgac S菌株的蛋白酶分泌完全消失,且3种运动能力相比HYS均显著下降(P<0.05)。此外,ΔgacS菌株的胞外脂多糖与氢氰酸的产量相比野生型HYS菌株也有不同程度的下降。而这些毒力因子表型在回补菌株Δgac S/p BBR2-gac SHYS中均可恢复至野生型水平,且回补PAO1的同源gac S基因甚至可在一定程度上回补gac S的正调控毒性物质的功能。研究表明,潜在的动物致病菌--P.donghuensis HYS通过Gac系统正调控产生毒性因子,从而发挥对秀丽隐杆线虫的致死效应,这为将来生物体抵御HYS感染提供了理论基础。

Bacterial pathogenic factors are an important part of bacterial toxicity.The Gac system is a global regulatory system of bacteria that regulates the production of various virulence factors in bacteria.In previous studies,a gram-negative bacterium,siderophore high-yielding Pseudomonas donghuensis(HYS),was isolated from the waters of East Lake in Wuhan for the first time.In order to explore the effect of Gac system on HYS toxicity,firstly,the position of Gac S protein from Gac system in HYS was located through amino acid sequence Blast P alignment.Through bioinformatics analysis,Gac system was determined to exist in HYS.Then gac S fragments derived from P.donghuensis HYS and P.aeruginosa PAO1 were respectively supplemented into gac S gene knockout HYS strainΔgac S through gene cloning,and the obtained strains were fed to Caenorhabditis elegans,and the toxicity of the strains were estimate by comparing the survival number and days of the nematodes.The results showed that the toxicity ofΔgac S strain was obviously weakened.However,the toxicity of the two complementary strains to nematodes recovered to different degrees,which indicated that Gac system in HYS participated in toxicity regulation pathway,and gac S fragment of P.aeruginosa PAO1 could also play a certain toxicity regulation role in HYS.In order to further explore whether the attenuated characteristics ofΔgac S strain were related to the loss of virulence factors in HYS,the activity of extracellular protease of the strain was detected by defatted milk plate,and the exercise capacity of the strain was detected by swimming motility plate,swarming motility plate and twitching motility plate.Meanwhile,metabolites such as lipopolysaccharide and hydrocyanic acid were also detected.The results showed that the protease secretion ofΔgac S strain completely disappeared compared with HYS,and the three kinds of exercise abilities ofΔgac S were significantly decreased compared with HYS(P<0.05).In addition,the yield of extracellular lipopolysaccharide ofΔgac S decreased to different degrees compared with that of wild type HYS.However,the phenotypes of these virulence factors could all be recovered to the wild type level in the complementary strainsΔgac S/p BBR2-gac SHYS,and the complementation of homologous gac S gene from PAO1 could even complement the function of gac S in regulating toxic substances to a certain extent.Studies have shown that the potential animal pathogenic bacteria,P.donghuesis HYS,was positively regulating and producing toxic factors through Gac system,thus exerting lethal effect on Caenorhabditis elegans,which provided theoretical basis for organisms to resist HYS infection in the future.