胸膜肺炎放线杆菌relA基因缺失株的构建及其生物学特性的研究

摘要

为探究信号分子4或5磷酸鸟苷[(p)ppGpp]对胸膜肺炎放线杆菌(APP)适应性及致病性影响,本研究以APP血清7型S8为亲本株,通过同源重组、卡那抗性及蔗糖负向筛选的方法,将(p)ppGpp的合成调控基因relA敲除,构建缺失突变株S8△relA.生物学特性鉴定结果表明,S8△relA具有良好的遗传稳定性,与亲本株增殖速度变化差异不显著,但平台期营养限制环境下,S8△relA存活能力降低,而且在Gly、Iie、Val营养缺失时,S8△rel生长能力显著抑制;S8△relA的持留菌形成能力、生物膜形成能力以及体外抗肺泡巨噬细胞(PAM)吞噬能力均有不同程度降低;对小鼠致病性试验结果显示,S8△relA相比亲本株S8毒力降低2.7倍.研究结果表明(p)ppGpp在环境胁迫下参与APP适应性调控,并对其毒力有一定的影响.本研究首次证明了(p)ppGpp作为一种信号分子,同样可以调控APP的生长,并且在宿主肺部弱营养环境条件下,APP与致病力相关的表型均受到影响,这为进一步阐明APP的致病机制提供实验依据.%To explore the effect of (p)ppGpp on the adaptability and pathogenicity of Actinobacillus pleuropneumoniae,the S8 △relA was constructed by deletion of (p)ppGpp synthesis regulator relA gene based on the A.pleuropneumoniae serotype 7(S8),using homologous recombination,kanamycin resistant characters and counter-selection of sucrose.The results of PCR and sequence showed that the S8 △relA was obtained successfully.The identification results suggested that the S8 △relA had high genetic stability and the growth rate did not significantly changed,while comparing with the parental strain,the viability of S8△relA was decreased on the static period.Simultaneously,the deficiency of the Gly,Ile,or Val in the medium,the growth ability of the S8△relA was dramatically inhibited.In addition,the ability of persisters formation,biofilm formation and the resistance to the phagocytosis by alveolar macrophages (PAM) in vitro all decreased in different degree.Animal experiment illustrated that the virulence of the S8△relA declined by 2.7 times compared with that of the parental strain S8 in mice.In conclusion,these results demonstrated that the (p)ppGpp regulated the adaptability of A.pleuropneumoniae and existed slight influence on virulence in the environmental stress.