胸膜肺炎放线杆菌自转运粘附素Apa1/Apa2基因敲除菌株的构建及其生物学特性研究

Construction and characterization of △Apa1/△Apa2 mutant of Actinobacillus pleuropneumoniae

为研究三聚体自转运粘附素(TAAs)对胸膜肺炎放线杆菌(APP)生物学特性的影响,本研究利用自杀性载体系统,通过正负向筛选标记的同源重组方法筛选APP血清5b型L20菌株的TAAs基因Apa1及Apa2的双基因敲除菌株,命名为△Apa1/△Apa2菌株,以野生菌株为对照,比较△Apa1/△Apa2菌株在体外的生物学特性变化,以小鼠为动物模型检测TAAs基因缺失对细菌毒力的影响。体外试验表明,△Apa1/△Apa2菌株在BHI液体培养基中生长速率与野生菌株无明显差异;电镜下观察△Apa1/△Apa2菌株菌体表面粗糙,有絮状物沉积;自凝、被膜形成能力下降;对RAW264.7细胞的粘附效率低于野生菌株。小鼠体内试验表明TAAs基因的缺失导致细菌毒力降低,在肺部和脾脏的定殖菌量明显低于野生菌株,然而两者的病理学差异不显著。本研究表明TAAs影响APP的形态、自凝、被膜形成等特性,并在APP粘附细胞以及对小鼠致病性方面发挥重要作用,为进一步研究TAAs在APP感染机制中的作用奠定了基础。

To further investigate the role of trimeric autotransporter adhesins （TAAs） in Actinobacillus pleuropneumoniae, a mutant of double genes deletion of A.pleuropneumoniae （△Apa1/△Apa2） was generated by homologous recombination utilizing high performance suicide vector system. The characterizations of the △Apa1/△Apa2 mutant and wild-type strains were compared in vitro and in mice. PCR confirmed the loss of TAAs genes in the △Apa1/△Apa2 mutant. The△Apa1/△Apa2 mutant showed growth deficiency in BHI media, rough and irregular surfaces; Bacterial aggregation and biofilm formation were decreased when compared to the wild-type strain. And in vitro adhesion rate, △Apa1/△Apa2 mutant reduced adherence to the RAW264.7 cells. For the mice, the virulence of the △pa1/△Apa2 mutant was reduced and showed the decrease ability to colonize in mice lungs and spleens, but no obvious difference in the histopathology of mice lungs were observed about different strains. This study illustrates the important role of TAAs in the bacterial growth, cell morphology, autoagglutination, biofilm formation, adherence, ability to colonize in mice. Further suggest a putative role of TAAs in the virulence of Actinobacillus pleuzopneumoniae.