鸡白痢沙门菌htrA基因缺失株的构建及其相关生物学特性研究

Construction and biological feature identification of htrA knock-out mutant of Salmonella pullorum

为研究丝氨酸蛋白酶HtrA对鸡白痢沙门菌在巨噬细胞内生存的影响,本研究利用λ-Red同源重组系统构建了鸡白痢沙门菌(ATCC19945) htra基因缺失突变株(ATCC19945Δhtra),通过测定其巨噬细胞内存活能力、应激条件实验,探究Htr A蛋白在鸡白痢沙门菌致病过程中的作用。结果显示,缺失株ATCC19945Δhtra与野生型菌株和回复菌株相比,其生长速度、生化特性无明显差异,但其巨噬细胞内存活能力显著降低。应激环境试验结果表明,htra基因缺失后鸡白痢沙门菌对pH、热和H_2O_2氧化应激的敏感性增加,在蛋清中生存能力下降,生物被膜形成能力显著下降。本研究为进一步阐释鸡白痢沙门菌的胞内存活机制、疫苗开发奠定基础。

To investigate the roles of HtrA protein played for intracellular survival of Salmonella in macrophage, the htrA gene deleted Salmonella pullorum was constructed with k-Red homologous recombination, followed by further assay of the bacterial intracellular survival in macrophage, biochemical characteristics and stress response. The results showed that HtrA knocked-out had no impact on growth and biochemical characteristics of S.pullorum strain ATCC 19945. While compared to the parent strain, the HtrA knock-out mutant displayed sharply impaired survival in macrophage and the persistence to acidic, alkalic, overheated and H202 treament. Moreover, the HtrA knock-out mutant reduced the ablities of biofilm formation and survival in egg albumen. In this study, a HtrA knock-out mutant of S.pullorum was successfully generated, providing a guidance and foundation for mechanism investigation of S.pullorum intracellular infection and vaccine development.