猪支气管败血波氏杆菌hcp基因缺失株构建及其生物学特性研究

Construction and characterization of an hcp mutant of swine Bordetella bronchiseptica

【目的】构建猪支气管败血波氏杆菌(Bordetella bronchiseptica,Bb)Ⅵ型分泌系统(T6SS)溶血素共调节蛋白hcp基因缺失株,并对其基本生物学特性进行初步的研究。【方法】使用自杀性质粒介导同源重组的方法敲除猪支气管败血波氏杆菌QH0814菌株hcp基因,并比较hcp基因缺失前后,菌体对细胞的黏附入侵、小鼠毒力及组织载菌量上的差异。【结果】成功构建支气管败血波氏杆菌hcp基因缺失株QH0814Δhcp,连续传50代且遗传稳定;缺失株与亲本株生长无明显差异;缺失株的黏附能力与亲本株差异不显著,但入侵能力显著降低(P<0.05);与亲本株相比,半数致死量提高,同时,缺失株对昆明鼠的感染能力也显著降低(P<0.05)。【结论】hcp基因的缺失对支气管败血波氏杆菌增殖无影响,但缺失后其入侵能力和定殖能力显著降低,由此推测hcp基因与支气管败血波氏杆菌的入侵和定殖相关。

[Objective] We constructed Bordetella bronchiseptica QH0814 hcp mutant to characterize its pathogenicity. [Methods] Through the homologous recombination mediated by a suicide plasmid pRE112, we acquired the mutant QHO814Ahcp successfully. Then, we evaluated the growth condition, the ability of adhesion and invasion, the median lethal dose （LDs0） and the infection capacity. [Results] There was no significant variation of the growth rate between the mutant and the parental strain. Compared with the parental strain, the adherence ability of the mutant did not change remarkably. However, the invasion ability decreased significantly. Mice lethal test showed that the LDso of the mutant was higher than that of the parental strain. Correspondingly, the bacterial colonization of the mutant in mice blood, lung and liver was much less than that of the parental strain. [Conclusion] The knocking-out of the hop gene had no influence on bacterial growth, but it could attenuate significantly the invasion and colonization of the bacterium. Therefore, the gene may play a role in the pathogenesis of Bordetella bronchiseptica.