Abstract：
Riemerella anatipestifer is a pathogenic bacterium that causes duck serositis and meningitis, leading to significant harm to the duck industry. To escape from the host immune system, the meningitis-causing bacteria must survive and multiply in the bloodstream, relying on specific virulence factors such as capsules. Therefore, it is essential to study the genes involved in capsule biosynthesis in R. anatipestifer. In this study, we successfully constructed gene deletion mutants Δ3820 and Δ3830, targeting the GE296_RS03820 and GE296_RS03830 genes, respectively, using the RA-LZ01 strain as the parental strain. The growth kinetics analysis revealed that these two genes contribute to bacterial growth. Transmission and scanning electron microscopy (TEM and SEM) and silver staining showed that Δ3820 and Δ3830 produced the altered capsules and compounds of capsular polysaccharides (CPSs). Serum resistance test showed the mutants also exhibited reduced C3b deposition and decreased resistance serum killing. In vivo, Δ3820 and Δ3830 exhibited markedly declining capacity to cross the blood-brain barrier, compared to RA-LZ01. These findings indicate that the GE296_RS03820 and GE296_RS03830 genes are involved in CPSs biosynthesis and play a key role in the pathogenicity of R. anatipestifer. Furthermore, Δ3820 and Δ3830 mutants presented a tendency toward higher survival rates from RA-LZ01 challenge in vivo. Additionally, sera from ducklings immunized with the mutants showed cross-immunoreactivity with different serotypes of R. anatipestifer, including 1, 2, 7 and 10. Western blot and SDS-PAGE assays revealed that the altered CPSs of Δ3820 and Δ3830 resulted in the exposure of some conserved proteins playing the key role in the cross-immunoreactivity. Our study clearly demonstrated that the GE296_RS03820 and GE296_RS03830 genes are involved in CPS biosynthesis in R. anatipestifer and the capsule is a target for attenuation in vaccine development.
摘要：
鼠疫Riemerella是一种引起鸭浆膜炎和脑膜炎的致病菌，对养鸭业造成重大危害。为了逃离宿主免疫系统，引起脑膜炎的细菌必须在血液中存活和繁殖，依赖于特定的毒力因子，如胶囊。因此,研究与厌食R.anatipestifer胶囊生物合成有关的基因至关重要。在这项研究中,我们成功构建了针对GE296_RS03820和GE296_RS03830基因的基因缺失突变体Δ3820和Δ3830，分别,使用RA-LZ01菌株作为亲本菌株。生长动力学分析表明，这两个基因有助于细菌生长。透射和扫描电子显微镜（TEM和SEM）以及银染表明，Δ3820和Δ3830产生了改变的胶囊和荚膜多糖（CPS）化合物。血清抗性测试显示突变体还表现出减少的C3b沉积和减少的抗性血清杀伤。在体内,Δ3820和Δ3830显示出穿过血脑屏障的能力明显下降，与RA-LZ01相比。这些发现表明，GE296_RS03820和GE296_RS03830基因参与CPSs的生物合成，并在抗药性R.anatipestifer的致病性中起关键作用。此外，Δ3820和Δ3830突变体在体内表现出更高的RA-LZ01攻击存活率的趋势。此外,用突变体免疫的鸭血清显示出与不同血清型的R.anatipestifer的交叉免疫反应性，包括1、2、7和10。Western印迹和SDS-PAGE测定表明，Δ3820和Δ3830的CPS改变导致一些保守蛋白的暴露在交叉免疫反应中起关键作用。我们的研究清楚地表明，GE296_RS03820和GE296_RS03830基因参与了厌食R.anatipestifer中的CPS生物合成，并且胶囊是疫苗开发中减毒的目标。
