The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis.
The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis.
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DOI：
10.1186/s13567-018-0589-8
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发表时间：
2018-09-17
影响因子：
4.4
通讯作者：
Yu S
中科院分区：
文献类型：
--
作者：
Dou Y;Yu G;Wang X;Wang S;Li T;Tian M;Qi J;Ding C;Yu S
本研究通过随机筛选Tn4351转座子突变体库获得鸭疫里默氏菌突变株RA1062。酶联免疫吸附试验证明，该突变株与抗CH3脂多糖单克隆抗体不发生反应，并且其M949_RS01035基因失活。当在胰蛋白酶大豆肉汤中培养时，突变体RA1062的后期生长显着下降。突变体RA1062经结晶紫染色，呈现出粗糙的脂多糖表型，与野生型菌株CH3不同，表明M949_RS01035基因的缺失导致脂多糖缺陷。银染色和蛋白质印迹分析进一步证实，与野生型CH3菌株的脂多糖相比，RA1062脂多糖在梯状结合模式方面存在缺陷。此外，与野生型菌株 CH3 相比，突变体 RA1062 对补体依赖性杀伤的敏感性更高，细菌对 Vero 细胞的粘附和侵袭能力增强，血液细菌载量降低，并且感染鸭的毒力减弱。此外，RNA-Seq和实时聚合酶链式反应分析表明，突变体RA1062基因组中有两个基因上调，两个基因下调。此外，一项动物保护实验表明，用灭活的 RA1062 菌苗对鸭进行免疫可提供有效的交叉保护，以抵抗有毒力的鸭疫立克氏菌血清型 1、2 和 10 的攻击。这项研究提供了 M949_RS01035 基因参与鸭疫立克氏菌的细菌表型、毒力和基因调控的证据。突变株 RA1062 可用作交叉保护性候选疫苗。本文的在线版本 (10.1186/s13567-018-0589-8) 包含补充材料，可供授权用户使用。
In this study, the Riemerella anatipestifer mutant strain RA1062 was obtained by screening a random Tn4351 transposon mutant library. The mutant strain was unreactive with the anti-CH3 lipopolysaccharide monoclonal antibody, as demonstrated with an enzyme-linked immunosorbent assay, and its M949_RS01035 gene was inactivated. When cultured in trypticase soy broth, the late stage growth of the mutant RA1062 was significantly decreased. The mutant RA1062 was stained with crystal violet and presented a rough lipopolysaccharide phenotype, which differed from that of the wild-type strain CH3, suggesting that deletion of the M949_RS01035 gene resulted in defective lipopolysaccharide. Silver staining and Western blot analyses further confirmed that the RA1062 lipopolysaccharide had a deficiency in ladder-like binding pattern, as compared to lipopolysaccharide of the wild-type CH3 strain. In addition, the mutant RA1062 showed a higher susceptibility to complement-dependent killing, increased bacterial adhesion and invasion capacities to Vero cells, decreased blood bacterial loads, and attenuated virulence in infected ducks, when compared to the wild-type strain CH3. Moreover, RNA-Seq and real-time polymerase chain reaction analyses indicated that two genes were up-regulated and two were down-regulated in the mutant RA1062 genome. Furthermore, an animal protection experiment showed that immunization of ducks with inactivated RA1062 bacterin conferred effective cross-protection against challenge with the virulent R. anatipestifer serotypes 1, 2, and 10. This study presents evidence that the M949_RS01035 gene is involved in bacterial phenotype, virulence, and gene regulation in R. anatipestifer. The mutant strain RA1062 could be used as a cross-protective vaccine candidate. The online version of this article (10.1186/s13567-018-0589-8) contains supplementary material, which is available to authorized users.
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