Disruption of the M949_RS01915 gene changed the bacterial lipopolysaccharide pattern, pathogenicity and gene expression of Riemerella anatipestifer.
Disruption of the M949_RS01915 gene changed the bacterial lipopolysaccharide pattern, pathogenicity and gene expression of Riemerella anatipestifer.
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M949_RS01915基因的破坏改变了鸭疫里默氏菌的细菌脂多糖模式、致病性和基因表达
DOI：
10.1186/s13567-017-0409-6
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发表时间：
2017-02-06
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;Wang X;Yu G;Wang S;Tian M;Qi J;Li T;Ding C;Yu S
鸭疫里默氏杆菌是引起鸭败血症和血清渗出的重要病原菌。脂多糖被认为是鸭疫杆菌的主要毒力因子。为了鉴定与内毒素生物合成相关的基因，我们用抗鸭疫杆菌血清1型内毒素的单抗(抗内毒素单抗)筛选了Tn4351转座子的随机突变文库。获得了一株在间接酶联免疫吸附试验中失去反应性的突变株RA1067。Southern杂交和测序分析表明，在RA1067染色体DNA的M949_RS01915基因的116个碱基上插入了一个Tn4351。银染和Western印迹分析表明，RA1067脂多糖与野生型CH3脂多糖相比存在缺陷。与CH3相比，RA1067在TSB生长后期的生长速率显著降低。此外，与CH3相比，RA1067对补体依赖致死的敏感性更高，毒力降低360倍以上，对Vero细胞的细菌黏附和侵袭能力增加，血液细菌负荷显著降低。动物实验表明，灭活的RA1067细胞对鸭疫杆菌WJ4(血清1型)、Yb2(血清2型)和HXb2(血清10型)的攻击具有交叉保护作用，进一步证实了RA1067抗原性的改变。此外，RNA-Seq分析和实时定量聚合酶链式反应证实了RA1067中两个上调基因和三个下调基因。我们的研究结果表明，M949_RS01915基因与鸭疫杆菌的抗原性、致病性和基因调控有关。
Riemerella anatipestifer is an important pathogen that causes septicemia anserum exsudativa in ducks. Lipopolysaccharide (LPS) is considered to be a major virulence factor of R. anatipestifer. To identify genes involved in LPS biosynthesis, we screened a library of random Tn4351 transposon mutants using a monoclonal antibody against R. anatipestifer serotype 1 LPS (anti-LPS MAb). A mutant strain RA1067 which lost the reactivity in an indirect ELISA was obtained. Southern blot and sequencing analyses indicated a single Tn4351 was inserted at 116 bp in the M949_RS01915 gene in the RA1067 chromosomal DNA. Silver staining and Western blot analyses indicated that the RA1067 LPS was defected compared to the wild-type strain CH3 LPS. The RA1067 displayed a significant decreased growth rate at the late stage of growth in TSB in comparison with CH3. In addition, RA1067 showed higher susceptibility to complement-dependent killing, more than 360-fold attenuated virulence based on the median lethal dose determination, increased bacterial adhesion and invasion capacities to Vero cells and significantly decreased blood bacterial loads in RA1067 infected ducks, when compared to the CH3. An animal experiment indicated that inactivated RA1067 cells was effective in cross-protecting of the ducks from challenging with R. anatipestifer strains WJ4 (serotype 1), Yb2 (serotype 2) and HXb2 (serotype 10), further confirming the alteration of the RA1067 antigenicity. Moreover, RNA-Seq analysis and real-time PCR verified two up-regulated and three down-regulated genes in RA1067. Our findings demonstrate that the M949_RS01915 gene is associated to bacterial antigenicity, pathogenicity and gene regulation of R. anatipestifer.