鸭源鸡杆菌ompW、gtxA双基因缺失株的构建及其相关特性分析

Construction and characterization of Gallibacterium anatis ΔompWΔgtxA double deletion strain

摘要:

鸭源鸡杆菌（Gallibacterium anatis，G.anatis）是家禽中常见的条件性致病菌，主要引起蛋鸡生殖道疾病，造成产蛋量下降。[目的] 为探究RTX样毒素GtxA及外膜蛋白（OmpW）对鸭源鸡杆菌生物学特性和致病力的影响。[方法] 本研究采用自然转化法对突变株RZΔompW进一步缺失gtxA构建突变株RZΔompWΔgtxA，通过分析其生长特性、黏附能力、引起细胞凋亡程度及对小鼠致病性等，探究其与生物学特性及致病性可能的关系。[结果] 结果显示，RZΔompWΔgtxA能稳定遗传gtxA的缺失；单双基因突变株溶血活性均消失、与RZ株相比菌落形态及生长速率并未出现显著改变；相比RZ、RZΔompW和RZΔgtxA，双基因缺失株RZΔompWΔgtxA在不同时段对鸡原代输卵管上皮细胞的黏附能力显著降低（P<0.05），诱导鸡输卵管上皮原代细胞发生凋亡的能力明显减弱，对小鼠的致病力显著降低。[结论] GtxA毒素和外膜蛋白OmpW在鸭源鸡杆菌毒力、黏附宿主细胞及诱导其细胞凋亡中起重要作用，且可能存在明显的协同关系。本研究为鸭源鸡杆菌感染机制的阐明奠定基础。

关键词:鸭源鸡杆菌;RZΔompWΔgtxA缺失株;生物学特性;致病性

Abstract:

Gallibacterium anatis is a conditional pathogen common in poultry production, and causes the laying hen genital tract diseases, resulting in decreased egg production. [Objective] To understand the biological characteristics and pathogenicity of the virulence factor RTX-like toxin GtxA and outer membrane protein W of G. anatis. [Methods] Based on the G. anatis OmpW gene deletion strain, we successfully constructed the OmpWgtxA double gene deletion strain by natural transformation. The differences in biological characteristics and virulence between single and double gene deletion strains and parental strain were investigated. [Results] The hemolytic activity of the OmpWgtxA double gene deletion strain was inactivated, while the deletion was stable in vitro and in vivo. Compared with gtxA, ompW single gene deletion strain and parental strain Yu-PDS-RZ-1-SLG, the growth of the double gene deletion strain was not significantly altered, while its adhesion ability and lesions of primary chicken oviduct epithelial cells were decreased to some extent; and its ability to induce apoptosis in cells of chicken oviduct epithelium was lessened; its pathogenicity was also reduced. [Conclusion] GtxA toxin and outer membrane protein W were suggested to play important roles in G. anatis virulence, adhesion to host cells and induction of apoptosis and there may be synergies. This study laid the foundation for further revealing the pathogenesis of G. anatis.

Key words:Gallibacterium Anatis;RZΔompWΔgtxA gene deletion strain;biological character;pathogenic