美人鱼发光杆菌美人鱼亚种dly基因缺失株构建及其生物学特性研究

Construction and biological characteristics of the dly-deleted mutant strain of Photobacterium damselae subsp

关键词:  美人鱼发光杆菌美人鱼亚种  缺失株  胞外产物  生物学特性  致病性

Key words:  Photobacterium damselae subsp. damselae  Mutant strain  Extracellular products  Biological characteristics  Pathogenicity

摘要:

美人鱼发光杆菌美人鱼亚种(Photobacterium damselae subsp. damselae, PDD)是广泛分布于全球海洋环境中的一种致病菌。本研究以实验室保存的一株高致病性PDD菌株(PDD1608)作为对象，初步探究毒力基因dly对PDD菌株的生物学特性和致病力的影响。利用λRed重组技术成功构建毒力基因dly缺失株Δdly PDD1608::Cm，比较野生株和缺失株的生长、涌动性、药物敏感性、生理生化特性、生物被膜形成能力、菌株及胞外产物(extracellular products, ECP)的溶血性和磷脂酶活性等生物学特性。选用海水青鳉鱼(Oryzias melastigma)作为实验动物，通过人工感染实验测定野生株和缺失株及其ECP对海水青鳉鱼的致病性。结果显示，毒力基因dly缺失后导致PDD菌株生长变慢，涌动性、溶血性和磷脂酶活性均降低；野生株和缺失株的药物敏感性和生理生化特性未产生变化；与野生株相比，缺失株的生物被膜形成能力有显著差异(P<0.05)；人工感染实验表明，缺失株及其ECP对海水青鳉鱼的致病性降低。毒力基因dly影响PDD菌株的生长、涌动性、溶血性和磷脂酶活性等多种生物学特性，并且与PDD菌株及其ECP的致病力强弱密切相关。

Abstract:

As a pathogen, Photobacterium damselae subsp. damselae (PDD) is distributed widely in the marine environment, and the host species of PDD are diverse. Although in the past decade the number of reports on the pathogenicity of PDD to marine animals has gradually increased, it is necessary to study this further in order to establish targeted prevention and technical control measures to reduce harm to marine organisms. In this study, a high-virulence PDD strain (PDD1608) was selected to explore the effect of the virulence gene dly on the biological characteristics and pathogenicity. The dly-deleted mutant strain Δdly PDD1608::Cm was successfully constructed using the λRed recombination technique. The biological characteristics of the wild-type and mutant strains were compared, including growth, swarming motility, drug susceptibility, physiological and biochemical characteristics, biofilm formation ability, and hemolytic and phospholipase activity of extracellular products (ECP). Meanwhile, Oryzias melastigma was used as the target host, and the pathogenicity of wild-type and mutant strains and their ECPs to O. melastigma was detected by the artificial infection test. There was a positive correlation between the results of the bacterial solution and ECP challenge, indicating that deletion of the dly gene affects the virulence of the PDD strain. Deletion of the virulence gene dly resulted in slower growth of the PDD mutant strain; reduced swarming and hemolytic and phospholipase activity; no change in the drug susceptibility or physiological and biochemical characteristics of the wild type and mutant strains; compared with the wild type strain, significant different (P<0.05) biofilm formation ability of the mutant strain; the artificial infection test showed that the pathogenicity of the mutant strain and its ECP decreased. The results of this study revealed that the virulence gene dly affects many biological characteristics of PDD strains, such as growth, swarming motility, and hemolytic and phospholipase activity, and it was found to be closely related to the pathogenicity of the PDD strain and ECP. The results of this study contribute to further study of the influence of the virulence gene dly on the biological characteristics of the PDD strain in order to determine the appropriate prevention and control measures to curtail its spread and infection. The artificial infection test of the model organism O. melastigma helps provide an animal reference model for further exploration of the pathogenic mechanism and infection process of the PDD strain on the host. The results of the ECP experiments provide a theoretical reference for the research and development of PDD subunit vaccines.