质粒介导的美人鱼发光杆菌美人鱼亚种理化特性与毒力差异

Physiological and biochemical characteristics and virulence of Photobacterium damselae subsp. damselae mediated by a plasmid

摘要:

【背景】美人鱼发光杆菌美人鱼亚种(Photobacterium damselae subsp. damselae, PDD)是一种可导致多种海洋生物患病的病原菌，比较基因组测序分析表明，具有高致病性的PDD菌株内携带大质粒pPDD1608，基因注释分析表明该质粒携带多种毒力基因和分泌系统相关基因，而对于其功能机制缺乏报道。【目的】以一株高致病性PDD为研究对象，通过诱导缺失突变获得质粒缺失菌株，比较分析野生菌株与质粒缺失株的表型与致病性相关特征差异，明确该质粒对PDD理化表型和致病力的影响。【方法】建立变温-SDS质粒去除方法，构建菌株PDD1608的质粒缺失株ΔpPDD1608；利用TSB固体培养基及扫描电镜观察分析菌株PDD1608与ΔpPDD1608的表观形态特征；通过细菌微量生化鉴定管测定菌株PDD1608与ΔpPDD1608的生理生化表型；利用K-B纸片扩散法测定菌株PDD1608与ΔpPDD1608的药物敏感性；使用指示培养基测定菌株PDD1608与ΔpPDD1608的体外磷脂酶活性和溶血性；通过注射感染许氏平鲉测定菌株PDD1608与ΔpPDD1608的致病力。【结果】变温-SDS法可有效去除菌株PDD1608的大质粒，并基于全基因组序列信息建立了特异位点PCR质粒去除效果检测方法；理化检测结果表明，质粒缺失株与野生株的药物敏感性与生理生化特性并无差异，但质粒缺失株的溶血性与磷脂酶活性显著降低；注射感染试验表明，野生株对许氏平鲉表现为高致病性，实验鱼在24 h内全部死亡，而质粒缺失株对许氏平鲉致病性较低，24 h死亡率仅为56.6%。【结论】通过对野生株与质粒缺失株溶血能力、磷脂酶活性及对许氏平鲉致病力的研究证实，毒力质粒pPDD1608是介导菌株PDD1608具有高致病力的重要元件。为后续深入分析该毒力质粒及染色质介导的PDD致病和毒力蛋白分泌机制提供了技术支撑与理论基础。

Abstract:

[Background] Photobacterium damselae subsp. damselae (PDD) can infect a variety of marine animals to cause diseases. Comparative genome analysis revealed that PDD strains harboring a large plasmid pPDD1608 exhibit strong pathogenicity. Furthermore, gene annotation analysis unveiled the presence of virulence genes and secretion system-related genes in this plasmid. However, there is a dearth of studies examining the functions of this plasmid. [Objective] In this study, a highly pathogenic PDD strain was selected and its plasmid-deleted mutant was generated through induced mutagenesis. The phenotypic characteristics and pathogenicity were compared between the wild type and the mutant to reveal the role of this plasmid in the phenotype and pathogenicity of PDD. [Methods] The plasmid-deleted strain ΔpPDD1608 was successfully constructed by the temperature variation-SDS method. Then, the phenotypes of PDD1608 and ΔpPDD1608 cultured on the TSB plates were observed by scanning electron microscopy. The physiological and biochemical characteristics were measured by the microbiochemical tests and compared between PDD1608 and ΔpPDD1608. The antibiotic susceptibility of PDD1608 and ΔpPDD1608 was determined by the K-B disk diffusion method. The phospholipase and hemolytic activities of the two strains were measured by the indicator media in vitro. The pathogenicity of PDD1608 and ΔpPDD1608 was assessed through the artificial challenge test by injection in Sebastes schlegeli. [Results] The large plasmid of PDD1608 was effectively eliminated by the temperature variation-SDS method, and a gene-specific PCR method was established based on the whole genome sequence of PDD1608. The experimental results revealed no discernible disparities in antibiotic susceptibility or physiological and biochemical characteristics between PDD1608 and ΔpPDD1608. However, ΔpPDD1608 showed reduced hemolytic and phospholipase activities. Furthermore, the artificial challenge test demonstrated that PDD1608 displayed higher pathogenicity towards S.schlegeli, resulting in the death of all the tested fish within 24 h, whereas ΔpPDD1608 exhibited lower pathogenicity towards S.schlegeli, causing a 24-h mortality rate of 56.6%. [Conclusion] The comparison on hemolytic and phospholipase activities and pathogenicity between ΔpPDD1608 and PDD1608 confirmed that the plasmid pPDD1608 played a crucial role in mediating the high pathogenicity of PDD1608.