flgK 对美人鱼发光杆菌美人鱼亚种生理生化和分子调控机制

Physio-biochemical and molecular regulation mechanism of flgK on Photobacterium damselae subsp. damselae

摘要:

美人鱼发光杆菌美人鱼亚种( Photobacterium damselae subsp. damselae, PDD)是一种广泛存在于海水中的致病菌，能够感染多种经济鱼类，对全球水产养殖业造成巨大经济损失。鞭毛基因 flgK可编码鞭毛钩蛋白FlgK，该蛋白对细菌鞭毛的正常形成至关重要。 目的 系统解析 flgK基因对PDD感染宿主毒力作用的影响机制。 方法 采用高效自杀质粒介导的同源重组方法构建PDD的 flgK基因缺失突变株(Δ flgK-PDD)，并通过基因测序证实突变成功。对Δ flgK-PDD在生物学特性、毒力基因表达及致病性等方面与野生株(WT-PDD)的差异进行比较分析。 结果 Δ flgK-PDD的生长能力、溶血活性、磷脂酶活性与WT-PDD无显著差异。然而，Δ flgK-PDD的运动能力和生物被膜形成能力较WT-PDD显著下降。透射电子显微镜观察显示，Δ flgK-PDD未能形成鞭毛结构。通过人工感染实验发现Δ flgK-PDD对许氏平鲉的LD ₅₀为WT-PDD的557%，致病性显著降低。实时荧光定量PCR结果进一步表明，与WT-PDD株相比，Δ flgK-PDD的鞭毛相关基因 fliK和 flgL，II型分泌系统(type II secretion system, T2SS)相关基因 gspC和 gspD以及毒力基因 hlyA _pl表达显著下调；而鞭毛相关基因 fliH，T2SS相关基因 gspE，外膜相关基因 ompP、 flhB和脂多糖相关基因 lapB表达显著上调，其余基因未见显著变化。 结论 flgK基因的突变可导致Δ flgK-PDD无法形成完整的鞭毛结构，并显著改变鞭毛相关基因的相对表达水平，从而降低其运动性和定殖能力，最终导致其致病性显著下降。

Abstract:

Photobacterium damselae subsp. damselae (PDD), a pathogenic bacterium widely found in seawater, can infect a variety of economic fish and cause huge economic losses to the global aquaculture industry. The flagellar gene flgK encodes the flagellar hook protein FlgK, which is essential for the normal formation of bacterial flagella. Objective To systematically analyze the influencing mechanism of flgK on the virulence of PDD. Methods The flgK-deleted mutant of PDD (Δ flgK-PDD) was constructed by homologous recombination mediated by a high-efficiency suicide plasmid, and the mutation was confirmed by gene sequencing. The biological characteristics, virulence gene expression, and pathogenicity were compared between Δ flgK-PDD and the wild-type strain (WT-PDD). Results There was no significant difference in the growth ability, hemolytic activity or phospholipase activity between Δ flgK-PDD and WT-PDD. However, the motility and biofilm formation of Δ flgK-PDD were significantly lower than those of WT-PDD. Transmission electron microscopy showed that Δ flgK-PDD failed to form a flagellar structure. The artificial infection experiments showed that the LD ₅₀ of Δ flgK-PDD in Sebastes schlegelii was 557% that of WT-PDD, and the pathogenicity was significantly reduced. Real-time quantitative PCR results showed that compared with WT-PDD, Δ flgK-PDD demonstrated significantly down-regulated expression of the flagellar-related genes fliK and flgL, the type II secretion system (T2SS)-related genes gspC and gspD, and the virulence gene hlyA _pl. The expression levels of flagellar-related gene fliH, T2SS-related gene gspE, outer membrane-related genes ompP, lapB, and flhB were significantly up-regulated, and those of the remaining genes did not change significantly. Conclusion The mutation of flgK can lead to the failure of Δ flgK-PDD to form a complete flagellar structure and significantly change the relative expression levels of flagellar-related genes, thereby reducing the motility and colonization ability and ultimately weakening the pathogenicity of PDD.