EvpP对迟缓爱德华氏菌生物学特性及感染巨噬细胞的影响

Impacts of EvpP on the biological characteristics of Edwardsiella tarda and infection of macrophages

摘要:

VI型分泌系统(type VI secretion system, T6SS)是迟缓爱德华氏菌( Edwardsiella tarda)的一种新型毒力因子。迟缓爱德华氏菌毒力蛋白P ( E. tarda virulence protein P, EvpP)是T6SS的效应蛋白，但目前对EvpP功能机制的研究仍十分有限。 目的 解析EvpP的生物学功能，深入探究T6SS在 E. tarda致病过程中的作用。 方法 构建 E. tarda的 evpP基因缺失株(Δ evpP)和回补株(Δ evpP-C)，在此基础上开展 evpP基因缺失对 E. tarda生物学特性以及巨噬细胞感染影响的研究。 结果 E. tarda野生株、Δ evpP和Δ evpP-C在生长曲线和生理生化特性方面并无显著差异。相较于野生株，Δ evpP的运动性、生物被膜生成能力以及感染RAW264.7巨噬细胞的黏附率、胞内增殖率和触发细胞自噬的能力均显著下降，但能诱发巨噬细胞分泌更多的肿瘤坏死因子α (tumor necrosis factor-α, TNF-α)。Δ evpP-C的胞内增殖能力未完全恢复至野生株水平，但其他表型均得到完全恢复。 结论 EvpP不影响 E. tarda的生长和生理生化特性，但能不同程度地增强该菌的运动性、生物膜形成能力以及对巨噬细胞的黏附、胞内增殖能力和自噬活性，且能抑制 E. tarda感染的巨噬细胞分泌更多TNF-α。研究结果进一步证实EvpP参与 E. tarda 的致病过程，并在其中发挥重要作用。

Abstract:

Objective The type VI secretion system (T6SS) is a novel virulence factor of Edwardsiella tarda. E. tarda virulence protein P (EvpP) is an effector of the T6SS. To date, the functional mechanisms of EvpP are still poorly understood. This study aimed to comprehensively investigate the biological functions of EvpP and elucidate the roles of T6SS in the pathogenicity of E. tarda. Methods We constructed the evpP-deleted mutant (Δ evpP) and complemented strain (Δ evpP-C) to study the effects of evpP deletion on the biological characteristics of E. tarda and infection in macrophages. Results No significant differences were observed in the growth curves or physiological and biochemical properties among the wild-type (WT), Δ evpP, and Δ evpP-C. However, compared with WT, Δ evpP exhibited significantly reduced motility, biofilm formation, adhesion rate to RAW264.7 macrophages, intracellular proliferation capacity, and ability to induce host cell autophagy, while triggering increased secretion of tumor necrosis factor-α (TNF-α) by macrophages. The complementation of evpP-C did not fully restore the intracellular proliferation capability, but completely rescued the other phenotypic defects. Conclusion EvpP does not affect the growth or physiological and biochemical properties of E. tarda. However, it could enhance the bacterial motility, biofilm formation, adhesion to macrophages, intracellular proliferation, and autophagy, while suppressing TNF-α secretion in E. tarda-infected macrophages. These findings confirm that EvpP plays a critical role in the pathogenicity of E. tarda.