Li Hui, Fang Qingjian, Zhang Shanshan, Wang Yan, Ma Xiang, Hu Yonghua, Sui Zhihai, Gu Hanjie
School of Life and Health, Hainan University, Haikou, 570228, China; Institute of Tropical Bioscience and Biotechnology & Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences, Sanya, 572024, China.
Institute of Tropical Bioscience and Biotechnology & Sanya Research Institute, Chinese Academy of Tropical Agricultural Sciences, Sanya, 572024, China.
Microb Pathog. 2025 Oct;207:107915. doi: 10.1016/j.micpath.2025.107915. Epub 2025 Jul 16.
Edwardsiella piscicida is a serious aquatic pathogen that infects a diverse spectrum of economically valuable fish species, inflicting substantial economic damage on the aquaculture industry. DegP, a periplasmic protein endowed with dual chaperone-protease activity, is vital for bacteria to preserve homeostasis and acclimate to environmental stresses. Nevertheless, studies about DegP in E. piscicida have been relatively limited. In this study, we determined that the DegP of E. piscicida shared high sequence identity with DegP proteins from diverse bacterial species and comprised conserved functional domains. The azocasein hydrolysis assays showed that the recombinant protein rDegP exhibited significant proteolytic activity in a substrate concentration-dependent manner with the optimal enzyme activity at 48 °C and pH 9.0. To probe the impact of DegP on stress adaptation and virulence, we constructed an in-frame deletion strain, TH1ΔdegP, through homologous recombination. Under normal conditions, TH1ΔdegP exhibited no discernible growth defects. However, TH1ΔdegP caused the significantly decreased survival in various forms of adversity including high temperature, alkalinity, acidity, oxidation stress, and high osmolarity. Additionally, TH1ΔdegP impaired the motility, but had no effect on biofilm formation and cell membrane integrity. Pathogenicity assays revealed that TH1ΔdegP significantly reduced adhesion to FG cells and impaired intracellular proliferation in RAW264.7 macrophages in vitro. Moreover, TH1ΔdegP displayed decreased resistance to tilapia serum, diminished capacity for tissue colonization, and attenuated overall virulence towards tilapia. The complementary strain TH1ΔdegPC successfully restored all the lost capabilities of TH1ΔdegP. To clarify the regulatory mechanism of DegP, the EMSA, β-galactosidase detection and qRT-PCR demonstrated that the Cpx envelope stress system regulated the expression of degP through direct binding of CpxR to the degP promoter. These findings underscore that DegP, a protease regulated by the Cpx system, is indispensable for the adaptation to environmental stresses and virulence in E. piscicida. This study provides a comprehensive elucidation of the multifaceted roles of DegP and enhances our comprehension of its contributions to stress tolerance and pathogenicity in E. piscicida.
杀鱼爱德华氏菌是一种严重的水生病原菌，可感染多种具有经济价值的鱼类，给水产养殖业造成巨大经济损失。DegP是一种具有双重伴侣-蛋白酶活性的周质蛋白，对细菌维持体内平衡和适应环境压力至关重要。然而，关于杀鱼爱德华氏菌中DegP的研究相对有限。在本研究中，我们确定杀鱼爱德华氏菌的DegP与来自不同细菌物种的DegP蛋白具有高度的序列同一性，并包含保守的功能域。酪蛋白水解试验表明，重组蛋白rDegP在底物浓度依赖性方式下表现出显著的蛋白水解活性，最佳酶活性在48°C和pH 9.0。为了探究DegP对应激适应和毒力的影响，我们通过同源重组构建了一个框内缺失菌株TH1ΔdegP。在正常条件下，TH1ΔdegP没有明显的生长缺陷。然而，TH1ΔdegP在包括高温、碱性、酸性、氧化应激和高渗透压等各种逆境中的存活率显著降低。此外，TH1ΔdegP损害了运动能力，但对生物膜形成和细胞膜完整性没有影响。致病性试验表明，TH1ΔdegP显著降低了对FG细胞的粘附，并损害了RAW264.7巨噬细胞在体外的细胞内增殖。此外，TH1ΔdegP对罗非鱼血清的抵抗力降低，组织定植能力减弱，对罗非鱼的总体毒力减弱。互补菌株TH1ΔdegPC成功恢复了TH1ΔdegP所有丧失的能力。为了阐明DegP的调控机制，电泳迁移率变动分析、β-半乳糖苷酶检测和定量逆转录-聚合酶链反应表明，Cpx包膜应激系统通过CpxR直接结合到degP启动子上来调节degP的表达。这些发现强调，DegP是一种受Cpx系统调控的蛋白酶，对杀鱼爱德华氏菌适应环境压力和毒力不可或缺。本研究全面阐明了DegP的多方面作用，并增强了我们对其在杀鱼爱德华氏菌应激耐受性和致病性方面贡献的理解。
