State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Sciences, Hunan Normal University, Changsha, 410081, China.
Department of Nutrition, National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, 410008, China.
Fish Shellfish Immunol. 2022 Aug;127:530-541. doi: 10.1016/j.fsi.2022.06.068. Epub 2022 Jul 4.
Aeromonas hydrophila is a Gram-negative bacterial pathogen with a broad host range, including fish and humans. In this study, we examined the function of a secretory serine protease (named Ssp1) identified in pathogenic A. hydrophila CCL1. Ssp1 possesses a trypsin-like serine protease domain and contains two conserved PDZ domains. Recombinant Ssp1 protein (rSsp1) treatment increased intestinal permeability by downregulating and redistributing tight junction protein Occludin in intestinal Caco-2 cells in vitro. Western blot demonstrated that rSsp1 treatment in Caco-2 cells resulted in marked increases in the expressions of myosin light chain kinase (MLCK) and phosphorylated myosin light chain (p-MLC). For virulence analysis, an isogenic CCL1 mutant ΔSsp1 was created. ΔSsp1 bears an in-frame deletion of the Ssp1 gene. A live infection study in crucian carps showed that, compared to CCL1, ΔSsp1 infection exhibited increased Occludin expression, reduced intestinal permeability and tissue dissemination capacity, and attenuated overall virulence in vivo. However, ΔSsp1 showed no differences in the biofilm formation, swimming motility, and resistance to environmental stress. These lost virulence capacities of ΔSsp1 were restored by complementation with the Ssp1 gene. Global transcriptome analysis and quantitative real-time RT-PCR showed that compared to CCL1 infection, ΔSsp1 promoted the expressions of antimicrobial molecules (MUC2, LEAP-2, Hepcidin-1, and IL-22). Finally, CCL1 infection caused significant dysbiosis of the gut microbiota, including increased Vibrio and Deefgea compared to ΔSsp1 infected fish. Taken together, these results indicate that Ssp1 is essential for the virulence of A. hydrophila and is required for the perturbation of intestinal tight junction barrier.
嗜水气单胞菌是一种具有广泛宿主范围的革兰氏阴性细菌病原体，包括鱼类和人类。在本研究中，我们研究了在致病性嗜水气单胞菌 CCL1 中鉴定的一种分泌丝氨酸蛋白酶（命名为 Ssp1）的功能。Ssp1 具有胰蛋白酶样丝氨酸蛋白酶结构域，并包含两个保守的 PDZ 结构域。重组 Ssp1 蛋白（rSsp1）处理在体外通过下调和重新分布紧密连接蛋白 Occludin 增加了肠道 Caco-2 细胞的通透性。Western blot 表明，rSsp1 处理 Caco-2 细胞导致肌球蛋白轻链激酶（MLCK）和磷酸化肌球蛋白轻链（p-MLC）的表达显著增加。为了进行毒力分析，构建了一个同源 CCL1 突变体ΔSsp1。ΔSsp1 具有 Ssp1 基因的框内缺失。在鲫鱼的活体感染研究中，与 CCL1 相比，ΔSsp1 感染表现出 Occludin 表达增加、肠道通透性和组织扩散能力降低以及体内整体毒力减弱。然而，ΔSsp1 在生物膜形成、游泳运动和对环境胁迫的抵抗力方面没有差异。用 Ssp1 基因互补恢复了ΔSsp1 的这些丧失的毒力能力。全转录组分析和定量实时 RT-PCR 显示，与 CCL1 感染相比，ΔSsp1 促进了抗菌分子（MUC2、LEAP-2、Hepcidin-1 和 IL-22）的表达。最后，CCL1 感染导致肠道微生物群发生显著失调，与ΔSsp1 感染的鱼相比，增加了 Vibrio 和 Deefgea。总之，这些结果表明 Ssp1 是嗜水气单胞菌毒力所必需的，并且需要扰动肠道紧密连接屏障。
