Edwardsiellaictaluri是一种革兰氏阴性兼性细胞内鱼病原体,可引起cat鱼(ESC)的肠败血症。虽然各种分泌系统有助于艾氏大肠杆菌的毒力,VI型分泌系统(T6SS)仍然知之甚少。在这项研究中,我们通过重叠延伸PCR进行剪接,构建了13个伊氏大肠杆菌T6SS突变体,并对其进行了表征,评估它们在通道cat鱼(马尾Ictaluruspunctatus)腹膜巨噬细胞中的摄取和存活,通道鲶鱼卵巢(CCO)细胞的附着和侵袭,体外抗逆性,和毒力和功效的通道鲶鱼。在变种人中,EiΔevpA,EΔevpH,EΔevpM,EiΔevpN,和EiΔevpO在腹膜巨噬细胞内表现出减少的复制。EΔevpM,EiΔevpN,EiΔevpO显示对CCO细胞的附着显着降低,而EiΔevpN和EiΔevpO也显示出降低的CCO细胞侵袭(p<0.05)。总的来说,与基本培养基相比,T6SS突变体在营养丰富的培养基中表现出对氧化和硝化胁迫的抵抗力增强。然而,EiΔevpA,EΔevpH,EΔevpM,EiΔevpN,和EiΔevpO在营养丰富的培养基和基本培养基中均容易受到氧化应激。在鱼类挑战中,EΔevpD,EiΔevpE,EiΔevpG,EiΔevpJ,和EiΔevpK表现出减弱作用,并提供了有效的保护作用,以抵抗鲶鱼鱼种中的艾氏大肠杆菌野生型(EiWT)感染。然而,它们的减毒和保护效力在鲶鱼鱼苗中较低。这些发现揭示了T6SS在伊氏大肠杆菌发病机制中的作用,强调它在细胞内生存中的重要性,宿主细胞附着和侵袭,抗应力,和毒力。减毒的T6SS突变体有望成为cat鱼种保护性免疫策略的潜在候选者。
Edwardsiella ictaluri is a Gram-negative facultative intracellular fish pathogen causing enteric septicemia of catfish (ESC). While various secretion systems contribute to E. ictaluri virulence, the Type VI secretion system (T6SS) remains poorly understood. In this study, we constructed 13 E. ictaluri T6SS mutants using splicing by overlap extension PCR and characterized them, assessing their uptake and survival in channel catfish (Ictalurus punctatus) peritoneal macrophages, attachment and invasion in channel catfish ovary (CCO) cells, in vitro stress resistance, and virulence and efficacy in channel catfish. Among the mutants, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO exhibited reduced replication inside peritoneal macrophages. EiΔevpM, EiΔevpN, and EiΔevpO showed significantly decreased attachment to CCO cells, while EiΔevpN and EiΔevpO also displayed reduced invasion of CCO cells (p < 0.05). Overall, T6SS mutants demonstrated enhanced resistance to oxidative and nitrosative stress in the nutrient-rich medium compared to the minimal medium. However, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO were susceptible to oxidative stress in both nutrient-rich and minimal medium. In fish challenges, EiΔevpD, EiΔevpE, EiΔevpG, EiΔevpJ, and EiΔevpK exhibited attenuation and provided effective protection against E. ictaluri wild-type (EiWT) infection in catfish fingerlings. However, their attenuation and protective efficacy were lower in catfish fry. These findings shed light on the role of the T6SS in E. ictaluri pathogenesis, highlighting its significance in intracellular survival, host cell attachment and invasion, stress resistance, and virulence. The attenuated T6SS mutants hold promise as potential candidates for protective immunization strategies in catfish fingerlings.