Edwardsiella piscicida (formerly included in E. tarda) is the leading pathogen threatening worldwide fresh and seawater aquaculture industries and has been considered as a model organism for studying intracellular and systemic infections. Toxin-antitoxin (TA) systems are omnipresent in bacteria and participate widely in stress responses, biofilm formation and pathogenicity. However, the roles of TA systems are completely unknown in aquatic pathogenic bacteria. In this study, we identified and characterized a type II TA system HigBA in E. piscicida, where HigB is the toxin and HigA is the antitoxin. higB and higA are expressed in a bicistronic operon. To examine the biological role of HigBA, two markerless higB and higBA in-frame mutant strains, TX01ΔhigB and TX01ΔhigBA, were constructed. Compared to the wild strain TX01, TX01ΔhigB exhibited markedly reduced resistance against oxidative stress, but TX01ΔhigBA remained unchanged; the biofilm growth of TX01ΔhigB was decreased, but that of TX01ΔhigBA was increased. The deletion of higB enhanced bacterial motility but impaired bacterial resistance against host serum killing. In vitro infection experiment showed that the inactivation of higB dramatically abated bacterial capability of adhesion, invasion, and propagation in host cells. Consistently, in vivo experiment suggested that the higB mutation had an obvious negative effect on bacteria dissemination of fish tissues and general virulence. Introduction of a trans-expressed higB restored the lost virulence of TX01ΔhigB. However, the virulence of TX01ΔhigBA was compared to that of TX01. These findings indicate that HigB is essential for responding adverse circumstance and pathogenicity of E. piscicida. Based on the attenuated virulence of TX01ΔhigB, we further investigated its potential as a live vaccine. Vaccination experiment showed that TX01ΔhigB induced effective protection against lethal E. piscicida infection. In addition, we found that antitoxin HigA negatively autoregulated the expression of higBA by binding directly with own promoter, and toxin HigB promoted the regulatory function of HigA. This study provides new insights into the biological activity of type II TA system HigBA in aquatic pathogenic bacteria and contributes to understand the pathogenesis of E. piscicida.

Edwardsiella piscicida（以前包括在E. tarda中）是威胁全球淡水和海水养殖业的主要病原体，已被视为研究细胞内和全身感染的模型生物。毒素-抗毒素（TA）系统在细菌中无处不在，并且广泛参与应激反应，生物膜形成和致病性。但是，TA系统的作用在水生致病细菌中是完全未知的。在这项研究中，我们确定了E. piscicida中的II型TA系统HigBA并对其进行了表征，其中HigB是毒素，HigA是抗毒素。higB和higA在双顺反子操纵子中表达。为了检查HigBA的生物学作用，两个无标记higB和higBA在框内突变体菌株，TX01Δ higB和TX01Δ higBA，构建。相比于野生株TX01，TX01Δ higB表现出抗氧化应激显着降低阻力，但TX01Δ higBA不变; TX01Δ的生物膜生长higB下降，但该TX01Δ的higBA增加。higB的缺失增强了细菌的运动性，但削弱了细菌对宿主血清杀伤的抵抗力。体外感染实验表明，higB失活大大减弱了细菌在宿主细胞中的粘附，侵袭和繁殖能力。一致的是，体内实验表明，higB突变对鱼的细菌传播和一般的毒力具有明显的负面影响。反式表达的介绍higB恢复TX01Δ失去的毒力higB。然而，TX01Δ的毒力higBA是相比TX01的。这些发现表明HigB对于应对大肠杆菌的不利环境和致病性至关重要。基于TX01Δ的毒力减弱higB，我们进一步研究了其作为活疫苗的潜力。接种试验表明，TX01Δ higB诱导对抗致死有效保护E. piscicida感染。此外，我们发现，抗毒素比嘉负面的表达autoregulated higBA用自己的启动子结合，直接和毒素HigB促进比嘉的调节功能。该研究为II型TA系统HigBA在水生致病菌中的生物学活性提供了新的见识，并有助于了解大肠杆菌的发病机制。