Edwardsiella piscicida YefM-YoeB: A Type II Toxin-Antitoxin System That Is Related to Antibiotic Resistance, Biofilm Formation, Serum Survival, and Host Infection

Abstract

The emergence of drug resistant bacteria is a tricky and confronted problem in modern medicine, and one of important reasons is the widespread of toxin-antitoxin (TA) systems in pathogenic bacteria. <i>Edwardsiella piscicida</i> (also known as <i>E. tarda</i>) 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. However, the role of type II TA systems are completely unknown in aquatic pathogenic bacteria. In this study, we identified and characterized a type II TA system, YefM-YoeB, of <i>E. piscicida</i>, where YefM is the antitoxin and YoeB is the toxin. <i>yefM</i> and <i>yoeB</i> are co-expressed in a bicistronic operon. When expressed in <i>E. coli</i>, YoeB cause bacterial growth arrest, which was restored by the addition of YefM. To investigate the biological role of the TA system, two markerless <i>yoeB</i> and <i>yefM-yoeB</i> in-frame mutant strains, TX01Δ<i>yoeB</i> and TX01Δ<i>yefM-yoeB</i>, were constructed, respectively. Compared to the wild strain TX01, TX01Δ<i>yefM-yoeB</i> exhibited markedly reduced resistance against oxidative stress and antibiotic, and markedly reduced ability to form persistent bacteria. The deletion of <i>yefM-yoeB</i> enhanced the bacterial ability of high temperature tolerance, biofilm formation, and host serum resistance, which is the first study about the relationship between type II TA system and serum resistance. <i>In vitro</i> infection experiment showed that the inactivation of <i>yefM-yoeB</i> greatly enhanced bacterial capability of adhesion in host cells. Consistently, <i>in vivo</i> experiment suggested that the <i>yefM-yoeB</i> mutation had an obvious positive effect on bacteria dissemination of fish tissues and general virulence. Introduction of a trans-expressed <i>yefM-yoeB</i> restored the virulence of TX01Δ<i>yefM-yoeB</i>. These findings suggest that YefM-YoeB is involved in responding adverse circumstance and pathogenicity of <i>E. piscicida</i>. In addition, we found that YefM-YoeB negatively autoregulated the expression of <i>yefM-yoeB</i> and YefM could directly bind with own promoter. This study provides first insights into the biological activity of type II TA system YefM-YoeB in aquatic pathogenic bacteria and contributes to understand the pathogenesis of <i>E. piscicida</i>.