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. Edwardsiella piscicida (also known as 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. 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 E. piscicida, where YefM is the antitoxin and YoeB is the toxin. yefM and yoeB are co-expressed in a bicistronic operon. When expressed in E. coli, YoeB cause bacterial growth arrest, which was restored by the addition of YefM. To investigate the biological role of the TA system, two markerless yoeB and yefM-yoeB in-frame mutant strains, TX01ΔyoeB and TX01ΔyefM-yoeB, were constructed, respectively. Compared to the wild strain TX01, TX01ΔyefM-yoeB exhibited markedly reduced resistance against oxidative stress and antibiotic, and markedly reduced ability to form persistent bacteria. The deletion of yefM-yoeB 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. In vitro infection experiment showed that the inactivation of yefM-yoeB greatly enhanced bacterial capability of adhesion in host cells. Consistently, in vivo experiment suggested that the yefM-yoeB mutation had an obvious positive effect on bacteria dissemination of fish tissues and general virulence. Introduction of a trans-expressed yefM-yoeB restored the virulence of TX01ΔyefM-yoeB. These findings suggest that YefM-YoeB is involved in responding adverse circumstance and pathogenicity of E. piscicida. In addition, we found that YefM-YoeB negatively autoregulated the expression of yefM-yoeB 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 E. piscicida.
Highlights
Edwardsiella was isolated from infected humans and animals and identified as a new genus of Enterobacteriaceae in 1965 (Ewing et al, 1965)
Sequence alignment showed that the YefM of E. piscicida shares 77.78, 55.26, and 38.75% sequence identities with YefM from E. coil, S. aureus, and S. pneumoniae, respectively (Figure 1B), while YoeB of E. piscicida shares 64.04, 44.44, and 45.28% sequence identities with YefM from E. coil, S. aureus, and S. pneumoniae, respectively (Figure 1C)
Bioinformatics analysis showed that YefM and YoeB share high homology with type II toxin-antitoxin system Phd/YefM family antitoxin and toxin YoeB, respectively. yefM and yoeB are transcriptionally organized in a bicistronic operon, and yefM locates upstream of the yoeB, which is one of typical characteristic of most of type II TA system (Yamaguchi et al, 2011)
Summary
Edwardsiella was isolated from infected humans and animals and identified as a new genus of Enterobacteriaceae in 1965 (Ewing et al, 1965). The Edwardsiella genus was classified into five species, including E. piscicida, E. anguillarum, E. ictaluri, E. tarda, and E. hoshinae (Abayneh et al, 2013; Leung et al, 2019). E. piscicida (formerly included in E. tarda) is recognized as one of the most severe pathogens in cultivating fishery (Leung et al, 2012). It can infect freshwater and marine fish at the same time, causing a large number of infections and deaths of fish, and a zoonotic pathogen with a wide host range, including mammals and reptiles (Janda and Abbott, 1993; Mohanty and Sahoo, 2007; Park et al, 2012). Toxin-antitoxin (TA) system, an important stress and antibiotic resistance factor/system, are totally unknown in E. piscicida
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