Unraveling the biofilm armor: How ROS drives biofilm formation in aquatic pathogen Edwardsiella piscicida

Excessive reactive oxygen species (ROS) in the environment and within host cells present a serious threat to the survival and infectivity of pathogenic bacteria. Bacterial biofilm is recognized as a crucial resistance strategy against ROS. However, the mechanisms by which pathogenic bacteria respond to ROS to facilitate biofilm formation, particularly in aquatic pathogens, remain poorly understood. In our prior study, we generated a mutant (designated as ΔtrxAC) with elevated ROS levels by knocking out trxA and trxC of the thioredoxin system (Trx) in Edwardsiella piscicida, a ubiquitous pathogen causing serious economic losses in aquaculture. In this study, we observed that, compared to the wild type (WT), the biofilm growth and expression of cellulose biosynthesis genes in ΔtrxAC were significantly enhanced, while complementation strain of ΔtrxAC by expressing grx1 exhibited restored redox homeostasis and biofilm formation, indicating that elevated ROS promotes E. piscicida's capability of biofilm formation. We also observed a substantial increase in the levels of c-di-GMP, a vital signaling molecule involved in cellulose production and biofilm formation, in ΔtrxAC. The expression of phosphodiesterase YhjH, which degrades c-di-GMP, was decreased in ΔtrxAC and negatively correlated with biofilm formation. Consistently, ΔtrxAC exhibited a reduction in the expression of the regulatory factor FliA, which positively regulates the expression of yhjH. Regarding pathogenicity, the Trx mutation diminished the proliferation of E. piscicida in host cells and tissues, but it enhanced bacterial hemolytic activity, a virulence trait closely associated with biofilm formation, suggesting that the impact of ROS on bacterial virulence is multifaceted and intricate. In summary, this study elucidates for the first time the pathway through which E. piscicida responds to ROS to promote biofilm formation, as well as its implication for pathogenicity. These findings provide insights into the pathogenic mechanisms of E. piscicida and inform the development of novel strategies for preventing infections caused by this pathogen.