Characterization of the interactions between fish serum proteins and pathogenic bacteria by using label-free quantitative proteomics

Abstract Fish blood is a valuable source of antimicrobial proteins which form a crucial part of the innate immune system, but the types and functions of these proteins are still not fully understood. The objectives of this study are to systematically characterize the serum proteins from fish that stably bind to bacteria and to investigate how bacteria respond to these interactions. Serum from turbot, but not the heat-inactivated control, significantly decreased the number of E. tarda bacteria. By conjugating fish serum proteins with fluorescent dye, we showed that E.tarda were coated with multiple labeled proteins. In order to systematically identify these bacteria-binding proteins, we used E.tarda cells to capture turbot serum proteins and subjected the samples to shotgun proteomic analysis, followed by label-free quantitation. A total of 76 fish proteins have been identified, including known antimicrobial proteins such as complement components and Wap65-2. 34 proteins with no previously known immunological function were also identified to bind to E.tarda. This approach also allowed the study of the proteomic changes in E.tarda exposed to turbot serum. Our data indicated a rapid decrease of translation factors and an upregulation of membrane components in response to serum treatment. Interestingly, fish serum induced a rapid suppression of the expression of bacterial antioxidant enzymes like catalase and led to an accumulation of reactive oxygen species, suggesting that fish serum can override a bacterial self-defence mechanism. Taken together, this work offers a comprehensive view of the interactions between fish serum proteins and pathogenic bacteria, and reveals previously unknown factors and mechanisms in fish innate immunity.