The Mechanism Of Recognition And Inhibition Of Bacterial Infection In Shrimp And Crayfish

The research of shrimp immune defense mechanism is important for disease prevention in shrimp aquaculture.Shrimp lacks adaptive immunity,it is critical to accurately recognize and initiate an innate immune response to exogenous bacterial pathogens in response to their invasion and to generate an effective antimicrobial response.The discovery of key antimicrobial molecules and the uncovering of their antimicrobial mechanisms will help develop sustainable disease prevention.In this thesis,we selected the important aquaculture shrimps Marsupenaeus japonicus and Procambarus clarkii as experimental subjects to investigate the key molecules and their mechanisms of bacterial recognition and suppression.We obtained the following results:1.Shoc2 recognizes bacterial flagellin and mediates antibacterial Erk/Stat signalingFlagellin is a key bacterial virulence factor that can stimulate molecular immune signaling in both animals and plants.The detailed mechanisms of recognizing flagellin and mounting an efficient immune response have been uncovered in vertebrates;however,whether invertebrates can discriminate flagellin remains largely unknown.In the present study,the homolog of human SHOC2 leucine rich repeat scaffold protein in kuruma shrimp(M.japonicus),designated MjShoc2,was found to interact with Vibrio anguillarum flagellin A(FlaA)using yeast twohybrid and pull-down assays.MjShoc2 plays a role in antibacterial response by mediating the FlaA-induced expression of certain antibacterial effectors,including lectin and antimicrobial peptide.FlaA challenge,via Mj Shoc2,led to phosphorylation of extracellular regulated kinase(Erk),and the subsequent activation of signal transducer and activator of transcription(Stat),ultimately inducing the expression of effectors.Therefore,by establishing the FlaA/MjShoc2/Erk/Stat signaling axis,this study revealed a new antibacterial strategy in shrimp,and provides insights into the flagellin sensing mechanism in invertebrates.2.Maturation of an antimicrobial peptide to inhibit bacterial infections in crayfishRapid synthesis and release of active antimicrobial peptides(AMPs)is an important strategy in innate immune.Processing of the precursor into the active form is a common posttranslational modification of AMPs in mammals.However,in invertebrates,the mechanism of AMP maturation is largely unknown.In the present study,a novel potential AMP,designated as PcnAMP,was identified because of its significant induction by bacterial infection in the red swamp crayfish(P.clarkii).PcnAMP was cleaved into a short fragment after infection.Using the purified native peptide,this cleavage was found to be mediated by trypsin after synthesis.Proteolysis produced an N-terminal peptide that exerted the antibacterial function.Although the N-terminal peptide did not show significant similarity to any other sequences,it was predicted to have an overall helical structure and high amphipathicity,both of which are typical features of many AMPs.The N-terminal active peptide exhibited a wide spectrum of antimicrobial activity.Atomic force microscope imaging and flow cytometry analysis showed that treatment with the active form of PcnAMP led to the collapse of the bacterial cell wall and permeabilization of the bacterial cell membrane.Thus,this study provided a new candidate for therapeutic agent development,and revealed new insights into the maturation of AMPs in invertebrates.Both M.japonicus and P.clarkii are important aquaculture species with close kinship and similar bacterial pathogens.Through research on immune recognition and immune effects in two separate species,we have identified key recognition and effector molecules in antimicrobial immunity in shrimp and revealed their functions and mechanisms.The above research has deepened the understanding of the immune response process in shrimp in response to bacterial infection.