Antimicrobial Mechanism Of MMR1L In Zebrafish Immunity

Mannose receptor（MR）is a multi-lectin receptor containing multiple lectin-like domains.It can recognize a variety of carbohydrate molecules with residues such as mannose,trehalose and N-acetylglucosamine,bind and remove pathogens and endogenous molecules,and participate in receptor-mediated phagocytosis.It plays an important role in participating in innate immunity,inducing adaptive immunity,regulating immune response and maintaining the stability of the internal environment of the body.As a model animal,zebrafish（Danio rerio）plays an increasingly important role in immune research.A variety of Mannose receptors have been found in zebrafish,such as MRC1b（Mannose receptor C type 1b）,which may be related to the immune response to bacterial infection,but the understanding is not comprehensive.In this study,we have cloned a novel mannose receptor gene,Macrophage mannose receptor 1-like（MMR1L）,and investigated its innate immune mechanism.The expression of MMR1L was detected in all the normal tissues of zebrafish,and the relative expression in hepatopancreas was the highest.After the stimulation by Aeromonas hydrophila,the relative expression of MMR1L in spleen,gills and muscle of zebrafish was significantly upregulated,suggesting that MMR1L may be involved in the innate immunity caused by bacteria.Different domains of MMR1L recombinant protein were expressed and purified in prokaryotic expression.According to the characteristics of the domains,they were named as CLECT 1-2,CLECT 3-5,GLD,CLECT 6-8,CLECT 9-10 and CLECT 11-12 respectively.The bacterial agglutination and binding experiments of different domain proteins showed that MMR1L could bind and agglutinate bacteria,suggesting that MMR1L plays a role in the resistance to bacterial immune response.In order to further study the mechanism of antibacterial function of MMR1L,the String website was used to predict the interacting protein of MMR1L,and it was found that TLR2 may be the interacting protein of MMR1L.Pull-down and cell colocalization experiments proved that there was interaction between MMR1L and TLR2.MMR1L was overexpressed in ZF4 cell lines of zebrafish,and the relative expression levels of inflammatory cytokines（IL-1β,IL-6,IL-8）,important molecules of TLR pathway（My D88,TRAF6）,NF-κB（P65/P50）and antimicrobial peptides（Hamp,Defbl1,Defbl3）were up-regulated.It is speculated that it may interact with TLR2 and induce the activation of NF-κB signaling pathway through the transduction of TLR signaling pathway,promote the expression and secretion of antimicrobial peptides,and play antibacterial immune function through antimicrobial peptides.The deletion mutant MMR1L^Δ757-978was constructed,and the overexpression detection of the deletion of"GLD"domain showed that the expression of molecules in the pathway was upregulated less than that in the control group,indicating that"GLD"domain plays an important role in the antimicrobial peptide expression induced by MMR1L.The antimicrobial peptide has a broad spectrum of antibacterial activity.The deletion mutant Hamp was constructed.The liquid antibacterial experiment showed that"KRQS"may play an important role in the inhibition of bacterial growth by Hamp.Hamp^Δ65-68（deletion of"KRQS"）can reduce the combining ability of bacteria and weaken the agglutinative activity of bacteria.Scanning electron microscope was used to observe the surface structure of A.hydrophila incubated with Hamp and Hamp^Δ65-68,and it was found that Hamp^Δ65-68had less damage to the bacterial surface structure than Hamp,indicating that"KRQS"played an important role in the bactericidal process of Hamp.In conclusion,this study found that MMR1L can agglutinate and bind bacteria,activate the NF-κB signaling pathway through collaboration with TLR2,and induce the expression and secretion of Hamp and other antimicrobial peptides downstream of the pathway,and further realize the anti-bacterial immune response.The"GLD"domain plays an important role in the expression of antimicrobial peptides induced by MMR1L.The"KRQS"sequence may be an important functional site for Hamp to clear bacteria.