Study On The Mechanism Of Antibacterial Immunity Of Ficolin Protein In Eriocheir Sinensis

Ficolin,a member of the fibrinogen-related protein family(FREPs),functions as a pattern recognition receptor(PRR)as a novel lectin in vertebrates and invertebrates.In vertebrates,ficolins are oligomeric proteins composed of three polypeptide chains,subunits formed by the assembly of their collagen-like domains into homotrimers.Some of these ficolins are serum lectins that can form complexes with mannan-binding lectin-associated serine proteases(MASPs)and small mannan-binding lectin(MBL)-associated proteins(s MAPs),which activate complement through the lectin pathway.In invertebrates,Ficolin has a C-terminal conserved fibrinogen-related domain(FRe D),but its N-terminal structure is quite different or even unknown.It is known that part of Ficolin has a coiled coil structure,which may be able to It promotes the formation of polypeptide chains similar to the role of mammalian Ficolin trimer oligomeric protein.Invertebrate Ficolin functions functionally in two ways.One is to act as a lectin similar to pattern recognition receptors,which can recognize PAMPs of invading pathogens to promote the clearance of invading pathogens,thereby protecting the host from infection;The original opsonophagocytosis,as an opsonin,promotes the phagocytosis of pathogenic microorganisms by blood cells to achieve the purpose of eliminating bacteria.This paper takes Chinese mitten crab(Eriocheir sinensis)as the research object,and explores the mechanism of Ficolin in regulating the innate immunity of Chinese mitten crab.Based on the whole genome database of Eriocheir sinensis and the Ficolin gene sequence of crustaceans,a Ficolin homologue was searched and compared,and it was named EsFicolin.Through bioinformatics analysis,it was found that it has a signal peptide,a highly conserved C-terminal fibrinogen-related domain(FRe D)and a coiled-coil structure for trimer formation.The results of multiple sequence alignment confirmed that the C-terminal FRe D domain is highly similar to other crustacean FRe D domains,indicating that it is highly conserved in evolution and is an important functional domain structure.The results of the phylogenetic tree showed that EsFicolin was clad with mammalian Ficolin but not with crustaceans,implying that EsFicolin is functionally similar to mammalian Ficolin.The results of RT-q PCR analysis of tissue distribution showed that EsFicolin was expressed in muscle,hepatopancreas,intestinal tract,stomach and blood cells,indicating that EsFicolin was involved in a wide range of physiological functions.After stimulation with Staphylococcus aureus and Vibrio parahaemolyticus,EsFicolin was significantly upregulated in blood cells compared with controls,suggesting a potential association with antimicrobial immune responses.The recombinant protein r EsFicolin was expressed and purified using the Escherichia coli prokaryotic expression system,and subsequent chemical cross-linking experiments confirmed that the recombinant r EsFicolin protein was in bis(3-sulfo-N-hydroxysuccinimide ester)suberate sodium salt(BS~3)can form a trimer oligomeric structure,Westren blot results show that the band corresponding to the trimer protein is obviously thicker and darker than the band corresponding to the monomer protein,indicating that the trimer oligomerization The number of proteins is greater than the number of monomeric proteins,and most proteins exist in a trimer structure.In order to explore whether r EsFicolin can play the role of lectin,our agglutination test shows that r EsFicolin has strong agglutinating activity against S.aureus and V.parahaemolyticus,and has stronger agglutinating activity against Gram-negative bacteria.Binding experiments showed that r EsFicolin had binding ability to five Gram-negative bacteria and three Gram-positive bacteria,and the binding ability to Gram-negative bacteria was stronger.Subsequent polysaccharide binding experiments showed that r EsFicolin combined with cell-expressed pathogen-associated molecular patterns(PAMPs)in a dose-dependent manner,revealing the specific mechanism of its recognition and binding to bacteria.In-depth research found that the recombinant r EsFicolin protein can effectively remove bacteria and exhibit direct antibacterial activity.r EsFicolin also promotes phagocytosis of blood cells to enhance bacterial clearance.In summary,EsFicolin,as a pattern recognition receptor in the innate immune system of Chinese mitten crab,can recognize pathogen-associated molecular patterns(PAMPs)expressed by cells and agglutinate and bind bacteria.There are two main ways to fight against invading microorganisms:on the one hand,EsFicolin has the activity of directly inhibiting the growth of bacteria;The purpose of protecting the host from pathogen infection.These findings indicate that EsFicolin plays an important role in the antibacterial immune response of Chinese mitten crab.This paper enriches our functional research on invertebrate Ficolin and enhances our understanding of invertebrate innate immune system.