The Antibacterial Immune Mechanism Research Of Myeloid Differentiation Factor-2-related Lipid Recognition (ML) Protein In Eriocheir Sinensis

The myeloid differentiation factor 2(MD-2)-related lipid-recognition(ML)domain exists in various proteins,including MD-2,MD-1,Niemann–Pick disease type C2,GM2 activator protein(GM2A),and various proteins of unknown function.The main functions of ML proteins are involved in regulating intracellular lipid metabolism,immune responses mediated by the LPS pathway,and other functions related to lipid recognition.ML proteins are relatively clear in the functional studies of vertebrates are MD-2 and NPC2.MD-2 was identified as an accessory receptor for TLR4 in LPS sensing.Mutations in the NPC2 gene cause impairment of cholesterol excretion from the lysosome,a neurodegenerative lysosomal lipid storage disorder.In invertebrates,the research of ML proteins mainly focuses on the interaction between host and virus and antibacterial response,but the specific mechanism is not clear.This thesis takes Chinese mitten crab as the research object,and uses the relevant methods and means of molecular biology to explore the function of ML protein in the crab and the mechanism of its involvement in immunity.Based on the Chinese mitten crab genome and transcriptome data,three ML genes(Es ML1-3)were identified.Each protein includes a signal peptide and a canonical MD-2-related lipid recognition(ML)domain.The ML domain covers almost the entire coding regions of Es ML1(aa 33?156),Es ML2(aa 31?153)and EsML3(aa 23?144)proteins.Although all three ML genes contain ML domains,their sequence similarity at the amino acid level is low.The above three genes are widely expressed in various tissues of Chinese mitten crab,and their expression levels are upregulated to varying degrees after bacterial infection,but the expression of EsML3 is extremely significantly up-regulated,reaching about 35 times.Therefore,the function of EsML3 was further explored.Using the expressed recombinant EsML3 protein(r EsML3)to conduct bacterial binding experiments,it was found that r EsML3 showed binding activity to all eight tested bacteria,but the binding ability to Gramnegative bacteria was stronger.Given that EsML3 showed strong binding activity to the tested microorganisms,we speculated that some components on the cell surface were recognized by EsML3.ELISA results showed that r EsML3 combined with peptidoglycan and lipopolysaccharide in a dose-dependent manner,indicating that EsML3 has a broad binding activity to microbial cell wall components.Meanwhile,bacterial agglutination experiments showed that EsML3 effectively promoted bacterial agglutination.Although r EsML3 did not show direct bacteriostatic or bactericidal activity,in further bacterial clearance experiments,pre-incubating bacteria with r EsML3 significantly promoted bacterial clearance in vivo.In addition to agglutination,phagocytosis is another key mechanism for clearing invading pathogens,and r EsML3 has also been shown to significantly promote the phagocytic activity of hemocytes in vivo.Moreover,knockdown of EsML3 downregulated the expression levels of several antimicrobial peptides(Es ALF3,Es Crus1,Es Crus2 and Es DWD)after V.parahaemolyticus infection,indicating that EsML3 promoted the expression of antimicrobial peptides in crabs.In summary,EsML3 has the recognition activity of diverse PAMPs and microorganisms.EsML3 can mediate cellular immune response by identifying PAMPs,agglutination of invasive microorganisms and promoting bacterial clearance and phagocytosis in hemocytes.EsML3 also modulates the host immune response to bacteria by regulating the expression of some AMPs.This study enriches the functional diversity of the invertebrate ML family and provides a theoretical reference for in-depth exploration of the immunological function of ML.