Study On The Antibacterial Function And Mechanism Of Teleost-specific CXCL20a

Teleost serve as a bridge connecting higher vertebrates and invertebrates,which plays an important role in the study of immune evolution.As the effector of innate immune response,chemokines mainly promote the activation and differentiation of leukocytes,induce migration of immune cells,and regulate the immune response.Chemokines also exert function of antibacterial peptide like and immunoregulation effect to constrain microbial pathogenic infection.CXCL20 is a teleost-specific member of the CXC type chemokine subfamily and its function and mechanism remain largely unknown.In this study,direct antibacterial activity and immunoregulation function of Ci CXCL20 a were investigated.The main results are as follows:1.CXCL20 a,a teleost-specific chemokine that orchestrates direct bactericidal,chemotactic,and phagocytosis-killing-promoting functions,contributes to clearance of bacterial infectionsIn the current study,combined in vitro direct bactericidal experiment,in vivo antiCi CXCL20 a antibody blocked experiment with recombinant Ci CXCL20 a injection experiment,we found that Ci CXCL20 a is a potent and broad-spectrum antibacterial protein and play an important role in host aganist aquatic pathogenic bacteria A hydrophilia infections.Ci CXCL20 a binds PAMPs(LPS,LTA or PGN)on the surface of bacteria,mainly targets acid lipids,perforates bacterial membrane,and results in bacterial cytoplasm leakage and death.Ci CXCL20 a aggregates LPS and neutralizes LPS endotoxin.Ci CXCL20 a can induce native LPS with 100 nm to form LPS aggregation with the diameter of 7000 nm.Ci CXCL20 a also has broad-spectrum ability to aggregate bacteria in concentration dependent manner,but it just aggregates Gram-negative bacteria,not Grampositive bacteria.Ci CXCL20 a mainly concentrates in immune organs or tissues with rich immune cells,and can be quickly induced after bacterial infection.It may be to maintain the homeostasis of immune cells or regulate immune cells to resist bacterial invasion.The results of chemotactic experiments on leukocytes showed that low concentration of Ci CXCL20 a has no chemotactic activity,but chemotaxis was observed to myeloid and lymphoid leukocytes under high concentration of Ci CXCL20 a.The results of fluorescent microsphere phagocytosis showed that Ci CXCL20 a promotes the phagocytosis of myeloid leukocytes,but not lymphoid leukocytes through Ci CXCR3.1b1.Ci CXCL20 a can also facilitate phagocytic leukocytes to kill intracellular A.hydrophila through Ci CXCR3.1b1.In addition,the results showed that the structural characteristic,bactericidal activity and mechanism of CXCL20 are conservative in teleost fish.2.CXCL20 a,a bactericidal chemokine,consists of four structural fragments with potent bactericidal activityWe truncated Ci CXCL20 a into four segments based its secondary structure.There are different from primary protein structures in classical chemokines-mediated antimicrobial activity that attribute to the N-terminal peptides or C-terminal regions of peptides chemokines.All the four truncated peptides from Ci CXCL20 a have antibacterial activity and neligible toxicity to eukaryotic cells.All the four truncated peptides from Ci CXCL20 a cause bacterial death by disrupting bacterial membrane.The aggregation activity result showed that Ci CXCL20 a aggregates Gram-negative bacteria.When screening its aggregation activity of the primary Ci CXCL20 a structure,we found that only the Cterminal coli of Ci CXCL20 a has weak aggregation activity of bacteria,but not other Ci CXCL20 a structure.These results that the ability of Ci CXCL20 a to aggregate bacteria might come from the C-terminal coli region.In addition,we also found that the truncated peptides from Ci CXCL20 a play key roles in restricting pathogenic infection and attenuate pro-inflammatory cytokines epression and increase anti-inflammatory cytokines expression caused by pathogen.So,these peptides can be used as effective candidates of therapeutic agents for bacterial diseases and inflammatory disorders.The above results showed that Ci CXCL20 a can enhance anti-infection abilities of host by directly killing bacteria,recruiting immune cells and promoting the phagocytic activity of recruited immune cells.In addition,Ci CXCL20 a can also aggregate LPS and Gramnegative bacteria to limit the invasion of bacteria.All secondary structures of Ci CXCL20 a have antibacterial activity,but its aggregation activity mainly located in its C-terminal coli.These results are the first systematic exposition of a chemokine from teleost with direct bactericidal,chemotactic,and phagocytosis-killing-promoting activities to mediate host resistance to microbial infection,and this is the first report that all secondary structures of chemokines have antibacterial activity.This will provide further evidence to expand the close functional and evolutionary relationship between chemokines and AMPs,and also provide theoretical support for the screening of fish immunopotentiators,feed additives,vaccine adjuvants and new antibacterial drugs.