| Cytokines are a class of small molecular proteins with various biological activities,which function by binding to corresponding receptors on the surface of target cells.In recent years,many studies have shown that cytokines not only play biological functions through the ligand-receptor pathway,but also a few of them have direct antibacterial activities.These special cytokines are defined as“kinocidins",which can help body to effectively clear pathogenic microorganisms at the infection sites.The study of kinocidins is one of the research focuses in new anti-infective drugs.At present,a variety of kinocidins have been reported in higher mammals,mainly include several CXC and CC chemokines and Interleukin 26(IL-26).However,there are few reports on kinocidins in fish.Fish is an important group of vertebrates,the study of kinocidins in fish is helpful to deepen understanding of fish innate immune system.Moreover,it can provide theoretical support for the genetic improvement of cultured fish species and the development of new anti-infective drugs with prominent practical significance.In this thesis,we made a deep study on the biological activities and mechanisms of grass carp(Ctenopharyngodon idella)Interferon 1(IFN1),CXC-chemokine ligand 20b(CXCL20b)and helix E peptides derived from IFN1.The main research results are as follows:1.IFN1 is a cationic amphiphilic protein which could kill various bacteria by disrupting the outer cell membraneWe analyzed the three-dimensional structures and physicochemical properties of IFN1.The results showed that IFN1 molecule is composed of six α-helix elements(helix A-F),and its positive charges are mainly distributed on the adjacent helix E and helix F,while the helix D element on the back of these two helices are rich in hydrophobic amino acids.We produced the recombinant IFN1.Through the colony forming units plate(CFU)method,we found that the recombinant IFNI with concentration of 0.5-1μM could kill more than 90%of Escherichia coli(E.coli),Staphylococcus aureus(S.aureus),Aeromonas hydrophila(A.hydrophila),Streptococcus agalactiae(S.agalactiae)and Pseudomonas aeruginosa(P.aeruginosa)in 1 h compared with the control group.We found that the recombinant IFN1 could cause depolarization of the outer membrane of E.coli by flow cytometry(FCM)analysis,and the destruction of outer membrane of E.coli was observed by scanning electron microscope(SEM)and atomic force microscope(AFM).In additions,we detected the binding of recombinant IFN1 with lipopolysaccharide(LPS)and peptidoglycan(PGN)by enzyme linked immunosorbent assay(ELISA).These data indicated that IFN1 could kill bacteria by disrupting the integrity of bacterial outer membrane.2.Helix E peptides exerted direct and broad-spectrum bactericidal activity in vitro and in mice modelWe found that helix E peptide adopt facially amphipathic a-helical conformations,in which cationic and hydrophobic amino acids residues are segregated to opposite faces along the helical axis.We synthesized helix E peptide in vitro,and the results of CFU plate method showed that this peptides can effectively kill various multiple drug resistance bacteria.Through in vivo antimicrobial assays,we found that helix E peptides has outstanding antibacterial activity against two kinds of Extraintestinal pathogenic Escherichia coli(ExPEC)PCN033 and RS218 in mice.Injection of 0.5 mg/kg helix E peptide into mice could significantly improve the survival rate of mice and reduce the total amount of bacteria in mouse tissues.We compared the hemolytic toxicity of helix E and human LL-37 peptides on the erythrocytes of grass carp and mice,as well as the effects on the proliferation of human Human embryonic kidney(HEK239T)cell-line and grass carp kidney(CIK)cell-line by Thiazolyl blue tetrazolium bromide(MTT)assay.The results showed that the cytotoxicity of helix E was peptides for eukaryotic cells much lower than that of LL-37.3.Helix E peptides could kill bacteria through membrane disruption mechanism or targeting intracellular bacterial DNAThrough SYTOX Green assay,we found that helix E peptides could destroy the integrity of outer membrane of Gram-positive bacteria,but could not cause damage to the outer membrane of Gram-negative bacteria.Similarly,SEM results showed that there were obvious pore structures in S.aureus treated with helix E peptides,but they did not significantly affect the morphology of E.coli.Further using structured illumination microscopy(SIM)and electrophoretic mobility shift assay(EMSA),we found that the location of helix E peptides in E.coli and S.aureus was different,and helix E peptides could enter the cell through the outer membrane of E.coli and bind to the intracellular DNA of the bacteria.This indicated that helix E peptides could kill bacteria through two mechanisms:membrane damage and targeting intracellular bacterial DNA.4.Helix E peptides exerted anti-inflammation activity in vitro and in mice modleWe found that helix E peptides could inhibit the Tumor necrosis factor-α(TNF-α)and Nitric oxide(NO)content produced by LPS-induced RAW264.7 cells.In the LPS-induced sepsis model of mice,injection of 0.5 mg/ml LPS could significantly reduce the content of TNF-α and IL-6 in the blood of mice.After injection of LPS into mice for 24 hours,the degree of lung injury in mice treated with helix E peptides was significantly lower than that in PBS group,indicating that helix E can be developed as a drug against gram-negative bacterial infection.5.Helix E peptides neutralize LPS by inducing LPS to form aggregatesWe found that helix E peptides could bind to bacterial LPS through electrostatic interactions by isothermal titration calorimetry(ITC)and Zeta potential analysis(ZETA).Through dynamic light scattering(DLS),we found that the combination will eventually affect the structure of LPS and promote the formation of larger aggregates of LPS molecules.Finally,through Limulus Amebocyte Lysate(LAL)test,we found that this special aggregate changed the normal structure of LPS,and ultimately affected the binding activity of LPS to LAL.6.CXCL20b is a cationic amphiphilic protein which could kill various bacteria by disrupting the outer cell membraneWe produced the recombinant CXCL20b.Through the CFU assay,we found that recombinant CXLC20b has an inhibitory effect on both Gram-positive and Gram-negative bacteria.Through homologous modeling,we found that CXCL20b and mammalian kinocidin molecule CXCL1 have similar cationic amphiphilic structure.Using AFM and FCM,we found that recombinant CXCL20b could cause depolarization and damage of S.aureus membranes in a short period of time,which indicated that recombinant CXCL20b could kill bacteria by destroying the integrity of the bacterial membrane surface.The above results confirmed that grass carp IFN1 and CXCL20b had previously unknown antibacterial function,which laid a foundation for further study on fish kinocidins.At the same time,we found that helix E peptides,derived from IFN1,had potent antibacterial and anti-sepsis effects,which showed a promising prospect for drug development.These findings broadened the understanding of fish antibacterial immunity,and provided some theoretical supports for the development of new anti-infective drugs. |
