Mechanism Study Of PcALF5,PcLysi4 And PcLysi5 Involved In Immune Responses In Procambarus Clarkii

With the extensive use of antibiotic in the animal and plant breeding industry and clinical trials,many antibiotics are discharged into the water environment after being recycled by animals and humans.Bacteria have gradually developed antibiotic resistance due to long-term exposure to the water environment containing low concentrations of antibiotics.Bacterial resistance is an unavoidable major scientific problem of human beings.Antimicrobial peptides(AMPs)are a class of small molecular peptides widely distributed in nature.It is a new type of antibacterial and antiviral drug with great potential.With the increase in the scale of Procambarus clarkii breeding,the improvement of the cultured density is gradually increased by bacterial viruses and fungi.Study on antimicrobial mechanism of AMPs is helpful for people to solve the scientific problem of bacterial resistance,and at the same time helps the research and development of antiviral drugs in the aquatic industry.1.PcALF5 inhibits the proliferation of microbiota by binding to RPS4 and MscL of E.coliAntimicrobial peptides(AMPs),most of which are small proteins,are necessary for innate immunity against pathogens.Anti-lipopolysaccharide factor(ALF)with a conserved lipopolysaccharide binding domain(LBD)can bind to lipopolysaccharide(LPS)and neutralize LPS activity.The antibacterial mechanism of ALF,especially its role in bacteria,needs to be further investigated.In this study,the antibacterial role of an antilipopolysaccharide factor(PcALF5)derived from crayfish was analyzed.PcALF5 could inhibit the replication of the microbiota in vitro and enhance the bacterial clearance ability in crayfish in vivo.Far-western blot assay results indicated that PcALF5 bound to two proteins of E.coli(approximately 25 kDa and 15 kDa).Mass spectrometry(MS),farwestern blot assay,and pulldown results showed that 30 S ribosomal protein S4(RPS4,25 k D)interacted with PcALF5.Further studies revealed that another E.coli protein binding to PcALF5 could be the large mechanosensitive channel(MscL),which is reported to participate in the transport of peptides and antibiotics.Additional assays showed that PcALF5 inhibit protein synthesis and promote the transcription of ribosomal component genes in E.coli.Overall,these results indicate that PcALF5 could transfer into E.coli by binding to MscL and inhibit protein synthesis by interacting with RPS4.This study reveals the mechanism underlying ALF involvement in the antibacterial immune response and provides a new reference for the research on antibacterial peptides drugs.2.Identification and characterization of two highly homologous lysozymes from red swamp crayfishLysozyme is widely distributed in both eukaryotes and prokaryotes,and play an important role in innate immunity against pathogen invasion.But the active region of lysozyme is largely unknown.In this study,two highly homologous lysozymes were identified from crayfish(designated as PcLysi4 and PcLysi5).The molecular structures of PcLysi4 and PcLysi5 were predicted by using SWISS-MODEL with the structure of Lysozyme(PDB accession No.4PJ2.2.B)as model.The results suggested that the structure of PcLysi4 and PcLysi5 were highly similar,but there were more α-helices at positions(127-139)and longer β-sheet at positions(49-57)in the structure of PcLysi5.The antibacterial and antiviral functions of the two isoforms lysozymes were investigated.PcLysi4 and PcLysi5 enhanced bacterial clearance ability of crayfish,and enhanced the survival rate of Vibrio-infected crayfish.Further results suggested that PcLysi5 inhibited WSSV replication,and enhanced the survival rate of WSSV-infected crayfish.There was no evidence that PcLysi4 has an influence on WSSV replication.Furthermore,PcLysi5 was detected to interact with envelope protein VP24 of WSSV.Our results would provide a new reference for viral protein binding AMPs.