Antimicrobial Activity And Mechanism Of Scorpion Venom-derived Peptides

The alarming rise in the prevalence of antibiotic resistance among pathogenic bacteria poses a severe challenge for the development of effective therapeutic agents.Antimicrobial peptides(AMPs)have attracted a great deal of attention as a potential solution to the problem of antibiotic-resistant bacteria.The large inventory of host defense peptides from different organisms have the advantages of broad spectrum of antimicrobial activity,rapid action and low target-based resistance,which can open up new avenues for the development of new anti-infective agents.The scorpions appeared and evolved as very complex animals around 430 million years ago.Scorpion has developed a variety of venom peptides for successful survival during its long-term evolution.As one kind of arachnid animals,scorpions mainly rely on innate immune system against infectious microorganisms.Many AMPs have been characterized from scorpion venoms as an important component of their innate immune system.They often kill microorganisms such as bacteria rapidly by disrupting the integrity of the cytoplasmic membrane.Some of them can also interfere with the intracellular physiological processes,such as affecting cell-wall biosynthetic pathway,inhibiting protein biosynthesis,or interacting with nucleic acids.The scorpion Mesobuthus martensii is an important raw material in Chinese Traditional Medicine for the treatment of some nervous system and infection system-related diseases,which has been used for thousands of years in China.Scorpion venom-derived peptides have potential medical prospects and a potential as a source of antimicrobial drug candidates.This study systematically identified four venom-derived AMPs from M.martensii and screened a new AMP from Heterometrus petersii to unravel their mechanism of action.Firstly,this study systematically identified four AMPs Bm Kb1,Bm Kn2,Marcin-18 and Bm Kbpp from the venom of the scorpion M.martensii at the genomic,transcriptional and proteic levels.Based on the genome and transcriptome of M.martensii previously decoded by our group,Bm Kb1,Bm Kn2 and marcin-18 genes were amplified by PCR.Only two fragments of Bm Kbpp gene were amplified and the fulllength genomic sequence of Bm Kbpp was not obtained.These four genes share the same genomic organization and structure and are composed of two exons and an intron with a GT-AG splice junction.The introns are rich in A+T,which locate in the DNA region encoding N-terminal part of signal peptide.Real-time PCR was performed to detect the m RNA expression of these four AMPs in the venom gland of M.martensii.The relative m RNA expression of Bm Kb1 and Bm Kbpp were obviously higher than Bm Kn2 and marcin-18.By establishing a model of intact scorpion infected by bacteria,real-time PCR was performed to detect the m RNA expression of these four AMPs in the venom gland of M.martensii after bacterial challenge.The relative m RNA expression of Bm Kb1 and Bm Kn2 were significantly up-regulated after bacterial infection,while the m RNA expression of marcin-18 were significantly increased after Staphylococcus aureus infection,which indicated inducible expression at the transcriptional level of Bm Kb1,Bm Kn2 and marcin-18.There was no apparent difference in the m RNA expression of Bm Kbpp after challenged by S.aureus or Escherichia coli,which indicated a constitutive expression at the transcriptional level of Bm Kbpp.After cloning and analyzing the promoter regions of these four genes,it was found that the promoter of Bm Kbpp lacked several typical immune response elements,especially NF-?B,compared with the promoters of the other three genes.RPHPLC and 2-DE combined with MALDI-TOF MS and LC/ESI-Q-TOF MS were used to investigate the venom of M.martensii.It has been successfully demonstrated the existence of these four AMPs in the natural venom of M.martensii.Then the antibacterial activity and mechanism of marcin-18 and its homologs(meucin-18 and megicin-18)were clarified in this study.According to the precursor nucleotide sequence of marcin-18 registered into Gen Bank(No.ADT89762.1)and the draft genome of M.martensii decoded by our group,marcin-18 was identified at both genomic and transcriptional levels,and the existence of marcin-18 in the venom of M.martensii was confirmed by proteomic approaches.A protein sequence homology search revealed that marcin-18 shares extremely high sequence identity to the AMPs meucin-18 and megicin-18.Their ORFs were predicted to encode precursors that consist of putative signal peptide,propeptide and mature peptide.The helical wheel projection revealed that marcin-18 and its homologs adopted an amphipathic structure,where hydrophilic(polar)residues are on one side while hydrophobic residues are found on the other side.This structure was confirmed by circular dichroism spectroscopy.These three chemically synthetic peptides exhibited random coil structures in water;however,they showed high percentages of ?-helix in either 30% or 70% aqueous trifluoroethanol.In vitro,chemically synthetic marcin-18 and its homologs showed strong inhibitory activity against Gram-positive bacteria,including some clinical antibiotic-resistant strains.In addition,three peptides retained stable antibacterial activity in 0?250 m M Na Cl.Meanwhile,marcin-18 and megicin-18 exhibited stable antibacterial activity in an acidic environment(p H 3.0?5.0),but it was reduced in neutral and alkaline environments(p H 6.0?10.0).Meucin-18 has great tolerance to the acidic,neutral and alkaline environments.In a mouse acute peritonitis model,these peptides rescued nearly all mice heavily infected with clinical methicillinresistant Staphylococcus aureus(MRSA)from lethal bacteremia and significantly decreased the bacterial load in ascites of infected mice,which suggested that marcin-18 and its homologs still have good antibacterial activity in vivo.Time-killing assay showed that these peptides could kill bacteria rapidly.Competitive binding assay showed that lipopolysaccharide(LPS)and lipoteichoic acid(LTA)didn't significantly affect the antibacterial activity of the three peptides.Fluorescence measurements showed that these peptides killed bacteria by rapidly destroying the cell membrane.Transmission electron microscopy showed that disruption of the cell envelope was observed,together with the appearance of a granulated cytoplasm.These peptides exerted antibacterial activity via a bactericidal mechanism and killed bacteria through membrane disruption.Finally,a novel AMP named hp1473 was identified and characterized from the venom gland c DNA library of the scorpion H.petersii and its antibacterial mechanism was explored.The ORF of hp1473 was predicted to encode a precursor that consists of a putative 23-residue signal peptide,a 13-residue mature peptide and a 32-residue propeptide.Secondary structure analysis showed that hp1473 is an amphipathic ?-helical peptide.In vitro,chemically synthetic hp1473 has inhibitory activity against Gram-positive bacteria including clinical isolates of antibiotics-resistant bacteria,and it can also inhibit the growth of Candida albicans.Hp1473 was further confirmed to have good biological activity in vivo by a mouse peritonitis model.Time-killing assay showed that the viable colony number of S.aureus didn't decrease compared with the initial colony number in the process of 3 hours post treatment with different concentrations of hp1473.Hp1473 is a bacteriostatic peptide,which may have a different mechanism of action from the existing scorpion venom-derived AMPs.Fluorescence measurements showed that the fluorescent intensity didn't significantly change during treatment with different concentrations of hp1473 for 30 minutes,which indicated that the bacterial cell membrane was not lysed by hp1473.Transmission electron microscopy and scanning electron microscopy showed that bacterial cells treated with hp1473 at 4×MIC were round and had intact cell wall and cell membrane,which indicated that hp1473 didn't damage the ultrastructure of bacterial cells.Confocal laser-scanning microscopy showed that FITC-hp1473 could aggregate into a point at the edge of a bacterial cell.By detecting the GTPase activity of the recombinant Fts Z protein after incubation with hp1473,it was found that hp1473 might not interact with Fts Z.Competitive binding assay showed that the antibacterial activity of hp1473 gradually decreased with the increasing concentration of LPS or LTA.Thus,hp1473 may have an unknown target related to bacterial cell wall.In addition,the hemolytic activity and physiological stability of hp1473 was examined.Hp1473 could lyse about 55% of human erythrocytes at the concentration of 100 ?g/m L.After incubation with serum,hp1473 immediately lost its antibacterial activity,which indicated that the serum stability of hp1473 was poor.Hp1473 has great tolerance to the acidic,neutral and alkaline environments.Hp1473 remained stable antibacterial activity in 0?150 m M Na Cl.Once Na Cl concentration was greater than 200 m M,the minimum inhibitory concentration(MIC)of hp1473 against bacteria would increase with the increasing Na Cl concentration.Taken together,this study gained insight into the genomic organization and structure of scorpion venom-derived AMPs and lays a basis for characterizing related components involved in regulation of scorpion venom gland AMP gene expression.This work also enriches the peptide composition information of scorpion venom.The functional characterization and mechanism of marcin-18 and its homologs and a new AMP hp1473 screened from H.petersii were also studied,providing a new molecular template for the development and design of antimicrobial drugs.