Structural Optimization And Antibacterial Activity Of Porcine Antimicrobial Peptide PMAP-36

Antibiotic resistance and veterinary drug residues have become increasingly widespread,causing a serious threat to the global public health system.Therefore,it is urgent to develop new antibacterial drugs to replace traditional antibiotics to resist bacterial infection.Antimicrobial peptides(AMPs)are kinds of small molecule polypeptide active substances,which can be isolated in many kinds of organisms and play an important role in the innate immunity.Natural AMPs have the advantages of small molecular weight,high stability,wide antimicrobial spectrum and low induced resistance,etc.,and are new antibacterial drugs with great development prospect.However,compared with traditional antibiotics,natural AMPs have low yield,high extraction cost,low antibacterial activity,cytotoxicity and hemolytic activity.Therefore,it is very important to develop safe,stable and efficient new antibacterial drugs by structural modification of natural AMPs.PMAP-36(porcine myeloid antimicrobial peptides-36)is a high cationic amphiphilic α-helix antimicrobial peptide with 36 amino acids isolated from porcine bone marrow cells.In this study,a series of structural optimization was carried out on PMAP-36.The antibacterial activity,stability,cytotoxicity and antibacterial mechanism of PMAP-36 derivatives were evaluated to provide reference for the research and application of novel antimicrobial peptides.In this study,two analogues of PMAP-36,PMAP-36 PW and PMAP-36 PK,were designed and synthesized by amino acid substitution with hydrophobic tryptophan(WW)and positively charged lysine(KK)to replace proline(PP)at position 25 and 26 of PMAP-36,respectively.The results showed that PMAP-36 PW and PMAP-36 PK increased the antibacterial spectrum and antibacterial activity compared with PMAP-36.PMAP-36 PW showed significantly enhanced antibacterial activity against seven species of bacteria.PMAP-36 PK showed significantly enhanced antibacterial activity against five species of bacteria.In addition,PMAP-36 PW showed significantly enhanced p H stability and good biosafety for mammalian cells.In vivo treatment experiments in mice infected with Listeria monocytogenes CICC 21533,PMAP-36 PW significantly reduced the number of bacteria in liver and lung tissues,reduced inflammatory damage,and reduced mortality in mice,showing impressive therapeutic effects.Based on the PMAP-36 PW,a new fatty acid modified peptide Myr-36 PW was designed and synthesized by coupling myristic acid at the N-terminal of PMAP-36 PW through an amide bond.Compared with PMAP-36 PW,Myr-36 PW showed good stability and higher antibacterial activity in vitro.Myr-36 PW also exhibited effective anti-biofilm activity against Gram-negative bacteria and may kill bacteria by improving bacterial cell membrane permeability.In addition,Myr-36 PW could inhibit the bacterial growth of Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa GIM1.551 to target organs and reduce tissue inflammatory damage in mice infection model in vivo,show significant therapeutic effects on pneumonia and peritonitis experiments.Myr-36 PW also promoted abscess reduction and wound healing in infected mice.Based on the peptide Myr-36 PW,sodium alginate,calcium carbonate and glucolactone were mixed evenly to form Myr-36 PW hydrogel with sustained release of hydrogel and antibacterial activity of Myr-36 PW.The results show that Myr-36 PW hydrogel had uniform texture,smooth surface and uniform three-dimensional network space structure.Moreover,the high swelling rate,water content and slow release of hydrogel allowed Myr-36 PW hydrogel to load and release Myr-36 PW slowly and continuously.Besides,Myr-36 PW hydrogel had similar antibacterial activity to peptide Myr-36 PW in vitro.In the mouse wound infection and treatment experiment,Myr-36 PW hydrogel as wound dressing significantly reduced bacterial infection in the wound site,and effectively promoted the wound healing,which showed that hair follicles in the wound site were densely distributed and highly differentiated.In conclusion,amino acid substitution,fatty acid modification and preparation of antibacterial hydrogel can improve the antibacterial activity of antimicrobial peptide PMAP-36,which are effective strategies for structural optimization of antimicrobial peptide.