Prediction And Bioactivity Of Small Molecule Antimicrobial Peptides From Protaetia Brevitarsis Lewis Larvae

In recent years,due to the widespread use of antibiotic drugs,the development of drug-resistant pathogens has been extremely rapid,and the reduction in the approval rate of new antibiotic molecules has led to an increase in mortality worldwide.Therefor e,it is urgent to find new antimicrobial substances that are not easy to cause drug resistance.Antimicrobial peptides(AMPs)are a class of peptide substances produced by autoimmune organisms that are infected by microorganisms and are capable of self-defense against the whole body.Compared with conventional antibiotics that target specific intracellular enzymes or DNA,due to its short length,simple composition,protease stabilization and membrane targeting,this unique mechanism of action physically and rapidly penetrates and destroys the plasma membrane,leading to difficult to repair damage,is less prone to drug resistance,and does not normalize to mammals.The cells cause damage.Therefore,microbes are much less likely to develop resistance than antibiotics.It suggests that AMPs can be developed as new therapeutic solutions by overcoming the inherent shortcomings of antibiotics.It has been reported that the antimicrobial substance is extracted and isolated from Protaetia brevitarsis Lewis larvae,which belongs to Coleoptera.We used its genome-wide sequence as an experimental sequence resource and compared it with the antimicrobial peptides collected by the antimicrobial peptide database(APD3)to obtain natural AMPs as templates.Combined with the characteristics of the AMPs sequence,we used the amino acid substitution method and a series of bioinformatics website software to predict the possibility of screening candidate small molecule AMPs,and then sent them to the company to make all AMPs mo re than 95% pure.By analyzing the structure of the template peptide,we listed 30 candidate small molecule AMPs.By comparing physical and chemical parameters,we screened 16 peptides with better parameters as candidate peptide sequences.We found that the best peptide VFRLKKWIQKVI has the highest positive charge(+4)and a relatively high hydrophobic residue ratio with a very low instability coefficient(-4.98)and the secondary structure dominated by ?-helix.We carried out a series of bioassay experiments on small molecule antimicrobial peptides.The agar perforation method,micro-liquid dilution method and colony counting method were used to determine the antimicrobial activity of the designed small-molecule peptides against five kinds of bacteria.The MIC and MBC was determined of Escherichia coli,Pseudomonas aeruginosa,Staphylococcus aureus,Bacillus thuringiensis and Canidia aibicans.And we analyzed the effect of AMPs on the growth of bacteria,and plotted the time-sterilization curve to determine the antibacterial effect.The preliminary screening of the activity experiment showed that 5 small molecule AMPs with high antimicrobial activity,among which VFRLKKWIQKVI has the most antimicrobial effect.Excellent,not only has good bacteriostatic effect on two Gram-negative and positive bacteria,but also has obvious antimicrobial effect on fungus Candida albicans.Its minimum inhibitory concentration against Escherichia coli and Pseudomonas aeruginosa is 16 ?g/mL,the MIC is 32 ?g/mL,the MIC of Bacillus thuringiensis and Staphylococcus aureus is 16 ?g/mL and 32 ?g/mL,the MBC is 64 ?g/mL and 128 ?g/mL,the MIC of Candida albicans is 64 ?g/mL,and the MBC is 128 ?g/mL.The dynamic bacteriostatic experiment showed that the AMPs treatment group could better delay the growth of microorganisms.After 1 h of 1/4 MIC in ampicillin group,Escherichia coli and Staphylococcus aureus began to grow compared with 1/4 MIC.In contrast,at the concentration of 1/4 times MIC,it was found that each antibacterial peptide group began to grow after 3 h,indicating that the antibacterial peptide treatment group can better delay the growth of microorganisms;The bactericidal curve showed that after the antimicrobial peptide treatment of 2 times MIC,the five antimicrobial peptides were immediately sterilized at the beginning of the action,and the number of colonies was significantly reduced.As time increased,the sterilization speed gradually decreased,and the curve showed a gentle trend;salt stability Sex experimen ts showed that the same concentration of peptide in PBS treated Escherichia coli and Staphylococcus aureus,the antibacterial activity was significantly reduced;the enzymatic stability experiment showed that all the antimicrobial peptides were greatly red uced after being treated with trypsin for 1 min.Antibacterial activity.This indicates that the five antimicrobial peptides designed in this study are easily decomposed by trypsin,which reduces the original activity.In this study,five AMPs with antimicrobial activity were screened by designing AMPs.One of them had good antimicrobial activity against bacteria and fungi,which laid a foundation for the design and research of novel peptide antimicrobial substances.