The Antibacterial Activity And Mechanism Of Antimicrobial Peptides W3R6 And Its Analogs Against Mycobacterium Smegmatis

Tuberculosis(TB)is a chronic infectious disease caused by Mycobacterium tuberculosis.TB still poses a permanent threat to global health.At present,nearly 1.8 million people die of TB every year,and nearly 1/3 of them suffer from latent TB.In the past 40 years,there have been no new drugs except Bedaquiline and Delamanid,it is urgent to develop new drugs with novel mechanism.Antimicrobial peptides,which have unique bactericidal mechanism and are not easy to produce drug resistance,are expected to be potential drugs against Mycobacterium infection.Screening the existing antibacterial peptides in the laboratory,antibacterial peptides W3R6,D-W3R6,L-W3R6 and D/L-W3R6,have exhibited antibacterial activity against Mycobacterium smegmatis(ATCC 700084)(M.s)with MICs at 6.25 μM,12.5 μM and 6.25μM,respectively.Antimicrobial peptide D-W3R6 was modified to enhance its hydrophobicity by conjugating acetic acid,butyric acid,n-octanoic acid,lauric acid and palmitic acid at the N-terminal of D-W3R6.The results showed that the MIC values of both AC-D-W3R6 and BA-D-W3R6 decreased to 3.13 μM,but with the increase of hydrophobicity,the cytotoxicity of OA-D-W3R6,LA-D-W3R6 and PA-D-W3R6 are also increased.In addition,the mechanism of action of antimicrobial peptides W3R6 and its analogs against smegmatis was explored in order to provide reference for the development and utilization of antimicrobial peptides in clinic.Antimicrobial peptides W3R6 and its analogs shows good antibacterial activity on Mycobacterium and Mycobacterium intracellulare,and they have synergistic effect with first-line anti tuberculosis drugs,the development of drug resistance result shows that such antimicrobial peptides are less likely to develop drug resistance than Rifampin.In addition,D-W3R6 antimicrobial peptides show strong stability in human serum environment,even if they are incubated with human serum for 96 h,there is only slight degradation.The results of scanning electron microscopy and Llive/Dead staining show that the influence of antimicrobial peptides W3R6 and its analogs on Mycobacterium cell membrane.Zeta potential shows that antimicrobial peptides W3R6 and its analogs combine with the surface charge of Mycobacterium smegmatis.AC-D-W3R6 and BA-D-W3R6 are neutralized at the concentration of 16 μM,D-W3R6,L-W3R6 and D/L-W3R6 are neutralized at the concentration of 32 μM,NPN and depolarization experiment confirm that antimicrobial peptides W3R6 and its analogs increase the permeability of the inner and outer membrane ofMycobacterium smegmatis.The calcein release experiment demonstrate that the antimicrobial peptides have a destructive effect on the membrane of Mycobacterium smegmatis.When the concentration of AC-D-W3R6 and BA-D-W3R6 was 12.5 μM,the calcein release rate reaches 100%,when the concentration of D-W3R6 and D/L-W3R6 is 25 μM,the release rate reaches 100%,when the concentration of L-W3R6 is 50 μM,the calcein release rate reaches100%.The results show that antimicrobial peptides W3R6 and its analogs target on cell membrane to exert antibacterial activity;Confocal laser scanning microscopy is used to observe the binding of antimicrobial peptides W3R6 and its analogs to the cytoplasm of Mycobacterium smegmatis.The experiment of DNA agarose gel electrophoresis shows that antimicrobial peptides W3R6 and its analogs can bind to the genome DNA of Mycobacterium smegmatis.L-W3R6,which has the strongest binding ability to DNA,binds at a ratio of0.0156/1,D/L-W3R6 binds at a ratio of 0.032/1,and AC-D-W3R6 and BA-D-W3R6 bind at a ratio of 0.0625/1,and D-W3R6 binds at a ratio of 0.125/1.The binding of antimicrobial peptides W3R6 and its analogs to the genomic DNA of Mycobacterium smegmatis takes place in a chromogenic effect,obstructs Mycobacterium genomic DNA synthesis.The above results show that antimicrobial peptides W3R6 and its analogs have good stability and certain antibacterial activity on Mycobacterium.they can change the permeability of mycobacterial membrane,destroy its membrane structure and combine with its genomic DNA.