Screening And Therapeutic Mechanism Of Brevinin-2 Family Antimicrobial Peptides Based On MRSA Infection Model Of Caenorhabditis Elegans

Since the discovery of penicillin by British biologist Fleming in 1929,many kinds of antibiotics have been developed.At present,a variety of antibiotics have been applied in clinic.More and more infected patients have been cured.At the same time,more and more pathogenic bacteria have strong resistance to antibiotics.The discovery and research of new drugs to combat multidrug-resistant bacteria has become one of the important issues in the field of life science research.In this study,the classical model organism Caenorhabditis elegans(C.elegans)was used as the experimental object.Based on high throughput screening of database information,85 polypeptides of Brevinin-2 family and their antimicrobial activities against MRSA were showed.Meanwhile,the therapeutic effects of typical antimicrobial peptides of Brevinin-2 family on MRSA infection model of Caenorhabditis elegans,the toxicity of inhibiting MRSA and the activation of DAF-2/DAF-16 of C.elegans were systematically studied from behavioral level,reproductive development,genetic variation,physiology and biochemistry,gene transcription,protein expression and mechanism.It was found that the typical antimicrobial peptides of Brevinin-2 family had therapeutic effects on MRSA infection model of C.elegans,inhibited the virulence of MRSA and activated the innate immune regulatory pathway of C.elegans DAF-2/DAF-16.This study is significance to the screening,evaluation,mechanism of action and clinical research and development of new antibiotics.The main results are as follows:1.Brevinin-2 family have great inhibitory activity and self-characteristics against MRSAThrough large data,bioinformatics analysis and high throughput screening of antimicrobial activity,it was found that the antimicrobial activity of MRSA of Brevinin-2 family antimicrobial peptides varied greatly in vitro.The MIC range of 33 antimicrobial peptides ranged from 4.9 to 135.5 ugM,and that of MBC ranged from 23.4->500 ?M.Brevinin-2,Brevinin-2DYc/2RNa?Brevinin-2HS2,Brevinin-2ISb,Brevinin-2LF2,Brevinin-2-OA3,Brevinin-2-OR5,Brevinin-2-OW2,Brevinin-2RE1,Brevinin-2-related,Brevinin-2SKa,Brevinin-2TP1 and Brevinin-2TSa were found for stability,hemolysis rate and cytotoxicity.These peptides that exhibit better characterization.The Minimum Inhibitory Concentration(MIC)of many antimicrobial peptides is less than 10 ?M,which is the lowest MIC concentration family of antimicrobial peptides found so far.However,in the present Brevinin-2 family,52 antimicrobial peptides showed lower or no inhibitory activity against MRSA,and showed similar evolutionary distances in phylogenetic evolution.Brevinin-2 polypeptide with small difference in amino acid sequence is often found.Its bacteriostatic activity also has great difference.2.MRSA is harmful to C elegans in different levelMRSA was used to study the behaviour,development,reproduction.survival and inheritance of multi-strain C elegans.It was found that MRSA had a significant effect on the physiological status of each level of C elegans.At the same time,the study found that MRSA significantly reduced the movement parameters(except backward movement frequency)of C.elegans.It also significantly inhibited the growth of adult Caenorhabditis elegans,i.e.body length(0.687(±0.024)?Control:0.988(±0.008).The oviposition rate of C elegans was significantly reduced(257+0.024,Control:355.7±15.2).It also had a significant lethal effect on C elegans,and all the nematodes in MRSA treatment group died within 96 hours.Six genes,Cep-1,Ced-3?Daf-2,Daf-16,Pmk-1 and Skn-1.were found to play a major role in resisting MRSA infection in nematodes.At the same time,comet electrophoresis experiments showed that MRSA had strong genotoxicity.Sum up,the results confirmed that the model organism C elegans is suitable for the study of MRSA infection because of its various characteristics.On the other hand,the strong toxicity of pathogenic microorganisms such as MRSA to C elegans was confirmed from various angles and levels.3.Brevinin-2 family can effectively cure MRSA infected C.elegansIn order to obtain more accurate treatment data in the experimental study of treatment,on the one hand,MRSA infection model of Caenorhabditis elegans was constructed.On the other hand,healthy adults of Caenorhabditis elegans were cultured directly on a MRSA culture plate for 96 hours and treated with antimicrobial peptides.In addition,in terms of screening criteria for therapeutic active antimicrobial peptides,a more rigorous new evaluation system was constructed.Firstly,the pathogenic microorganism-sensitive Caenorhabditis elegans was used instead of the pathogenic microorganism-resistant wild type N2.Secondly,the survival rate of the experimental group after 96 hours was significantly different from that of the positive infection control group,and the survival rate of the positive infection group was more than 60%,and the survival rate of the positive infection group was less than 20%as a polypeptide with better therapeutic effect.Four antimicrobial peptides,Brevinin-2,Brevinin-2-OA3,Brevinin-2 ISb and Brevinin-2 TSa,have been found to be effective under the above rigorous screening conditions.Brevinin-2-OA3 of 1/2 MIC,Brevinin-2ISb of 1/4 MIC and Brevinin-2TSa of 1/2 MIC were found to have good therapeutic activity in subsequent low-concentration infection treatment experiments.At the same time,a polypeptide Brevinin-2ISb was finally determined in the hemolytic activity test.The results showed that the survival rate of infected model was 90.02%±1.23%.The survival rate of persistent infection was 81.72%±2.62%.The hemolysis rate was very low.The concentration of 1/4 MIC was 5.03%±0.35%.It had certain beneficial effects on nematodes.Compared with the control,it accelerated the development of larvae to adults by 24.1 h and prolonged the life span by 7 days.It can be said that Brevinin-2ISb is an important discovery in the research and development of new drugs as an alternative.It accelerates the clinical research and application of Brevinin-2ISb and its related antimicrobial peptides,and also promotes the value and significance of the overall study of this paper.4.Brevinin-2ISb inhibits the virulents in MRSA by intro-cellular binding nucleic acidIn order to explore the underlying mechanism of low dose Brevinin-2ISb producing effective infective therapeutic activity,relevant studies have carried out.It was found that low concentration of Brevinin-2ISb antimicrobial peptide(1/4MIC)could significantly inhibit the activity of several virulence factors which produced by MRSA in a certain time range.After 2 hours of Brevinin-2ISb at 1/4(2.175 ?M)concentration,the activity of MRSA bacteria decreased significantly(17.54%)compared with that of MRSA positive control group(50.62%).After 4 hours?Brevinin-2ISb treatment with 1/4 MIC showed complete coagulation,which inhibited plasma coagulase activity in MRSA.The total enterotoxin content of MRSA in 1/4 MIC Brevinin-2ISb treatment group decreased significantly from 12 hours to 48 hours,and reached the lowest level.It also inhibited the release of TNF-? from lymphocytes stimulated by MRSA supernatant:Brevinin-2ISb of 1/4 MIC had the best effect of inhibiting the release of TNF-? from lymphocytes stimulated by MRSA supernatant at 8 h of co-culture,reaching 32.38 pg/mL,and then showed a stable inhibition state.In addition,the expression of virulence genes and proteins in MRSA was significantly inhibited:the expression level of a-hemolysin gene was 1.96?5.23 times lower than that in the control group;the expression level of coagulase gene in MRSA plasma was significantly lower than that in the control group;the expression level of coagulase gene in MRSA plasma was lowest in 3?4 hours(5.207 times);and the expression level of various toxins genes in MRSA was significantly lower 48 hours before treatment.The transcription of positive transcription-related regulatory genes such as Tsst-1?FnbA,FnbB and agrA was significantly reduced 2.23-5.31 times.At the same time,the expression of alpha-hemolysin protein,enterotoxin A,B,G and TSST-1 protein was significantly inhibited in the first 48 hours of Brevinin-2ISb action.Subsequently,the circular dichroism experiment of Brevinin-2ISb antimicrobial peptide and the prediction of its tertiary structure showed that the above inhibition was due to the special structure of its a-helix and random curl.This inhibition was also confirmed by Brevinin-2ISb fluorescence localization and gel retardation experiments.The inhibition was caused by the nucleic acid binding of Brevinin-2ISb antimicrobial peptide into the membrane.It was also found that Brevinin-2ISb changed its secondary structure after binding with the DNA of MRSA.The clarification of Brevinin-2ISb antimicrobial peptide on the important mechanism of MRSA inhibition has laid a solid foundation for further research and development of new drugs against multiple drug-resistant bacteria and pathogenic microorganisms.5.Brevinin-2ISb inhanced the pathway of DAF-2/DAF-16 in C elegansTo explore the relationship between Brevinin-2ISb and DAF-2/DAF-16 innate immune regulatory pathway of Cryptorhabditis elegans,the relevant research had carried out.The expression of DAF-2/DAF-16 pathway-related immune genes was inhibited after 24 hours.After 24 hours of up-regulation of C29F3.7 and K08D8.5 genes,the expression level in each treatment group returned to normal and was not inhibited by long-term treatment with MRSA.The main reason is that these two genes are early expressed genes,rather than the antimicrobial genes encoding proteins in DAF-2/DAF-16 pathway.However,under the action of Brevinin-2ISb,Lys-7 and Spp-1 showed persistent high expression within 48 hours,especially Lys-7(up-regulated 8-25 times within 48 hours).Due to the significant increase of Lys-7 gene expression level,the protein level was further verified by immunoblotting and laser confocal localization.By measuring the expression intensity of LYS-7 protein,which is the activation of key antimicrobial proteins in the DAF-2/DAF-16 immune pathway(4.37-7.22 times up-regulated by in vitro Western blotting and 2.97-4.62 times up-regulated by in vivo fluorescent protein)was fully confirmed under the action of low concentration Brevinin-2ISb.This further confirmed the important role of Brevinin-2ISb in activating DAF-2/DAF-16 immune pathway.