Discovery And Antibacterial Mechanism Of Novel Nonribosomal Peptide Derivatives

The emergence and rapid spread of multi-drug resistant bacteria pose a serious threat to public health.Discovery of new antibiotics or antibiotics adjuvants are urgently needed.Nonribosomal peptide antibiotics are a group of antibacterial peptides widely found in nature's organisms,and bacterial secondary metabolites are one of the important sources of such antibiotics.Nonribosomal peptide antibiotics and their derivatives draw more attention due to a wide range of sources,good security,not prone to produce resistance and other advantages.Therefore,this study aims to screen novel non-ribosomal peptide antibiotics and antibiotic adjuvants,further systematically evaluate the structure-activity relationship and elucidate the potential antibacterial mechanism.In this study,a strain of Bacilus subtilis CAU21 was isolated from soil-sourced samples.The antibacterial activity test showed that its secondary metabolites had an excellent inhibitory effect on Staphylococcus aureus.By separation and purification by gel chromatography and high performance liquid chromatography,we have obtained the antibacterial active substance.Its structure was further identified by UV,IR,MS and 1H NMR,which was a cyclic lipopeptide consisting of fatty acid chain and seven amino acid residues,named bacaucin.Bacaucin has a broad spectrum of antibacterial activity against Gram-positive resistant bacteria,but has moderate hemolytic(60%hemolytic rate at 100 ?g/mL)and cytotoxic(IC50 = 68.3-184 ?g/mL).Through the modification of its chemical structure,the fatty acid chain that may cause nonspecific toxicity was removed,and the structure of the cyclic peptide was opened and the influence of the peptide chain length and other factors on its antibacterial activity was explored,and various peptide derivatives were obtained.Among them,bacaucin-1 has a specific bactericidal effect against Staphylococcus aureus and MRSA(MIC = 4-16 ?g/mL),which can destroy bacterial cell membranes and induce loss of membrane potential,further kill bacteria.In order to improve the antibacterial activity of bacaucin-1 and obtain better pharmacokinetic parameters,we further optimized the structure of bacaucin-1 and obtained its derivatives bacaucin-1a(MIC = 2 ?g/mL),a linear non-cationic peptide antibiotic with increased stability and reduced cytotoxicity.The structure-activity relationship study shows that the guanidine group on arginine at position 5 is the active site,and the presence of serine and valine at both ends of arginine contributes to its trypsin tolerance.In order to study its possible antimicrobial targets,we continuously subcultured the drug-resistant mutants in sub-inhibitory concentrations and performed genome sequencing and SNP analysis.As a result,non-synonymous mutations were observed on the GMP synthetase and the RNA polymerase SigB,and therefore we concluded that both of these may be responsible for the resistance of bacaucin-1 a.In Vero cell and mouse infection models,bacaucin-1 a can effectively control MRSA T144-associated infections in vitro and in vivo.After screening for linear antibacterial peptides antibiotics,we obtained a novel broad-spectrum antilbiotic adjuvant SLAP-S25,can enhance the antibacterial activity of multiple antibiotics against E.coli B2(blaNDM-5 + mcr-1),including tetracycline,ofloxacin,rifampicin and vancomycin.SLAP-S25 exerts its bactericidal activity through targeting lipopolysaccharide in bacterial outer membrane and phosphatidylglycerol in bacterial plasma membrane.SLAP-S25 and colistin have complementary effect in the loss of membrane potential and the generation of reactive oxygen species.In addition,SLAP-S25 can significantly inhibit the transcription and translation of the colistin resistance gene mcr-1,thereby enhancing the antibacterial activity of colistin.The combination of SLAP-25 and colistin effectively improves the survival rate of the Galleria mellonella and mice.These results indicate that SLAP-S25 can effectively reverse the colistin resistance in vivo.The emergence of bacterial resistance crisis calls for the discovery of novel antibiotics or antibiotic adjuvants.In this study,the discovery of novel non-ribosomal peptide antibiotics bacaucin and its derivatives provide new antibiotic candidates against MRSA.And,the discovery of the broad-spectrum antibiotic adjuvant SLAP-S25 opens new avenue for improving the efficiency of existing antibiotics and mitigating multidrug resistance problems.