Regulation Of The Trp-containing Antimicrobial Peptides On Quorum Sensing System Of Drug-resistant Pseudomonas Aeruginosa

Abuse of antibiotics results in emergence of multiple-drug resistant(MDR)bacteria in clinical therapy.Among MDR bacteria,Pseudomonas aeruginosa is knovm to be an important human pathogen,responsible for various infections,and is very difficult to eradicate as its high intrinsic resistance to a wide variety of antibiotics and secondary adaptive resistance mechanisms.In this case,traditional drugs have not solved the current problem,and there is therefore an urgent need to discover novel and more effective drugs.Quorum sensing system as a communication mechanism between bacterium,is closely related to the serious infectivity and pathogenicity of Pseudomonas aeruginosa.Antimicrobial peptides(AMPs)are a fundamental component of innate immunity and exhibit broad spectrum antimicrobial activities against Gram-negative and Gram-positive bacteria,viruses and fungi.Their membrane active mode might become a possibly be the most promising way to solve bacterial resistance.In our laboratory,a series of Trp-containing antimicrobial peptides were designed and synthesized by replacing with tryptophan residues from L-K6,an antibacterial peptide rich in Iysine/leucine residues.The previous results showed that the Trp-containing antimicrobial peptides had high antibacterial activity against a variety of multi-drug resistant bacteria isolated from the clinical samples.They could insert into the outer membrane of MRPA0108 cells by interacting with LPS,the main component of outer membrane.In addition,the Trp-containing antimicrobial peptides were able to inhibiting the expression of outer membrane pump-associated genes in MRPA0108 cells,resulting in the reduction the efflux of MRPA0108 against antibiotics.The formation of efflux pump and biofilm is related to the bacterial quorum sensing system.Therefore,this paper focuses on the action mechanism of the Trp-containing antimicrobial peptides on quorum sensing system in the clinically isolated multi drug-resistant Pseudomonas aentginosa(MRPA0108),and hopes to give an approach for treatment of infection caused by multidrug resistant bacteria.The Trp-containing antimicrobial peptides were found to penetrate the bacterial cell membrane and accumulate in the nuclear region.A DNA gel retardation assay indicated that the Trp-containing antimicrobial peptides were able to bind with the genomic DNA of MRPA0108 cells,L5W exhibited a stronger DNA-binding ability than that of the other peptides,and the ratio of peptide to DNA was 0.62/1,and I4W and I1WL5W had DNA-binding at the ratio of peptide to DNA 2.5/1.Next,the UV absorption spectrum of DNA and circular dichroism(CD)assays showed that the Trp-containing antimicrobial peptides could interact with the MRPA0108 genomic DNA,resulting in the hyperchromic effect.The results suggested that insertion of the peptide into the groove of the DNA molecule made loosening of the double-helical structure of the originally contracted DNA.However,when the ratio of L5W/DNA and I1WL5W/DNA was 1.25/1 and 2.5/1,the hypochromic effect occurred and accompanied by a red shift,suggesting that the peptides at high concentrations,the electrostatic interactions between positively-charged peptides and negatively-charged DNA made an axial spatial contraction of the DNA molecules,leading to the conformational changes.RT-qPCR experiments showed that the Trp-containing antimicrobial peptides could down-regulate the expression of DNA replication-initiation genes dnaA,dnaB and dna.in MRPA0108 cells.The result suggested that the Trp-containing antimicrobial peptides may kill bacteria by targeting intracellular DNA.RT-qPCR results showed that the Trp-containing antimicrobial peptides at low concentration could inhibit the expression of virulence factor regulatory genes lasA,lasB,rhlA and rhfB in MRPA0108 quorum sensing system.The effects of the Trp-containing antimicrobial peptides on elastase and alkaline protease activities,the virulence factor regulated by the las system in the MRPA0108 quorum sensing system were examined by using elastin-Congo red method and azocasein method,respectively.The results showed that the Trp-containing antimicrobial 1 peptides at a concentration of 1/4 MIC could inhibit the activity of two proteases with 12-28%of the inhibition for elastase activity and 18-44%of the inhibition for alkaline protease activity.In addition,the effects of the Trp-containing antimicrobial peptides on the production of pyocyanin and rhamnolipid,the virulence factors regulated by the rhl system in the MRPA0108 quorum sensing system were examined by using chloroform extraction and orcinol method.The results showed that the Trp-containing antimicrobial peptides inhibited the release of pyocyanin and rhamnolipids.IIW,L5W and L12W at a concentration of 1/4 MIC inhibited 73.7%,69.4%and 63.1%of pyocyanin production,respectively,while the others had about 55%of inhibition for pyocyanin.Sililarly,I1W and I4W inhibited the release of rhamnolipids with 45.2%and 43.9%of inhibition.This result is consistent with RT-qPCR,suggesting that the inhibitory effect of the Trp-containing antimicrobial peptides on production of the virulence factors regulated by the rhl system is better than by the las system.The rhamnose and pyocyanin regulated by the rhl system in the P.aemginosa quorum sensing system are responsible to the formation of biofilm.We tested the effect of the Trp-containing antimicrobial peptides I1W>I4W and L5W on the morphology of MRPA0108 biofilm by laser confocal microscopy.The results showed that thickness of lxMRPA0108 biofilm significantly was reduced from 6 ?M to 1.5 uM after MRPA0108 cells were treated with IIW?I4W and L5W for 24 h,suggesting that IIW?I4W and L5W can inhibit the formation of MRPA0108 biofilm.Then,the formation of MRPA0108 biofilm was quantitatively exanined by using crystal violet staining and RT-qPCR.The results showed that the Tip-containing antimicrobial peptides could significantly inhibit the formation of MRPA0108 biofilm with a concentration-dependent manner.IIW and 14W at a concentrations of 1/4 MIC inhibited 33.9%and 40.7%of biofilm formation,respectively.Nevertheless,They had low effect on the decomposition of one-day-old biofilm.Only 21.9%and 21.5%of one-day-old biofilm could be decomposd by IIW and I4W at the concentration of 1/4 MIC.Next,biofilm component including extracellular polymer(EPS),psl and alginate were quantitatively examined.The Trp-containing antimicrobial peptides could inhibit the synthesis of EPS with a concentration-dependent manner.I1W at the concentration of 1/4 MIC inhibited 38%of MRPA0108 EPS production.IIW and I4W could significantly reduce the production of alginate in the biofilm with inhibition of 53%and 47%,respectively.RT-qPCR results showed that the Trp-containing antimicrobial peptides inhibited the expression of genes related with formation of biofilm.