| Currently,polymyxins have been considered as the “last resort” antibiotics for the treatment of multidrug-resistant Gram-negative bacteria.However,due to the unreasonable use of antibiotics,the number of polymyxin-resistant Gram-negative bacteria is growing.Therefore,it is urgent for us to know about the resistance mechanism of polymyxin,so as to provide new ideas and drugs for drug discovery against bacterial infection.Studies have shown that the primary target of polymyxin is the lipopolysaccharide(LPS) in the outer membrane of Gram-negative bacteria.After binding to the phosphate group of lipid A in LPS,polymyxin enters the periplasm of the cell,and plays an antimicrobial role.There are three hypotheses about the antibacterial mechanism of polymyxins,membrane cleavage hypothesis,phospholipid exchange hypothesis and hydroxyl radical lethal hypothesis: membrane lysis death pathway,vesicle-vesicle contact pathway,hydroxyl radical death pathway.The resistance mechanism of polymyxin has been gradually reported in Gram-negative bacteria.The major polymyxin resistance mechanisms include(i)alteration of the LPS moiety;(ii)mutations in genes;(iii)increased drug efflux;(iv)reduced porin pathway,(v)formation of capsules.Among them,the modification of LPS by 4-amino-4-deoxy-Larabinose(L-Ara4N),which is regulated by arn operon,is the most common in Pseudomonas aeruginosa.This thesis is intended to take Pseudomonas aeruginosa as the main model for further research on the resistance mechanism of polymyxins.In this thesis,Pseudomonas aeruginosa DK2(MIC = 256 μg/ml),which was highly resistant to polymyxin,was used as the research model.I screened the mutants sensitive to polymyxin and compounds that enhanced the antibacterial activity of polymyxin from both the mutant library and the drug library.DK2 is a multi-drug resistant bacterium,which makes genetic manipulation difficult.Firstly,I modified the transposable plasmid pBT20 successfully,and obtained a transposable plasmid pBT20-TET-1 that can be used in DK2.After the successful establishment of the mutant library(about 20000 insertion mutants),I screened the insertion mutants that were sensitive to polymyxin,and finally got insertion mutants with 15 genes,which resulted in a significant reduction in polymyxin resistance to DK2(MIC ≤ 4 μg/ml).The 15 genes were identified as the genes in the arn operon,the genes associated with LPS synthesis,PA4029,PA1064,and PA3800(bamB).Through functional complementarity experiments,I confirmed the role of bam B in polymyxin resistance,which encodes an outer membrane protein assembly factor.Both in DK2 and MPAO1 that was commonly used in the laboratory,the loss of bam B did not affect the production of LPS,but could increase the permeability of the outer membrane.In addition,the loss of bam B did not cause changes in the outer membrane protein in DK2,while the corresponding outer membrane protein changed in MPAO1.For secreted proteins,the loss of bam B resulted in the changes of secreted proteins in DK2 and MPAO1 to varying degrees.In addition to screening the mutant library,I also screened the drug library to find drugs that could enhance the antimicrobial effect of polymyxin.It was found that niclosamide,rifampicin,rifabutin,rifaximin,NH125,IMD-0354 could significantly increase the antibacterial effect of polymyxin.At the same time,I tested the effect of the compounds combined with polymyxin on the expression of arn operon,and found that NH125 combined with polymyxin could effectively inhibit the expression of arn operon.I also found that,except for niclosamide,the other 5 compounds combined with polymyxin could reduce the production of LPS to a certain extent.In summary,I analyzed the genetic basis of polymyxin highly resistant Pseudomonas aeruginosa DK2 at the genomic level,and screened out some compounds that could enhance the antibacterial effect of polymyxin,which laid a foundation for further study of the molecular mechanism of polymyxin resistance. |
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