Study On The Anti-quorum Sensing Activity And The Self-assembly Behavior Of Tryptophan-containing Cyclic Dipeptides

At present,the bacterial resistance has become an urgent problem to be solved.Gram-negative bacteria cause more bacterial infections and diseases among these,which can produce broad-spectrumβ-lactamases,and hydrolyze antibiotics,thus causing the drugs to lose their efficacy.Novel antimicrobial treatment strategies are urgently needed to compensate for the shortcomings associated with conventional antibiotic therapy.Evidences have been reported that QS system can regulate bacterial biofilm and virulence factor genes,subsequently prompting the infection of host tissue cells.Therefore,new drugs which targeted the inhibition of QS pathway can slow down the development process of drug resistance and disarm pathogens.Natural small-molecule cyclic dipeptides（CDPs）have gradually entered the vision of researchers because of their advantages,such as good biocompatibility and anti-QS activity.Cyclo（Trp-Ser）,abbreviated as c（WS）,was isolated from marine bacteria in the early stage of our group,and the results demonstrated that it has a superior anti-QS activity against both C.violaceum CV026 and P.aeruginosa PAO1.In this study,we designed and synthesized c（WS）and its isomers by replacing serine sites with different amino acids as well as stereoiso meric transformations,then evaluated the effects of these CDPs on the release of virulence factors and the ability of biofilm disruption against C.violaceum CV026 and P.aeruginosa PAO1.Their cytotoxic and self-assembly behavior were assessed,too.The main work of this thesis is as follows:Firstly,we evaluated the antibacterial activity of tryptophan-containing cyclic dipeptide on C.violaceum CV026 and P.aeruginosa PAO1 in two medium.The results showed that the minimum inhibitory concentration of most of the CDPs was greater than15 m M for PAO1 strain,indicating that these CDPs inhibit the bacteria in a way which different from the direct bactericidal approach of traditional antibacterial drugs.By measuring the effect of violet pigment production secreted by CV026,we found that all CDPs inhibited violet pigment production,with a range of 40-60%inhibition while at a bacterial survival rate of more than 90%;further,the molecular docking experimental technique was used to investigate the possible interaction between CDPs and CV026QS-associated receptor protein Cvi R,the results showed that all CDPs were easier to bind the Cvi R protein than C₆HSL with lower binding energy.Then a series of virulence factors assays of P.aeruginosa PAO1 were evaluated by chloroform extraction,elastin-congo red substrate binding and RT-q PCR.The results showed that the c（WS）isomer and its analogs significantly inhibited the production of pyocyanin in a concentration-dependent way;the enzyme activity assay results showed that CDPs containing tryptophan inhibited elastase production by 20-40%and proteolytic enzymes production by 10-20%at 1 m M.RT-q PCR data showed that these CDPs affected the expression of different QS genes.CDPs containing D-Ser have better biological activity which inhibited the expression of rhl I by 69%and 72%,respectively.However,the four analogs had different effects on gene expression.They have a significant inhibitory effect mainly on the pqs system.The results of the mammalian cytotoxicity and hemolytic toxicity assays showed that all CDPs exhibited low cytotoxicity at the tested concentration of 1 m M.Subsequently,we evaluated the effects of CDPs on biofilm formation and EPS production of PAO1 by using crystalline violet staining and phenol-sulfate method.We concluded that all CDPs were able to significantly interfere with the biofilm formation of P.aeruginosa in a concentration-dependent manner;the SEM images showed that the biofilm density decreased by treated with c（WS）analogs and their isomers.The anti-adhesion rate of c（WS）,c（Ws）,c（w S）and c（ws）were 73%,81%,77%and 70%,respectively..Finally,the self-assembly behavior of CDPs was initially investigated in this research.Our experimental results show that the critical aggregation concentration values of all the CDPs were less than 1 m M,indicating that the self-assembly behavior can easily occur.Different assembly morphologies could be formed in water and methanol solvents by using SEM and AFM.In summary,this study was conducted to screen the inhibitors which can inhibit the QS system of CV026 and PAO1 from tryptophan-based cyclic dipeptides.And we investigated the mechanism of QS action by these CDPs.The experimental results showed that all tested CDPs could inhibit the QS system of P.aeruginosa PAO1 and its related phenotypes to some degrees;and had significant destructive effects on the biofilm of P.aeruginosa PAO1.These findings indicated that these CDPs have the potential to act as QSIs,and provide an important structural basis reference for the development of anti-QS drugs in the future.