| The biofilm formed by bacteria plays an important role in the persistent infection of bacteria.The resistance of bacteria in biofilm to antibiotics is several orders of magnitude higher than that of floating bacteria.It has been reported that more than 80%of malignant infections in clinic are related to the resistance of bacterial biofilm.3’,5’-cyclic guanosine(c-di-GMP)is a kind of second messenger widely existing in bacteria,which is involved in many biological functions including the regulation of bacterial biofilm formation and release of virulence factors.Studies have shown that high levels of c-di-GMP will induce bacteria to choose a way of living in clusters and significantly promote biofilm formation.Therefore,c-di-GMP pathway is an attractive drug target for controlling bacterial infection.The latest research found that under physiological conditions,c-di-GMP molecules can spontaneously form a G-quadruplex advanced structure,and once this structure is formed,c-di-GMP can not combine with its target protein and lose its biological function.At the same time,it has been reported that some aromatic compounds can induce and stabilize the structure of c-di-GMP G-quadruplex.Therefore,using small molecules to directly act on c-di-GMP itself and indirectly reduce its effective concentration by inducing it to form a functional G-quadruplex is a new potential anti-biofilm drug design direction.This paper puts forward a brand-new anti-biofilm drug design strategy based on the regulation of c-di-GMP advanced structure(G-quadruplex),and systematically studies the feasibility of this strategy,in order to find new anti-biofilm compounds based on this strategy and provide a new choice for overcoming bacterial biofilm resistance.The main contents of this thesis include:1、 Taking thiazole orange(TO),a reported inducer of c-di-GMP G-quadruplex,as a model molecule,this paper explores the feasibility of designing new anti-bacterial biofilm drugs targeting the regulation of c-di-GMP higher structure(Gquadruplex).The inhibitory effect of inducer TO on P.aeruginosa biofilm formation was systematically evaluated by various biological methods and means.The results showed that the inhibitory rate of inducer TO on P.aeruginosa biofilm could reach about 50% when the concentration of inducer TO was 2.5 μg/m L.Furthermore,it was proved by reporter strain PAO1-Δwsp F-mini-ctx-pel A-lac Z and reverse transcription-polymerase chain reaction(RT-PCR)that inducer TO had a significant effect on the expression of Pel,Psl operon and its downstream related genes regulated by c-di-GMP.the above results verified that the target of inducer TO was c-di-GMP signaling molecular pathway.At the same time,the fluorescence of c-di-GMP-TO complex in bacteria was observed by c-di-GMP biosensor,which proved the interaction between compound to and c-di-GMP in bacteria.And by detecting the influence of inducer TO on the activity of c-di-GMP metabolic enzymes,it was proved that the compound could directly interact with c-di-GMP instead of affecting the activity of related metabolic enzymes,thus realizing the influence on the biological function of c-di-GMP pathway.Based on the above results,the feasibility of the new strategy proposed in this paper is preliminarily proved.2、 In order to overcome the DNA embedding toxicity of TO and find a low-toxic and effective inducer with clinical application prospect,the activity screening and structure-activity relationship of a new type of c-di-GMP G-quadruplex inducer designed and synthesized by our research group were studied.Firstly,the induced activities of the compounds were tested by circular dichroism(CD)experiment,and the compounds 2a,2e,2f and 5h with better induced activities than the positive drug TO were screened out.At the same time,the anti-P.aeruginosa biofilm formation effects of all the compounds were preliminarily screened,and it was found that the inhibition rate of inducer 5h on P.aeruginosa biofilm at 1.25 μM was over 60%.In addition,the fluorescence changes of the compound interacting with c-di-GMP and other nucleic acid analogues were measured by fluorescence spectrophotometry,which proved that the compound can selectively act on c-diGMP.The stoichiometric ratio of the compound interacting with c-di-GMP for 5h was calculated to be 1:4.5 by continuous titration.The stoichiometric ratio of the compound 5h interacting with c-di-GMP was calculated to be 1:4.5 by continuous titration.At last,the structure-activity relationship of related compounds was summarized by systematically analyzing the induction activity,selectivity specificity and screening results of P.aeruginosa biofilm inhibition ability of all inducers,which provided theoretical guidance for rational design of new c-diGMP G-quadruplex inducers in the future.Finally,a new anti-bacterial biofilm precursor compound 5h based on this strategy was discovered.3、 Study on the action mechanism of C-di-GMP G-quadruplex inducer for 5h.By testing the effect of compound 5h on bacterial swarming movement and exopolysaccharide secretion,it was confirmed that 5h can effectively regulate the related phenotype regulated by c-di-GMP molecule.The effect of compound 5h on the production of reporter strain PAO1-Δwsp F-mini-ctx-pel A-lac Z β-galactosidase was tested,and the effect of compound 5h on the expression of related genes regulated by c-di-GMP in bacteria was proved from the gene expression level by transcriptome data analysis.In addition,by testing the effect of compound 5h on the activity of c-di-GMP-related metabolic enzymes,it was proved that the effect of compound 5h on the relative content of c-di-GMP in bacteria was not to change the activity of its metabolic enzymes.The above results reveal that the action mechanism of inducer 5h is to induce c-di-GMP signal molecules to form G-quadruplex structure with loss of biological function,which is consistent with the new strategy proposed in this paper.Finally,MTT and gel electrophoresis experiments proved that compound 5h did not have the reported DNA insertion toxicity of inducer TO,and its biological safety could be guaranteed within a certain concentration range.Therefore,compound 5h is expected to be a potential lead compound for treating chronic infection of P.aeruginosa biofilm. |
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