| Objective:Drug-resistant diseases has already caused at least 700,000 deaths each year and has become a very serious public health problem in the world.Methicillin-resistant Staphylococcus aureus(MRSA)is the main cause of healthcare-associated and community-associated infections.Persister is the cause of chronic and recurrent nature of many infections,and is the major culprit of treatment failure.The emergence of drug-resistant bacteria and persister make traditional antibiotics unable to continue to be effective,which are two major problems in the treatment of S.aureus infections.Persister is considered to be drug storage reservoirs where bacteria have developed into drug resistance.Persister is incurable in clinics at present.Therefore,research on new anti-persister drugs is of great significance to overcome antibiotic resistance.The previous research of our research group found that cajaninstilbene acid derivatives have good antibacterial activity against persister.In this thesis,we screened the new cajaninstilbene acid derivatives designed by our group to investigate their anti-persister activity and to explore the mechanism of active compound,as well as the anti-persister ability in mouse infectious model.It can be further studied as a potential novel lead compound for the treatment of persister infection.Methods:1.Screening the active compound.The MIC values of the new cajaninstilbene acid derivatives were determined by the standard microdilution method.These derivatives were evaluated for antibacterial activities against 12 Gram-positive strains and 3 Gram-negative strains.The in vitro cytotoxicity of the cajaninstilbene acid derivatives was determined against two mammalian cell lines(mouse macrophages RAW264.7 and normal human hepatocytes LO2).MRSA ATCC43300 strain was selected to evaluate the anti-persister activity of cajaninstilbene acid derivatives,combined with MIC and MTT activity data to select an active compound for further mechanism research.2.Action Mechanism Investigations.The derivatives were designed to target the bacterial cell membrane.To verify our conjecture,membrane permeability experiment,membrane depolarization experiment and confocal laser scanning microscopy were carried out to explore the damaging effect of active compounds on bacterial cell membranes.Transmission Electron Microscopy(TEM)was used to confirm the damage of cell membrane on the morphology.The absorption of 260 nm and a kit were used to verify the damage of bacterial cell membranes by detecting leakage of nucleic acid and ATP.3.Evaluation of anti-MRSA activityThe effect of active compound combined with antibiotics on MRSA was further evaluated by the checkboard method.The ability to eradicate adherent biofilm of MRSA was evaluated using a 96-well plate biofilm formation experiment.In addition,a cutaneous mouse infection model was established and inoculated with MRSA persister.The ability to reduce MRSA persister in vivo was evaluated.Results:1.Result of screening the active compound.Synthetic cajaninstilbene acid derivatives showed potential antibacterial activity against 12 Gram-positive bacteria and showed no effective against Gram-negative bacteria.MTT cytotoxicity evaluation revealed that compound Y7 and Y8 exhibited low-toxicity on mouse macrophages RAW264.7 and normal human hepatocytes LO2,Anti-persister bacteria screening showed that 23 compounds out of 37 synthetic cajaninstilbene acid derivatives existed a good inhibitory effect on MRSA persister..Combined with MTT toxicity data,Y8 was selected as our active compound for further studies.The persister kill-time curves of Y8 showed that 4 times,6 times MIC can remove the persister bacteria in 4 hours,2 times MIC can remove the persister bacteria in 8 hours compared with DMSO.4.Mechanism research ResultsMembrane permeability experiment,depolarization experiment,and confocal laser scanning microscopy all proved that the active compound Y8 showed a damaging effect on the membrane in time-dependent.The TEM experiment verified the damaging effect on the membrane morphologically.Nucleic acid leakage experiments and ATP leakage results both proved that the active compound Y8 damaged the bacterial cell membrane and caused the content to leak,further verifying the damage effect on the membrane.All experiment proved that Y8 acted on the cell membrane of bacteria and damaged the complete membrane structure,which made the contents of the bacteria leaking further,thus killing the bacteria.3.Further activity evaluation resultsa.The combinational antibacterial results showed that Y8 combined with paracillin exhibited a synergistic effect,and showed partially synergistic effect with gentamicin.b.Biofilm eradication experiment proved that the Y8 showed a good activity to eradicate MRSA biofilms in concentration-dependent effect,which was significantly better than the positive control vancomycin.It proved that the active compound Y8 was not only effective against planktonic bacteria,but also can remove adhesion mature biofilms.c.Result of cutaneous mouse infection model: Compared with the normal saline group,the compound group can reduce the load of MRSA persister.It showed compound Y8 can reduce persister in vivo.Pathological sections showed no harm to heart,liver,spleen,lung and kidney.Conclusion:The thesis screened two series of cajaninstilbene acid derivatives synthesized by our group,a low-toxic and highly effective compound Y8 was screened out.Further study on the mechanism of bacterial membranes had clarified that compound Y8 exerted its effect mainly by destroying bacterial cell membranes.Finally,further activity evaluation showed that compound Y8 had a synergistic effect combining with ineffective antibiotic piperacillin and can eradicate the adherent biofilm.The cutaneous mouse infection model showed better load-reducing activity of MRSA persister.This study suggests that the active compound Y8,an antibacterial agent acting on bacterial cell membranes,can be used as a lead compound against persister in chronic infection. |
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