High-throughput Screening And Verification Of Quorum Sensing Inhibitors Of Salmonella Typhimurium LT2

Salmonella,as one of the common pathogenic bacteria in human body,is easy to cause diseases such as acute gastroenteritis in humans,and plays an important role in host food poisoning and disease.In traditional treatment regimens,antibiotics can treat infections caused by Salmonella,but the abuse of antibiotics can cause Salmonella to develop multiple drug resistance,and the resistance will migrate and mutate at the genetic level.In recent years,more and more studies have shown that the quorum sensing（QS）system can be manipulated to regulate the formation of bacterial biofilms,the production of virulence factors,and the inhibition of drug resistance.Therefore,based on the important role of the QS system in regulating bacterial virulence and drug resistance,the development of new QS inhibitors can open up new ideas for solving the problem of bacterial drug resistance.The laboratory is based on the idea of screening quorum sensing inhibitors.First,molecular docking technology,molecular docking of metabolite molecules in the GMD database with Salmonella typhimurium LT2 quorum sensing receptor Lsr B protein,and screening a batch of putative quorum sensing inhibitor molecules.The binding ability of metabolite molecules with Lsr B protein was verified by Surface Plasmon Resonance（SPR）technology.According to the affinity constant KD,the order is:gallic acid(KD=6.14×10^-6)<salicylic acid(KD=8.686×10^-6)<cinnamaldehyde(KD=1.14×10^-5)<benzoquinone(KD=1.96×10^-5)<indole-3-acetic acid(KD=8.93×10^-5)<Mandelic acid(KD=9.81×10^-5).According to the principle that the smaller the affinity constant,the greater the affinity.The affinity is:gallic acid>salicylic acid>cinnamaldehyde>benzoquinone>indole-3-acetic acid>mandelic acid.This article then verifies the quorum sensing inhibition performance of small molecule inhibitors on the basis of screening.Firstly,we studied the MIC,inhibitory growth curve,inhibition zone experiments of small molecules against Salmonella.It was found that the antibacterial ability of 2m M salicylic acid and 0.5m M cinnamaldehyde increased with the increase of the concentration of salicylic acid and cinnamaldehyde.Scanning electron microscope,confocal fluorescence microscope,and optical microscope were used to observe the biofilm formation of Salmonella on the surface of the cover glass.It was found that small molecule inhibitors inhibited the ductility and thickness of Salmonella biofilm at a sub-MIC concentration of 2/5 MIC,salicylic acid and cinnamaldehyde reduced the biofilm formation ability by 71.5%and 16.4%,and the biofilm inhibitory effect increased with the increase of concentration.In vivo fluorescence intensity characterizes the inhibitory effect of Salmonella’s internal QS system.The addition of salicylic acid and cinnamaldehyde leads to an increase in the concentration of phosphorylated AI-2 molecules in the cell,and enhances the expression of fluorescent protein by P_lsr.The fluorescence intensity rises,and the salicylic acid is 2/5 MIC.Salicylic acid and cinnamaldehyde increased the fluorescence intensity of LT2-p SC101-P_lsr-e GFP by 7%and 13%respectively,and increased the fluorescence intensity of LT2-p SC101-P_J23119-Lux S-P_lsr-e GFP by 76%and 46%respectively.Fluorescence quantitative PCR results showed that salicylic acid and cinnamaldehyde significantly down-regulated the invasion ability of Salmonella and the expression of genes related to the T3SS transport system（sop B down-regulated 2.9 times,inv H down-regulated 6.36 times,hil A down-regulated 3times,sip A down-regulated 3 times）,affecting Salmonella’s toxicity and infection ability.The results of this study show that salicylic acid and cinnamaldehyde can inhibit the biofilm formation and virulence factor production of Salmonella through the quorum sensing system.Salicylic acid can also inhibit the division of bacteria to inhibit Salmonella’s quorum sensing system.The methods and processes of developing and improving quorum sensing inhibitors provide a reference for the later development of new quorum sensing inhibitors to reduce the resistance of bacteria.