Inhibitory Activity Study Of Three Kinds Of Natural Product Derivatives On The Type Ⅲ Secretion System Of Salmonella

Antibiotics are drugs used to treat or inhibit microbial infections and antimicrobial therapy is one of the most important medical achievements of the 20th century,saving millions of lives.However,along with the mass use of antibiotics,the spread of antibiotic resistance worldwide has become an enormous public health challenge.Virulence represents the pathogenic capacity of the pathogen,and the substances that exert bacterial virulence are called virulence factors.Compared with conventional antibiotics,antivirulence therapy prevents bacteria from harming hosts by inhibiting bacterial virulence,with the advantages of low survival stress on pathogenic bacteria and low risk of drug resistance,and has become an important design strategy for antimicrobial drugs.Type Ⅲ secretion system(T3SS)is a kind of complex protein secretion apparutuses,common in most gram-negative pathogens,including Salmonella.T3SS functions by directly injecting effector proteins into host cells and is a key factor for many pathogens,which also makes T3SS a high-profile target in the field of antivirulence research.It was found that cytosporone B,fusaric acid,licoflavonol and myricanol were potent inhibitors of Salmonella T3SS,and according to their structural characteristics and rational drug design,71 compounds of stilbenes,cinnamamides and diphenyl sulfides were synthesized.In this thesis,the 71 compounds were screened for T3SS inhibitory activity and studied for structure-activity relationship by SDS-PAGE analysis.The results showed that stilbene compounds SS-A6,SS-A13 and SS-A14,cinnamamide compound SY-20 and diphenyl sulfide compounds SL-8 and SL-19 had better inhibitory activity against T3SS than other compounds.Subsequently,western blot,growth curve determination,gentamicin protection experiment,cytotoxicity test,internal and extracellular protein level detection,real-time quantitative PCR and other experiments were carried out to comprehensively evaluate the T3SS inhibitory activity and preliminarily investigate the mechanism of action of these six preferred compounds.The results showed that the six compounds inhibited SPI-1 effector protein secretion of S.typhimurium in a dose-dependent manner and did not inhibit bacterial growth and reproduction.The six compounds inhibited the invasion of S.typhimurium to human adenocarcinoma Caco-2 cells and also showed no obvious toxicity to Caco-2 cells.Primary studies on action mechanism suggested that SS-A6 might inhibit the transmembrane secretion of SipC protein,SY-20 might inhibit the HilD-HilC-RtsA transcriptional regulation loop,and SL-8 and SL-19 might bind to SipC protein to inhibit its transmembrane transport.Furthermore,the tag fusion protein SipC-HA was attempted to construct and express,which laid the experimental foundation for the study of protein-small molecule interaction.In summary,this thesis screened,evaluated and studied the anti-T3SS activity and preliminary action mechanism of natural product derivatives of stilbenes,cinnamamides and diphenyl sulfides,and then identified six T3SS inhibitors with novel structures,which facilitated their in-depth discussion on action mechanism and had positive significance for the development of new T3SS inhibitors.