Screening Of Type Ⅲ Secretion System Inhibitors,Mechanism And The Heterologous Expression And Crystallization Of Related Proteins

The problems caused by pathogen to human social life cannot be ignored and antimicrobial drugs played a major role in fighting against the harm.At the same time,it brings trouble.The abuse of antibacterial drugs is so common in the community or in the hospital,resulting in that the bacteria become resistant to these drugs,which have caused great threat to human public security.The strong pressure of survival leads to the resistant strains,so seeking novel antibacterial drugs that can reduce the pathogenicity and have no effect to the growth of pathogen is urgent.Type Ⅲsecretion system(T3SS)is a crucial virulence,which is highly conserved in Gram-negative pathogenic bacterium.In addition,T3SS is a specialized virulent protein nano-injector that transports effector proteins from bacterial cytoplasm to the host cell cytosol and promotes bacterial invasion and dissemination.Salmonella enterica serovar Typhimurium is an infectious bacteria and used in this experiment.Firstly,twenty prenylated flavonoids were preliminary screened for their effects on the secretion of the SPI-1 effector proteins by SDS-PAGE.Then several compounds were chosen to confirm by western blot and analysis the structure-activity relationship,showing no significant common structure features.Among them,LCF exhibited strong inhibitory effects on the secretion of the SPI-1 effectors SipA/B/C/D and have no effect on FliC proteins.Furthermore,LCF showed potent inhibitory activity on the T3SS in a dose-dependent manner and had no effects on the growth of Salmonella.Thus,it was selected for further investigation.After the co-culture of Salmonella with HeLa cells,Western Blotting analysis showed that LCF had effects on the transportation of the SPI-1 effector SipC.Subsequently,RT-PCR was used to determine whether LCF had any effects on the transcription of T3SS related genes.We found that LCF could inhibit the secretion of SipC protein by inhibiting the transcription of the SicA/InvF genes.To detect whether LCF could have any effects on the assembly of Needle Complex(NC),and the results showed that LCF have no effects on the assembly of PrgH NC.Meanwhile,the synthesis of Csn-B,which was used as the positive control on T3SS,was designed to seek the binding sites of Csn-B and related proteins.The synthesis was prepared from the raw material 3,5-dimethoxyphenylacetic acid followed by Friedel-Crafts reaction to connect the acyl group to the aromatic ring.Then it was demethylated by using AICl3 in toluene under reflux.Finally,Csn-B was obtained after esterification in acid medium with ethanol.Furthermore,the chaperone SicA and transcription factor InvF were cloned,which belong to S.enterica serovar Typhimurium.After continuous trying and different constructs’ building,we obtained the columnar crystal of SicA.InvF is soluble from the initial inclusion in the end.In conclusion,we found a novel natural compound LCF in the T3SS inhibitors screening experiments and explored its mechanism of action,which is significant for the development of new anti-resistant antibiotics.