| Infectious diseases are the mainly cause of death in the worldwide.At present,the traditional antibiotics effect the growth and survival of pathogens.Under survival pressure,pathogens are easy to be drug-resistant.With the emergence of drug-resistant strains,traditional antibiotics cannot completely inhibit the growth of pathogens.The targets and mechanisms of newly discovered antibiotics are similar to those of traditional antibiotics.Therefore,we need to look for new strategies and methods to inhibit drug-resistant bacteria.The virulence indicates the strength of the pathogen,and the substance that constitutes the bacterial virulence is called the virulence factor.The secretion system of bacteria is one of important virulence factors,among which Type III Secretion System(T3SS)is one of the most mature and well-studied systems.Because inhibiting T3SS don’t kill bacteria and it reduce the production of drug-resistant bacteria,T3SS is potential to be antimicrobial target.Myanmar has a wealth of plant resources and traditional knowledge of the treating various diseases,which provide a wealth of samples and new ideas for this study.93 species of traditional Myanmar medicinal plants were screened for antibacterial activity by disc diffusion method.The extracts of Mesua ferrea L(My10),Curcuma comosa Roxb.(My67)and Coptis teeta Wall.(My 109)inhibited the growth of Staphylococcus aureus.The extracts of Mansonia gagei J.R.Drumm(My3)and Mesua ferrea L.(My10)inhibited the growth of Proteusbacillus vulgari.In this study,the SDS-PAGE was used to screen the inhibitors of T3SS of Salmonella enterica Serovar Typhimurium UK-1.The extracts of Luvunga scandens(Roxb.)Buch.-Ham.exWight&Arn(My7),Myrica nagi Thunb.(Myll),Terminalia citrina Roxb.ex Fleming(My21),Tymus vulgaris L.(My49)and Cinnamomum bejolghota(Buch.-Ham.)Sweet(My104)had strong inhibitory effects on the secretion of T3SS virulence protein SipA,SipB,SipC and SipD,and had no significant effect on the growth of Salmonella.From T.citrina(My21),My21-1 that inhibited the secretion of SipA,SipB,SipC and SipD was obtained by bioassay-guided method.The inhibitory mechanism of My21-1 on T3SS was preliminarily studied.The results showed that My21-1 could inhibit the secretion of virulent proteins in a dose-dependent manner and didn’t affect the growth of Salmonella.The invasion experiment showed that the secretion levels of total protein(Total),intracellular protein(Intracellular)and supernatant protein(Supernatant)in My21-1 treated group decreased,which indicated that My21-1 can inhibit the invasion of HeLa cells by Salmonella by inhibiting the transcription and translation of virulent proteins.The hilD and hilA genes were highly expressed,respectively.The SipC level in the supernatant of the overexpressional strain was detected by SDS-PAGE,and the target of My21-1 was determined.The results showed that overexpressional strains of pWSK29-hilD-pBAD χ8956 and pWSK29-hilA-pBAD x8956 were able to promote the secretion of SipC compared with x8956.In pWSK29-hilD-pBAD χ8956,My21-1 inhibited the secretion of SipC.My21-1 regulated the transcriptional regulator of HilD downstream.In pWSK29-hilA-pBAD x8956,My21-1 had not significant effect on the secretion of SipC.HilA can directly bind to the promoter and activate the expression of prg-org and inv-spa operons.My21-1 may inhibit the transcription of hilA,thereby inhibiting the secretion of T3SS virulence protein. |
