The Plant Phenolic Compounds That Suppress The Virulence Of Xanthomonas Campestris Pv.campestris Via The Type ? Secretion System

Traditional antibiotic therapy,which mainly interferes with cell primary metabolism,hinders their life activities to stops bacterial growth.This ?live or death? selection pressure leads to rapid emergence of the bacterial resistance problem that becomes more and more common and severe.So,development of new antimicrobial agents which target the bacterial virulence factors and do not affect the bacterial growth has become particularly important.Xanthomonas campestris pv.campestris,(Xcc)is a gram negative pathogen,which can cause black rot of a wide range of cruciferous crops,leading to significant economic losses in agricultural production around the world.The previous results showed that the type III secretion system(T3SS)is a major virulence factor in many bacterial pathogens.This secretion system translocates effectors directly into the cytosol of eukaryotic host cells,where the effector proteins facilitate bacterial pathogenesis by interfering with host cell signal transduction and other cellular processes.Thereofre,T3 SS can be used as an ideal target for new drug design and development.Phenolic compounds of plant secondary metabolites are widespread in plants,some of which play a key role for resisting pathogen infection.The plant phenolic compounds that suppress the virulence of Xanthomonas campestris pv.campestris via the type III secretion system has not yet been reported.To identify potential antivirulence drugs against this pathogens,we screened a library of plant phenolic compounds and derivatives for their effects on the Xcc T3 SS by a high-throughput approach.The promising inhibitors were further tested for their impact on the role of T3 SS,and provide a theoretical basis for its further application in disease prevention and treatment.This study first used a green fluorescent protein(GFP)reporter system by fusing to the promoter of the hpa1 gene,which encodes a T3 SS translocon protein and contains complete PIP-box.The promoter activity of the harpin gene hpa1 was assayed in the presence or absence of each compound and the average fluorescence intensity were detected by flow cytometry.The results showed that 7 compounds inhibited the hpa1 promoter activity by at least 64% among the 42 tested compounds.Then,we investigated their effect on bacterial growth at different stages by using a Growth curve instrument.TS008,TS011,TS015,TS019,TS021 and TS110 were added to the media at a concentration of 200 ?M.The results showed that in comparison with the non-treatment control,except for TS012,the other 5 compounds did not show statistically significant changes at different time points.In order to further test the impact of the 6 inhibitors on T3 SS,we selected the promoters of the key hrp regulatory genes hrpG and hrpX to detect the influence by the 6 inhibitors.The results showed that all the 6 inhibitors could inhibit the expression of hrpG and hrpX.In previous studies,almost all of the studies were on creening of single small molecule inhibitors against bacterial T3 SS.Based on the structure of the compounds with inhibitory activity,we found that the phenolic compounds could be classified into 3 categories:(1)The substitution of different sites and functional groups on the benzene ring with cinnamic acid as framework.(2)hydrocinnamic acid.(3)trans-3-(2-thienyl)acrylic acid.Considering that different molecular structures may have different inhibitory mechanisms or different targets,we mixed 6 small molecules in various combinations.The promoter activity of the harpin gene hpa1 was assayed in presence of two mixed compounds and the average fluorescence intensity were detected by flow cytometry.The results showed that the inhibitory rates of 10 combinations were higher than that of single component and the inhibition rates of 8 combinations were higher than 80%.Among the 8 combinations with over 80% inhibitory activity,4 combinations were formed using the category(1)compounds,the other 4 combinations were made using compounds from categories(1)and(2),and(1)and(3),respectively.The above results suggest that substitution at the different sites on a molecule such as benzene rine can improve the inhibitory activity,and that combination of compounds with different chemical structures may improve inhibitory activity through interacting with different sites of same target protein or acting on different target proteins.In summary,the results from this study identified a few chemical inhibitors with promising activity against the T3 SS of X.campestris pv.campestris.In addition,it is also found that combination of inhibitors with different substitution or different chemical structures could further improve the T3 SS inhibitory activity.The findings from this study provide useful lead compounds for further optimization and practical control of the bacterial diseases.