亚抑菌浓度下血根碱对IncX3型质粒接合转移的抑制作用及机制研究OA

To identify inhibitors of IncX3 plasmid conjugative transfer and investigate their mechanisms of action,an IncX3 plasmid conjugation model was established using Escherichia coli BW25113-IncX3 as the donor strain and MG1655 as the recipient strain,followed by high-throughput screening.The effects of candidate compounds were evaluated by calculating inhibition folds at sub-inhibitory concentrations,assessing bacterial growth,and validating their applicability in different recipient strain models.Furthermore,the mechanisms underlying the regulation of IncX3 plasmid conjugative transfer were investigated by measuring bacterial respiratory activity,the NAD⁺/NADH ratio,proton motive force,intracellular ATP content,cell membrane permeability,intracellular reactive oxygen species(ROS)levels,and gene expression using real-time fluorescence quantitative polymerase chain reaction(qPCR).The results showed that sanguinarine(SAN)was identified from a library of 1 191 natural compounds.Compared with the Control group,SAN at one-half of the minimum inhibitory concentration(1/2 MIC)had no significant effect on the growth of donor or recipient strains(P>0.05),but significantly inhibited the conjugative transfer efficiency of IncX3 plasmids in different recipient strains(MG1655 and EC600)(P<0.000 1).Mechanistic studies demonstrated that,compared with the Control group,treatment with 1/2 MIC SAN significantly reduced iodonitrotetrazolium chloride(INT)reduction capacity,the NAD ⁺/NADH ratio,transmembrane proton gradient(ΔpH),intracellular ATP content,and ROS levels in both donor and recipient strains(P<0.000 1).In contrast,cell membrane permeability in recipient strains was significantly increased(P<0.000 1).In addition,treatment with 1/2 MIC SAN significantly downregulated the mRNA expression levels of genes associated with the type Ⅳ secretion system and energy metabolism(P<0.01 or P<0.000 1).These results indicate that SAN inhibits the conjugative transfer of IncX3 plasmids at sub-inhibitory concentrations by suppressing electron transport in the bacterial respiratory chain,disrupting the proton motive force,reducing intracellular energy supply,and downregulating the expression of conjugation-related genes.