Influence of the tolC gene on Escherichia coli membrane integrity and antibacterial activity of traditional Chinese medicine monomers
Objective To investigate the effect of tolC gene deletion on Escherichia coli membrane integrity and antibacterial activity of traditional Chinese medicine monomers.Methods E.coli MG 1655 and DH5a were used to construct tolC knockout strains by Red homologous recombination.The permeability of the outer membrane of the strain was determined using the NPN method.The effects of tolC knockout on the inner membrane integrity and membrane potential of E.coli were investigated by flow cytometry combined with fluorescent dyes,propidium iodide(PI)and DiBAC4(3).The drug sensitivity was detected by microdilution method using 96-well plate.Results The tolC knockout strain and tolC complementary strain were successfully constructed.The NPN assay showed that tolC knockout significantly enhanced the outer membrane permeability of E.coli,indicating the structure of the outer membrane was severely damaged.PI and DiBAC4(3)assays indicated that tolC knockout did not affect the integrity of the E.coli cytoplasmic membrane,but dissipated the cell membrane potential.Drug susceptibility analysis revealed that tolC deletion significantly increased the susceptibility of E.coli to herbal monomers,such as cryptotanshinone,sanguinarine,berberine,thymol and carvacrol.It was noteworthy that tolC knockout increased the susceptibility of E.coli to cryptotanshinone up to more than 128-fold.In contrast,acrB knockout did not significantly affect the susceptibility of E.coli to cryptotanshinone.This suggested that TolC defection might reduce the susceptibility of E.coli to cryptotanshinone independently of the efflux effect,potentially through the enhancement of the permeability of the outer membrane.Consistent with this speculation,the outer membrane permeabiliser significantly enhanced the antibacterial activity of cryptotanshinone against wild-type E.coli.Conclusion The deletion of the tolC gene not only impacted the efflux function of efflux pumps,such as AcrB,but also enhanced the permeability of the outer membrane of E.coli and depolarized the membrane potential.This resulted in a significant increase in the susceptibility of E.coli to multiple herbal monomers,including cryptotanshinone.
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