Effects of sub-inhibitory concentrations of alkaloids from Macleaya cordata on the expression of outer membrane protein and type II toxin-antitoxin systems of ExPEC

Abstract:

[Background] Protopine, sanguinarine, and chelerythrine are main components of the new veterinary drugs Boluohui powder and Bopuzongjian powder in China, which have multiple pharmacological effects. [Objective] To preliminarily reveal the influence and mechanisms of the alkaloids from Macleaya cordata on the physiological activities of extraintestinal pathogenic Escherichia coli (ExPEC), we evaluated the effects of sub-inhibitory concentrations of sanguinarine, chelerythrine, and protopine on the expression of outer membrane protein (OMP), their regulatory genes, and type II toxin-antitoxin (T-A) systems of ExPEC. [Methods] We measured the minimum inhibitory concentrations (MICs) of protopine, sanguinarine, chelerythrine, and antibiotics on the ExPEC mutants with the deletion of yafON, hicAB, and prlF-yhaV. We then compared the expression of OMP genes (ompC,ompX,tolC, and ompF), their regulatory genes (acrR,rob,marR,rpoS, andsoxS), and type II T-A systems (yafON,hicAB, and prlF-yhaV) between WT and the tolC-deleted strain (ΔtolC) exposed to 1/2 MIC of protopine, sanguinarine, and chelerythrine. [Results] The sensitivity ofhicAB-and prlF-yhaV-deleted strains to chloramphenicol increased by 2–4 times, and that of hicAB-deleted strain to sanguinarine also increased by 2 times. Chelerythrine at 1/2 MIC significantly up-regulated the expression of ompX and tolC in WT and ΔtolC, while sanguinarine inhibited the expression of tolC in WT. Protopine, sanguinarine, and chelerythrine at 1/2 MIC up-regulated the expression of ompX and ompC while down-regulated that ofompF in ΔtolC. Sanguinarine and chelerythrine at 1/2 MIC significantly promoted the marR expression in WT and ΔtolC. Moreover, sanguinarine improved the expression of rpoS. Protopine at 1/2 MIC significantly lowered the expression of yafN in WT while significantly increased that in ΔtolC. Protopine, sanguinarine, and chelerythrine at 1/2 MIC down-regulated the expression of hicA and hicB in WT while up-regulated that in ΔtolC. They improved the expression of prlF in WT and ΔtolC, while sanguinarine significantly inhibited the expression of yhaV. [Conclusion] Type II T-A systems and OMPs participate in the responses of ExPEC to alkaloids. The integrity of OMPs (such as TolC) affects the role of T-A systems in bacterial responses to stress, the mechanisms of which remain unclear.

Key words:extraintestinal pathogenic Escherichia coli (ExPEC);alkaloids of Macleaya cordata;sub-inhibitory concentration;outer membrane protein (OMP);toxin-antitoxin (T-A) system