亚抑菌浓度博落回生物碱对ExPEC主要外膜蛋白和Ⅱ型T-A系统表达的影响

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

【背景】血根碱、白屈菜红碱、原阿片碱等生物碱是我国二类新兽药博落回散和博普总碱散的主要成分,具有广泛的药理作用。【目的】研究亚抑菌浓度血根碱、白屈菜红碱、原阿片碱对肠外致病性大肠杆菌(extraintestinal pathogenic Escherichia coli,ExPEC)主要外膜蛋白及其调控基因和Ⅱ型毒素-抗毒素(toxin-antitoxin,T-A)系统基因表达的影响,初步探讨博落回生物碱对ExPEC细菌生理活动影响的可能机制。【方法】比较不同Ⅱ型T-A系统基因yafON、hicAB、prlF-yhaV缺失的ExPEC对血根碱、白屈菜红碱、原阿片碱及抗生素的最小抑菌浓度;在1/2MIC亚抑菌浓度血根碱、白屈菜红碱、原阿片碱条件下,比较它们对ExPEC野生型(wild type,WT)菌株和外膜蛋白tolC缺失菌株(ΔtolC)的不同外膜蛋白基因ompC、ompX、tolC、ompF和调控基因acrR、rob、marR、rpoS、soxS表达的影响,以及对T-A系统基因yafON、hicAB、prlF-yhaV表达的影响。【结果】T-A系统hicAB和prlF-yhaV缺失菌株对氯霉素的敏感性提高2–4倍,而且hicAB缺失菌株对血根碱的敏感性也提高2倍;1/2 MIC白屈菜红碱显著促进WT和ΔtolC菌株的ompX和tolC基因表达,而血根碱抑制WT菌株tolC基因的表达;在ΔtolC菌株中1/2MIC血根碱、白屈菜红碱、原阿片碱不同程度地促进ompX和ompC的基因表达,而对ompF存在着不同程度的抑制;1/2 MIC血根碱和白屈菜红碱显著促进WT和ΔtolC菌株的marR基因表达,并且血根碱还可以促进rpoS基因的表达。1/2 MIC原阿片碱显著降低WT菌株的yafN基因表达,而显著增加ΔtolC菌株的yafN基因表达水平;1/2 MIC血根碱、白屈菜红碱、原阿片碱不同程度地抑制WT菌株hicA和hicB基因表达,而促进ΔtolC菌株hicA和hicB的基因表达;1/2MIC血根碱、白屈菜红碱、原阿片碱不同程度地促进WT和ΔtolC菌株的prlF基因表达,而血根碱显著抑制yhaV基因表达。【结论】Ⅱ型T-A系统和外膜蛋白参与ExPEC对不同生物碱的应激反应,外膜蛋白TolC的完整性对于T-A系统参与细菌应激具有一定的影响,并且其机制可能存在不同。

[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 typeⅡtoxin-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,and soxS),and typeⅡT-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 of hicAB-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 andΔtolC,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 of ompF inΔtolC.Sanguinarine and chelerythrine at 1/2 MIC significantly promoted the marR expression in WT andΔtolC.Moreover,sanguinarine improved the expression of rpoS.Protopine at 1/2 MIC significantly lowered the expression of yaf N in WT while significantly increased that inΔtolC.Protopine,sanguinarine,and chelerythrine at 1/2 MIC down-regulated the expression of hicA and hicB in WT while up-regulated that inΔtolC.They improved the expression of prlF in WT andΔtolC,while sanguinarine significantly inhibited the expression of yhaV.[Conclusion]TypeⅡT-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.