伤寒沙门菌Ⅲ型分泌系统效应蛋白SopE对晚期沙门菌液泡稳定性的影响

Effect of SopE on stability of late Salmonella-containing vacuole

为探讨伤寒沙门菌Ⅲ型分泌系统效应蛋白SopE对其入侵巨噬细胞后形成的晚期沙门菌液泡(Salmonella-containing vacuole,SCV)稳定性的影响,以伤寒沙门菌GIFU10007野生株(wild type,WT)为研究对象,利用自杀质粒pGMB151介导的同源重组法构建sopE突变株(ΔsopE);以pBAD33为载体,利用质粒回补法构建ΔsopE回补株(C-ΔsopE);利用绿色荧光蛋白(green fluorescent protein,GFP)原核表达质粒pET28-sfGFP构建GFP表达菌株WT/pBAD33∷pET28-sfGFP、ΔsopE/pBAD33∷pET28-sfGFP和C-ΔsopE/pET28-sfGFP。通过巨噬细胞THP-1胞内生存能力实验,研究SopE对伤寒沙门菌在巨噬细胞内生存能力的影响;利用免疫荧光和实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR),探究SopE对晚期SCV稳定性的影响;进一步采用qRT-PCR和蛋白免疫印迹法探究SopE与自噬的关系。结果显示,ΔsopE/pBAD33∷pET28-sfGFP菌株在巨噬细胞内的生存能力显著低于WT/pBAD33∷pET28-sfGFP和C-ΔSopE/pET28-sfGFP菌株,sopE缺失导致晚期SCV的稳定性降低,且SopE能够抑制自噬相关基因的转录和表达。以上结果提示,SopE可能通过影响自噬而调节晚期SCV的稳定性。

The present paper was aimed to study the effect of SopE,a typeⅢsecretory system effector of Salmonella typhi(S.typhi),on the stability of late Salmonella-containing vacuole(SCV)induced by S.Typhi after invasion of macrophages.S.Typhi GIFU10007 was used as the wild type(WT)strain to construct the sopE mutant(ΔsopE)by homologous recombination using the suicide plasmid pGMB151.TheΔsopE complementation strain(C-ΔsopE)was constructed by plasmid complementation method using pBAD33.The recombinant plasmid pET28-sfGFP was introduced into the WT,ΔsopE and C-ΔsopE strains,respectively,to yield WT/pBAD33∷pET28-sfGFP,ΔsopE/pBAD33∷pET28-sfGFP and C-ΔsopE/pET28-sfGFP strains.The effect of SopE on the intracellular viability of S.Typhi in THP-1 macrophages was studied.The effect of SopE on the stability of late SCV was investigated by immunofluorescence and quantitative real-time polymerase chain reaction(qRT-PCR).Furtherly,the relationship between SopE and autophagy was investigated by qRT-PCR and Western blotting.The results showed that the intracellular viability ofΔsopE/pBAD33∷PET28-sfGFP strain was significantly lower than that of WT/pBAD33∷PET28-sfGFP and C-ΔsopE/PET28-sfGFP strains in THP-1 macrophages.The deletion of sopE decreased the stability of late SCV.SopE inhibited the expressions of autophagy-related genes.It is suggested that SopE could regulate the stability of late SCV by affecting autophagy.