SopE对晚期伤寒沙门菌液泡（SCV）稳定性的影响

SopE对晚期伤寒沙门菌液泡（SCV）稳定性的影响

翁轶玮

微生物与感染 ›› 2022, Vol. 17 ›› Issue (5) : 297-304.

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微生物与感染 ›› 2022, Vol. 17 ›› Issue (5) : 297-304.

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SopE对晚期伤寒沙门菌液泡（SCV）稳定性的影响

翁轶玮

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Effect of SopE on stability of advanced Salmonella-containing vacuole (SCV)

Yiwei Weng

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摘要

为研究伤寒沙门菌III型分泌系统效应子SopE对其入侵巨噬细胞后形成的沙门菌液泡（SCV）稳定性的作用。以伤寒沙门菌GIFU1007（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的回补株（C-ΔsopE）；通过原核表达绿色蛋白质粒pET28-sfGFP，构建伤寒沙门菌绿色荧光蛋白表达菌株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对伤寒沙门菌胞内生存能力的影响；通过免疫荧光和实时荧光定量PCR（qRT-PCR）实验，探究SopE对晚期SCV稳定性的影响；进一步用qRT-PCR和蛋白印迹法（Western Blot），探究SopE与自噬的关系。巨噬细胞THP-1胞内生存能力实验结果显示，ΔsopE/pBAD33::pET28-sfGFP的胞内生存能力显著低于WT/pBAD33::pET28-sfGFP和C-ΔSopE/pET28-sfGFP的；细胞免疫荧光实验和qRT-PCR结果表明，sopE的缺失会导致晚期SCV的稳定性降低；qRT-PCR和Western Blot结果表明，SopE能够抑制自噬相关基因的转录和表达。以上结果显示SopE通过影响自噬而调节晚期SCV的稳定性。

Abstract

To study the effect of SopE, a type III secretory system effector encoded by Salmonella enterica serovar Typhi, on SCV induced by S. Typhi after invasion of macrophages. S.Typhi GIFU1007was used as the wild type (WT) strain to construct the sopE mutant (ΔsopE) by homologous recombination using the suicide plasmid pGMB151. The ΔsopE complementary strain (C-ΔsopE) was constructed with the plasmid complementary method by using pBAD33.The recombinant plasmid pET28-SFGFP was introduced into the WT, ΔsopE and C-ΔsopE , respectively, to yield WT/pBAD33::pET28-SFGFP, ΔsopE/pBAD33::pET28-SFGFP and C-ΔsopE/pET28-SFGFP. The effect of SopE on the intracellular viability of S. Typhi was studied by THP-1 intracellular viability assay of macrophages. The effect of SopE on the stability of late SCV was investigated by immunofluorescence and real-time quantitative PCR (qRT-PCR). The relationship between SopE and autophagy was further investigated by qRT-PCR and Western blot. The intracellular viability of THP-1 macrophages with ΔsopE/pBAD33::PET28-SFGFP was significantly lower than that of WT/pBAD33::PET28-SFGFP and C-ΔsopE/PET28-SFGFP. The immunofluorescence assay and qRT-PCR results showed that deletion of sopE decreased the stability of late SCV. The qRT-PCR and Western blot results showed that SopE inhibited the expression of autophagy related genes. The data presented here showed that SopE regulated the stability of late SCV by affecting autophagy.

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翁轶玮. SopE对晚期伤寒沙门菌液泡（SCV）稳定性的影响[J]. 微生物与感染. 2022, 17(5): 297-304

Yiwei Weng. Effect of SopE on stability of advanced Salmonella-containing vacuole (SCV)[J]. Journal of Microbes and Infections. 2022, 17(5): 297-304

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参考文献

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