摘要
【目的】比较重组耻垢分枝杆菌Ms-PPE61与Ms-Vec在不同体外应激条件下的抗压能力,研究二者感染巨噬细胞后MAPK/NF-κB信号通路的激活/抑制水平,并探究感染RAW264.7细胞后炎症细胞因子表达差异。【方法】构建Ms-Vec和Ms-PPE61菌株,培养至对数生长期后分别置于酸性、SDS、H2O2条件下处理,检测不同时间点的菌落形成单位(colony-forming unit,CFU);收集感染后1-48 h的细胞,提取蛋白,用Western blotting检测信号通路标志分子表达水平;用Ms-Vec和Ms-PPE61菌株分别感染RAW264.7细胞24 h和48 h,采用酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)检测上清中IL-6、 TNF-α及IL-1β的浓度;数据用GraphPad Prism 7.0分析,进行ANOVA方差分析,P<0.05为显著。【结果】PCR结果显示,Ms-PPE61有目的条带,Ms-Vec无目的条带;考马斯亮蓝染色显示蛋白上样水平一致,Western blotting显示Ms-PPE61表达约42 kDa的Flag融合蛋白,Ms-Vec无融合蛋白。采用超高速离心分离耻垢分枝杆菌组分,Western blotting结果显示细胞质标记蛋白GroES在Ms-Vec和Ms-PPE61细胞质中均有表达,含Flag标签的目的蛋白仅存在于Ms-PPE61细胞壁。在酸性条件(pH 3.0)下处理3 h时Ms-PPE61的存活率显著高于Ms-Vec(P<0.000 1),6 h和9 h时无显著差异(P>0.05);在0.2%SDS条件下处理3、6和9 h时Ms-PPE61的存活率均显著高于Ms-Vec(P<0.000 1);H2O2处理后3 h和6 h,Ms-PPE61的存活率也显著高于Ms-Vec(P<0.000 1)。Western blotting检测显示,Ms-PPE61组p-p38和p-ERK在感染48 h时显著降低,IκB-α在各时间点均高于Ms-Vec组。ELISA检测显示,Ms-PPE61组TNF-α的分泌水平在24 h和48 h时无显著差异(P>0.05),IL-6在24 h和48 h时显著低于Ms-Vec组(P<0.000 1),IL-1β在48 h时显著低于Ms-Vec组(P<0.01)。【结论】PPE61可增强重组耻垢分枝杆菌对酸性、SDS及H2O2胁迫的耐受能力,能通过下调p-p38和p-ERK的表达、上调IκB-α的表达来抑制MAPK和NF-κB信号通路,还可减少巨噬细胞中IL-6的分泌(24 h和48 h均显著)及IL-1β的分泌(48 h显著),但对TNF-α的分泌无显著影响。
[Objective]To compare the stress tolerance of recombinant Mycobacterium smegmatis strains Ms-PPE61 and Ms-Vec under different external stress conditions,investigate the activation/inhibition levels of the mitogen-activated protein kinase(MAPK)/nuclear factor(NF)-κB signaling pathway following their infection of macrophages,and explore differences in inflammatory cytokine expression after infection of RAW264.7 cells.[Methods]Ms-Vec and Ms-PPE61 were constructed and cultured to the logarithmic growth phase before being subjected to acidic,SDS,and H2O2 conditions.Colony-forming units(CFUs)were measured at different time points.Proteins were extracted from cells collected 1−48 h post-infection(hpi),and the expression levels of signaling pathway marker molecules were determined by Western blotting.The interleukin(IL)-6,tumor necrosis factor(TNF)-α,and IL-1β concentrations in the supernatants of RAW264.7 cells infected with Ms-Vec and Ms-PPE61 were measured by ELISA at 24 hpi and 48 hpi.GraphPad Prism 7.0 was used for analysis of variance of the data,and P<0.05 was considered significant.[Results]PCR revealed the presence of a target band in Ms-PPE61 but not in Ms-Vec.Coomassie brilliant blue staining confirmed consistent protein loading.Western blotting showed that Ms-PPE61 expressed a~42 kDa Flag fusion protein,while Ms-Vec did not.Ultra-high-speed centrifugation was performed to separate the components of M.smegmatis.Western blotting revealed that the cytoplasmic marker protein GroES was expressed in the cytoplasmic fractions of both Ms-Vec and Ms-PPE61,while the Flag-tagged target protein was exclusively present in the cell wall of Ms-PPE61.After treatment under acidic conditions(pH 3.0)for 3 h,the survival rate of Ms-PPE61 was higher than that of Ms-Vec(P<0.0001),while the survival rate showed no significant difference after treatment for 6 h and 9 h(P>0.05).After treatment with 0.2%SDS for 3,6,and 9 h,the survival rate of Ms-PPE61 was higher than that of Ms-Vec(P<0.0001).Similarly,after H2O2 treatment for 3 h and 6 h,the survival rate of Ms-PPE61 was higher than that of Ms-Vec(P<0.0001).Western blotting showed that the Ms-PPE61 group had significantly lower p-p38 and p-ERK levels at 48 hpi and higher IκB-α levels at all time points than the Ms-Vec group.ELISA results indicated no differences in TNF-α secretion between the Ms-PPE61 and Ms-Vec groups at 24 hpi and 48 hpi(P>0.05),while the Ms-PPE61 group had lower IL-6 levels at 24 hpi and 48 hpi(P<0.0001)and lower IL-1β level at 48 hpi(P<0.01)than the Ms-Vec group.[Conclusion]PPE61 can enhance the tolerance of recombinant Mycobacterium smegmatis to acidic,SDS and H2O2 stress,inhibit the MAPK and NF-κB signaling pathways by down-regulating the expression of p-p38 and p-ERK and up-regulating the expression of IκB-α,and reduce the secretion of IL-6(significantly at both 24 h and 48 h)and IL-1β(significantly at 48 h)in macrophages,but has no significant effect on the secretion of TNF-α.
作者
宋丽
梁志航
毕静
郭庆龙
张国良
SONG Li;LIANG Zhihang;BI Jing;GUO Qinglong;ZHANG Guoliang(Department of Infectious Diseases,The Affiliated Hospital of Southwest Medical University,Luzhou,Sichuan,China;National Clinical Research Center for Infectious Diseases,Shenzhen Third People’s Hospital,Shenzhen,Guangdong,China;Department of Laboratory Medicine,The First Affiliated Hospital of Guangdong Pharmaceutical University,Guangzhou,Guangdong,China)
出处
《微生物学报》
北大核心
2025年第12期5380-5391,共12页
Acta Microbiologica Sinica
基金
国家自然科学基金(82102403)
深圳市科技计划(KCXFZ20211020163545004,RCJC20221008092726022)
深圳市“医疗卫生三名工程”项目(SZZYSM202311009)。
