单增李斯特菌溶血素O的PEST序列激活ERK1/2磷酸化的作用

Roles of the PEST-like sequence of Listeriolysin O from Listeria monocytogenes in activating ERK1/2 phosphorylation

[目的]本研究旨在构建单核细胞增多性李斯特菌(Listeria monocytogenes,简称单增李斯特菌)溶血素O(Listeriolysin O,LLO)的关键结构域PEST序列(包含S44、S48和T51关键磷酸化位点)突变体,并针对其生物学功能展开研究。[方法]以李斯特菌参考菌株EGD-e为模板扩增编码LLO的hly基因,克隆至pET30a(+)原核表达载体,在此基础上利用氨基酸突变技术获得表达PEST突变体(LLO△PEST、LLOS44A、LLOS48A和LLOT51A)的重组质粒,转入E.coli Rosetta感受态细胞中,诱导表达重组蛋白经镍离子亲和层析纯化后进行SDS-PAGE分析。利用红细胞裂解试验检测重组蛋白的溶血活性,并通过Western blotting检测重组突变蛋白刺激Caco-2细胞后对MAPK关键信号分子ERK1/2磷酸化水平变化的影响。[结果]结果显示,本研究成功获得重组LLO及其突变体蛋白LLO△PEST、LLOS44A、LLOS48A和LLOT51A。在pH5.5和7.4条件下,LLO△PEST、LLOS44A、LLOS48A和LLOT51A均具有和LLO相当的溶血活性,说明PEST序列缺失或突变并不影响LLO的膜裂解活性。研究进一步发现,重组LLO及其突变蛋白刺激Caco-2细胞后均能激活ERK1/2的磷酸化。[结论]研究表明LLO的关键结构域PEST序列对于维持该蛋白的膜裂解能力及穿孔活性并非必需,且该结构域的缺失不影响李斯特菌在感染宿主时依赖LLO介导ERK1/2磷酸化的生物学过程。本研究将为进一步探索细菌感染过程中PEST序列对于LLO发挥生物学功能的潜在作用及分子机制奠定基础。

[Objective]The determinant virulence factor Listeriolysin O(LLO)of Listeria monocytogenes,a foodborne pathogen,contains a unique N-terminal amino acid sequence that is absent in other cytolysins and was previously referred as the PEST-like sequence(containing three putative phosphorylation sites,S44,S48,and T51).We here,therefore,aimed to explore the biological roles of the PEST-like sequence in LLO-induced ERK1/2 kinases phosphorylation in human epithelial cells(Caco-2).[Methods]The plasmid for expressing the recombinant LLO was constructed and transformed into E.coli Rosetta,and the his-tagged soluble protein was purified using the nickel-chelated affinity column chromatography.The LLO variants(LLOΔPEST,LLOS44A,LLOS48A,and LLOT51A)were then obtained by using the site-directed mutagenesis strategy and expressed as above for LLO.The hemolytic activity for these recombinant proteins was assessed by lysis the erythrocytes,and moreover,effects of LLO or its variants on ERK1/2 kinases phosphorylation in Caco-2 cells was detected by using the Western blotting method.[Results]The results in the present study showed that the recombinant LLO,as well as the four LLO variants were able to lysis the erythrocytes at p H 5.5 and p H 7.4,suggesting that the PEST-like sequence was not required for the pore-forming activity of LLO.Besides,treatment of the LLO or its variants at the cytolytic concentration of 5 nmol/L could significantly induce ERK1/2 kinases phosphorylation in Caco-2 cells.[Conclusion]Our data collectively showed that the PEST-like sequence was not necessary for the LLO-mediated perforation ability on host membranes and not required for the LLO-triggered ERK1/2 signaling,which laid the foundation for further exploration of the potential roles of this motif during L.monocytogenes infection.