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

摘要:

[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_ΔPEST、LLO_S44A、LLO_S48A和LLO_T51A）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_ΔPEST、LLO_S44A、LLO_S48A和LLO_T51A。在pH5.5和7.4条件下，LLO_ΔPEST、LLO_S44A、LLO_S48A和LLO_T51A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。

关键词:单核细胞增多性李斯特菌;溶血素O;PEST序列;膜裂解活性;ERK1/2磷酸化

Abstract:

[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 (LLO_ΔPEST, LLO_S44A, LLO_S48A, and LLO_T51A) 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 pH 5.5 and pH 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.

Key words:Listeria monocytogenes;Listeriolysin O;the PEST-like sequence;pore-forming activity;ERK1/2 kinases phosphorylation