单增李斯特菌毒力因子溶血素关键氨基酸位点在感染过程中的作用研究

【目的】探究单增李斯特菌溶血素O (listeriolysin O, LLO)中D3区域β8折叠片上第253位氨基酸(谷氨酰胺，Q)和第254位氨基酸(异亮氨酸，I)对单增李斯特菌(Listeria monocytogenes)感染生物学功能的影响。【方法】构建LLO_Q253A和LLO_I254A突变蛋白的原核表达菌株，以及利用同源重组方法构建hly_Q253A和hly_I254A突变株；通过表达纯化突变蛋白，测定溶血活性；比较LLO第253位Q和第254位I均突变成丙氨酸(A)后，对细菌体外生长能力、黏附侵袭、胞内迁移和增殖能力的影响。【结果】相应位点突变后，LLO蛋白均能够正常表达。在pH 6.5条件下，所有突变蛋白和突变株的溶血活性丧失。然而，在pH 5.5条件下，LLO_I254A和hly_I254A恢复了溶血活性。与野生株相比，突变株的体外生长、黏附能力和胞内增殖能力均无明显差异；突变株的侵袭能力和胞间迁移能力显著低于野生株。【结论】本研究证实第253位Q和第254位I均突变成A后，单增李斯特菌在pH 6.5条件下丧失溶血活性，并降低了感染宿主细胞的能力，但具体机制还有待进一步探索。本研究为深入探究LLO结构对单增李斯特菌生物学功能的影响奠定基础，对单增李斯特菌点突变株的构建具有一定参考意义。

Roles of key amino acid residues of the virulence factor listeriolysin O in Listeria monocytogenes infection

[Objective] To investigate the impacts of the amino acid residues at position 253 (glutamine, Q) and 254 (isoleucine, I) in the β8 sheet of the D3 domain of listeriolysin O (LLO) on the biological functions of Listeria monocytogenes. [Methods] We constructed the mutant proteins LLO_Q253A and LLO_I254A and the mutant strains hly_Q253A and hly_I254A by homologous recombination. After the expression and purification, the mutant proteins examined for the hemolytic activity. Furthermore, the growth, adhesion, invasion, intracellular migration, and proliferation were compared between the mutant strains hly_Q253A and hly_I254A. [Results] After the mutation of the corresponding sites, LLO proteins could be expressed normally. However, the mutant proteins and strains lost hemolytic activity at pH 6.5, and the hemolytic activities of LLO_I254A and hly_I254A were restored at pH 5.5. The mutant strains showed no significant differences in extracellular growth, adhesion, and intracellular proliferation compared with the wild-type strain. However, the invasion and intercellular migration of the mutant strains were significantly lower than that of the wild-type strain. [Conclusion] The mutations of Q253A and I254A in LLO cause the loss of hemolytic activity at pH 6.5 and a reduction in the bacterial infection, the specific mechanisms of which remain to be explored. This study establishes a foundation for deeply understanding the impact of LLO structure on the biological function of L. monocytogenes and holds significance for the construction of point-mutated strains of L. monocytogenes.