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  

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

作　　者：冯昱超 徐加利 朱斌杰 吴泽斌 王雯静 丁桉 蔡孜萌 陈绵绵 孙静 江铃丽 宋厚辉 夏菁 程昌勇 FENG Yuchao;XU Jiali;ZHU Binjie;WU Zebin;WANG Wenjing;DING An;CAI Zimeng;CHEN Mianmian;SUN Jing;JIANG Lingli;SONG Houhui;XIA Jing;CHENG Changyong(Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province,Zhejiang Provincial Engineering Research Center for Animal Health Diagnostics&Advanced Technology,Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management,China-Australia Joint Laboratory for Animal Health Big Data Analytics,College of Animal Science and Technology&College of Veterinary Medicine,Zhejiang A&F University,Hangzhou 311300,Zhejiang,China;Ningbo College of Health Sciences,Ningbo 315100,Zhejiang,China)

机构地区：[1]浙江农林大学动物科技学院·动物医学院、浙江省畜禽绿色生态健康养殖应用技术研究重点实验室、动物健康互联网检测技术浙江省工程研究中心、浙江省动物医学与健康管理国际科技合作基地、中澳动物健康大数据分析联合实验室,浙江杭州311300 [2]宁波卫生职业技术学院,浙江宁波315100

出　　处：《微生物学报》2024年第4期1219-1232,共14页Acta Microbiologica Sinica

基　　金：国家自然科学基金(32172849,32302961);浙江省自然科学基金(LQ22C180001,LY23C180002);宁波市公益类科技项目(2022S006);浙江农林大学人才启动项目(2023LFR013)。

摘　　要：【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。[Objective]To investigate the impacts of the amino acid residues at position 253(glutamine,Q)and 254(isoleucine,I)in theβ8 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.

关 键 词：单增李斯特菌 溶血素O 感染 点突变缺失株 

分 类 号：S852.6[农业科学—基础兽医学]