基于比较基因组分析研究嗜热链球菌抗过氧化物胁迫的分子机制

Streptococcus thermophilus is an important strain widely used in yogurt and cheese manufactures. It is facultative anaerobic and lacks catalase. But the mechanism that this strain adapts to the peroxide stress remains unknown. The predicted sequence encoded a peroxidase was identified in the genome of S. thermophilus by comparative genome analysis. Strikingly, two components (TatA, TatC) of twin- arginine translocation (Tat) pathway were also found locating at the same cluster with the predicted peroxidase, which suggests that the predicted peroxidase should be presented in S.thermophilus strain and dedicates to the secretion by Tat pathway. But all of those predictions lack the experimental evidences. Here we focus on the predicted peroxidase EfeB of S. thermophilus ST15, and investigate the function and the expression regulation of the predicted peroxidase EfeB by comparative genome analysis and antioxidant stress assay. The function of TatA and TatC to secrete folded proteins will be also detected by measuring the subcellular localization in intracellular, cellular membrane and extracellular, and secretion efficiency of the fused green fluorescence protein (GFP) as a reporter. We would also study the model of TatA and TatC complexes by cross-linking of proteins in vitro and ITC analysis, and try to find out the molecular chaperones of the protein EfeB. The aim of this project is to provide a new enzymetic defense against the peroxide stress in S.thermophilus.

嗜热链球菌是重要的工业菌株，兼性厌氧、过氧化氢酶阴性，如何应对过氧化物胁迫一直未见报道。最近，我们发现嗜热链球菌基因组存在携带双精氨酸(Tat)信号肽的过氧化物酶编码序列，以及对应Tat系统的结构元件TatA和TatC，且两者位于同一操纵子内，这种独特的遗传结构预示菌株内存在过氧化物酶且可能通过Tat途径分泌，但目前缺乏实验证实。本项目聚焦S.thermophilus ST15的过氧化物酶EfeB，通过比较基因组分析和抗氧胁迫能力测定，确定efeB基因的功能及表达调控；以荧光蛋白为报告基因，利用缺失突变、融合表达Tat系统的结构元件，检测荧光蛋白在细胞内、细胞膜及细胞外的定位及分泌效率；通过体外化学交联和ITC分析，探究TatA和TatC蛋白在细胞膜上的聚集形式及与EfeB蛋白的结合能力；结合转录组分析，寻找分子伴侣，阐明过氧化物酶的分泌机制，最终揭示嗜热链球菌抗过氧化物胁迫的分子机制。

嗜热链球菌是重要的工业菌株，兼性厌氧、H2O2酶阴性，但是该菌株在低H2O2浓度下仍能存活，预示着细胞内存在抵抗H2O2胁迫的生物机制。本项目在对Streptococcus thermophiles CGMCC7.179全基因组测序的基础上，在其基因组上鉴定到一个过氧化物酶基因efeB，其编码蛋白的氨基端携带双精氨酸易位（twin-arginine translocation, Tat）保守基序的信号肽，与Tat系统的结构元件基因tatA和tatC一起位于efeOBU-tatCA操纵子中。.在摇动培养时，efeB基因缺失导致S. thermophilus CGMCC7.179的生物量减少和活菌数下降，预示EfeB赋予菌株抵抗氧胁迫能力，而且这种抗氧胁迫需要Tat系统的协助。通过氯霉素抗性实验和在Escherichia coli DE3中的亚细胞定位实验，进一步证实了TatCA组分在EfeB分泌中协助作用。.氨基酸序列比对分析发现，EfeB与肉金黄色葡萄球菌编码的过氧化物酶FepB的相似性高达48.13%，且具有染料过氧化物酶家簇的结构特征，即保守结构域G-X-X-D-G和heme结合位点。将EfeB在大肠杆菌中表达并纯化，该纯化蛋白在H2O2存在条件下使活性蓝5褪色，证实了EfeB属于染料过氧化物酶。通过测定S. thermophilus CGMCC7.179 及其efeB缺失株在摇动培养物中的H2O2浓度， 证实了EfeB具有消除生存环境中H2O2的能力，并且这种消除与二价铁离子的摄取有关，该结果与其基因的遗传结构相吻合。.目前嗜热链球菌是唯一保留有Tat系统的乳酸菌，为验证Tat系统在乳酸菌中作用，分别将金黄色葡萄球菌核酸酶基因nuc和绿色荧光蛋白基因gfp与EfeB的信号肽序列sp融合，再与tatCA一起转化到Lc. lactis MG1363中，结果表明，S. thermophilus CGMCC7.179的TatCA系统在Lc. lactis MG1363中可以发挥作用，但是对于不同的底物蛋白有不同的效果。.最后还首次证实了过氧化物酶EfeB介导的嗜热链球菌通过激活Nrf2-ARE途径实现对肠上皮细胞的抗氧化保护作用。