双组分信号转导系统pnpRS调控肺炎链球菌氧化应激的分子机制

Streptococcus pneumoniae is a highly invasive human pathogen. Owing to its serious drug resistance and the poor vaccine protection, it is urgent to develop new methods for treating pneumococcal disease. The mechanism of reactive oxygen resistance is one of the hottest aspects of pneumococcal biology. Although proteins involved in scavenging H2O2 and other reactive oxygen species are constantly being identified, the regulatory mechanism is not yet clear. In our previous study, we found that the two-component signal transduction system pnpRS of S. pneumoniae is closely related to its resistance to reactive oxygen species, but the regulatory mechanism needs to be further studied. In this study, we will construct the strain of pneumococcal pnpRS mutant and compare the difference between wild-type and deletion mutants under H2O2 treatment by growth curve method. Combining RNA-seq and proteomics to analyze the differences in gene and protein expression. The results will be further analyzed by bioinformatics based on the KEGG database. And the potential key genes will be screened and identified by real-time RT-PCR. The gene deletion strain will be constructed to verify its role in toxicology and infection. Meanwhile, the protein PnpR and PnpS will be expressed and purified by Escherichia coli expression system. Electrophoretic mobility shift assay, SPR, ITC, and other technologies will be used to analyze the DNA sequence characteristics recognized by PnpR and the signal molecules recognized by PnpS. Our study attempts to reveal the molecular mechanism that pnpRS regulates its oxidative stress and provide theoretical support for the development of new methods to treat pneumococcal diseases.

肺炎链球菌是一种高侵袭性人类致病菌，其耐活性氧机制一直是研究的热点。尽管肺炎链球菌中参与清除H2O2等活性氧的蛋白不断被鉴定，然而调控机制尚未明确。我们在前期研究中发现肺炎链球菌双组分信号转导系统pnpRS与其耐活性氧密切相关，但调控机制有待深入研究。本课题拟通过构建肺炎链球菌∆pnpRS突变株，比较野生型和突变株在H2O2处理下的差异，结合RNA-seq及蛋白组学分析基因和蛋白表达差异。利用KEGG数据库等对结果进行生物信息学分析，筛选潜在调控关键基因并用荧光定量RT-PCR予以鉴定；构建该基因缺失株，验证其在毒理和侵染中的作用。表达、纯化蛋白PnpR和PnpS，结合凝胶阻滞实验、SPR、ITC等技术分析PnpR识别的DNA序列特征和PnpS识别的信号分子。本研究尝试揭示pnpRS调控其氧化应激的分子机制，为研发治疗肺炎链球菌疾病的新方法提供理论支持。

肺炎链球菌是一种重要的人类致病菌，作为一种缺乏过氧化氢酶的耐氧厌氧菌，但其能产生高浓度H2O2并在此环境下存活。因此，肺炎链球菌耐受氧化应激的分子机制一直是悬而未决的问题。已有研究表明，肺炎链球菌双组分信号转导系统pnpRS与其氧化应激相关，但具体机制尚不清楚。在我们前期研究的基础上，本项目的研究结果揭示肺炎链球菌双组分信号转导系统pnpRS对肺炎链球菌应对氧化应激是必需的，且双组分信号转导系统pnpRS可通过调控①磷酸转移酶系统，②脂肪酸代谢通路和③苯丙氨酸、酪氨酸和色氨酸生物合成通路等代谢通路来应对活性氧作用。主要的研究进展总结如下：第一，双组分信号转导系统pnpRS对肺炎链球菌应对羟基自由基、超氧自由基等活性氧作用是必需的，且基因pnpR在调控中发挥功能更重要。第二，双组分信号转导系统pnpRS也通过调控肺炎链球菌细胞内Mn2+、Cu2+和Mg2+等金属离子浓度来响应氧化应激作用。第三，双组分信号转导系统pnpRS主要通过调控①磷酸转移酶系统，②苯丙氨酸、酪氨酸和色氨酸生物合成通路，③脂肪酸合成和④脂肪酸代谢通路等响应氧化应激作用。综上所述，肺炎链球菌以双组分信号转导系统pnpRS为枢纽综合调控多种代谢途径来响应氧化应激作用。我们的研究结果有助于更好地理解肺炎链球菌响应氧化应激的机制，可为研制防治肺炎链球菌疾病的新方法提供理论依据。