一种肺炎链球菌磷壁酸合成相关蛋白的功能鉴定

Teichoic acid is an important virulence factor in Streptocccus pneumoniae. Our previous results showed that rafX is involved in the biosynthesis of pneumococcal teichoic acids; however, rafX inactivation did not lead to complete loss of the teichoic acids. We thus hypothesized the existence of an alternative pathway that is involved in the attachment of teichoic acids to the cell wall. To this end, we have performed some basic experiments and we found that the deletion of SPD_1741, a LytR family member, also led to a significant decrease of teichoic acids in Streptococcus penumoniae D39 strain. However, the physiological and biological functions of SPD_1741 are required to be further investigated. Based on our preliminary data and literary evidence, we proposed that SPD_1741 may act as a ligase responsible for the attachment of teichoic acids to the cell wall and hence contribute to pneumococcal virulence. To address this hypothesis, recombinant SPD_1741 will be expressed in E.coli BL21 and purified by Ni2+ affinity chromatography; The cellular localization will be revealed by use of cell fractionation and Western blotting; Gel shift assays will be performed to reveal whether SPD_1741 could bind to the promoter region of lytA gene, and hence its transcriptional regulation on LytA; In vitro ligation assays are used to reveal the potential ligase activity of the SPD_1741; SPD_1741 deletion mutants and SPD_1741 complemented strains will be generated; The influence of SPD_1741 on bacterial growth, teichoic acid production, and bacterial morphology will be defined. Finally, whole-blood-mediated killing assays and infection models will be carried out to address the molecular mechanism of SPD_1741-mediated teichoic acid production in pneumococcal virulence. The experiments outlined in this proposal will provide fundamental information for understanding the biosynthesis of teichoic acids in Streptococcus pneumoniae, and provide a novel strategy for the design of novel antibiotics and vaccines.

磷壁酸是肺炎链球菌重要毒力因子。我们前期研究发现rafX参与磷壁酸胞外连接过程，但rafX基因缺失并未造成肺炎链球菌胞外磷壁酸完全缺失，我们猜测还存在替代途径参与这一过程。为此，我们前期进行了相应的探讨，预实验结果提示LytR蛋白家族成员SPD_1741在磷壁酸的胞外连接中发挥了重要作用，但其功能还有待进一步研究。我们猜测SPD_1741可能通过作为胞外连接酶或转录因子参与磷壁酸的胞外连接过程，影响细菌毒力。因此，本项目拟利用亚组分分离技术对其定位分析，凝胶阻滞等实验明确其是否具有转录调控lytA的作用，进行体外连接酶活性实验证实其磷壁酸连接作用；并构建SPD_1741基因缺陷株和回补株，对比分析细菌生长特征、磷壁酸含量和细菌形态，利用体外全血吞噬杀伤实验和体内攻毒实验分析其毒力机制。本研究将从SPD_1741蛋白这个新视点为揭示磷壁酸合成机制奠定基础，为新型抗菌素和疫苗研制提供新的思路。

磷壁酸是肺炎链球菌重要毒力因子。我们前期研究发现RafX参与磷壁酸胞外连接过程，但rafX基因缺失并未造成肺炎链球菌胞外磷壁酸完全缺失，我们猜测还存在替代途径参与磷壁酸胞外连接过程。总体来说，该项目是希望发现肺炎链球菌磷壁酸合成的替代途径。为了明确磷壁酸胞外连接的替代途径，我们预设了三个研究目标，分别为：1、明确肺炎链球菌磷壁酸可能的替代途径；2、明确LytR_cps_psr（LCP）蛋白家族中的LytR蛋白的功能和发挥作用的机制；3、明确肺炎链球菌LytR蛋白做细菌毒性的作用。在项目的支持下，我们对肺炎链球菌基因组进行了分析，发现了一个名为LytR_cps_psr（LCP）蛋白家族，我们对其中的LytR蛋白（SPD_1741）进行了深入研究，发现该蛋白定位于细菌表面，含有一个跨膜结构，该蛋白在肺炎链球菌中非常保守，在细菌生长的各阶段均稳定表达；LytR功能缺陷后，肺炎链球菌自溶加快，这与其影响细胞壁磷壁酸合成导致菌体对LytA敏感性增加有关；我们首次发现LytR蛋白与肺炎链球菌磷壁酸和荚膜多糖的胞外连接有关，主要通过酶学活性发挥其生物学功能；最后，动物实验显示LytR蛋白与肺炎链球菌致病性密切相关。本研究揭示了SPD_1741蛋白与磷壁酸和荚膜多糖胞外连接中的重要作用，回答了肺炎链球菌中磷壁酸合成存在替代途径的科学问题，为了理解磷壁酸合成机制提供了有力实验支撑，为肺炎链球菌防治提供了新的思路。目前该项目已发表论文3篇，申请国家发明专利1项，培养硕士研究生2名，在读硕士研究生2名。总体来说，项目执行人已经顺利完成了项目设定的目标。