葡萄糖促进葡萄球菌黄素生物合成的基因调控分析

Hyperglycaemia caused by diabetes or other acute diseases can increase the risk of Staphylococcus aureus infection, and its mechanism is still unclear. Staphyloxanthin is a triterpenoid, membrane-bound carotenoid which helps the microbe escape from host’s innate immune clearance via detoxifying reactive oxygen species produced by phagocytes and stabilizing the S. aureus membrane during infection and pathogenesis. In the previous work, we found that glucose could promote the synthesis of staphyloxanthin in S. aureus in a dose-dependent manner and S. aureus cells cultivated in culture medium containing relatively high concentration of glucose were more resistant to oxidative damage. Hence, we speculated that staphyloxanthin could play an important role in the increased risk of S. aureus infection in hyperglycaemia patients. This project aims to reveal the gene regulation network involved in the mechanism whereby glucose promotes the synthesis of staphyloxanthin in S. aureus. Transcriptomic analysis will be performed on the S. aureus cells grown in the culture medium containing no glucose, low and high concentration of glucose by using microarray and qRT-PCR techniques. The differentially expressed genes will be determined and the mutant strains be constructed by deleting the genes or introducing foreign genes into S. aureus genome. The contents of staphyloxanthin and its precurosors in wild type and mutant strains will be measured by laser tweezers Raman spectroscopy and high performance lipid chromatography. In addition, the resistance to oxidative damage and host’s innate immune clearance will be compared between wild type and mutant strains. By this means, the function of the genes involved will be determined. The implementation of the project will help to understand the regulation of staphyloxanthin biosynthesis in S. aureus, and reveal the interaction between the pathogens and their hosts when diabetic patients are complicated with S. aureus infection.

糖尿病和其他急性疾病所致的高血糖会增加金黄色葡萄球菌感染风险，其机制仍未明确。葡萄球菌黄素是金葡菌合成的一种类胡萝卜素，通过中和活性氧、稳定细胞膜帮助金葡菌逃避宿主固有免疫的清除。我们前期研究中发现一定浓度的葡萄糖可剂量依赖性地促进葡萄球菌黄素合成，且高糖浓度下生长的金葡菌对氧化损伤有更强的耐受能力，据此我们推测该色素在介导高血糖患者增加的金葡菌感染风险中可能发挥重要作用。为了阐明葡萄糖促进葡萄球菌黄素合成涉及的基因调控机制，本项目拟利用基因芯片和qRT-PCR对无糖，低糖，高糖条件培养的金葡菌进行转录组分析，对差异表达显著的基因采用基因敲除或引入外源基因的方法构建突变株，比较突变株和野生株在色素及其前体合成水平以及对氧化损伤、宿主免疫清除的耐受能力的差异，以此获知相关基因的功能。项目的实施有助于加深对葡萄球菌黄素生物合成调控规律的了解，揭示糖尿病并发金葡菌感染中病原菌与宿主的相互作用。

金黄色葡萄球菌是引起社区和医院内感染的重要致病菌。毒力因子是病原菌产生的用于帮助其逃避宿主免疫清除的一类生物分子，研究环境因素对金葡菌毒力因子表达调控的影响将深化对金葡菌致病分子机制的认识。在本项目中，我们研究了葡萄糖对金葡菌转录组的影响，发现葡萄糖促进葡萄球菌黄素合成是因为葡萄糖的代谢产物乙酰辅酶A是色素合成的前体，前体物质的堆积会促进产物的合成。葡萄糖自身不但不促进反而会抑制色素合成相关酶的表达。我们还利用RNA-Seq研究了黄酮对金葡菌转录组的影响，发现黄酮可以下调金葡菌多种毒力因子表达，而这种对毒力因子表达的抑制是通过金葡菌SaeRS双组份系统介导的。我们还利用qPCR，GFP报道系统，ELISA验证了部分毒力因子基因的差异表达。此外，我们还建立了毒力因子抑制化合物的筛选系统，能够高效筛选对金葡菌SaeRS和AgrCA的转录调控具有抑制活性的化合物，并利用SaeS和AgrA敲除的金葡菌和一些阳性化合物验证了该系统的筛选能力。另外，我们还改进了一个金葡菌基因编辑载体，使其对难转化的金葡菌菌株有较高的基因编辑效率。