二氢杨梅素靶向Sortase A抑制金黄色葡萄球菌生物膜形成的分子机制

Staphylococcus aureus is an important foodborne pathogen. Biofilm formation is the main cause of the persistent food contamination provoked by S. aureus. It is of great significance for food safety to develop safe and efficient biofilm inhibitor. Previously, we have demonstrated that 2R,3R-dihydromyricetin (DMY) could significantly inhibit the biofilm formation by S. aureus. However, the biofilm inhibitory mechanism of DMY is still unclear. In the continuous study, we found that DMY could interact with the sortase A (SrtA) of S. aurues, inhibit the activity of SrtA and down-regulate its expression. These results implied that DMY probably achieved its inhibitory effect on the biofilm formation of S. aureus by targeting SrtA. This project will combine in vitro and in vivo studies, to investigate the interaction mode between DMY and S. aureus SrtA, and to clarify the effect of DMY on the transpeptidation and protein anchoring activity of SrtA. Furthermore, the protein interaction network of SrtA will be identified through co-immunoprecipitation combined with mass spectrometry. The interaction between interacting proteins and SrtA and their functions in biofilm formation will be confirmed by bacterial two-hybrid system and gene deletion mutants. This project will elucidate the molecular mechanism of the inhibitory effect of DMY on the biofilm formation of S. aureus by targeting SrtA, together with providing a theoretical basis for the application of DMY in the prevention and control of biofilm formed by foodborne pathogens.

金黄色葡萄球菌是一种重要的食源性致病菌，生物膜的形成是其持续污染食品的主要因素。开发安全高效的生物膜抑制剂对保障食品安全具有重要意义。前期我们发现，二氢杨梅素（DMY）能显著抑制金黄色葡萄球菌生物膜的形成，但其作用机制尚不清楚。后续研究发现，DMY可与金黄色葡萄球菌sortase A（SrtA）相互作用，抑制其酶活并下调其表达，提示DMY可能通过靶向SrtA抑制金黄色葡萄球菌生物膜形成。本项目拟结合菌体细胞体内外实验，研究DMY与金黄色葡萄球菌SrtA的相互作用；明确DMY对SrtA转肽作用及蛋白锚定活性的影响；通过免疫共沉淀联合质谱技术构建SrtA蛋白互作网络，利用细菌双杂交体系与基因缺失突变株，验证互作蛋白与SrtA的互作关系及其在生物膜形成中的功能。本项目的顺利实施将阐明DMY靶向SrtA抑制金黄色葡萄球菌生物膜形成的分子机制，为DMY在食源性致病菌生物膜防控中的应用提供理论基础。

本项目针对金黄色葡萄球菌食品污染防控需求和生物膜抑制剂开发的关键问题，以二氢杨梅素（DMY）与金黄色葡萄球菌sortase A（SrtA）的相互作用为切入点，探究DMY抑制金黄色葡萄球菌生物膜形成的分子机制。本项目采用计算机分子模拟及光谱学技术分析发现，DMY能够通过氢键和疏水作用与金黄色葡萄球菌SrtA的活性位点结合，形成DMY-SrtA复合体，抑制其转肽活性，并进一步影响其蛋白锚定活性。多组学分析结果表明，DMY抑制了金黄色葡萄球菌中与细菌附着和积累阶段相关的大量基因和蛋白的表达水平，导致多种金黄色葡萄球菌表面蛋白，包括凝集因子A（ClfA）、铁调节表面决定因子（IsdA, IsdB, IsdC）、表面蛋白（SasG）、纤维蛋白原结合蛋白（FnbA, FnbB）及丝氨酸蛋白酶下调，从而阻碍金黄色葡萄球菌生物膜形成。本项目的实施为食源性致病菌生物膜抑制剂的开发提供了新靶点，同时为DMY作为一种潜在的新型天然抑菌剂应用于食品工业中的生物膜防控奠定了理论基础。