以壁磷壁酸为靶点抗金黄色葡萄球菌的复合杀菌体系设计研究

Staphylococcus aureus is a common pathogen, and the clinical treatment of S. aureus infections usually relies on antibiotics. However, antibiotics are wide-spectrum antimicrobial reagents, and their use always leads to many side effects including microbial imbalance in human bodies. A potential anti-S. aureus reagent with high activity and high specificity is lysostaphin (Lst). However, S. aureus is resistant to Lst in complicated environments such as blood and intestine. The underlying mechanism remains elusive, thus hindering the clinical application of Lst. It has been found in our earlier studies that S. aureus resistance to Lst is related to wall teichoic acid (WTA), the glycopolymer present on S. aureus cell surface. Based on this, we plan to study Lst resistance in S. aureus by focusing on WTA structure and by using a simulated small intestine environment. Through detailed investigations on the impact of WTA structure on Lst functionality and S. aureus tolerance of Lst, we will elucidate at the molecular level the relationship between WTA structure and S. aureus resistance to Lst and the underlying mechanism. According to such relationship, a complex anti-S. aureus system will be designed by targeting particular WTA structures and by combining the staphylolytic effect of Lst to achieve highly efficient, highly specific eradication of S. aureus in simulated small intestine environment. This study will shine light on the understanding of the mechanisms related to S. aureus resistance to Lst in actual human environments such as blood, intestine, etc., and will provide guidance to the development of potent and narrow-spectrum anti-S. aureus treatment.

金黄色葡萄球菌（金葡菌）是常见致病菌，其感染的治疗主要依靠抗生素。但抗生素是广谱抗菌药，易导致人体菌群失调，副作用严重。一种高效特异性的替代方法是使用溶葡球菌酶（Lst）。然而在真实使用环境如血液、小肠中，金葡菌耐受Lst，其机理尚不明确，限制Lst的实际应用。申请人前期研究发现，金葡菌对Lst的耐受性与细胞表面多糖复合物壁磷壁酸有关。据此，本项目将以壁磷壁酸的化学结构为切入点，建立小肠模拟环境，详细考察不同化学结构的壁磷壁酸对Lst的效用、以及对金葡菌Lst耐受性的影响，在分子层面阐明壁磷壁酸化学结构与金葡菌耐受性的关联机制。依此设计以壁磷壁酸为靶点、联合Lst的复合杀菌体系，在小肠模拟环境中高效专一地杀灭金葡菌。本项目有望为金葡菌在真实人体环境中耐受Lst的机理研究提供参考，为高效窄谱抗金葡菌疗法的开发提供新思路。

金黄色葡萄球菌是常见致病菌，易产生耐药性。溶葡球菌酶可高效杀灭金黄色葡萄球菌的普通菌株及耐药菌株，具有良好的临床应用前景。然而，在丰富营养环境中，代谢旺盛的金黄色葡萄球菌耐受溶葡球菌酶，作用机制不明。现有研究发现，金黄色葡萄球菌的耐药性与壁磷壁酸密切相关。在此基础上，本项目以壁磷壁酸的具体结构为切入点，具体研究壁磷壁酸对溶葡球菌酶的效用以及菌体耐受溶葡球菌酶的影响。首先通过CRISPR-dCas9的方法，针对壁磷壁酸合成途径各基因，构建了基因抑制突变菌株。通过体外酶活动力学测试发现，壁磷壁酸与溶葡球菌酶之间无明显结合作用，但壁磷壁酸的存在可影响溶葡球菌酶的功能。通过流式细胞实验分析发现，壁磷壁酸可阻止溶葡球菌酶与菌体表面的肽聚糖进行结合。对于代谢旺盛的菌体，壁磷壁酸的存在可显著增强菌体对溶葡球菌酶的耐受性，且壁磷壁酸的丰度、链长、胞内外分布对这一耐受过程的贡献程度相当，而壁磷壁酸的糖基化修饰对菌体耐受溶葡球菌酶的过程影响有限。抑制这些关键基因的表达，可显著提高代谢旺盛的菌体对溶葡球菌酶的敏感性。该研究可为新型抗金黄色葡萄球菌药物的开发提供新靶点和新思路，为其它革兰氏阳性致病菌的耐药机制研究提供参考。