壳寡糖对菌膜多糖结构破坏作用的机制研究

Antimicrobial resistance is an increasingly serious threat to global public health. Most case of pathogen (bacteria and fungi) persistent infection diseases is related to the drug-resistance of the biofilm formed by the pathogen. Forming biofilm will 10-1000 fold enhance the drug-resistance of pathogen and help them escape from host immune system attack and drug treatment. Developing anti-biofilm compounds as new antimicrobial drugs or drug synergist will be the key to resolve the drug-resistance problem caused by the biofilm in the persistent disease. In recent studies, we found that chitosan oligosaccharides showed a strong effect on breaking down biofilm structure and increasing of drug-sensitivity of pathogenic bacteria and fungi. Chitosan oligosaccharide is bio-safe bio-degradable functional oligosaccharide produced from nature materials. Thus, it is an intriguing target for the development of potential antimicrobial drugs. In this application, we project a systematic research on the molecular mechanism of anti-biofilm activity of chitosan oligosaccharides, using major human fungal pathogen Candida albicans as the research model. We will study the influence of chitosan oligosaccharides on the activity and transcriptional level of key biofilm factors. Based on these studies, we will focus on the disruption of biofilm structure as well as the influence on fungal cells in the biofilm, to discover the target of the chitosan oligosaccharide and the molecular mechanism of its anti-biofilm activity. Furthermore, we will evaluate the structure-activity relationship in the anti-biofilm function of chitosan oligosaccharides. Studies in this project will provide a theoretical basis for the application of chitosan oligosaccharides on biofilm-related infections treatment and the development of the new antimicrobial drug and drug synergist.

病原菌耐药性问题日趋严重，严重威胁人类社会健康安全。病原菌持续性感染疾病，与病原菌形成菌膜产生的耐药性密切相关。病原菌形成菌膜可以10-1000倍的提高耐药性，躲避宿主细胞免疫系统和药物的攻击。开发抗菌膜活性物质作为新型抗菌药物或药物增效剂，是解决持续性病原感染治疗问题的关键。申请团队近期研究发现，壳寡糖可有效破坏病原真菌菌膜的多糖结构，降低其耐药性。壳寡糖是一种天然、安全、生物可降解的功能寡糖，具备良好的药物开发潜力。本项目拟以人类主要病原白色念珠菌为对象，系统研究壳寡糖抗菌膜活性的分子机制，并探讨壳寡糖结构与抗菌膜活性的构效关系。通过研究壳寡糖对菌膜多糖结构关键因子活性及转录水平的影响，从破坏菌膜多糖结构和调控菌膜细胞活性两方面，分析壳寡糖作用靶点及作用机理。本项目的开展将为壳寡糖应用于病原菌感染治疗及新型抗菌药物及增效剂的开发奠定理论基础和参考依据。

念珠菌是一种寄生在人体皮肤、黏膜、肠道中的条件致病真菌，易发于艾滋病等严重免疫功能低下患者。随着抗菌药物在临床上的广泛应用，真菌常常出现耐药性,为临床治疗带来困难。因此，研发新的抗真菌策略显得尤为重要。壳聚糖又称脱乙酰甲壳素，是由自然界广泛存在的几丁质经过脱乙酰作用得到的，是天然糖中唯一带正电荷的多糖，具有良好的天然抗菌活性。壳寡糖是将壳聚糖经过特殊的生物酶技术或化学降解、微波降解等技术得到的寡糖，具有分子量低、水溶性好、生物活性高、生物相容性好等优势。前期研究已发现壳寡糖具有抗真菌活性，但目前对于其抗真菌尤其是抗真菌生物被膜活性的构效关系及分子机制尚不明确。.本研究开展壳寡糖破坏念珠菌生物被膜作用构效关系及机制研究，系统研究了壳寡糖抗真菌作用构效关系与分子机制。结果表明，壳寡糖的结构对于其抗真菌及生物被膜活性影响巨大。高分子量壳寡糖可以定位于真菌细胞壁并发挥抗真菌活性，而低分子量壳寡糖进入真菌细胞内，抗真菌活性不显著。通过多种实验，明确了壳寡糖抗真菌及生物被膜的作用位点在真菌细胞壁，发现了其作用的关键靶蛋白Phr家族蛋白。壳寡糖是通过干扰细胞壁葡聚糖酶家族Phr1的转糖苷酶活性，从而影响了细胞壁葡聚糖的重组延伸，进一步造成细胞壁完整性的丧失，从而发挥其抗真菌效果。因为Phr1表达水平随着生物被膜成熟而逐渐升高，壳寡糖对于真菌生物被膜的活性也逐渐升高。因此，壳寡糖是一种针对生物被膜态真菌具有特别作用的抗真菌活性物质，且其显示出与其他抗真菌药物有效的复配增效效果。因此，本研究表明壳寡糖具有作为抗真菌生物被膜药物或药物增效剂开发的良好前景，为抗真菌药物的开发提供了新思路，为其应用开发奠定了理论基础。