抗菌肽与铜绿假单胞菌生物被膜的相互作用及促抗菌肽渗透因子的筛选

Multi-drug resistant Pseudomonas aeruginosa (P.a) causes life threatening infections in several scenarios, especially in acute and chronic lung infection. Antimicrobial peptides (AMPs) are promising drugs to treat multi-drug resistant bacteria like P. aeruginosa as alternative to antibiotics due to its broad spectrum antimicrobial activity and less induced resistance. However, the formation of mucoid biofilm, i.e. over-production of alginate in the extracellular polymeric substances (EPS), became a significant barrier against the penetration of AMPs..In the proposed project, to achieve fast screening of penetration promoters for AMPs (AA-230, LL-37 and DPK-060), we established PA biofilm infection models of different complexity, including alginate gel, EPS gel and EPS-mucin gel. Candidates of penetration promoters can be selected through three strategies: to degrade the macromolecules in the biofilm model, to shield the negative charge of the EPS, or to shield the positive charge of the AMPs. The preliminary results showed enhanced penetration of AMPs in EPS biofilm model and corresponding enhanced bactericidal effect of AMPs in the 24-hour-old mucoid PA biofilm. In the most significant case, while 200mM DPK-060 could achieve only 38% killing of 24-hour-old PA biofilm, addition of 20mM citric acid in DPK-060 formulation lead to 96% killing of the biofilm. The completion of the project is expected to give us the suggestions about the most effective penetration promoters for each AMP and the synergy effect of combining different penetration promoters in AMP formulations.

铜绿假单胞菌（P.a）是最难治愈的多重耐药菌之一，常见于肺部感染，并伴随生物被膜（biofilm）形成。抗菌肽被寄予治疗耐药菌感染的厚望。然而，文献和项目预实验均表明P.a biofilm的胞外基质（EPS）对抗菌肽有显著的电荷吸引作用，从而阻止其渗透。我们据此提出：通过EPS降解，电荷屏蔽等策略选择促抗菌肽渗透因子，可以增强抗菌肽的渗透，提高杀菌效率。预实验的初步筛选中，柠檬酸、氯化铁、氯化铝等因子的添加使抗菌肽渗透显著增强，对真实biofilm的杀菌率也相应提高。如，DPK-060抗菌肽制剂中柠檬酸的添加使杀菌率从38%提高到96%。项目拟通过复杂度递增的三种biofilm模型继续进行高效、大规模的促渗透因子筛选，并探索抗菌肽-促渗透因子组合在P.a肺炎患者的咳出粘液样本中的渗透和杀菌作用的最优化，及对人体细胞安全性。该研究有望提高P.a感染的治疗效率，减轻患者痛苦，挽救患者生命。

铜绿假单胞菌（P.a）是最难治愈的多重耐药菌之一，常见于肺部感染，并伴随生物.被膜（biofilm）形成。抗菌肽被寄予治疗耐药菌感染的厚望。然而，文献和项目预实验.均表明P.a biofilm的胞外基质（EPS）对抗菌肽有显著的电荷吸引作用，从而阻止其渗透.。我们据此提出：通过EPS降解，电荷屏蔽等策略选择促抗菌肽渗透因子，可以增强抗菌.肽的渗透，提高杀菌效率。预实验的初步筛选中，柠檬酸、氯化铁、氯化铝等因子的添加.使抗菌肽渗透显著增强，对真实biofilm的杀菌率也相应提高。如，DPK-060抗菌肽制剂中.柠檬酸的添加使杀菌率从38%提高到96%。项目拟通过复杂度递增的三种biofilm模型继续.进行高效、大规模的促渗透因子筛选，并探索抗菌肽-促渗透因子组合在P.a肺炎患者的咳.出粘液样本中的渗透和杀菌作用的最优化，及对人体细胞安全性。该研究有望提高P.a感.染的治疗效率，减轻患者痛苦，挽救患者生命。