基于酰化高丝氨酸内酯降解酶的钛合金植入体表面抑制细菌生物膜研究

Bacteria sense and regulate the growth of bacterial group via quorum sensing effects and form biofilm on implant surface. It makes the major contribution to the antimicrobial resistance that leads to the failure of anti-infection treatment after implant surgery. The importance of inhibiting and removing biofilm is significant for preventing antimicrobial resistance in anti-infection treatment. Most of the current strategies for preventing bacteria on implant surface rely on anti-adhesive and anti-bacterial treatments, which are limited by the contradiction between biocompatibility and antimicrobial ability of biomaterials as well as affected by environmental factors. This project aims to inhibit and remove bacterial biofilm on titanium alloy implant surface by designing and constructing recombinant enzymes to degrade the quorum molecules acyl-homoserine lactones and block quorum sensing pathways. The concentration and activity of acyl-homoserine lactone degradases will be maintained on titanium alloy implant surface via enzyme immobilizing technology. It provides innovative theory and technology for preventing and treating continuing infections after implant surgery. Research issues to be addressed include (1) screening, structural and functional optimizing, expressing and purifying recombinant enzymes for the degradation of quorum molecules acyl-homoserine lactones; (2) direct anchoring enzymes and indirect immobilizing enzymes via carriers on titanium alloy implant surface; (3) qualitative and quantitative evaluating biofilm formation on implant surface. The ultimate objective is to establish the anti-biofilm (bacteria) surface of titanium alloy implants based on blocking quorum sensing effects.

细菌通过群感效应感知和调控群体的生长，在植入体表面形成生物膜，是细菌产生耐药性并导致植入手术术后抗感染治疗失败的主要原因。抑制和清除生物膜对于预防细菌耐药性和抗感染治疗具有重要意义。目前，植入体表面抗菌的基本思路是针对细菌个体的抗粘附和抑菌处理，在指导思想上存在生物相容性和抗菌性之间的矛盾，并容易受到环境因素的影响。本项目以钛合金植入体为研究对象，针对生物膜的形成机制以及群感效应，设计和制备降解群感信号分子酰化高丝氨酸内酯的基因重组酶，通过固定化技术维持降解酶在基体表面的有效浓度和活性，阻断群感效应，抑制植入体表面的生物膜，为植入手术术后持续性感染的预防和治疗提供新方法和新理论。研究内容包括：酰化高丝氨酸内酯降解酶的筛选、结构和功能优化、表达、纯化；降解酶在钛合金表面的直接固定以及载体介导的间接固定；细菌生物膜的定性和定量评价。最终建立基于阻断群感效应的抗生物膜（细菌）钛合金植入体表面。

细菌通过群感信号分子的调控，在植入体表面形成生物被膜，阻止抗生素和杀菌药物的渗透以及免疫细胞对细菌的吞噬，是细菌产生抗药性并导致植入术后抗感染治疗失败的主要原因。 本项目针对植入体表面细菌生物被膜的形成和清除，主要取得了以下方面的研究成果。（1）在载体介导的酶固定化抑制和清除细菌生物被膜方面，构建了含有磁性蛋白标签MagR的重组群感信号分子水解酶AiiA和重组生物膜分散酶DspB，并实现了二者在磁性纳米粒子上的在固定化以及对细菌生物被膜的抑制和清除。（2）在酶的直接固定化方面，构建了含有金属结合多肽标签的重组群感信号分子降解酶AiiA，并实现了其在金属表面固定化和抑制细菌生物被膜的功能。（3）针对纳米载体的合成技术和机理进行了探讨，包括酶无机载体和磁性纳米粒子的生物合成机制和功能。这些研究有利于进一步开发适于临床应用的具有抗细菌生物膜功能的酶试剂或植入体表面。截止目前为止，依托本项目共发表期刊论文10篇（其中SCI收录7篇），申报专利12项（其中4项已经获得授权）；培养博士后1人，研究生5人。