靶向破坏铜绿假单胞菌生物被膜的定向蛋白投放技术

An engineered bacterium for the targeted delivery of proteins to destroy Pseudomonas aeruginosa biofilms

摘要:

[目的] 随着合成生物学的发展，通过在细菌体内设计合成复杂、多功能的基因线路进行靶向治疗已经取得巨大进展。虽然这种使用细菌作为治疗传递系统，选择性地在体内释放有效治疗成分的方式具有极大优势，但是如何使细菌在代谢负荷增加较低的情况下有效地分泌功能蛋白并发挥作用依旧是一个难题。[方法] 针对这一难题，本研究提供了一种新的策略，即以细菌中广泛存在的蛋白类杀菌素和丝状噬菌体等相关编码基因作为生物模块，通过对铜绿假单胞菌的这些内源生物模块的重新编排和组装，构建了一种能在特定条件下裂解并投放功能蛋白的工程菌。为了评价工程菌中构建的生物模块能否工作，本研究选择胞外多糖水解酶PelA和PslG作为工程菌投放的功能蛋白，以此构建了工程菌PAO1102。通过对铜绿假单胞菌生物被膜的破坏实验、抑制形成实验以及抗生素耐药性实验，检验PAO1102对铜绿假单胞菌生物被膜的破坏和预防效果。[结果] 与对照组相比，工程菌PAO1102的处理可以显著破坏已形成的生物被膜并抑制生物被膜的形成，同时还可显著增强生物被膜中的细菌对妥布霉素的敏感性，且这些功能主要通过外界Pf4丝状噬菌体侵染并使工程菌裂解而释放功能蛋白这一途径实现的。[结论] 本研究所构建的工程菌可以作为一种微生物工具，用于靶向破坏铜绿假单胞菌生物被膜。在后续的研究中可根据不同的需求，在工程菌中表达不同的功能基因并实现功能蛋白的定向投放，从而执行不同的生物学功能。

Abstract:

[Objective] With the development of synthetic biology, great progress has been made in targeted therapy by designing and synthesizing complex, multifunctional gene circuits in bacteria. Although using bacteria as a therapeutic delivery system to selectively release effective therapeutic components in vivo has great advantages, how to make bacteria secrete functional proteins effectively and play a role under low metabolic load remains a challenge.[Methods] To solve this problem, this study provided a new strategy. In this strategy, protein germicide and filamentous phage encoding genes widely existed in bacteria were used as biobricks, and through the rearrangement and assembly of these endogenous biobricks of P. aeruginosa, an engineered bacterium capable of lysis and release of functional proteins under specific conditions was constructed. In order to evaluate whether the biobricks constructed in the engineered bacterium could work, the extracellular polysaccharides hydrolase PelA and PslG were selected as functional proteins to construct the engineered bacterium PAO1102. The destruction and prevention effects of PAO1102 on P. aeruginosa biofilm were tested by the biofilm destruction experiment, biofilm inhibition experiment and antibiotic resistance experiment.[Results] Compared with the control group, the treatment of PAO1102 could not only significantly destroy the mature biofilm and inhibit the biofilm formation, but also significantly enhance the sensitivity of the bacteria in the biofilm to tobramycin. Moreover, these functions of PAO1102 were mainly dependent on the release of functional proteins by phage Pf4 induced cell lysis.[Conclusion] The engineered bacterium could be used as a microbial tool for the targeted delivery of proteins. In the follow-up study, different functional genes will be expressed in the engineered bacterium according to different needs, and the targeted delivery of functional proteins will be realized, to perform different biological functions.