高效递送趋化因子CXCL12工程益生菌的构建及其在糖尿病慢性创面愈合中的应用

Construction of an engineered probiotic strain for efficiently delivering chemokine CXCL12 and application of the strain in diabetic chronic wound healing

摘要:

糖尿病慢性创面具有难愈性、反复进展性和较高的致残/死亡率等特点，其临床治疗是亟待解决的医学难题之一。因其复杂的局部微环境，如高蛋白酶活性和持续的炎症反应等，使外源性因子(如趋化因子CXCL12)在局部慢性创面的生物利用率非常低，限制了外源性因子在慢性创面的局部应用。本研究利用合成生物学技术原理，以植物乳杆菌WCFS1为底盘菌，旨在开发一种高效的CXCL12递送系统，并评估其在糖尿病慢性创面修复中的作用。采用分子克隆技术将植物乳杆菌内源性分泌信号肽lp_3050和乳酸菌常用分泌信号肽usp45分别与gusA报告基因融合，插入pTRK892-P_32(pgm)质粒中，制备相应的工程菌，并通过GusA酶活分析表征了该菌株的蛋白分泌表达效率。结果表明，与usp45信号肽相比，lp_3050信号肽在植物乳杆菌WCFS1中能高效驱动GusA的分泌表达，其发酵上清中GusA酶活力提高了近5倍。基于此，将lp_3050分泌信号肽与密码子优化后的CXCL12基因进行融合表达，构建了高效分泌表达CXCL12的工程益生菌Lpw-CXCL12，其发酵上清中CXCL12的含量高达(13.40±0.20) μg/mL。随后，在慢性创面小鼠模型中发现Lpw-CXCL12工程益生菌可加速糖尿病慢性创面愈合。本研究构建了一株高效分泌表达趋化因子CXCL12的工程益生菌，并揭示了其在糖尿病慢性创面小鼠模型中的促创面愈合功效，为未来糖尿病慢性创面的治疗提供了新的思路和技术支撑。

Abstract:

Diabetic chronic wounds are characterized by difficult healing, recurrent progression, and high rates of disability and mortality, which make their clinical treatment a medical challenge urgent to be addressed. However, the complex local microenvironment conditions of chronic wounds, such as high protease activity and persistent inflammatory responses, result in low bioavailability of exogenous cytokines (e.g., chemokine CXCL12) at the wound site, limiting their clinical application. In this study, we utilized Lactobacillus plantarum WCFS1 as the chassis to develop an efficient CXCL12 delivery system based on synthetic biology. Subsequently, we evaluated the role of the engineered probiotic strain in promoting the chronic wound healing in diabetic mice. Firstly, we fused the endogenous secretion signal peptide lp_3050 (SP_{lp_3050}) of L.plantarumWCFS1 and the commonly used secretion signal peptide usp45 (SP_usp45) of lactic acid bacteria with the reporter gene gusA and inserted them into the pTRK892-P_32(pgm) plasmid by molecular cloning. Then, we prepared the engineered strains and characterized the efficacy of the two signal peptides in driving the secretion of GusA. The results showed that SP_{lp_3050} efficiently drove the secretion of GusA inL.plantarum WCFS1, increasing the activity of GusA in the culture supernatant by nearly five times compared with that of SP_{lp_3050}. Further, we fused SP_{lp_3050} and codon-optimized CXCL12 gene to construct an engineered probiotic strain Lpw-CXCL12 for CXCL12 delivery. The results demonstrated that the content of CXCL12 in the culture supernatant reached (13.40±0.20) μg/mL. Finally, we found that the engineered probiotic strain Lpw-CXCL12 accelerated chronic wound healing in a diabetic mouse model. In conclusion, these results support an engineered probiotic strain in promoting diabetic chronic wound healing, providing a new strategy and technological foundation for the management of diabetic chronic wounds in the future.