Pérez-Lorente Alicia Isabel, Araujo-Garrido Mario, de Vicente Antonio, Romero Diego, Molina-Santiago Carlos
Departamento de Microbiologia, Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora", Universidad de Málaga - Consejo Superior de Investigaciones Cientificas (IHSM-UMA-CSIC), Universidad de Málaga, Bulevar Louis Pasteur 31 (Campus Universitario de Teatinos), Málaga, 29071, Spain.
Departamento de Microbiología, Universidad de Málaga, Bulevar Louis Pasteur 31 (Campus Universitario de Teatinos), Málaga, 29071, Spain.
J Biol Eng. 2025 Apr 3;19(1):28. doi: 10.1186/s13036-025-00497-w.
Bacteria employ diverse molecular systems, such as the type VI secretion system (T6SS) to outcompete other microorganisms and adapt to ecological niches. The T6SS is a versatile nanomachine capable of delivering toxic effectors into neighboring cells, providing advantages in bacterial interactions. In recent years, T6SSs have been proposed as promising tools for engineering selective antimicrobial platforms.
In this study, we successfully engineered Pseudomonas putida KT2440 to heterologously express and release T6SS effectors. The expression of Tse1, an effector from Pseudomonas chlororaphis, induced sporulation in plant-beneficial Bacillus strains via a T6SS-dependent mechanism, particularly when Tse1 was paired with a PAAR protein. Similarly, the engineered strain effectively inhibited Aeromonas hydrophila growth using the phospholipase toxin TplE from Pseudomonas aeruginosa. Furthermore, antifungal activity was achieved by coexpressing Tfe2, an effector from Serratia marcescens, with VgrGs, resulting in increased reactive oxygen species levels and cellular damage in Botrytis cinerea. Importantly, the T6SS was also employed to deliver non-T6SS effectors such as chitosanase, demonstrating its versatility in degrading fungal cell walls.
Our findings demonstrate that the T6SS can be engineered to deliver both canonical and noncanonical effectors, providing a robust platform for targeted antibacterial and antifungal applications. The modularity of the system enables precise pairing of effectors with structural components such as VgrG and PAAR proteins, optimizing delivery efficiency. These engineered systems provide new opportunities for the development of biocontrol strategies in agriculture, microbiome modulation, and potential therapeutic applications. Future advancements in bioinformatics and protein engineering will further increase the specificity and functionality of T6SS-based delivery systems, offering innovative tools for managing microbial ecosystems and addressing global challenges in health and agriculture.
细菌利用多种分子系统，如VI型分泌系统（T6SS）来胜过其他微生物并适应生态位。T6SS是一种多功能纳米机器，能够将毒性效应蛋白输送到邻近细胞中，在细菌相互作用中具有优势。近年来，T6SS被认为是构建选择性抗菌平台的有前途的工具。
在本研究中，我们成功地对恶臭假单胞菌KT2440进行了工程改造，使其异源表达并释放T6SS效应蛋白。来自绿针假单胞菌的效应蛋白Tse1的表达通过T6SS依赖机制诱导了植物有益芽孢杆菌菌株的孢子形成，特别是当Tse1与PAAR蛋白配对时。同样，工程菌株利用来自铜绿假单胞菌的磷脂酶毒素TplE有效抑制了嗜水气单胞菌的生长。此外，通过共表达来自粘质沙雷氏菌的效应蛋白Tfe2和VgrG实现了抗真菌活性，导致灰葡萄孢中的活性氧水平升高和细胞损伤。重要的是，T6SS还被用于递送非T6SS效应蛋白，如壳聚糖酶，证明了其在降解真菌细胞壁方面的多功能性。
我们的研究结果表明，T6SS可以被工程改造以递送经典和非经典效应蛋白，为靶向抗菌和抗真菌应用提供了一个强大的平台。该系统的模块化能够使效应蛋白与VgrG和PAAR蛋白等结构成分精确配对，优化递送效率。这些工程系统为农业生物防治策略、微生物群落调节及潜在治疗应用的发展提供了新机会。生物信息学和蛋白质工程的未来进展将进一步提高基于T6SS的递送系统的特异性和功能，为管理微生物生态系统以及应对健康和农业领域的全球挑战提供创新工具。
