PDF(Pubmed)
Abstract：
Genetic editing has revolutionized biotechnology, but delivery of endonuclease genes as DNA can lead to aberrant integration or overexpression, leading to off-target effects. Here, we develop a mechanism to deliver Cre recombinase as a protein by engineering the bacterial type six secretion system (T6SS). Using multiple T6SS fusion proteins, Aeromonas dhakensis or attenuated Vibrio cholerae donor strains, and a gain-of-function cassette for detecting Cre recombination, we demonstrate successful delivery of active Cre directly into recipient cells. The most efficient transfer was achieved using a truncated version of PAAR2 from V. cholerae, resulting in a relatively small (118-amino-acid) delivery tag. We further demonstrate the versatility of this system by delivering an exogenous effector, TseC, enabling V. cholerae to kill Pseudomonas aeruginosa. This implies that P. aeruginosa is naturally resistant to all native effectors of V. cholerae and that the TseC chaperone protein is not required for its activity. Moreover, it demonstrates that the engineered system can improve T6SS efficacy against specific pathogens, proposing future application in microbiome manipulation or as a next-generation antimicrobial. Inexpensive and easy to produce, this protein delivery system has many potential applications, ranging from studying T6SS effectors to genetic editing. IMPORTANCE Delivery of protein-based drugs, antigens, and gene-editing agents has broad applications. The type VI protein secretion system (T6SS) can target both bacteria and eukaryotic cells and deliver proteins of diverse size and function. Here, we harness the T6SS to successfully deliver Cre recombinase to genetically edit bacteria without requiring the introduction of exogenous DNA into the recipient cells. This demonstrates a promising advantage over current genetic editing tools that require transformation or conjugation of DNA. The engineered secretion tag can also deliver a heterologous antimicrobial toxin that kills an otherwise unsusceptible pathogen, Pseudomonas aeruginosa. These results demonstrate the potential of T6SS-mediated delivery in areas including genome editing, killing drug-resistant pathogens, and studying toxin functions.
摘要：
基因编辑彻底改变了生物技术，但将核酸内切酶基因作为DNA递送可导致异常整合或过表达，导致脱靶效应。这里,我们开发了一种通过工程化细菌六型分泌系统（T6SS）将Cre重组酶作为蛋白质递送的机制。使用多个T6SS融合蛋白，达克气单胞菌或减毒霍乱弧菌供体菌株，和用于检测Cre重组的功能增益盒，我们证明了活性Cre直接成功递送到受体细胞中。最有效的转移是使用霍乱弧菌的截短版本的PAAR2实现的，导致相对较小（118个氨基酸）的递送标签。我们通过提供外源性效应子进一步证明了该系统的多功能性，TseC,使霍乱弧菌杀死铜绿假单胞菌.这意味着铜绿假单胞菌对霍乱弧菌的所有天然效应子具有天然抗性,并且TseC伴奏蛋白不是其活性所必需的。此外，这表明工程系统可以提高T6SS对特定病原体的功效，提出了在微生物组操作或作为下一代抗菌剂的未来应用。价格低廉，易于生产，这种蛋白质递送系统具有许多潜在的应用，从研究T6SS效应子到遗传编辑。基于蛋白质的药物的重要性，抗原，和基因编辑剂具有广泛的应用。VI型蛋白质分泌系统（T6SS）可以靶向细菌和真核细胞，并提供不同大小和功能的蛋白质。这里,我们利用T6SS成功地将Cre重组酶递送到基因编辑细菌，而不需要将外源DNA引入受体细胞。这证明了优于需要DNA转化或缀合的当前遗传编辑工具的有希望的优势。工程化的分泌标签还可以递送异源抗微生物毒素，该毒素杀死原本不敏感的病原体，铜绿假单胞菌.这些结果证明了T6SS介导的传递在包括基因组编辑在内的领域的潜力。杀死耐药病原体，研究毒素的功能.
