Pinheiro Bruno, Moura Ana C, Oliveira Pedro, Azevedo Jorge E, do Vale Ana, Dos Santos Nuno M S
Fish Immunology and Vaccinology, Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal.
Fish Immunology and Vaccinology, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal.
Front Cell Infect Microbiol. 2025 Aug 6;15:1596550. doi: 10.3389/fcimb.2025.1596550. eCollection 2025.
The intracellular delivery of biologics, particularly large cargoes like proteins, remains a challenge in biotechnology and biomedicine. The modular structure of well-characterized AB toxins allows different cargoes to be grafted, creating a target-specific biotechnological tool capable of cytosolic delivery.
In this study, we employed protein-protein fusion strategies-SpyCatcher003, SnoopCatcher, and SnoopLigase-to generate chimeras between the delivery region of AIP56 (AIP56) and β-lactamase and performed functional delivery assays.
The chimeras were successfully obtained using these strategies and were all able to deliver β-lactamase into the cytosol of J774.A1 macrophages. Cellular fractionation showed that, although most of the β-lactamase remains associated with the endosomal compartment, an active portion is released into the cytosol.
AIP56 delivery region transporting other cargo directly to the cytosol of antigen-presenting cells might be a promising platform for antigen/cargo delivery. This study highlights the potential of protein-protein fusion strategies to create versatile, antigenically distinct toxin-based delivery systems for therapeutic applications.
生物制剂的细胞内递送，尤其是像蛋白质这样的大分子货物，在生物技术和生物医学领域仍然是一项挑战。特征明确的AB毒素的模块化结构允许嫁接不同的货物，从而创建一种能够进行胞质递送的靶向特异性生物技术工具。
在本研究中，我们采用蛋白质-蛋白质融合策略——SpyCatcher003、SnoopCatcher和SnoopLigase——在AIP56（AIP56）的递送区域和β-内酰胺酶之间生成嵌合体，并进行功能递送测定。
使用这些策略成功获得了嵌合体，并且它们都能够将β-内酰胺酶递送至J774.A1巨噬细胞的细胞质中。细胞分级分离表明，尽管大多数β-内酰胺酶仍与内体区室相关，但有一部分活性部分被释放到细胞质中。
将其他货物直接运输到抗原呈递细胞细胞质中的AIP56递送区域可能是用于抗原/货物递送的一个有前景的平台。本研究突出了蛋白质-蛋白质融合策略在创建用于治疗应用的通用、抗原性不同的基于毒素的递送系统方面的潜力。
