ORIGINAL RESEARCH article
Front. Cell. Infect. Microbiol.
Sec. Bacteria and Host
Volume 15 - 2025 | doi: 10.3389/fcimb.2025.1596550
This article is part of the Research TopicBacterial Exotoxins: Exploring Pathogenesis and Therapeutic PotentialsView all articles
Exploring protein-protein ligation approaches for the cytosolic delivery of antigens using AIP56
Provisionally accepted
Bruno Pinheiro1,2,3
Ana C Moura3,4
P. Oliveira5
Jorge E. Azevedo5,6,7
Ana do Vale3,4
Nuno M. S. dos Santos3,4*- 1Fish Immunology and Vaccinology Group, Fish Immunology and Vaccinology Group, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
- 2Doctoral Program in Molecular and Cell Biology, ICBAS, Universidade do Porto, Porto, Portugal
- 3Fish Immunology and Vaccinology Group, i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal
- 4Fish Immunology and Vaccinology Group, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
- 5Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal
- 6Organelle Biogenesis and Function Group, i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal
- 7Organelle Biogenesis and Function, IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
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Introduction: 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. Methods: In this study, we employed protein-protein fusion strategies - SpyCatcher003, SnoopCatcher, and SnoopLigase - to generate chimeras between the delivery region of AIP56 (AIP56L258-N497) and β-lactamase and performed functional delivery assays. Results: 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. Conclusion: AIP56 delivery region transporting other cargo directly to the cytosol of antigen-presenting cells might be a promising platform for antigen/cargoes delivery. This study highlights the potential of protein-protein fusion strategies to create versatile, antigenically distinct toxin-based delivery systems for therapeutic applications.
Keywords: AB toxins, biologics, Cytosolic delivery, protein-protein fusion, AIP56
Received: 19 Mar 2025; Accepted: 07 Jul 2025.
Copyright: © 2025 Pinheiro, Moura, Oliveira, Azevedo, do Vale and dos Santos. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Nuno M. S. dos Santos, Fish Immunology and Vaccinology Group, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
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