Exploring protein–protein ligation approaches for the cytosolic delivery of antigens using AIP56
IntroductionThe 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...
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Frontiers Media S.A.
2025-08-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2025.1596550/full |
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| author | Bruno Pinheiro Bruno Pinheiro Bruno Pinheiro Ana C. Moura Ana C. Moura Pedro Oliveira Jorge E. Azevedo Jorge E. Azevedo Jorge E. Azevedo Ana do Vale Ana do Vale Nuno M. S. dos Santos Nuno M. S. dos Santos |
| author_facet | Bruno Pinheiro Bruno Pinheiro Bruno Pinheiro Ana C. Moura Ana C. Moura Pedro Oliveira Jorge E. Azevedo Jorge E. Azevedo Jorge E. Azevedo Ana do Vale Ana do Vale Nuno M. S. dos Santos Nuno M. S. dos Santos |
| author_sort | Bruno Pinheiro |
| collection | DOAJ |
| description | IntroductionThe 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.MethodsIn 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.ResultsThe 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.ConclusionAIP56 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. |
| format | Article |
| id | doaj-art-0a0ebfa53b004ca89104349428b76b29 |
| institution | Kabale University |
| issn | 2235-2988 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj-art-0a0ebfa53b004ca89104349428b76b292025-08-20T03:39:09ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882025-08-011510.3389/fcimb.2025.15965501596550Exploring protein–protein ligation approaches for the cytosolic delivery of antigens using AIP56Bruno Pinheiro0Bruno Pinheiro1Bruno Pinheiro2Ana C. Moura3Ana C. Moura4Pedro Oliveira5Jorge E. Azevedo6Jorge E. Azevedo7Jorge E. Azevedo8Ana do Vale9Ana do Vale10Nuno M. S. dos Santos11Nuno M. S. dos Santos12Fish Immunology and Vaccinology, Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, PortugalFish Immunology and Vaccinology, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, PortugalDoctoral Program in Molecular and Cell Biology (MCBiology), Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, PortugalFish Immunology and Vaccinology, Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, PortugalFish Immunology and Vaccinology, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, PortugalInstituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, PortugalInstituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, PortugalOrganelle Biogenesis and Function, Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, PortugalOrganelle Biogenesis and Function, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, PortugalFish Immunology and Vaccinology, Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, PortugalFish Immunology and Vaccinology, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, PortugalFish Immunology and Vaccinology, Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, PortugalFish Immunology and Vaccinology, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, PortugalIntroductionThe 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.MethodsIn 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.ResultsThe 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.ConclusionAIP56 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.https://www.frontiersin.org/articles/10.3389/fcimb.2025.1596550/fullAB toxinsbiologicscytosolic deliveryprotein-protein fusionAIP56 |
| spellingShingle | Bruno Pinheiro Bruno Pinheiro Bruno Pinheiro Ana C. Moura Ana C. Moura Pedro Oliveira Jorge E. Azevedo Jorge E. Azevedo Jorge E. Azevedo Ana do Vale Ana do Vale Nuno M. S. dos Santos Nuno M. S. dos Santos Exploring protein–protein ligation approaches for the cytosolic delivery of antigens using AIP56 Frontiers in Cellular and Infection Microbiology AB toxins biologics cytosolic delivery protein-protein fusion AIP56 |
| title | Exploring protein–protein ligation approaches for the cytosolic delivery of antigens using AIP56 |
| title_full | Exploring protein–protein ligation approaches for the cytosolic delivery of antigens using AIP56 |
| title_fullStr | Exploring protein–protein ligation approaches for the cytosolic delivery of antigens using AIP56 |
| title_full_unstemmed | Exploring protein–protein ligation approaches for the cytosolic delivery of antigens using AIP56 |
| title_short | Exploring protein–protein ligation approaches for the cytosolic delivery of antigens using AIP56 |
| title_sort | exploring protein protein ligation approaches for the cytosolic delivery of antigens using aip56 |
| topic | AB toxins biologics cytosolic delivery protein-protein fusion AIP56 |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2025.1596550/full |
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