Following translocation, autocatalytic cleavage in the β-domain interior results in cleavage of the passenger from its β-domain and release into the extracellular environment ( Barnard et al., 2007). The latter process also involves the host-derived β-barrel assembly machinery (Bam) complex ( Ieva and Bernstein, 2009 Sauri et al., 2009). To cross the complex Gram-negative bacterial cell envelope, autotransporters are organized in three domains ( Pohlner et al., 1987 Figure 1A): (i) an N-terminal signal peptide that targets the protein to the Sec translocon for translocation across the inner membrane, (ii) a secreted passenger domain that carries the effector function, and (iii) a C-terminal β-domain that integrates into the outer membrane (OM) and facilitates translocation of the passenger from the periplasm into the extracellular space. We have previously engineered a protein display system based on the bacterial autotransporter Hemoglobin protease (Hbp Daleke-Schermerhorn et al., 2014) to allow surface decoration of bacterial cells and derived OMVs with recombinant proteins. One effective strategy involves the display of heterologous antigens on the surface of OMVs for which various concepts have been described ( Gerritzen et al., 2017). This makes OMVs an attractive platform for the development of recombinant vaccines ( Bitto and Kaparakis-Liaskos, 2017 Gnopo et al., 2017). They are non-replicating and non-invasive, hence safe, but still contain the immunostimulatory properties that are a facsimile of the parental cells ( Alaniz et al., 2007). Outer membrane vesicles (OMVs) are nanoparticles (20–100 nm) that shed from the outer membrane of Gram-negative bacteria in a natural continuous process. In conclusion, we constructed a versatile modular platform for the development of bivalent recombinant OMV-based vaccines and therapeutics. Since coupling occurs to Hbp that is already exposed on the OMVs, there are no limitations to the size and complexity of the partner proteins. This allowed the coupling of two heterologous proteins to a single Hbp carrier molecule without obvious steric hindrance effects. Next, we combined distally placed SnoopCatcher or SnoopTag sequences with internal SpyTags in a single Hbp molecule. Inserted SpyTags were shown to be accessible at the Salmonella OMV surface and to efficiently couple Sp圜atcher-equipped fusion proteins. Here, we aimed to further expand the versatility of the Hbp platform by enabling the coupling of heterologous proteins to internal sites of the Hbp passenger. Moreover, we implemented Tag-Catcher protein ligation technology, to obtain dense display of single heterologous antigens and nanobodies on the OMVs through coupling to the distal end of the Hbp passenger domain. We previously engineered the autotransporter protein Hemoglobin protease (Hbp) into a surface display carrier that can be expressed to high density on the surface of Salmonella OMVs. 3Medical Microbiology and Infection Control, Amsterdam Institute of Infection & Immunity, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlandsīacterial outer membrane vesicles (OMVs) attract increasing interest as immunostimulatory nanoparticles for the development of vaccines and therapeutic agents.2Department of Molecular Microbiology, Amsterdam Institute of Molecular and Life Sciences, Vrije Universiteit Amsterdam, Amsterdam, Netherlands.Bart van den Berg van Saparoea 1, Diane Houben 1,2, Coen Kuijl 3, Joen Luirink 1,2 and Wouter S.
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