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Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach
Vise andre og tillknytning
2014 (engelsk)Inngår i: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, nr 18, s. 5854-5865Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Outer membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of the Mycobacterium tuberculosis antigens ESAT6, Ag85B, and Rv2660c were targeted to the surface of Escherichia coli OMVs upon fusion to Hbp. Furthermore, a hypervesiculating Delta tolR Delta tolA derivative of attenuated Salmonella enterica serovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by the M. tuberculosis antigens and epitopes from Chlamydia trachomatis major outer membrane protein (MOMP). Also, we showed that delivery of Salmonella OMVs displaying Ag85B to antigen-presenting cells in vitro results in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.

sted, utgiver, år, opplag, sider
2014. Vol. 80, nr 18, s. 5854-5865
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-107981DOI: 10.1128/AEM.01941-14ISI: 000341488200032OAI: oai:DiVA.org:su-107981DiVA, id: diva2:753849
Merknad

AuthorCount:19;

Tilgjengelig fra: 2014-10-09 Laget: 2014-10-06 Sist oppdatert: 2018-08-17bibliografisk kontrollert
Inngår i avhandling
1. Protein production in the E. coli cell envelope
Åpne denne publikasjonen i ny fane eller vindu >>Protein production in the E. coli cell envelope
2018 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Proteins fulfil essential functions in every cell and malfunctioning proteins are often the cause of diseases. On the other hand, proteins like antibody fragments or hormones can be used to treat diseases. Proteins are often produced in the bacterium Escherichia coli so that they can be studied to understand their (mal)function or so that they can be used to treat a disease. Unfortunately, producing proteins in the cell envelope of E. coli, like integral membrane proteins, which are important drug targets, and secretory proteins like antibody fragments and hormones, often results in unsatisfactory yields. Therefore, the objectives of this doctoral thesis were to identify bottlenecks that can limit the production of recombinant proteins in the cell envelope of E. coli and to try to overcome these bottlenecks. In the first study, we isolated and characterized the E. coli membrane protein production strain Mt56(DE3). This strain, in which the target gene expression intensity is strongly reduced, outcompetes the standard E. coli membrane protein production strains for most targets tested. In the second and third study we focused on the production of secretory proteins, i.e., proteins that are translocated across the inner membrane into the periplasm of E. coli. First, we investigated the impact of the targeting pathway used to direct a secretory protein to the translocation machinery on the cell physiology and protein production yields. We found that the co-translational targeting of a produced protein saturates the capacity of the translocation machinery resulting in heavily impaired biomass formation and low protein production yields. In contrast, post-translational targeting of a produced protein did not saturate the capacity of the protein translocation machinery resulting in hardly affected biomass formation and high protein production yields. In the third study we investigated how optimizing the production of a co-translationally targeted protein, by harmonizing its production rate with the capacity of the protein translocation machinery, affects the physiology of the cell. We found that, in stark contrast to the non-optimized condition, the optimized production did not affect the composition of the E. coli proteome. This surprising finding indicates that a protein can be produced efficiently in the periplasm of E. coli without compromising the physiology of the cell. In the last study we aimed at developing an outer membrane vesicle-based tuberculosis vaccine. To this end, an E. coli strain was created that produced outer membrane vesicles coated with different tuberculosis antigens. It was shown that a homogenous population of vesicles was produced, which will hopefully facilitate the isolation of these vesicles on an industrial scale.

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018. s. 91
Emneord
E. coli, protein biogenesis, recombinant proteins, membrane proteins, secretory proteins, protein displays, outer membrane vesicles
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
urn:nbn:se:su:diva-158451 (URN)978-91-7797-402-4 (ISBN)978-91-7797-403-1 (ISBN)
Disputas
2018-10-08, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (engelsk)
Opponent
Veileder
Merknad

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Tilgjengelig fra: 2018-09-13 Laget: 2018-08-15 Sist oppdatert: 2018-09-04bibliografisk kontrollert

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