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Improved cell surface display of Salmonella enterica serovar Enteritidis antigens in Escherichia coli
KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.ORCID-id: 0000-0002-3314-6060
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2015 (Engelska)Ingår i: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, nr 1, artikel-id 47Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Background: Salmonella enterica serovar Enteritidis (SE) is one of the most potent pathogenic Salmonella serotypes causing food-borne diseases in humans. We have previously reported the use of the β-autotransporter AIDA-I to express the Salmonella flagellar protein H:gm and the SE serotype-specific fimbrial protein SefA at the surface of E. coli as live bacterial vaccine vehicles. While SefA was successfully displayed at the cell surface, virtually no full-length H:gm was exposed to the medium due to extensive proteolytic cleavage of the N-terminal region. In the present study, we addressed this issue by expressing a truncated H:gm variant (H:gmd) covering only the serotype-specific central region. This protein was also expressed in fusion to SefA (H:gmdSefA) to understand if the excellent translocation properties of SefA could be used to enhance the secretion and immunogenicity. Results: H:gmd and H:gmdSefA were both successfully translocated to the E. coli outer membrane as full-length proteins using the AIDA-I system. Whole-cell flow cytometric analysis confirmed that both antigens were displayed and accessible from the extracellular environment. In contrast to H:gm, the H:gmd protein was not only expressed as full-length protein, but it also seemed to promote the display of the protein fusion H:gmdSefA. Moreover, the epitopes appeared to be recognized by HT-29 intestinal cells, as measured by induction of the pro-inflammatory interleukin 8. Conclusions: We believe this study to be an important step towards a live bacterial vaccine against Salmonella due to the central role of the flagellar antigen H:gm and SefA in Salmonella infections and the corresponding immune responses against Salmonella.

Ort, förlag, år, upplaga, sidor
2015. Vol. 14, nr 1, artikel-id 47
Nyckelord [en]
AIDA-I, Autotransport, Escherichia coli, Live vaccines, Salmonella enterica, Surface expression, Bacteria (microorganisms), Salmonella, Salmonella enteritidis
Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Identifikatorer
URN: urn:nbn:se:kth:diva-167747DOI: 10.1186/s12934-015-0227-3ISI: 000353617100001Scopus ID: 2-s2.0-84928551436OAI: oai:DiVA.org:kth-167747DiVA, id: diva2:815516
Forskningsfinansiär
Sida - Styrelsen för internationellt utvecklingssamarbete
Anmärkning

QC 20150601

Tillgänglig från: 2015-06-01 Skapad: 2015-05-22 Senast uppdaterad: 2017-12-04Bibliografiskt granskad
Ingår i avhandling
1. Influence of recombinant passenger properties and process conditions on surface expression using the AIDA-I autotransporter
Öppna denna publikation i ny flik eller fönster >>Influence of recombinant passenger properties and process conditions on surface expression using the AIDA-I autotransporter
2013 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Surface expression has attracted much recent interest, and it has been suggested for a variety of applications. Two such applications are whole-cell biocatalysis and the creation of live vaccines. For successful implementation of these applications there is a need for flexible surface expression systems that can yield a high level of expression with a variety of recombinant fusion proteins. The aim of this work was thus to create a surface expression system that would fulfil these requirements.

 

A novel surface expression system based on the AIDA-I autotransporter was created with the key qualities being are good, protein-independent detection of the expression through the presence of two epitope tags flanking the recombinant protein, and full modularity of the different components of the expression cassette. To evaluate the flexibility of this construct, 8 different model proteins with potential use as live-vaccines or biocatalysts were expressed and their surface expression levels were analysed.

 

Positive signals were detected for all of the studied proteins using antibody labelling followed by flow cytometric analysis, showing the functionality of the expression system. The ratio of the signal from the two epitope tags indicated that several of the studied proteins were present mainly in proteolytically degraded forms, which was confirmed by Western blot analysis of the outer membrane protein fraction. This proteolysis was suggested to be due to protein-dependent stalling of translocation intermediates in the periplasm, with indications that larger size and higher cysteine content had a negative impact on expression levels. Process design with reduced cultivation pH and temperature was used to increase total surface expression yield of one of the model proteins by 400 %, with a simultaneous reduction of proteolysis by a third. While not sufficient to completely remove proteolysis, this shows that process design can be used to greatly increase surface expression. Thus, it is recommended that future work combine this with engineering of the bacterial strain or the expression system in order to overcome the observed proteolysis and maximise the yield of surface expressed protein.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2013. s. xi, 63
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2013:9
Nyckelord
AIDA-I, Autotransport, Biocatalysis, Escherichia coli, Live vaccines, Surface expression
Nationell ämneskategori
Bioprocessteknik
Identifikatorer
urn:nbn:se:kth:diva-122230 (URN)978-91-7501-770-9 (ISBN)
Disputation
2013-06-05, Sal FB42, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

QC 20130516

Tillgänglig från: 2013-05-16 Skapad: 2013-05-14 Senast uppdaterad: 2015-06-01Bibliografiskt granskad

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Gustavsson, MartinLarsson, Gen

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Gustavsson, MartinSamuelson, PatrikLarsson, Gen
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Industriell bioteknologi
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Microbial Cell Factories
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)

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