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Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus.

Nhan NT, Gonzalez de Valdivia E, Gustavsson M, Hai TN, Larsson G - Microb. Cell Fact. (2011)

Bottom Line: FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off.Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins.Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Vietnam Institute of Biotechnology (IBT), Vietnamese Academy of Science and Technology (VAST), Hanoi, Vietnam.

ABSTRACT

Background: Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in Escherichia coli by the β-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes.

Results: Both SefA and H:gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H:gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H:gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis.

Conclusion: Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the Escherichia coli AIDA autotransporter efficiency.

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Flow cytometry analysis of E. coli expression of salmonella antigens. E. coli O:17 expression of the fusion proteins. The negative control consists of cells carrying the empty plasmid. Fusion proteins were detected by a biotinylated His tag-specific antibody conjugated to Streptavidin-Alexa488.
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Figure 4: Flow cytometry analysis of E. coli expression of salmonella antigens. E. coli O:17 expression of the fusion proteins. The negative control consists of cells carrying the empty plasmid. Fusion proteins were detected by a biotinylated His tag-specific antibody conjugated to Streptavidin-Alexa488.

Mentions: Flow cytometry with binding to the reporter systems was used to confirm the orientation of the fusion proteins. Since the plasmid is designed with a His6-tag, it will consequently be expressed in the N-terminal of the protein on the cell surface in an orientation mostly outwards towards the environment. The His6 tag antibody binding was detected by the fluorescence of streptavidin-Alexa488. Data are presented in the histograms of Figure 4 and, compared with the control, there is a distinct signal showing that the SefA protein is oriented outwards in contrast to the H:gm. The signal confirms a very narrow distribution of the fluorescence intensity from each cell, indicating that each cell expresses more or less the same amount of protein.


Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus.

Nhan NT, Gonzalez de Valdivia E, Gustavsson M, Hai TN, Larsson G - Microb. Cell Fact. (2011)

Flow cytometry analysis of E. coli expression of salmonella antigens. E. coli O:17 expression of the fusion proteins. The negative control consists of cells carrying the empty plasmid. Fusion proteins were detected by a biotinylated His tag-specific antibody conjugated to Streptavidin-Alexa488.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094208&req=5

Figure 4: Flow cytometry analysis of E. coli expression of salmonella antigens. E. coli O:17 expression of the fusion proteins. The negative control consists of cells carrying the empty plasmid. Fusion proteins were detected by a biotinylated His tag-specific antibody conjugated to Streptavidin-Alexa488.
Mentions: Flow cytometry with binding to the reporter systems was used to confirm the orientation of the fusion proteins. Since the plasmid is designed with a His6-tag, it will consequently be expressed in the N-terminal of the protein on the cell surface in an orientation mostly outwards towards the environment. The His6 tag antibody binding was detected by the fluorescence of streptavidin-Alexa488. Data are presented in the histograms of Figure 4 and, compared with the control, there is a distinct signal showing that the SefA protein is oriented outwards in contrast to the H:gm. The signal confirms a very narrow distribution of the fluorescence intensity from each cell, indicating that each cell expresses more or less the same amount of protein.

Bottom Line: FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off.Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins.Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Vietnam Institute of Biotechnology (IBT), Vietnamese Academy of Science and Technology (VAST), Hanoi, Vietnam.

ABSTRACT

Background: Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in Escherichia coli by the β-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes.

Results: Both SefA and H:gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H:gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H:gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis.

Conclusion: Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the Escherichia coli AIDA autotransporter efficiency.

Show MeSH
Related in: MedlinePlus