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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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Expression of the salmonella epitopes in E. coli. Western blot showing fusion proteins to the AIDAC translocator. Lane 1: Marker, Lane 2: SefA, Lane 3: H:gm, and Lane 4: Negative control (AIDAC without fusion partner). Arrows indicate the calculated sizes. AIDAC-specific antibodies were used for detection.
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Figure 2: Expression of the salmonella epitopes in E. coli. Western blot showing fusion proteins to the AIDAC translocator. Lane 1: Marker, Lane 2: SefA, Lane 3: H:gm, and Lane 4: Negative control (AIDAC without fusion partner). Arrows indicate the calculated sizes. AIDAC-specific antibodies were used for detection.

Mentions: E. coli O:17 was cultivated in a batch process with cells expressing the target proteins with the AIDA autotransport system. Samples of the cell extract were taken for Western blot analysis using AIDAC antibodies for target protein detection. The individual proteins were His6 tagged in the N-terminal and fused to the AIDAC translocator in the C-terminal (Figure 1) and the resulting molecular weights of the fusion proteins were; H:gm (104 kDa), SefA (64 kDa) and AIDAC (51 kDa). From the blots shown in Figure 2, it can be seen that SefA was located at approximately 64 kDa and was thus produced at the correct size. The H:gm-AIDAC, on the other hand, was detected with a molecular weight somewhat lower than expected. Protein fragments of the fusion protein, binding AIDAC, were also detected in this preparation. In the empty vector containing only AIDAC and no passenger the protein was detected at full length, but also here smaller proteins with AIDAC affinity were seen. These data show that the passenger proteins were produced as fusions to the translocation unit of AIDAC although they were also partly degraded, particularly in the case of the H:gm production. We have used an OmpT background which we have previously shown will reduce the cleavage from the surface and also of the AIDAC unit. Since a lower molecular weight than that of AIDAC was detected, it is evident that the mutant cannot altogether prevent this degradation.


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)

Expression of the salmonella epitopes in E. coli. Western blot showing fusion proteins to the AIDAC translocator. Lane 1: Marker, Lane 2: SefA, Lane 3: H:gm, and Lane 4: Negative control (AIDAC without fusion partner). Arrows indicate the calculated sizes. AIDAC-specific antibodies were used for detection.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Expression of the salmonella epitopes in E. coli. Western blot showing fusion proteins to the AIDAC translocator. Lane 1: Marker, Lane 2: SefA, Lane 3: H:gm, and Lane 4: Negative control (AIDAC without fusion partner). Arrows indicate the calculated sizes. AIDAC-specific antibodies were used for detection.
Mentions: E. coli O:17 was cultivated in a batch process with cells expressing the target proteins with the AIDA autotransport system. Samples of the cell extract were taken for Western blot analysis using AIDAC antibodies for target protein detection. The individual proteins were His6 tagged in the N-terminal and fused to the AIDAC translocator in the C-terminal (Figure 1) and the resulting molecular weights of the fusion proteins were; H:gm (104 kDa), SefA (64 kDa) and AIDAC (51 kDa). From the blots shown in Figure 2, it can be seen that SefA was located at approximately 64 kDa and was thus produced at the correct size. The H:gm-AIDAC, on the other hand, was detected with a molecular weight somewhat lower than expected. Protein fragments of the fusion protein, binding AIDAC, were also detected in this preparation. In the empty vector containing only AIDAC and no passenger the protein was detected at full length, but also here smaller proteins with AIDAC affinity were seen. These data show that the passenger proteins were produced as fusions to the translocation unit of AIDAC although they were also partly degraded, particularly in the case of the H:gm production. We have used an OmpT background which we have previously shown will reduce the cleavage from the surface and also of the AIDAC unit. Since a lower molecular weight than that of AIDAC was detected, it is evident that the mutant cannot altogether prevent this degradation.

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