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Surface display of the cholera toxin B subunit on Staphylococcus xylosus and Staphylococcus carnosus.

ABSTRACT: The heterologous surface expression of the cholera toxin B subunit (CTB) from Vibro cholerae in two staphylococcal species, Staphylococcus xylosus and Staphylococcus carnosus, has been investigated. The gene encoding native CTB (103 amino acids) was introduced into gene constructs encoding chimeric receptors designed to be translocated and anchored on the outer cell surface of the staphylococci. Since functionality of CTB is correlated with its ability to form pentamers and the capacity of the pentameric CTB to bind the GM1 ganglioside, both the surface accessibility and the functionality of the surface-displayed CTB receptors were evaluated. It could be concluded that the chimeric receptors were targeted to the cell wall of the staphylococci, since they could be released by lysostaphin treatment and, after subsequent affinity purification, identified as full-length products by immunoblotting. Surface accessibility of the chimeric receptors was demonstrated by a colorimetric assay and by immunofluorescence staining with a CTB-reactive rabbit antiserum. Pentamerization was investigated by using a monoclonal antibody described to be specific for pentameric CTB, and the functionality of the receptors was tested in a binding assay with digoxigenin-labelled GM1. It was concluded that functional CTB was present on both types of staphylococci, and for S. carnosus, the reactivity to the pentamer-specific monoclonal antibody and in the GM1 binding assay was indeed significant. The implications of the results for the design of live bacterial vaccine delivery systems intended for administration by the mucosal route are discussed.

SUBMITTER: Liljeqvist S

PROVIDER: S-EPMC168546 | biostudies-other | 1997 Jul

REPOSITORIES: biostudies-other

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Surface display of the cholera toxin B subunit on Staphylococcus xylosus and Staphylococcus carnosus.

Liljeqvist S S Samuelson P P Hansson M M Nguyen T N TN Binz H H Ståhl S S

Applied and environmental microbiology 19970701 7

The heterologous surface expression of the cholera toxin B subunit (CTB) from Vibro cholerae in two staphylococcal species, Staphylococcus xylosus and Staphylococcus carnosus, has been investigated. The gene encoding native CTB (103 amino acids) was introduced into gene constructs encoding chimeric receptors designed to be translocated and anchored on the outer cell surface of the staphylococci. Since functionality of CTB is correlated with its ability to form pentamers and the capacity of the p ...[more]

PMID: 9212399

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Project description: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 ?-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.

Dataset Information

Surface display of the cholera toxin B subunit on Staphylococcus xylosus and Staphylococcus carnosus.

Publications

Surface display of the cholera toxin B subunit on Staphylococcus xylosus and Staphylococcus carnosus.

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