SURFACE DISPLAY OF THE CHOLERA-TOXIN-B SUBUNIT ON STAPHYLOCOCCUS-XYLOSUS AND STAPHYLOCOCCUS-CARNOSUS

The heterologous surface expression of the cholera toxin B subunit (CT B) from Vibrio cholerae in two staphylococcal species, Staphylococcus xylosus and Staphylococcus carnosus, has been investigated. The gene e ncoding native CTB (103 amino acids) was introduced into gene construc ts encoding chimeric receptors designed to be translocated and anchore d on the outer cell surface of the staphylococci. Since functionality of CTB is correlated with its ability to form pentamers and the capaci ty of the pentameric CTB to bind the GM1 ganglioside, both the surface accessibility and the functionality of the surface-displayed CTB rece ptors were evaluated. It could be concluded that the chimeric receptor s were targeted to the cell wall of the staphylococci, since they coul d be released by lysostaphin treatment and, after subsequent affinity purification, identified as full-length products by immunoblotting. Su rface accessibility of the chimeric receptors was demonstrated by a co lorimetric assay and by immunofluorescence staining with a CTB-reactiv e rabbit antiserum. Pentamerization was investigated by using a monocl onal antibody described to be specific for pentameric CTB, and the fun ctionality of the receptors was tested in a binding assay with digoxig enin-labelled GM1. It was concluded that functional CTB was present on both types of staphylococci, and for S. carnosus, the reactivity to t he 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 administratio n by the mucosal route are discussed.