Expression of recombinant proteins on the surface of the coagulase-negative bacterium Staphylococcus xylosus

Abstract

An expression system to allow targeting of heterologous proteins to the cell surface of Staphylococcus xylosus, a coagulase-negative gram-positive bacterium, is described. The expression of recombinant gene fragments, fused between gene fragments encoding the signal peptide and the cell surface-binding regions of staphylococcal protein A, targets the resulting fusion proteins to the outer bacterial cell surface via the membrane-anchoring region and the highly charged cell wall-spanning region of staphylococcal protein A. The expression system was used to secrete fusion proteins containing sequences from a malaria blood-stage antigen and a streptococcal albumin-binding receptor to the cell surface of S. xylosus. Analysis of the recombinant cells by immunogold staining and immunofluorescence revealed that both the receptor and the malaria peptide were properly processed and exposed on the surface of the host cells. However, only approximately 40 to 50% of the recombinant cells were strongly stained with antiserum reactive with the albumin-binding receptor, while approximately 10 to 15% of the cells were stained with antiserum reactive with the malaria peptide. The incomplete staining of some of the cells suggests steric effects that make the recombinant fusion proteins inaccessible to the reactive antibodies because ofvariable cell wall structures. However, the results demonstrate for the first time that recombinant techniques can be used to express heterologous receptors and immunogens on the surface of gram-positive cells.

Surface display on staphylococci: a comparative study

“…xylosus and S. carnosus cells, wild type and those transformed with plasmid pSEGs~bABPXM or pSPPG~bABPXM, respectively, were subjected to an immunogold-electron microscopy analysis as described earlier [13], using an anti-ABP rabbit serum [10]. Briefly, the bacteria were incubated with rabbit anti-ABP or preimmune serum and subsequently with 10 nm colloidal gold conjugated to protein A (Aurion, Gent, Belgium).…”

“…A rabbit antiserum reactive with the serum ABP [13,36] present within the chimeric receptors was generated as described by Hansson and coworkers [10]. A G protein-reactive rabbit antiserum was generated as described by Nguyen and coworkers [12].…”

“…Two of the most promising Gram-positive bacteria include the staphylococcal host-vector systems which have recently been developed [10,12], one designed for targeting of heterologous proteins to the surface of S. xylosus, and the second suitable for surface display of hybrid receptors on S. carnosus [13]. Both these bacteria are described as non-pathogenic [11,27], and are indeed being used in starter cultures for meat-fermentation applications [28,29].…”

“…[3][4][5][6][7][8], and more recently certain Gram-positive bacteria have been investigated for surface display purposes. The Mycobacterium bovis strain, bacillus Calmette-Guerin (BCG) [9], as well as staphylococci [10][11][12][13][14] and streptococci [15 18] have been investigated. Most thoroughly evaluated are the staphylococcal systems which have been utilized for surface display of various heterologous proteins [10][11][12][13][14].…”

“…The Mycobacterium bovis strain, bacillus Calmette-Guerin (BCG) [9], as well as staphylococci [10][11][12][13][14] and streptococci [15 18] have been investigated. Most thoroughly evaluated are the staphylococcal systems which have been utilized for surface display of various heterologous proteins [10][11][12][13][14]. The different surface display systems have been extensively used to express heterologous antigenic determinants on the bacterial cells for the purpose of developing live bacterial vaccine vehicles, and the cell-surface presentation has for certain systems been considered advantageous to induce an antibody response to the exposed antigens using live recombinant bacteria for immunization [1,6,12,19].…”