Crystalline bacterial cell surface layers (S-layers) represent the outermost cell envelope component of many bacteria and archaea (35,37,38). S-layers are composed of identical protein or glycoprotein subunits, and they completely cover the cell surface during all stages of bacterial growth and division. The S-layer subunits assemble into either oblique, square, or hexagonal lattices. In the case of Bacillaceae, the N-terminal part is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer (SCWP) to the rigid cell wall layer (2,5,21,25,34,35).S-layers are unique biomaterials with properties most relevant for applications in molecular nanotechnology, nanobiotechnology, and biomimetics (38). Many S-layer proteins recrystallize into regularly structured monolayers on solid supports, such as silicon wafers, gold chips, and silanized glass or plastic materials, as well as on Langmuir lipid films, on liposomes (20,23), and at the air-water interface. Pores passing through S-layer lattices are of identical size and morphology, and functional groups show a regular distribution and high density. For production of S-layer-based biosensors (38), affinity microparticles (45), and solid-phase immunoassays (3,4,39), functional groups in the S-layer lattice were exploited as covalent binding sites for biologically active macromolecules, such as enzymes, antibodies, or ligands. As alternatives to the existing technology, namely, immobilization by chemical methods, genetic approaches are particularly attractive for incorporation of functional peptide sequences into S-layer proteins, which must be done at positions that do not interfere with their self-assembly properties and their interaction with SCWP.

No abstract

Counterregulating the disease-eliciting Th2-like immune response of allergen-specific Th lymphocytes by fostering an allergen-specific Th1-like response is a promising concept for future immunotherapy of type I allergy. The use of recombinant allergens combined with more functional adjuvants has been proposed. In this respect, we present a novel approach. The gene sequence encoding the major birch pollen allergen, Bet v 1, was fused with the gene encoding the bacterial cell surface (S-layer) protein of Geobacillus stearothermophilus, resulting in the recombinant protein, rSbsC-Bet v 1. rSbsC-Bet v 1 contained all relevant Bet v 1-specific B and T cell epitopes, but was significantly less efficient to release histamine than rBet v 1. In cells of birch pollen-allergic individuals, rSbsC-Bet v 1 induced IFN-γ along with IL-10, but no Th2-like response, as observed after stimulation with Bet v 1. Intracellular cytokine staining revealed that rSbsC-Bet v 1 promoted IFN-γ-producing Th cells. Moreover, rSbsC-Bet v 1 induced IFN-γ synthesis in Bet v 1-specific Th2 cell clones, and importantly, increased IL-10 production in these cells. In conclusion, genetic fusion of an allergen to S-layer proteins combined reduced allergenicity with immunomodulatory capacity. The strategy described in this work may be generally applied to design vaccines for specific immunotherapy of type I allergy with improved efficacy and safety.

The mature crystalline bacterial cell surface (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31-1099 and assembles into an oblique lattice type. As the deletion of up to 179 C-terminal amino acids did not interfere with the self-assembly properties of SbsC, the sequence encoding the major birch pollen allergen (Bet v1) was fused to the sequence encoding the truncated form rSbsC(31-920). The S-layer fusion protein, termed rSbsC/Bet v1, maintained the ability to self-assemble into flat sheets and open-ended cylinders. The presence and the functionality of the fused Bet v1 sequence was proved by blot experiments using BIP1, a monoclonal antibody against Bet v1 and Bet v1-specific IgE-containing serum samples from birch pollen allergic patients. The location and accessibility of the allergen moiety on the outer surface of the S-layer lattice were demonstrated by immunogold labeling of the rSbsC/Bet v1 monolayer, which was obtained by oriented recrystallization of the S-layer fusion protein on native cell wall sacculi. Thereby, the specific interactions between the N-terminal part of SbsC and a distinct type of secondary cell wall polymer were exploited. This is the first S-layer fusion protein described that had retained the specific properties of the S-layer protein moiety in addition to those of the fused functional peptide sequence.

In the present study, the applicability of crystalline bacterial cell-surface layers (Slayers)