Surface Display of Foreign Epitopes on the Lactobacillus brevis S-Layer

Abstract

So far, the inability to establish viable Lactobacillus surface layer (S-layer) null mutants has hampered the biotechnological applications of Lactobacillus S-layers. In this study, we demonstrate the utilization of Lactobacillus brevis S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system, L. brevis strains producing chimeric S-layers were obtained after testing of four insertion sites in the slpA gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-myc proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for L. brevis and used for the replacement of the wild-type slpA gene with the slpA-c-myc construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a Lactobacillus S-layer while still maintaining the S-layer lattice structure.Many organisms from the domains Bacteria and Archaea possess a surface layer (S-layer) as the outermost structure of the cell envelope. S-layers are composed of regularly arranged proteinaceous subunits of a single protein or glycoprotein species with molecular masses ranging from 40 to 170 kDa (43, 48). S-layer proteins represent 10 to 15% of the total protein of the bacterial cell, and S-layer lattices cover the cell surface during all stages of growth, which indicates that efficient gene expression, S-layer protein synthesis, and secretion take place (4). A high content of hydrophobic and acidic amino acids and a low theoretical isoelectric point (pI) are typical features of S-layer proteins (48). In contrast, very high pI values have been described for the S-layer proteins from various lactobacilli (4, 54) and Methanothermus fervidus (6). In general, S-layers have been considered to function as cell shape determinants, protective coats, promoters for cell adhesion and surface recognition, and molecular and ion traps; however, no general function found in all S-layers has been recognized (47, 48).S-layers have been found from many species of the genus Lactobacillus (31, 56). However, the S-layer protein genes have been cloned and sequenced only from Lactobacillus brevis ATCC 8287 (54) and from the closely related species L. acidophilus (3), L. helveticus (8), and L. crispatus (46). Th...

Surface Location of Individual Residues of SlpA Provides Insight into the Lactobacillus brevis S-Layer

“…Of these sites, only K251/A252 and D365/T366 yielded an antibody response (2). In contrast, our results suggest that A316C and T366C are among the most accessible residues studied.…”

“…The presence of two or more S-layer protein genes in the same strain is common in lactobacilli (5,6,11,28,35); however, only one S-layer protein gene, slpA, has so far been described to be present in the genome of L. brevis ATCC 8287. SlpA is a 435-amino-acid, 46-kDa S-layer protein that assembles into a lattice of oblique symmetry on the bacterial surface (2,36). L. brevis ATCC 8287 has GRAS (generally recognized as safe) status and has been shown to possess probiotic properties (21), which make SlpA a very attractive subject, e.g., in the development of live oral vaccines.…”

Receptor Binding Domain ofEscherichia coliF18 Fimbrial Adhesin FedF Can Be both Efficiently Secreted and Surface Displayed in a Functional Form inLactococcus lactis

“…The signal peptide of the L. lactis Usp45 protein (28) has been reported to be one of the most effective secretion signals among the known L. lactis signal peptides (34). We have demonstrated earlier that the L. brevis SlpA signal peptide (3,45) very efficiently directs the secretion of a reporter in L. lactis (40). Here, both signal peptides allowed secretion of all of the FedF-PrtP fusions tested but the quantity of fusion proteins found in the culture medium was throughout substantially higher in expression systems possessing the L. brevis S-layer protein signal peptide, confirming the superiority of SP SlpA over SP Usp , at least with these constructs.…”

“…The resulting construct (pKTH5138) was used to transform L. lactis NZ9000 cells, followed by chromosomal integration via a single crossover. The second crossover was obtained by cultivating the integrants essentially as described earlier (3). As a result, strain NZ9000⌬htrA, with a chromosomal deletion in the htrA promoter and the 5Ј-end region of htrA, was formed.…”

Gut Adhesive Bacillus subtilis Spores as a Platform for Mucosal Delivery of Antigens

“…For that purpose, the N-terminal region of CotB, a surface-exposed spore protein (6), was genetically fused with two different adhesins with receptors at gut epithelial cells: the S-layer protein (SlpA) from L. brevis, which binds to fibronectin, and the invasin protein (InvA) from Y. pseudotuberculosis, which binds to integrin. These adhesins have been demonstrated to be functional when expressed in heterologous bacterial hosts (26,27,37,38,44). The integrative vectors, named pLDV703 (encoding the cotB::slpA fusion) and pLDV704 (encoding the cotB::invA fusion), were introduced by transformation into B. subtilis strain 1012, and stable recombinants were selected for the integration of the recombinant gene cassettes at the thrC locus, as previously described (28) (Fig.…”