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.

FIG. 1.

PCR analysis of the slpA-c-myc gene replacement in L. brevis GRL1046. (A) Total chromosomal DNA of the GRL1046 (lanes 2, 4, 6, 7, and 10) and GRL1 wild-type (lanes 1, 3, 5, and 9) strains and pORI280 plasmid DNA (lane 8) were used as templates in PCR with the primer pairs p14 and p21 (lanes 1 and 2), p22 and p4 (lanes 3 and 4), p22 and p23 (lanes 5 and 6), p24 and p25 (lanes 7 and 8) and p26 and p13 (lanes 9 and 10). (B) Total DNA of GRL1 (lane 1) and GRL1046 (lane 2) amplified with primers p27 and p28. PCR products were separated by electrophoresis on a 0.8% (A) or 2% (B) agarose gel and stained with ethidium bromide. Values of the molecular size markers (SmartLadder; Eurogentec) are indicated on the left (A) or on the right (B).

FIG. 2.

Southern blot analysis of L. brevis strains GRL1046 and GRL1. Total DNA extracted from strains GRL1046 (lane1) and GRL1 (lane 2) was digested with EcoRI and hybridized with a 1,406-bp internal slpA fragment probe amplified with the primer pair p19 and p20. Values (in kilobase pairs) of the molecular size markers (SmartLadder) are indicated on the left.

FIG. 3.

SDS-PAGE analysis of L. brevis GRL1 and GRL1046 cells. Equal amounts of GRL1 (lane 1) and GRL1046 (lane 2) cells from an OD₆₀₀ of 0.8 were boiled in Laemmli sample buffer before being subjected to protein gel analysis. Values of the molecular mass marker proteins are indicated on the left.

FIG. 4.

Whole-cell ELISA for detection of surface-exposed c-Myc epitope in L. brevis GRL1046. Anti-Myc antibody was allowed to bind to lactobacillar cells, after the addition of a horseradish peroxidase conjugate, different lactobacillar cell densities were incubated with a chromogenic substrate. Symbols: •, GRL1046; ▴, GRL1 wild-type strain.

FIG. 5.

Immunofluorescence microscopy of L. brevis GRL1046 (A) and wild-type L. brevis GRL1 (B). Bacteria harvested from overnight cultures were treated with anti- Myc antibodies (diluted 1:10 in PBS) and FITC-conjugated secondary antibody. Both pictures were taken after 5-s exposures. Magnification, ×4,000.

FIG. 6.

Transmission electron microscopy of L. brevis GRL1046 and GRL1 S-layers. Negative staining was performed with cells of GRL1046 (A) and GRL1 (B) derived from aged colonies and with S-layer protein preparations from LiCl isolations of GRL1046 (C) and GRL1 (D). Bars, 0.1 μm.