The surface (S)-layer protein of Lactobacillus brevis was isolated, purified, and characterized. The S-layer protein is the major protein of the cell, with an apparent molecular mass of 46 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunogold electron microscopy with polyclonal antiserum against the isolated 46-kDa protein was used to confirm the surface location of this protein. N-terminal amino acid sequences of the intact 46-kDa protein and its tryptic peptides were determined. The gene of the S-layer protein was amplified from the genome of L. brevis by polymerase chain reaction with oligonucleotides, synthesized according to the N-terminal amino acid sequences, as primers. The polymerase chain reaction fragments containing the entire S-layer gene and its regulatory regions were sequenced. Nucleic acid sequence analysis revealed one open reading frame with a capacity to encode a protein of 48,159 Da. From the regulatory region of the gene, two subsequent promoters and a ribosome binding site, showing typical features of prokaryotic consensus sequences, were found. The coding region contained a characteristic gram-positive-type signal peptide of 30 amino acids. Removal of the signal peptide results in a polypeptide of 435 amino acids, which is in excellent agreement with the size of the S-layer protein determined by SDS-PAGE. The size and the 5' end analyses of the S-layer transcripts confirmed the monocistronic nature of the S-layer operon and the functionality of the two promoters found.

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Primarily arising from their well understood beneficial health effects, many lactobacilli strains are considered good candidates for use as probiotics in humans and animals. Lactobacillar probiosis can itself be best typified by the Lactobacillus rhamnosus GG strain, which, with its well-documented clinical benefits, has emerged as one of the most widely used probiotics in the food and health-supplement industries. Even so, many facets of its molecular mechanisms and limitations as a beneficial commensal bacterium still remain to be thoroughly explored and dissected. Because L. rhamnosus GG is one of only a few such strains exhibiting surface piliation (called SpaCBA), we sought to examine whether this particular type of cell-surface appendage has a discernible immunomodulating capacity and is able to trigger targeted responses in human immune-related cells. Thus, presented herein for this study, we recombinantly engineered Lactococcus lactis to produce native (and pilin-deleted) SpaCBA pili that were assembled in a structurally authentic form and anchored to the cell surface, and which had retained mucus-binding functionality. By using these recombinant lactococcal constructs, we were able to demonstrate that the SpaCBA pilus can be a contributory factor in the activation of Toll-like receptor 2-dependent signaling in HEK cells as well as in the modulation of pro- and anti-inflammatory cytokine (TNF-α, IL-6, IL-10, and IL-12) production in human monocyte-derived dendritic cells. From these data, we suggest that the recombinant-expressed and surface-anchored SpaCBA pilus, given its projected functioning in the gut environment, might be viewed as a new microbe-associated molecular pattern (MAMP)-like modulator of innate immunity. Accordingly, our study has brought some new insight to the molecular immunogenicity of the SpaCBA pilus, thus opening the way to a better understanding of its possible role in the multifaceted nature of L. rhamnosus GG probiosis within the human gut.