A Novel Rhamnose-Rich Hetero-exopolysaccharide Isolated from Lactobacillus paracasei DG Activates THP-1 Human Monocytic Cells

Abstract

Lactobacillus paracasei DG is a bacterial strain with recognized probiotic properties and is used in commercial probiotic products. However, the mechanisms underlying its probiotic properties are mainly unknown. In this study, we tested the hypothesis that the ability of strain DG to interact with the host is at least partly associated with its ability to synthesize a surface-associated exopolysaccharide (EPS). Comparative genomics revealed the presence of putative EPS gene clusters in the DG genome; accordingly, EPS was isolated from the surface of the bacterium. A sample of the pure EPS from strain DG (DG-EPS), upon nuclear magnetic resonance (NMR) and chemical analyses, was shown to be a novel branched hetero-EPS with a repeat unit composed of l-rhamnose, d-galactose, and N-acetyl-d-galactosamine in a ratio of 4:1:1. Subsequently, we demonstrated that DG-EPS displays immunostimulating properties by enhancing the gene expression of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), and particularly that of the chemokines IL-8 and CCL20, in the human monocytic cell line THP-1. In contrast, the expression of the cyclooxygenase enzyme COX-2 was not affected. In conclusion, DG-EPS is a bacterial macromolecule with the ability to boost the immune system either as a secreted molecule released from the bacterium or as a capsular envelope on the bacterial cell wall. This study provides additional information about the mechanisms supporting the cross talk between L. paracasei DG and the host.

Importance: The consumption of food products and supplements called probiotics (i.e., containing live microbial cells) to potentially prevent or treat specific diseases is constantly gaining popularity. The lack of knowledge on the precise mechanisms supporting their potential health-promoting properties, however, greatly limits a more appropriate use of each single probiotic strain. In this context, we studied a well-known probiotic, Lactobacillus paracasei DG, in order to identify the constitutive molecules that can explain the documented health-promoting properties of this bacterium. We found a novel polysaccharide molecule, named DG-EPS, that is secreted by and covers the bacterium. We demonstrated that this molecule, which has a chemical structure never identified before, has immunostimulatory properties and therefore may contribute to the ability of the probiotic L. paracasei DG to interact with the immune system.

Keywords: Capsular EPS; Enterolactis; Lactobacillus casei DG; Lactobacillus paracasei; MAMPs; THP-1; immunostimulation; probiotic.

FIG 1

Comparative genomic analysis of Lactobacillus paracasei DG and other L. paracasei strains with complete genome sequences. (A) Circular genome atlas of L. paracasei DG (reference genome) and six other publicly available L. paracasei genomes. Highlighted in the atlas are the two putative exopolysaccharide (EPS) regions of strain DG. (B) In silico-predicted functional organization of the EPS-b region of L. paracasei DG and BLASTN search results for the EPS-b region. A map of the putative EPS gene cluster is shown above a graphic representation of the BLASTN output for the genomes of 24 L. casei group strains. Arrows in the map represent ORFs. Open arrows, ORFs outside the putative EPS operon; shaded arrows, ORFs for which homologous sequences were found in GenBank; filled arrows, ORFs that do not share significant homology with other sequences in GenBank. D.R., direct repeat sequence.

FIG 2

NMR analysis of the EPS isolated from Lactobacillus paracasei DG. (A) Selected regions of the ¹H-NMR spectrum of DG-EPS recorded at 70°C in D₂O using acetone as an internal standard. Anomeric (H-1) resonances are labeled A to F in order of decreasing chemical shift. (B) Selected regions of overlaid COSY (black contours) and TOCSY (gray contours) spectra for DG-EPS recorded at 70°C. Top, ¹³C-methyl region; bottom, ¹³C region for anomeric and ring protons. The letters A to F designate individual sugars, and numbers 1 to 6 indicate the C/H ring position. (C) Selected regions of the HSQC spectrum of DG-EPS. (Top) Locations of the individual rings and H6 protons and carbons; (bottom) locations of the anomeric protons and carbons. The spectrum was recorded in D₂O at 70°C. (D) Anomeric region of a ROESY spectrum recorded for DG-EPS. Inter- and intraresidue NOEs from the anomeric hydrogens to ring protons are individually labeled.

FIG 3

Repeating unit structure of DG-EPS, the heteropolysaccharide isolated from Lactobacillus paracasei DG.

FIG 4

Gene expression analysis by qRT-PCR in THP-1 human macrophages after 4 h of stimulation with the purified DG-EPS molecule (0.1, 1, and 10 μg ml⁻¹), with or without the addition of LPS (1 μg ml⁻¹). Expression levels of TNF-α, IL-6, IL-8, CCL20, and COX-2 are shown as the fold change in induction (FOI) relative to expression by the control (unstimulated macrophages), which was set at a value of 1. Data are means of results from three independent experiments ± standard deviations. Asterisks indicate statistically significant differences (according to a two-tailed unpaired Student t test) from results for unstimulated (left y axis) or LPS-stimulated (right y axis) THP-1 cells. *, P < 0.05; **, P < 0.01.