Contribution of surface β-glucan polysaccharide to physicochemical and immunomodulatory properties of Propionibacterium freudenreichii

Abstract

Propionibacterium freudenreichii is a bacterial species found in Swiss-type cheeses and is also considered for its health properties. The main claimed effect is the bifidogenic property. Some strains were shown recently to display other interesting probiotic potentialities such as anti-inflammatory properties. About 30% of strains were shown to produce a surface exopolysaccharide (EPS) composed of (1→3,1→2)-β-D-glucan due to a single gene named gtfF. We hypothesized that functional properties of P. freudenreichii strains, including their anti-inflammatory properties, could be linked to the presence of β-glucan. To evaluate this hypothesis, gtfF genes of three β-glucan-producing strains were disrupted. These knockout (KO) mutants were complemented with a plasmid harboring gtfF (KO-C mutants). The absence of β-glucan in KO mutants was verified by immunological detection and transmission electron microscopy. We observed by atomic force microscopy that the absence of β-glucan in the KO mutant dramatically changed the cell's topography. The capacity to adhere to polystyrene surface was increased for the KO mutants compared to wild-type (WT) strains. Anti-inflammatory properties of WT strains and mutants were analyzed by stimulation of human peripheral blood mononuclear cells (PBMCs). A significant increase of the anti-inflammatory interleukin-10 cytokine production by PBMCs was measured in the KO mutants compared to WT strains. For one strain, the role of β-glucan in mice gut persistence was assessed, and no significant difference was observed between the WT strain and its KO mutant. Thus, β-glucan appears to partly hide the anti-inflammatory properties of P. freudenreichii; which is an important result for the selection of probiotic strains.

Fig 1

Modulation of the expression levels of the gtfF gene, which is responsible for the production of the β-glucan surface polysaccharide in P. freudenreichii strains CB1, LSP110, and LSP103 and their corresponding mutants. Bars: ■, WT strains, producing surface β-glucan; □, KO mutants where gtfF has been inactivated; ▩, KO-C mutants (KO strains reverted to WT). gtfF gene expression is expressed as the induction rate compared to the expression measured in the WT strain. The data were normalized with the software geNorm using groL1 as normalization gene. These results are the average of three independent experiments. Error bars indicate the standard deviation.

Fig 2

Inactivation of the gtfF gene led to the disappearance of the hairy surface. (A and B) Immunoagglutination test performed with a β-glucan-specific antiserum (A) and TEM observation (B) of P. freudenreichii WT strain CB1 (row 1), its abolished surface polysaccharide mutant CB1-KO (row 2), and the CB1-KO-C mutant (row 3), which was reverted to the original phenotype.

Fig 3

P. freudenreichii LSP103 and its surface polysaccharide-abolished mutant. AFM two-dimensional height (A), three-dimensional height (C), and amplitude (B) images were recorded with cells recorded at stationary growth phase and resuspended in HEPES buffered solution. (A2, B2, and C2) LSP103 strain, which produces a β-glucan surface polysaccharide; (A1, B1, and C1) its derivative strain LSP103-KO, wherein the gene responsible for the production of the surface polysaccharide has been inactivated.

Fig 4

Inactivation of the gtfF gene led to a higher adhesion of P. freudenreichii to abiotic surface. The adherence on polystyrene surface of strains CB1, LSP110, LSP103, and their mutants was evaluated. Bars: ■, WT strains, producing surface β-glucan; □, KO mutants, inactivated in the gtfF gene, responsible for the biosynthesis of the β-glucan polysaccharide; ▩, KO-C mutants (KO strains reverted to the WT). The values are the means for three independent experiments; the significance of the results was checked by using the Mann-Whitney test ( , P ≤ 0.01; , P ≤ 0.001).

Fig 5

Anti-inflammatory cytokine responses of human PBMCs for P. freudenreichii strains producing or not producing a surface β-glucan polysaccharide. Bars: black, WT strains that produce the surface β-glucan polysaccharide; white, KO mutant strains wherein gtfF, the gene responsible for synthesis of the β-glucan polysaccharide has been inactivated; dark gray, KO-C strains, KO strains reverted to WT, producing β-glucan; light gray, reference strains (Lactococcus lactis MG1363, Lactobacillus acidophilus NCFM, Lactobacillus salivarius LS33, and Bifidobacterium longum BB536). The data are expressed in pg/ml as means ± the standard error of the mean, with n = 3 distinct healthy blood donors. , P ≤ 0.05; , P ≤ 0.01; , P ≤ 0.001.

Fig 6

In vivo persistence of P. freudenreichii strains that produce or do not produce β-glucan surface polysaccharide. Mice were fed 3 days consecutively (G1, G2, and G3) either with the β-glucan-producing WT strain LSP103 (■), its non-β-glucan-producing mutant LSP103-KO (□), or LSP103-KO-C (LSP103-KO mutant reverted to WT phenotype) (▩). Viable counts of P. freudenreichii strains were measured on selective medium in feces 6 h and 24 h after the gavages and 48 and 72 h after the last gavage. ND, not detectable (<1E+02 CFU/g of stool).