Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection

Abstract

Bifidobacteria comprise a significant proportion of the human gut microbiota. Several bifidobacterial strains are currently used as therapeutic interventions, claiming various health benefits by acting as probiotics. However, the precise mechanisms by which they maintain habitation within their host and consequently provide these benefits are not fully understood. Here we show that Bifidobacterium breve UCC2003 produces a cell surface-associated exopolysaccharide (EPS), the biosynthesis of which is directed by either half of a bidirectional gene cluster, thus leading to production of one of two possible EPSs. Alternate transcription of the two opposing halves of this cluster appears to be the result of promoter reorientation. Surface EPS provided stress tolerance and promoted in vivo persistence, but not initial colonization. Marked differences were observed in host immune response: strains producing surface EPS (EPS(+)) failed to elicit a strong immune response compared with EPS-deficient variants. Specifically, EPS production was shown to be linked to the evasion of adaptive B-cell responses. Furthermore, presence of EPS(+) B. breve reduced colonization levels of the gut pathogen Citrobacter rodentium. Our data thus assigns a pivotal and beneficial role for EPS in modulating various aspects of bifidobacterial-host interaction, including the ability of commensal bacteria to remain immunologically silent and in turn provide pathogen protection. This finding enforces the probiotic concept and provides mechanistic insights into health-promoting benefits for both animal and human hosts.

Fig. 1.

Identifying and characterizing the EPS locus in B. breve UCC2003. (A) Schematic diagram of the B. breve UCC2003 EPS locus. For identification of promoters in the EPS: A “+” sign denotes a statistically significant (P < 0.02 by t test) difference in GUS activity between the generated promoter fusion constructs and the pNZ272 control in B. breve UCC2003 at the 12-h growth point, and a “−”sign denotes no statistically significant (P < 0.02 by t test) difference between the promoter fragment and the pNZ272 control in B. breve UCC2003 (n = 3). All cultures had similar growth rates. eps1 and eps2 refer to the two adjacent transcriptional units involved in EPS biosynthesis. represents a number of genes between Bbr_0451 and Bbr_0462 or between Bbr_463 and Bbr_0474. Transcriptional start sites, −10 and −35 sites of promoters are indicated; the color coding of the genes relates to their predicted function as indicated in the inset. (B) qRT-PCR analysis of the Bbr_0441 and Bbr_0442 transcriptional units. (I) Transcription of Bbr_0441 in strains UCC2003 and UCC2003-EPS Inv; (II) transcription of Bbr_0442 in UCC2003 and UCC2003-EPS Inv. (C) OD measurements (OD600_nm) of UCC2003 and its derivatives over a 5.5-h time period grown in batch culture without agitation; the observed drop in OD values for the EPS⁻ derivatives is because of cell sedimentation. (D) Transmission electron microscopy images of B. breve UCC2003 (I) and isogenic derivatives UCC2003-EPSInv (II), UCC2003-EPSdel (III), UCC2003::Bbr_0430 (IV). (Scale bars, 100 nm.) (E) High performance anion-exchange chromatography with pulsed amperometric detection profiles of acid-hydrolyzed surface EPS isolated from UCC2003 and isogenic derivatives (see D for strain coding).

Fig. 2.

B. breve surface EPS protects against acid and bile, facilitates in vivo persistence, and modulates cytokine expression from stimulated splenocytes. (A) Growth curve of UCC2003 (EPS⁺), UCC2003-EPSdel (EPS⁻), and UCC2003::Bbr_0430 (EPS⁻) in de Man-Rogosa-Sharpe (MRS) pH5.0 or in MRS supplemented with 0.3% bile over 24 h at 37 °C. Data represent mean ± SD (B) BALB/c mice were treated orally with ∼1 × 10⁹ UCC2003, UCC2003-EPSdel, or UCC2003::Bbr_0430 on 3 consecutive days and bacterial numbers (CFU) in feces determined (data represent log₁₀ CFU/g feces ± SD). (C) Organs were also removed on day 31 to determine CFU; columns show log₁₀ CFU/organ (± SD). (D) Spleens were harvested from naïve BALB/c mice and stimulated with wild-type (EPS⁺), deletion, and insertion mutants (EPS⁻) B. breve strains at 1:1 ratio for ∼20 h. Cells were also stimulated with ConA (dotted line). Columns represent the mean ± SD stimulation indices of splenocytes from 10 mice from two independent experiments. Significance was determined relative to mice treated with UCC2003 (EPS⁺) at the same time point using the Kruskal–Wallis test followed by Dunn's multiple comparison test; *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

B. breve surface EPS modulates recruitment and cytokine profile of immune cell populations in mice. (A) Cells were isolated from spleens of BALB/c mice 31 d after initial treatment, stained with fluorochrome-labeled mAb, and analyzed by flow cytometry. Columns represent the mean percentage ± SD of at least eight mice from two independent experiments. (B) Isolated cells were stimulated for 6 h with BD Leukocyte Activation Mixture plus GolgiPlug in vitro, stained with surface mAb to determine CD3⁺ and CD19⁺ populations, and then permeabilized and stained with anticytokine fluorochrome-labeled mAb. Data represent percent of cytokine-positive cells out of total specific cell population ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 between naïve and B. breve-treated mice; ^†P < 0.05; ^††P < 0.01, and ^†††P < 0.001 between EPS⁺ and EPS⁻ using one-way ANOVA followed by Bonferroni's multiple comparison test.

Fig. 4.

B. breve EPS influences B-cell responses. (A) Cells were isolated from spleens of B. breve-treated mice on day 31 and data represent total cell number of CD19^low/B220^low/CD138^high plasma cells. Columns represent means ± SD. Significant differences were determined by the one-way ANOVA followed by Bonferroni's multiple comparison test. ***P < 0.001. (B) Antibody titers were measured following treatment of BALB/c mice with UCC2003 (EPS⁺) and UCC2003-EPSdel (EPS⁻). Graphs show titers from individual serum and fecal samples (minus naïve titers) collected on day 31. Bars represent means from at least eight individual mice from two independent experiments. (C) Visualization of slide agglutination performed with the indicated antiserum and cultures of B. breve. Slides were counterstained with India ink and agglutination visualized by light microscopy (Original magnification, 20×). (Scale bars, 200 μm.) (D) B-cell–deficient mice and C57BL/6 controls were treated orally with ∼1 × 10⁹ EPS⁺ or EPS⁻ B. breve strains on 3 consecutive days and fecal CFU determined. (E) On day 14, organ counts were also determined. Data represent log₁₀ CFU (± SD) from eight individual mice. Significant differences determined by the Kruskal–Wallis test followed by Dunn's multiple comparison test. *P < 0.05; ***P < 0.001.

Fig. 5.

Presence of surface EPS-producing B. breve impacts on the colonization and persistence of the gut pathogen C. rodentium. BALB/c mice were treated with ∼1 × 10⁹ EPS⁺ or EPS⁻ B. breve strains on 3 consecutive days, before oral infection with ∼5 × 10⁸ C. rodentium. (A) Fecal CFU were determined every 3–4 d and data represent log₁₀ CFU/g feces (± SD). (B) Organs were also removed on day 14 to determine CFU. Significance was determined relative to mice infected with C. rodentium alone or mice treated with EPS⁻ B. breve and infected with C. rodentium at the same time point using the Kruskal–Wallis test followed by Dunn's multiple comparison test. n = 8 mice per group. *P < 0.05; ^†P < 0.05; ^††P < 0.001. (C) Images were acquired with the IVIS50 camera and are displayed as pseudocolor images, with variations in color representing light intensity at a given location. Red represents the most intense light emission, and blue corresponds to the weakest signal. For each time point three mice were imaged (whole body) and (D) the open colon and cecum of one representative mouse is shown.