Characterization of immunostimulatory CpG-rich sequences from different Bifidobacterium species

Abstract

The beneficial effects of Bifidobacterium are partly due to its immunostimulatory properties. These immunostimulatory properties may be linked to the presence of unmethylated CpG motifs specific to bacterial DNA, which may induce a TH1 response by activating Toll-like receptors (TLR). Using in silico analyses, PCR amplification, and dot blotting, we characterized the CpG content of various bifidobacterial strains and evaluated the immunostimulatory properties and genomic heterogeneity of these motifs in the genus. Our in silico study, based on entire genome sequences from five bifidobacterial strains, showed that Bifidobacterium genomes contain numerous CpG motifs, including 5'-purine-purine-CG-pyrimidine-pyrimidine-3' and 5'-purine-TCG-pyrimidine-pyrimidine-3' motifs, and biologically active sequences previously identified in lactic acid bacteria. We identified four CpG-rich sequences with Bifidobacterium longum NCC2705. Two sequences with a percent G+C of about 68% included 14 and 16 CpG motifs. Two sequences with a percent G+C of about 60% included 16 and 6 CpG motifs. These sequences induce the production of monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor alpha (TNF-alpha) through a pattern of TLR9 stimulation on RAW 264.7 macrophages. No link could be established between their immunostimulatory properties, the number of CpG motifs, and percent G+C. We investigated inter- and intraspecies heterogeneity in 71 strains of various origins. These sequences were highly conserved in the genus. No link was found between the presence of the CpG-rich sequence and the origin of the strains (healthy, allergic, or preterm infants). The high frequency of CpG motifs in the DNA of Bifidobacterium may play an important role in the immunostimulatory properties of commensal or probiotic bifidobacterial strains.

FIG. 1.

Location of putative CpG islands more than 200 bp in length and with a G+C content exceeding 80% within the Bifidobacterium genome. No sequences were found with B. animalis subsp. lactis AD011. Data were obtained with the cpgplot program.

FIG. 2.

Characteristics of CpG-rich sequences of Bifidobacterium longum NCC2705, including position, length, percent G+C, number of types 1 and 2 CpG motifs, and associated coding region. CoA, coenzyme A; Fas, fatty acid synthase.

FIG. 3.

Variability of H2, H3, N2, and N3 sequences from the Bifidobacterium strains for which entire genome sequences have been published. Comparisons with B. longum NCC2705 were performed with MultAlin by Florence Corpet (6). The overall percentage of identity to B. longum NCC2705 is shown in the last column. Percent identity for a specific fragment: ▪, >90%; , > 80%; , < 80%. , insertion into sequence; , deletion from sequence. Bli 15697, B. longum subsp. infantis ATCC 15697; Bal AD011, B. animalis subsp. lactis AD011.

FIG. 4.

(A) Secretion of cytokines and chemokines by RAW 264.7 macrophages after 12 h of stimulation with 10 μg/ml CpG-rich PCR products (H2, H3, N2, and N3), 250 ng/ml DNA from B. longum NCC2705, B. longum subsp. infantis ATCC 15697, and B. adolescentis ATCC 15703, 10 μg/ml CpG-ODN 1826 as a positive control, 100 ng/ml LPS as a positive control, or no stimulation as a negative control (NCT). ▪, TNF-α; □, MCP-1. (B and C) MCP-1 (B) and TNF-α (C) levels after 6 h of stimulation by N2 CpG-rich PCR product (10 μg/ml) with or without ODN 2088 (10 μg/ml), an inhibitor of TLR9 signaling. Cytokines and chemokines were dosed by the mouse inflammation cytometric bead array (CBA) kit (BD Biosciences, France). Data are presented as medians. *, P < 0.05.

FIG. 5.

Interspecies variability of CpG-rich sequences in the genus Bifidobacterium. The presence of each sequence was evaluated by dot blotting (A) or PCR (B). Bars indicate the number of strains in which the sequence is present (▪) or absent (▧). BL, B. longum group; BB; B. breve; BP, B. catenulatum/pseudocatenulatum group; BIF, B. bifidum; BA, B. adolescentis; BD, B. dentium.

FIG. 6.

Intraspecies variability of CpG-rich sequences in the genus Bifidobacterium. The presence of sequences was evaluated by dot blotting (A) or by PCR (B). Bars represent the number of strains containing 4 sequences (▪), 3 sequences (▥), 2 sequences (▨), 1 sequence (░⃞), or no sequence (□).