Project description:Bifidobacteria have been described as a key component of the human gut microbiota, and recently significant efforts have been made to investigate their genome contents and assess the genetic variability at inter- and intra-species level. In the current work we investigated genome diversity among representatives of bifidobacterial species, i.e., Bifidobacterium adolescentis. These analyses were performed with comparative genomic hybridization (CGH) experiments and they revealed the existence of a strictly conserved set of 685 gene families. Furthermore, CGH analyses showed that genetic regions of diversity included mobile elements and putative genomic life-style adaptation islands, such as loci that encode pili and capsular polysaccharides, and genes involved in carbohydrate metabolism. CGH analysis was performed with microarrays that were based on the genome sequences of B. adolescentis ATCC15703 (NC_008618) . A total of 39,249 probes of 35 bp in length were designed using OligoArray 2.1 software. Oligos were synthesized in triplicate on a 2 × 40-k CombiMatrix array (CombiMatrix, Mulkiteo, USA). Replicates were distributed on the chip at random, non-adjacent positions. A set of 74 negative control probes designed on phage and plant sequences was also included on the chip. Seventeen micrograms of purified genomic DNA was labeled with Cy5-ULS using the Kreatech ULS array CGH Labeling kit (Kreatech Diagnostics) according to the supplier’s instructions. Hybridization of labeled test DNA to these microarrays was performed according to CombiMatrix protocols.
Project description:In order to understand gene expression profile of Bifidobacterium adolescentis ATCC 15703, it was grown in minimal media upto late log phase in the presence of β-mannooligosaccharide from copra till OD A600 = 0.800
Project description:Bifidobacteria have been described as a key component of the human gut microbiota, and recently significant efforts have been made to investigate their genome contents and assess the genetic variability at inter- and intra-species level. In the current work we investigated genome diversity among representatives of bifidobacterial species, i.e., Bifidobacterium adolescentis. These analyses were performed with comparative genomic hybridization (CGH) experiments and they revealed the existence of a strictly conserved set of 685 gene families. Furthermore, CGH analyses showed that genetic regions of diversity included mobile elements and putative genomic life-style adaptation islands, such as loci that encode pili and capsular polysaccharides, and genes involved in carbohydrate metabolism. CGH analysis was performed with microarrays that were based on the genome sequences of B. adolescentis ATCC15703 (NC_008618) . A total of 39,249 probes of 35 bp in length were designed using OligoArray 2.1 software. Oligos were synthesized in triplicate on a 2 M-CM-^W 40-k CombiMatrix array (CombiMatrix, Mulkiteo, USA). Replicates were distributed on the chip at random, non-adjacent positions. A set of 74 negative control probes designed on phage and plant sequences was also included on the chip. Seventeen micrograms of purified genomic DNA was labeled with Cy5-ULS using the Kreatech ULS array CGH Labeling kit (Kreatech Diagnostics) according to the supplierM-bM-^@M-^Ys instructions. Hybridization of labeled test DNA to these microarrays was performed according to CombiMatrix protocols. We analysed seven strains belong to B. adolescentis species. Replicates were distributed on the chip at random, non-adjacent positions.
Project description:Emerging evidence highlights the critical role of the gut microbiota in maintaining intestinal homeostasis and modulating host immune responses. However, the mechanisms remain incompletely understood. In this study, we screened Lactobacillus and Bifidobacterium strains isolated from healthy individuals to identify symbionts capable of suppressing gut inflammation. Among them, Bifidobacterium adolescentis (Bifi-94) induced IL-10 production in mononuclear cells in vitro. Oral administration of Bifi-94 to mice treated with dextran sulfate sodium attenuated weight loss and reduced colonic inflammation scores. In wild-type C57BL/6 mice, Bifi-94 increased IL-10 levels in colonic tissue homogenates without altering the frequency of regulatory T cells. Instead, CD19+CD11b+ regulatory B (Breg) cells emerged as the primary source of IL-10, with their numbers significantly increasing in the peritoneal cavity (PEC) after treatment. IL-10 secretion by PEC cells was robustly activated by live, heat-killed, and formalin-fixed Bifi-94. Peptidoglycan (PGN) purified from Bifi-94 selectively stimulated IL-10 production in CD19+CD11b+ Breg cells and upregulated key PGN synthesis enzymes, including MurE, MurD, Alr, and UppP. Mechanistically, Bifi-94-derived PGN promoted Toll-like receptor 2 (TLR2)-dependent activation of ERK and p38 MAPK signaling in Breg cells. Notably, PGN similarly enhanced IL-10 production in CD19+ B cells from human colonic tissue. These findings demonstrate that Bifi-94-derived PGN promotes IL-10 production in Breg cells via TLR2-mediated signaling, thereby contributing to the attenuation of gut inflammation.