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Following birth, the human gastrointestinal tract is rapidly colonized by microorganisms that profoundly impact the physiology and health of the host by contributing to host nutrition and natural defense, amongst other activities. In the large intestine, bifidobacteria are especially predominant in infants, where they can comprise up to 90% of the total microbiota, and in adults still account for a significant several percent. In this thesis, we applied post-genomic technologies, including metaproteomics and bifidobacterial community transcript profiling, to deepen our understanding of the activity of commensal bifidobacteria in the human intestine. Proteins were extracted from total infant fecal microbiota that was dominated by bifidobacteria, and two-dimensional gel electrophoresis was used to visualize the metaproteomes. The succession of the proteins was studied in fecal microbiota of two infants during 40 days. This revealed that the number and intensity of protein spots changed in time, but the patterns remained similar and specific for each infant. In-gel digestion of protein-spots and sequencing of the peptides, revealed the presence of a bifidobacterial transaldolase supporting the future application of this approach. Global transcript profiling of the infant fecal bifidobacteria was also performed. Total RNA from the fecal microbiota of infants that were solely breast-fed or formula-fed was hybridized to a DNA microarray comprising clones covering the genomes of several bifidobacterial species. Significantly hybridizing clones were sequenced and compared with those in the public databases. While some sequences were found to be bifidobacterial ribosomal RNA, the majority showed similarity to protein-encoding genes predicted to be involved in carbohydrate metabolism, processing of information and housekeeping functions. Remarkably, significant similarity was observed to an operon involved in the utilization of specifically human milk oligosaccharides and mucin sugars, supporting the functionality of bifidobacteria in the infant intestine. Overall, transcript profiling revealed significant differences between breast-fed and formula-fed infants, which was also reflected in the diversity of fecal bifidobacterial species measured with quantitative real time PCR. In another study, a specific prebiotic mixture showed a shift in the dominant adult fecal microbiota as well as abundance of the different bifidobacterial species. Subsequent transcriptomics using the same bifidobacterial-targeted microarrays showed the activity of genes in a wide range of functional groups, the majority being involved in metabolism of carbohydrates of plant origin, house keeping functions, and membrane transport of a wide variety of substrates including sugars and metals. Furthermore, the transcriptome of Bifidobacterium longum was studied in vitro in human and formula milk and semi-synthetic medium with galactooligosaccharides which showed quite some differentially expressed genes for sugar utilization. Overall, the transcription of genes involved in carbohydrate metabolism and uptake were specifically induced. In conclusion, post-genomics studies of fecal bifidobacteria and batch cultures of B. longum resulted in enhanced understanding of the life style and generated important leads for further investigation of genes for metabolism and colonization of intestinal bifidobacteria within the human host. |
