1 Department of Systems Biology, Technical University of Denmark2 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark3 unknown
Bacterial communities (microbiota) of great diversity populate the large intestine of animals including humans and influence the physiology, biochemistry and immunology of the host. Microorganisms mainly bacteria that when administered in sufficient amounts promote a beneficial effect to the host are defined as probiotics. The positive clinical effects of probiotics, mainly belonging to the Bifidobacterium and Lactobacillus genera in treatments of irritable bowel syndrome, gut infections and lifestyle diseases are well documented. Compounds that selectively stimulate the beneficial effect of probiotics, primarily non-digestible carbohydrates, are termed prebiotics. The knowledge of prebiotic utilization and in particular the specificities of carbohydrate transport and metabolism are limited, hampering robust understanding for the basis of selective utilization of known prebiotics and the discovery and documentation of novel ones. In this project we set out to investigate the metabolism of carbohydrates that are prebiotic or potential prebiotic compounds utilized by the probiotic organisms Lactobacillus acidophilus NCFM (NCFM) and Bifidobacterium animalis subsp. lactis BL-04 (Bl-04). The aim of this Ph.D. thesis was the study of probiotic NCFM and Bl-04 interaction with prebiotic carbohydrates using differential proteomics and protein characterization. Proteomics is a potential omics tool to investigate probiotic bacteria and its response to prebiotic carbohydrates at the protein level. The reference proteome of NCFM and Bl-04 was established using 2D based proteomics. The whole cell extract proteome of NCFM grown on glucose until late exponential phase was resolved by two-dimensional gel electrophoresis (2-DE) (pH 3-7 and 6-11). A total of 507 and 150 protein identifications were made from the CBB stained 2D-gel using MALDI-TOF MS and/or MS/MS in the pH 3-7 and 6-11 respectively. While from the whole cell extract 2D proteome of Bl-04 (pH 3-7 and pH 3-10), 870 protein identifications were made using MALDI-TOF MS and/or MS/MS. Differential 2-DE (DIGE) of NCFM and Bl-04 grown on established and potential prebiotics revealed several proteins with differential abundance including the glycoside hydrolases involved in primary breakdown of oligosaccharides and changes in carbohydrate metabolic pathways. The potential carbohydrates transport systems involved in uptake of potential prebiotic carbohydrates, specifically the ABC-transporter associated solute binding protein BlXBP involved in the transport of XOS from Bl-04 was characterized. Surface plasmon resonance binding assays showed that BlXBP was specific for XOS with a degree of polymerization (DP) 2– 6 with optimal affinity (Kd ~50 nM) for xylotetraose followed by xylotriose. Differences in binding affinity was governed largely by the association rate (kon), while koff had a more modest contribution to binding affinities, except for xylobiose, which displayed the highest koff. The binding affinity and kinetics of arabinose decorated XOS were similar to undecorated counterparts, thus providing evidence that arabinoxylan fragments are efficiently captured by the ABC import system. The binding affinity towards xylotriose and xylotetraose was corroborated using fluorescence emission spectroscopy and isothermal titration. calorimetry that revealed that the binding free energy was dominated by a favourable enthalpy, which was off-set by a large unfavourable entropy change. The crystal structures of BlXBP in complex with xylotriose, xylotetraose and xylohexaose were determined and showed a binding cleft large enough to accommodate the preferred ligand xylotetraose. The helical structure of xylan fragments was recognized by aromatic stacking of xyloxyl rings 1, 3 and 4, in addition to several direct and solvent mediated hydrogen-bonds that contribute to the binding enthalpy. Binding of larger ligands requires conformational changes in a lid loop that contributes to the structural plasticity of BlXBP. This data bring novel insight into the molecular basis for XOS uptake by bifidobacteria, and xylan utilization by the gut microbiome.
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Abou Hachem, Maher, J. Slotboom, Dirk, Svensson, Birte, Jacobsen, Susanne