Andersen, Joakim Mark

Mikroorganismer, der når de tilføjes i tilstrækkelig dosis udviser en positiv effekt på modtageren, er defineret som probiotika. Det er dokumenteret, at probiotika, hovedsageligt fra genera Bifidobacterium og Lactobacillus, kan anvendes i behandlingen af irriteret tyktarm, tarminfektioner og livsstilssygdomme. Selektiv udnyttelse af primært ufordøjelige kulhydrater, kaldet præbiotika, er én virkningsmekanisme benyttet af probiotika, dog er de specialiserede molekylære interaktioner primært specificiteter af kulhydrattransportører og glykosid-hydrolaser stortset ukendt. Den manglende viden begrænser opdagelsen samt anvendelsen af nye præbiotika. Formålet med denne Ph.D. afhandling var at identificere de gener, som er involveret i optag og katabolisme af potentielle præbiotika i de probiotiske bakterier Lactobacillus acidophilus NCFM og Bifidobacterium animalis subsp. Lactis Bl-04, som model organismer, ved at benytte transkriptomanalyse og in silico rekonstruktion af metaboliske reaktionsveje for at kortlægge centrale gener til videre funktionel analyse ved hjælp af gen-deletioner og rekombinant proteinkarakterisering.Transkriptionsanalyse blev anvendt til at måle det globale gen-udtryk i begge bakterier, dyrket med glukose og en række præbiotika samt potentielle præbiotika dækkende en bred vifte af glykosid-bindinger og glykosid-kompositioner: ß-galacto-oligosakkarider, cellobiose, gentiobiose, isomaltose, panose, raffinose, stachyose samt udvalgte stamme-specifikke potentielle præbiotika – L. acidophilus NCFM: byg ß-glykaner, lactitol, isomaltulose og polydextrose, imens de følgende blev benyttet til B. lactis Bl-04: maltotriose, melibiose, xylobiose og xylo-oligosakkarider. Differential transkriptionsanalyse af L. acidophilus NCFM afslørede opregulering af gener kodende for phosphoenolpyrovatafhængige sukker-phospho-transferase systemer primært knyttet til optag af disakkarider. En galaktosid pentose hexuronid permease og ATP-bindende kasette transportører var opreguleret af hovedsageligt oligosakkarider. I tilknytning til de forskellige kulhydrattransportører var glykosid-hydrolaser involveret i kulhydratkatabolismen fra familierne 1, 2, 4, 13, 32, 36, 42 og 65 opreguleret. Differential transkriptionsanalyse af B. lactis Bl-04 identificerede kulhydrattransportører klassificeret som ’Major facilitator superfamily’ og galaktosid pentose hexuronid permeaser involveret i disakkaridtransport samt ATP-bindende kasette transportører primært for oligosakkaridoptag. Disse transportører blev fundet i genklynger med glykosid-hydrolaser fra familierne: 1, 2, 13, 36, 42, 43 og 77.Reaktionsveje for oligosakkaridudnyttelse blev genskabt in silico ud fra gen-landskabsanalyse og de transkriptionelle resultater. Betydningen af tre formodede essentielle gener kodende for en galaktosid pentose hexuronid permease, en ATP-bindende kasette transportør samt en a-galaktosidase blev undersøgt for deres rolle i optag af ß-galakto-oligosakkarider; for optag og katabolisme af raffinose-lignende oligosakkarider eftervist ved gen-deletioner i L. acidophilus NCFM.Det homologe B. lactis Bl-04 protein af det raffinose specifikke solute binding protein fra L. acidophilus NCFM udviste en dobbelt substratspecificitet for raffinose-lignende oligosakkarider og isomaltooligosakkarider. Bindingsaffiniteterne (KD) for et sæt af a-1,6-glykosider, der repræsentere begge typer af ligander, var i µM skala, hvilket er mærkbart lavere end typiske værdier for oligosakkarid binding til andre solute binding proteiner. Bindingen var entalpisk drevet og den lavere affinitet skyldtes en større ufavorabel entropi. Dette var muligvis resultat af ligang-bindingslommen ændrede form, som krævet for at binde de forskellige ligander varierende i længde og glykosid komposition, selvom a-D-(1,6) galaktose, eller den C-4 epimere glukose, blev genkendt i bindingslommen.Biokemisk karakterisering af rekombinant protein validerede den brede substratspecificitet, dog var affiniteten 100–1000 gange lavere for de fortrukne substrater panose og raffinose end for tidligere beskrevet mono-specifikke kulhydrattransportører omend det endnu ikke er undersøgt om de lavere affiniteter har biologisk relevans. Som konklusion har differentiel transkriptionsanalyse vist det globalt regulerede genudtryk af L. acidophilus NCFM og B. lactis Bl-04 i forhold til potentielle præbiotiske kulhydrater, hvorfra nye specificiteter bekræftet ved funktionel karakterisering er fundet for kulhydrattransportører og glykosid-hydrolaser. Dette studie tilføjer forståelse af hvordan probiotiske bakterier selektivt kan omsætte præbiotika samt hvordan nye præbiotika kan udvælges.Microorganisms that when administered in sufficient amounts exert 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 irritated bowel disorders, gut infections and lifestyle diseases are currently well documented. Selective utilization, of primarily non-digestible carbohydrates, termed prebiotics, by probiotics has been identified as an attribute of probiotic action, however the molecular mechanisms of prebiotics utilization and in particular the specificities of carbohydrate transporters and glycoside hydrolases that confer this remain largely unknown, limiting a robust understanding for the basis of selective utilization of known prebiotics and the discovery and documentation of novel prebiotics. The aim of this Ph.D. thesis was to identify the genes involved with uptake and catabolism of potential prebiotics by the probiotics Lactobacillus acidophilus NCFM and Bifidobacterium animalis subsp. lactis Bl-04 as model organisms, using DNA whole genome microarrays and by in silico pathway re-construction to identify key genes for further functional analysis by gene deletions and recombinant protein characterization.Transcriptional analysis was used to measure the global gene expression, in both bacteria, grown on glucose and various prebiotics and potential prebiotics covering diverse types of glycoside linkages and compositions: ß-galacto-oligosaccharides, cellobiose, gentiobiose, isomaltose, panose, raffinose, stachyose and selected strain-specific potential prebiotics – L. acidophilus NCFM: barley ß-glucan hydrolysate, lactitol, isomaltulose and polydextrose, while for B. lactis Bl-04: maltotriose, melibiose, xylobiose and xylo-oligosaccharides were used. The differential transcriptional analysis of L. acidophilus NCFM revealed upregulation of genes encoding phosphoenolpyruvate-dependent sugar phosphotransferase systems mainly associated with disaccharide uptake, galactoside pentose hexuronide permease and ATP-binding cassette transporters were upregulated by dominantly oligosaccharides. Glycoside hydrolases from families 1, 2, 4, 13, 32, 36, 42, and 65 were found associated with the various transporters for carbohydrate catabolism. The differential transcriptional analysis of B. lactis Bl-04 identified carbohydrate transporters of the major facilitator superfamily and galactoside pentose hexuronide permeases for disaccharide uptake and ATP-binding cassette transporters mainly for uptake of oligosaccharides. These transporters were found in gene clusters with glycoside hydrolases from families 1, 2, 13, 36, 42, 43 and 77.Based on gene landscape analysis and the transcriptional findings, reconstruction of utilization pathways were done in silico. Hereafter the role of essential gene products in uptake of ß-galacto-oligosaccharides putatively facilitated by a galactoside pentose hexuronide permease and the involvement of an ATP-binding cassette transporter and an a-galactosidase for uptake of raffinose family oligosaccharides and catabolism, respectively, were confirmed by gene deletion mutants in L. acidophilus NCFM. The B. lactis Bl-04 homologous protein of the L. acidophilus NCFM raffinose specific solute binding protein displayed dual substrate specificity for raffinose family oligosaccharides and isomalto-oligosaccharides. The binding affinities (KD) to a set of a-1,6 glycosides representing both classes of ligands were in the µM range, notably lower than typical values for oligosaccharide binding to solute binding proteins. The binding was enthalpically dominated and the lower affinity owed to a large unfavorable binding entropy suggestive of a high plasticity of the ligand binding site needed to accommodate different ligands varying in size, and monosaccharide composition, but recognizing a core structure comprising an a-D-(1,6)-linked galactose or its glucose C4 epimer. Biochemical characterization of the recombinant protein validated the broad substrate specificity, however the binding affinity was 100–1000 fold lower for the preferred substrates panose and raffinose, than seen for mono-specific carbohydrate transporters previously described although any biological implication of the weaken affinities is yet to be investigated. In conclusion, differential transcriptomics revealed the global regulated gene response of L. acidophilus NCFM and B. lactis Bl-04 to potential prebiotic carbohydrates from which novel specificities for carbohydrate transporters and glycoside hydrolases were identified and validated through functional characterization. The work adds to the understanding of how probiotic bacteria can selective utilize prebiotics and how novel prebiotics can be discovered

Online Research Database In Technology

A snapshot into the uptake and utilization of potential oligosaccharide prebiotics by probiotic lactobacilli and bifidobacteria as accessed by transcriptomics, functional genomics, and recombinant protein characterization

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A snapshot into the uptake and utilization of potential oligosaccharide prebiotics by probiotic lactobacilli and bifidobacteria as accessed by transcriptomics, functional genomics, and recombinant protein characterization

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Online Research Database In Technology