Prebiotic Utilization by Lactobacillus Acidophilus Ncfm and Framework for Protein Discovery

Prebiotic utilization by Lactobacillus acidophilus NCFM and framework for protein discovery.

Andersen, J.M.^1,4*; Majumder, A.¹; Fredslund, F.¹; Ejby, M. ¹; van Zanten, G.C.^1,2 ;Barrangou, R.³; Goh, Y.J.⁴; Lahtinen, S.J.⁵; Lo Leggio, L.⁶; Coutinho, P.M.⁷ Jacobsen, S.¹; Abou Hachem, M.¹; Klaenhammer, T.⁴; Svensson, B.¹

¹Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark. ² Department of Food Science, Faculty of Life Sciences, University of Copenhagen. ³DuPont Nutrition and Health, Madison, WI. ⁴North Carolina State University, Raleigh, NC. ⁵DuPont Nutrition and Health, Kantvik, Finland. ⁶Department of Chemistry, University of Copenhagen. ⁷CNRS, Université d’Aix Marseille, France.

*Contact: joa@bio.dtu.dk

Probiotic bacteria have been shown to positively modulate human chronic bowel disorders, immunity and life-style diseases. Carbohydrate utilization has been identified as an important determinant of probiotic action.   Utilization of non-digestible oligosaccharides (prebiotics) can selectively stimulate probiotics in the gastrointestinal tract.  However, only few carbohydrates are classified as prebiotics mainly due to limited experimental knowledge of in vivo studies and molecular understanding of their selective utilization.

The aim of the work was to use an array of omics technologies to provide a comprehensive and molecular understanding of prebiotic utilization, represented by the commercial Lactobacillus acidophilus NCFM.

Here we demonstrate the versatility of this approach by discerning the molecular basis of uptake and catabolism of galacto-oligosaccharides containing α- or β-linkages. Transcriptional analysis led to the discovery of the first β-galacto-oligosaccharide transporter within the Lactobacillus genus (1) which displayed extensive substrate specificity with respect to oligosaccharide size and composition. This work was integrated with the proteomic reference map and DIGE-proteomics of L. acidophilus NCFM (2), to reveal the catabolic pathways of potential galactoside prebiotics and identified the upregulated specific glycoside hydrolases of GH families 2, 42. Additionally transcriptional and functional genomics analysis of α-galacto-oligosaccharides identified a key catabolic α-galactosidase which was biochemically and structurally characterized, highlighting the specificity of this enzyme towards raffinose family oligosaccharides. Sequence motifs and other structural elements important for the molecular architecture were identified allowing us to propose a subdivision of GH36 (3) which differentiated sub-specificities within the family.

This work provides a more detailed view of key mechanisms underlying utilization of prebiotic galactosides within probiotic lactobacilli.

This work is supported by grants from Danish Council for Strategic Research, Committee for Health, Food and Welfare, Danish Council for Independent Research Natural Sciences, a HC Ørsted Postdoctoral Fellowship (to AM), PhD stipends from the Technical University of Denmark (to JMA and ME) and Danisco A/S (to JMA).

References: (1) Andersen et al. PNAS (2011) 108: 17785-17790 (2) Majumder et al. Proteomics (2011) 17: 3470–3481 (3) Fredslund et al. JMB (2011) 412: 466–480