Celebioglu H., Prehn K., Lahtinen S., Brix S., Abou Hachem M., Svensson B.
Department of Systems Biology, Technical University of Denmark

Lactobacillus acidophilus NCFM (NCFM) is a probiotic bacterium isolated from a human source and has been used extensively in foods and dietary supplements. It is a Gram-positive lactic acid-producing, well studied bacterium and its genome has been sequenced. Surface layer (S-layer) is a crystalline array of proteins forming the outermost part of the bacterial cell wall. In addition to S-layer proteins, extracellular proteins secreted by probiotics can have roles in interactions with the host as they are available for direct contacts with the mucosa and epithelial cells, which can involve bacterial adhesion to intestinal cells (HT-29 and Caco-2) and mucus layer.
Prebiotics are non-digestible components in food, which beneficially affect host health by selective stimulation of growth of probiotic bacteria in the gastrointestinal tract (GIT). Fructooligosaccharides (FOS), galactooligosaccharides (GOS), and lactulose fulfill prebiotic criteria as approved in clinical trials. By contrast, a number of emerging prebiotics; cellobiose; melibiose; isomaltulose; polydextrose; and trehalose await approval.

Following the growth of NCFM on different carbohydrates or supplemented with mucin and labeling with 5(6)-carboxyfluorescein diacetate, in vitro adhesion to mucin and HT-29 cells were performed by measuring the fluorescence. Surface proteomes were prepared by using 5 M lithium chloride, followed by 2-dimensional gel electrophoresis (2-DE), image analysis, and mass spectrometric identifications of differentially abundant proteins.

While growth on FOS only stimulated adhesion of NCFM to intestinal HT-29 cells, cellobiose and polydextrose also increased the adhesion to mucin. No significant change in adhesion to mucin and HT-29 cells was found for NCFM grown on GOS, lactulose, melibiose, palatinose, or trehalose. Remarkably addition of mucin to cultures on glucose only stimulated adhesion to HT-29 cells.
Comparative 2DE-based surface proteome analysis of NCFM showed higher relative abundance for cultures with added mucin of oxalyl-CoA decarboxylase co-migrating with pyruvate kinase and fructose-bisphosphate aldolase. Reduced relative abundant spots contained elongation factor G, phosphoglycerate kinase, BipAEFTU family GTP-binding protein, ribonucleoside triphosphate reductase, adenylosuccinate synthetase, 30S ribosomal protein S1, manganese-dependent inorganic pyrophosphatase. Comparative surface proteome analysis of cellobiose- and glucose-grown NCFM indicated phosphate starvation inducible protein stress-related, thermostable pullulanase, and elongation factor G to increase fold in abundance, while GAPDH, elongation factor Ts, and pyruvate kinase diminished fold in abundance when grown on cellobiose.

The alteration in adhesive properties can lead to easier bacterial colonization and thus exert more positive health effects. Higher abundant surface proteins in different conditions (i.e. growth on cellobiose or mucin-supplemented growth media) may indicate these proteins are responsible for the increased adhesion. Therefore, combination of NCFM with some carbohydrates gives insight into potential determinants of the synbiotic interactions.

Keywords: Subproteomes, Adhesion, Synbiotics, Probiotics, Mucin, HT-29 cells

Celebioglu H., et al. (2016). Modification of adhesion and surface proteome by carbohydrates and mucin-supplemented growth of probiotic bacterium Lactobacillus acidophilus NCFM.
Conference Proceedings of IPC2016. Paper presented at the International Scientific Conference on Probiotics and Prebiotics, Budapest (p. 87.). IPC2016

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