Exposure of Bifidobacterium longum subsp. infantis to Milk Oligosaccharides Increases Adhesion to Epithelial Cells and Induces a Substantial Transcriptional Response

Devon Kavanaugh is in receipt of a Teagasc Walsh Fellowship. The authors would also like to acknowledge the support of Science Foundation Ireland under Grant No. 08/SRC/B1393 and the Alimentary Glycoscience Research Cluster (AGRC).peer-reviewedIn this study, we tested the hypothesis that milk oligosaccharides may contribute not only to selective growth of bifidobacteria, but also to their specific adhesive ability. Human milk oligosaccharides (3′sialyllactose and 6′sialyllactose) and a commercial prebiotic (Beneo Orafti P95; oligofructose) were assayed for their ability to promote adhesion of Bifidobacterium longum subsp. infantis ATCC 15697 to HT-29 and Caco-2 human intestinal cells. Treatment with the commercial prebiotic or 3′sialyllactose did not enhance adhesion. However, treatment with 6′sialyllactose resulted in increased adhesion (4.7 fold), while treatment with a mixture of 3′- and 6′-sialyllactose substantially increased adhesion (9.8 fold) to HT-29 intestinal cells. Microarray analyses were subsequently employed to investigate the transcriptional response of B. longum subsp. infantis to the different oligosaccharide treatments. This data correlated strongly with the observed changes in adhesion to HT-29 cells. The combination of 3′- and 6′-sialyllactose resulted in the greatest response at the genetic level (both in diversity and magnitude) followed by 6′sialyllactose, and 3′sialyllactose alone. The microarray data was further validated by means of real-time PCR. The current findings suggest that the increased adherence phenotype of Bifidobacterium longum subsp. infantis resulting from exposure to milk oligosaccharides is multi-faceted, involving transcription factors, chaperone proteins, adhesion-related proteins, and a glycoside hydrolase. This study gives additional insight into the role of milk oligosaccharides within the human intestine and the molecular mechanisms underpinning host-microbe interactions.Science Foundation IrelandTeagasc Walsh Fellowship Programm

Molecular basis for intestinal mucin recognition by galectin-3 and C-type lectins

Intestinal mucins trigger immune responses upon recognition by dendritic cells via protein–carbohydrate interactions. We used a combination of structural, biochemical, biophysical, and cell-based approaches to decipher the specificity of the interaction between mucin glycans and mammalian lectins expressed in the gut, including galectin (Gal)-3 and C-type lectin receptors. Gal-3 differentially recognized intestinal mucins with different O-glycosylation profiles, as determined by mass spectrometry (MS). Modification of mucin glycosylation, via chemical treatment leading to a loss of terminal glycans, promoted the interaction of Gal-3 to poly-N-acetyllactosamine. Specific interactions were observed between mucins and mouse dendritic cell-associated lectin (mDectin)-2 or specific intercellular adhesion molecule–grabbing nonintegrin-related-1 (SIGN-R1), but not mDectin-1, using a cell-reporter assay, as also confirmed by atomic force spectroscopy. We characterized the N-glycosylation profile of mouse colonic mucin (Muc)-2 by MS and showed that the interaction with mDectin-2 was mediated by high-mannose N-glycans. Furthermore, we observed Gal-3 binding to the 3 C-type lectins by force spectroscopy. We showed that mDectin-1, mDectin-2, and SIGN-R1 are decorated by N-glycan structures that can be recognized by the carbohydrate recognition domain of Gal-3. These findings provide a structural basis for the role of mucins in mediating immune responses and new insights into the structure and function of major mammalian lectins.—Leclaire, C., Lecointe, K., Gunning, P. A., Tribolo, S., Kavanaugh, D. W., Wittmann, A., Latousakis, D., MacKenzie, D. A., Kawasaki, N., Juge, N. Molecular basis for intestinal mucin recognition by galectin-3 and C-type lectins

Exposure of bifidobacterium longum subsp. infantis to milk oligosaccharides increases adhesion to epithelial cells and induces a substantial transcriptional response

In this study, we tested the hypothesis that milk oligosaccharides may contribute not only to selective growth of bifidobacteria, but also to their specific adhesive ability. Human milk oligosaccharides (3\u27sialyllactose and 6\u27sialyllactose) and a commercial prebiotic (Beneo Orafti P95; oligofructose) were assayed for their ability to promote adhesion of Bifidobacterium longum subsp. infantis ATCC 15697 to HT-29 and Caco-2 human intestinal cells. Treatment with the commercial prebiotic or 3\u27sialyllactose did not enhance adhesion. However, treatment with 6\u27sialyllactose resulted in increased adhesion (4.7 fold), while treatment with a mixture of 3\u27- and 6\u27-sialyllactose substantially increased adhesion (9.8 fold) to HT-29 intestinal cells. Microarray analyses were subsequently employed to investigate the transcriptional response of B. longum subsp. infantis to the different oligosaccharide treatments. This data correlated strongly with the observed changes in adhesion to HT-29 cells. The combination of 3\u27- and 6\u27-sialyllactose resulted in the greatest response at the genetic level (both in diversity and magnitude) followed by 6\u27sialyllactose, and 3\u27sialyllactose alone. The microarray data was further validated by means of real-time PCR. The current findings suggest that the increased adherence phenotype of Bifidobacterium longum subsp. infantis resulting from exposure to milk oligosaccharides is multi-faceted, involving transcription factors, chaperone proteins, adhesion-related proteins, and a glycoside hydrolase. This study gives additional insight into the role of milk oligosaccharides within the human intestine and the molecular mechanisms underpinning host-microbe interactions

Down-regulated genes common to all treatments.

<p>3′SL –3′sialyllactose; 6′SL –6′sialyllactose; 3′+6′SL – combined treatment of 3′- and 6′-sialyllactose; FC – fold change; pval – p-value.</p

Screening oligosaccharides for their ability to influence adhesion of <i>B. longum</i> subsp. <i>infantis</i> ATCC 15697 to HT-29 cells.

<p>Abbreviations: P95 - Beneo Orafti P95; 3′SL - 3′sialyllactose; 6′SL –6′sialyllactose; Lac - lactose. Non-supplemented tissue culture media was used as control. Results are expressed as fold-change relative to control percent adhesion with error bars representing standard deviation. ^adenotes significant difference in relation to control; ^bdenotes significance in relation to 6′SL group; p<0.0001.</p

Effects of trypsin treatment on adhesion of <i>B. longum</i> subsp. <i>infantis</i> ATCC 15697 to HT-29 cells following exposure to 6′sialyllactose (1 mg/ml, 3 hours).

<p>Results are expressed as fold-change in percent adhesion in relation to the control with error bars representing standard deviation. ^adenotes significance in relation to the control, p<0.05; ^bdenotes significance in relation to Trypsin control and 6′SL/Trypsin groups; p = 0.0001.</p

Transcript levels for thirteen (13) selected genes as determined by qPCR (A) and microarray analysis (B).

<p>3′sialyllactose (white shading); 6′sialyllactose (black and white diagonal shading); 3′- and 6′-sialyllactose mixture (black shading). Blon_0029– Ferritin; Blon_0141 - Chaperonin protein DnaK; Blon_0156 - TadE family protein; Blon_0162– Sortase; Blon_0392 - Cation efflux protein;Blon_0459 - Glycoside hydrolase, family 20; Blon_0694– GroEL; Blon_0993 - Magnesium-translocating P-type ATPase; Blon_1687 - TfoX, C-terminal domain protein; Blon_1688 - Transcription activator, effector binding; Blon_1971 - High-affinity zinc ABC transporter; Blon_1990 - Glycine dehydrogenase; Blon_2061– Extracellular solute binding protein.</p

Up-regulated genes common to all three oligosaccharide treatments.

<p>3′SL –3′sialyllactose; 6′SL –6′sialyllactose; 3′+6′SL – combined treatment of 3′- and 6′-sialyllactose; FC – fold change; pval – p-value.</p

Bacterial influence on oligosaccharide concentrations during exposure.

<p>Aliquots of media prior to and following exposure with <i>B. longum</i> subsp. <i>infantis</i> ATCC 15697 for 3 hours were assessed by HPLC. Abbreviations: 6′SL - 6′Sialyllactose; 3′SL - 3′sialyllactose. Results are expressed as percentage of initial oligosaccharide concentration in non-exposed media for each treatment group with error bars representing standard deviation. *denotes p<0.005.</p