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

Point-of-Need DNA Testing for Detection of Foodborne Pathogenic Bacteria

Foodborne pathogenic bacteria present a crucial food safety issue. Conventional diagnostic methods are time-consuming and can be only performed on previously produced food. The advancing field of point-of-need diagnostic devices integrating molecular methods, biosensors, microfluidics, and nanomaterials offers new avenues for swift, low-cost detection of pathogens with high sensitivity and specificity. These analyses and screening of food items can be performed during all phases of production. This review presents major developments achieved in recent years in point-of-need diagnostics in land-based sector and sheds light on current challenges in achieving wider acceptance of portable devices in the food industry. Particular emphasis is placed on methods for testing nucleic acids, protocols for portable nucleic acid extraction and amplification, as well as on the means for low-cost detection and read-out signal amplification

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

Structural basis for the role of Serine-Rich Repeat Proteins from Lactobacillus reuteri in gut microbe-host interactions

Lactobacillus reuteri, a Gram-positive bacterial species inhabiting the gastrointestinal tract of vertebrates displays remarkable host adaptation. Previous mutational analyses of rodent strain L. reuteri 100-23C identified a gene encoding a predicted surface-exposed serine-rich repeat protein (SRRP100-23) that was vital for L. reuteri biofilm formation in mice. SRRPs have emerged as an important group of surface proteins on many pathogens but no structural information is available in commensal bacteria. Here we report the 2.00 Å and 1.92 Å crystal structures of the binding regions (BRs) of SRRP100-23 and SRRP53608 from L. reuteri ATCC 53608, revealing a unique “β-solenoid” fold in this important adhesin family. BRSRRP53608 boundto host epithelial cells and DNA at neutral pH and recognised polygalacturonic acid (PGA), rhamnogalacturonan I or chondroitin sulfate A at acidic pH. Mutagenesis confirmed the role of the BR putative binding site in the interaction of BRSRRP53608 with PGA. Long molecular dynamics simulations showed that SRRP53608 undergoes a pH-dependent conformational change. Together, these findings shed new mechanistic insights into the role of SRRPs in host-microbe interactions and open new avenues of research into the use of biofilm-forming probiotics against clinically important pathogens

Serine-Rich Repeat Protein adhesins from Lactobacillus reuteri display strain specific glycosylation profiles

Lactobacillus reuteri is a gut symbiont inhabiting the gastrointestinal tract of numerous vertebrates. The surface-exposed Serine-Rich Repeat Protein (SRRP) is a major adhesin in Gram-positive bacteria. Using lectin and sugar nucleotide profiling of wild-type or L. reuteri isogenic mutants, MALDI-ToF-MS, LC-MS and GC-MS analyses of SRRPs, we showed that L. reuteri strains 100-23C (from rodent) and ATCC 53608 (from pig) can perform protein O-glycosylation and modify SRRP100-23 and SRRP53608 with Hex-Glc-GlcNAc and di-GlcNAc moieties, respectively. Furthermore, in vivo glycoengineering in E. coli led to glycosylation of SRRP53608 variants with α-GlcNAc and GlcNAcβ(1→6)GlcNAcα moieties. The glycosyltransferases involved in the modification of these adhesins were identified within the SecA2/Y2 accessory secretion system and their sugar nucleotide preference determined by saturation transfer difference NMR spectroscopy and differential scanning fluorimetry. Together, these findings provide novel insights into the cellular O-protein glycosylation pathways of gut commensal bacteria and potential routes for glycoengineering applications

The influence of milk oligosaccharides on host-commensal interactions in the GI tract

This thesis sets out to explore the influence of milk oligosaccharides on the colonisation of the intestinal microflora and their potential downstream impact upon components of the gastrointestinal tract. Milk oligosaccharides are known to act as prebiotics for the early microflora, but it was not yet known if these molecules possessed alternative bioactivities relating to intestinal adhesion. Various oligosaccharides present in human and bovine milk were screened for their ability to influence the adhesion of an infant-associated commensal, Bifidobacterium longum subsp. infantis ATCC 15697, to in vitro intestinal models (Chapter II). Screening of individual oligosaccharides identified that exposure to 6'sialyllactose, but not 3'sialyllactose, could promote adhesion to the HT-29 cell line. Interestingly, exposure to a combination of 3'- and 6'-sialyllactose resulted in a dramatic increase in adhesion to the HT-29 cell line. 3'- and 6'-sialyllactose, alone or in combination, increased adhesion to the Caco-2 model, though to a reduced extent. Parameters such as oligosaccharide concentration, duration of oligosaccharide exposure, enzymatic treatments of the bacteria, and a screen of other bacterial strains were used to explore the activity of 6'sialyllactose to promote adhesion to the HT-29 cell line in greater detail. The capability of 6'sialyllactose to induce an adhesive phenotype was unique to Bifidobacterium longum subsp. infantis ATCC 15697, required a threshold oligosaccharide concentration above 0.5mg/ml, and likely involves a surface protein or combination of proteins. Following the discovery of the novel bioactivity of 6'sialyllactose, the genetic basis for the induced adhesive-phenotype was investigated. Oligosaccharide exposures were replicated as in the previous in vitro adhesion studies. Genetic expression was assessed through the use of whole genome DNA microarray analysis. Exposure to the combination of 3'- and 6'-sialyllactose resulted in both, the highest number of differentially transcribed genes and the greatest magnitude of transcription. Levels of gene transcription correlated with the trend of increased adhesion to the HT-29 cell line. The study identified several genes related to either stress-response or colonising factors (dnaK, groEL, sortase, dps-ferritin), which were further validated through the use of qPCR. Several of the genes have been confirmed as colonising factors in alternative strains of bacteria. This study demonstrates a connection between exposure to a combination of predominant sialylated milk oligosaccharides and an adaptive colonising response of B. longum subsp. infantis ATCC 15697. Milk oligosaccharides have demonstrated activity as both prebiotic and anti-infective molecules. Although human milk contains the highest concentration and greatest diversity of oligosaccharides, bovine milk and dairy whey streams contain several of the common acidic oligosaccharides and represent a scalable source of bioactive oligosaccharides. Bovine milk oligosaccharides (BMO) isolated from dairy whey streams and 6'sialyllactose were tested against an established commercial prebiotic, oligofructose, to assess their safety profile and impact on the microbial communities in the murine gut (Chapter III). Findings indicated that none of the treatments significantly affected markers of probiotic activity via short chain fatty acid production or altered IgA or cytokine profiles. Oligofructose demonstrated mixed activity to alter bacterial family proportions, while BMO and 6'sialyllactose were associated with significantly reduced proportions of bacterial families containing notable pathogens. The findings of chapter III demonstrate the overall safety of oligosaccharide supplementation and their ability to modulate the murine intestinal microbiome, with potential applications in infant formulations and functional foods. With the ability of milk oligosaccharides to promote the growth and adhesion of bifidobacteria, which are in turn decorated with numerous glycans on their cell walls, it is likely that altering the numbers of bifidobacteria will influence many lectin-mediated interactions in the gastrointestinal tract. Accordingly, a panel of commensal bacteria were screened for their ability to interact with galectin-3, a galactose-binding glycoprotein, which is notably expressed in the epithelial cells of the respiratory and gastrointestinal tract and can influence both metastasis and pathogenic bacterial colonisation (Chapter IV). Surface plasmon resonance was employed for the screening assay, identifying two strains of HMO-consuming commensal bacteria, B. longum subsp. infantis, which interacted with galectin-3 to a greater extent than the pathogenic positive control. The interaction was further validated and characterized through the use of agglutination and solid-phase binding experiments. The galectin-3-bacteria interaction is mediated through a carbohydrate moiety; however, the entire galectin-3 protein is required for optimal binding. The results demonstrate for the first time a novel interaction between galectin-3 and commensal bacteria. As the two strains can be influenced by HMOs via increased growth or colonisation, the findings present a potentially novel means to modulate the activity of galectin-3 in the GI tract. Overall these studies build a strong case for the use of milk oligosaccharides to modulate the intestinal microflora, with bovine milk and dairy whey streams presenting an abundant and yet untapped resource. Future research will aid in further elucidating the intricacies of the oligosaccharide-microflora-GI tract interaction, thereby identifying novel therapeutic targets and means by which to maintain or restore host health.2016-11-1

Mucin glycan foraging in the human gut microbiome

The availability of host and dietary carbohydrates in the gastrointestinal (GI) tract plays a key role in shaping the structure-function of the microbiota. In particular, some gut bacteria have the ability to forage on glycans provided by the mucus layer covering the GI tract. The O-glycan structures present in mucin are diverse and complex, consisting predominantly of core 1-4 mucin-type O-glycans containing α- and β- linked N-acetyl-galactosamine, galactose and N-acetyl-glucosamine. These core structures are further elongated and frequently modified by fucose and sialic acid sugar residues via α1,2/3/4 and α2,3/6 linkages, respectively. The ability to metabolize these mucin O-linked oligosaccharides is likely to be a key factor in determining which bacterial species colonise the mucosal surface. Due to their proximity to the immune system, mucin-degrading bacteria are in a prime location to influence the host response. However, despite the growing number of bacterial genome sequences available from mucin degraders, our knowledge on the structural requirements for mucin degradation by gut bacteria remains fragmented. This is largely due to the limited number of functionally characterized enzymes and the lack of studies correlating the specificity of these enzymes with the ability of the strain to degrade and utilize mucin and mucin glycans. This review focuses on recent findings unravelling the molecular strategies used by mucin-degrading bacteria to utilise host glycans, adapt to the mucosal environment, and influence human health

Sensitive Detection of E. coli in Artificial Seawater by Aptamer-Coated Magnetic Beads and Direct PCR

E. coli in seawater can be simply and efficiently detected by direct PCR. The sensitivity of detection is significantly improved using magnetic beads based bacterial pre-concentration.abstract Foodborne and waterborne E. coli remains a major economic burden worldwide. Assaying seawater for trace levels of E. coli is challenging since it applies time-consuming preparations, expensive instrumentation and complicated procedures. Therefore, there is a continued demand for new analytical technologies that can detect low bacterial concentrations in a more cost- and time-effective manner. In this study, combination of E. coli pre-concentration with a direct polymerase chain reaction (PCR) was shown to enable rapid bacterial detection without enrichment step or DNA extraction/purification. The E1 aptamer that targets E. coli surface epitope grafted onto magnetic beads efficiently concentrated E. coli from water samples containing high concentration of NaCl. When direct PCR was performed on bacteria attached to these aptamer-modified magnetic beads, a limit of 10(3) CFU/mL was obtained. The overall analysis was performed in less than 3 h. This approach may lead to a future PCR-based biosensor system for online monitoring of enteric bacteria in seawater

Use of Atomic Force Microscopy to Study the Multi-Modular Interaction of Bacterial Adhesins to Mucins

The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the molecular details of these interactions are not well understood. Here, we provide mechanistic insights into the adhesion properties of the canonical mucus-binding protein (MUB), a large multi-repeat cell–surface adhesin found in Lactobacillus inhabiting the GI tract. We used atomic force microscopy to unravel the mechanism driving MUB-mediated adhesion to mucins. Using single-molecule force spectroscopy we showed that MUB displayed remarkable adhesive properties favouring a nanospring-like adhesion model between MUB and mucin mediated by unfolding of the multiple repeats constituting the adhesin. We obtained direct evidence for MUB self-interaction; MUB–MUB followed a similar binding pattern, confirming that MUB modular structure mediated such mechanism. This was in marked contrast with the mucin adhesion behaviour presented by Galectin-3 (Gal-3), a mammalian lectin characterised by a single carbohydrate binding domain (CRD). The binding mechanisms reported here perfectly match the particular structural organization of MUB, which maximizes interactions with the mucin glycan receptors through its long and linear multi-repeat structure, potentiating the retention of bacteria within the outer mucus layer