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Lay Summary For the first time, fecal mucins of Crohn’s disease patients were analyzed by mass spectrometry. Compared with control subjects, Crohn’s disease patients showed a significant decrease in sialylated glycans that we propose as new noninvasive tool for screening of intestinal diseases.

Streptococcus gallolyticus subsp. gallolyticus (SGG), an opportunistic gram-positive pathogen responsible for septicemia and endocarditis in the elderly, is often associated with colon cancer (CRC). In this work, we investigated the oncogenic role ofSGGstrain UCN34 using the azoxymethane (AOM)-induced CRC modelin vivo, organoid formationex vivoand proteomic and phosphoproteomic analyses from murine colons. We showed thatSGGUCN34 accelerates colon tumor development in the murine CRC model. Full proteome and phosphoproteome analysis of murine colons chronically colonized bySGGUCN34 or the closely related non-pathogenicS. gallolyticus subsp. macedonicus(SGM) revealed that 164 proteins and 725 phosphorylation sites were differentially regulated following colonization bySGGUCN34. Ingenuity Pathway Analysis (IPA) indicates a pro-tumoral shift specifically induced following colonization bySGGUCN34, as most proteins and phosphoproteins identified were associated with digestive cancer. Comprehensive analysis of the altered phosphoproteins using ROMA software revealed significantly elevated activities in several cancer hallmark pathways affecting tumoral cells and their microenvironment, i.e. MAPK (ERK, JNK and p38), mTOR and integrin/ILK/actin signaling, inSGGUCN34 colonized colon. Importantly, analysis of protein arrays of human colon tumors colonized withSGGshowed up-regulation of PI3K/Akt/mTOR and MAPK pathways, providing clinical relevance to our findings. To testSGG’s capacity to induce pre-cancerous transformation of the murine colonic epithelium, we grewex vivoorganoids which revealed unusual structures with compact morphology following exposure toSGG. Taken together, our results reveal that the oncogenic role ofSGGUCN34 is associated with activation of multiple cancer-related signaling pathways.Author SummaryColorectal cancer is the third most common cause of cancer mortality worldwide. The colon is a very singular organ, colonized by a vast and complex community of microorganisms, known as the gut microbiota. Strong evidence supports a role of the microbiota in colon cancer development.Streptococcus gallolyticussubsp.gallolyticus(SGG), a gut commensal, was one of the first bacteria to be associated with colorectal cancer. A better understanding of the role ofSGGin colon cancer development is critical to developing novel strategies to improve clinical diagnosis and treatment of this disease. Here, using a global proteomic analysis of mouse colonic tissue colonized bySGG, we show that over 90% of the proteins with altered levels are involved in cancer.SGGcolonization promotes autocrine and paracrine pro-tumor signals contributing to transformation of the colonic epithelium but also modifying the stromal microenvironment, which in turn sustains tumor development. Importantly, two tumor hallmark pathways (PI3K/Akt/mTOR and MAPK) identified in our mouse model were also found in human colon tumor biopsies colonized bySGG, strengthening the clinical relevance to our study.

International audience; Silver biodistribution and gut toxicity of two commercially available colloidal silver products, Mesosilver™ and AgC, were evaluated in male mice. AgC is composed solely of ionic silver (Ag+) while Mesosilver™ contains a mix of silver nanoparticles (AgNPs) and Ag+ ions. After high-dose (approximately 3 mg per kg body weight (bw) per day) sub-chronic exposure, silver accumulation was close for Mesosilver™ and AgC. The combination of micro X-ray fluorescence and micro X-ray absorption spectroscopy showed that metallic AgNPs and Ag+ ions initially contained in Mesosilver™ were subjected to physicochemical modifications during their fate in the gut. In the colon, most Ag atoms were oxidized and dissolved to form Ag complexes with thiol groups (-SH) of proteins and/or peptides. Sub-chronic exposure at lower dose (150 μg per kg bw per day) led to a moderate impact on the gut barrier for both colloidal silver products. An increase in colonic LCN-2 was observed only after AgC exposure. For gut microbiota at the genus level, exposure to Mesosilver™ led to a decrease in Ruminococcus and Anaerosporobacter, while Intestinimonas increased. Exposure to AgC resulted in an increase in Clostridium sp. ASF356 and Tyzzerella, while the relative abundance of Anaerosporobacter decreased. In addition, the Saccharomycetes fungal population increased. Near-infrared spectroscopy was able to satisfactorily discriminate the Mesosilver™-vs. AgC-exposed mice for both exposure doses. This study highlights the applicability of biophysics-based methodologies for providing novel insights into colloidal silver uptake, fate and toxicological effects after oral exposure

International audience; Background: Bacterial colonization in cystic fibrosis (CF) lungs has been directly associated to the loss of CFTR function, and/or secondarily linked to repetitive cycles of chronic inflammation/infection. We hypothesized that altered molecular properties of mucins could contribute to this process.Methods: Newborn CFTR+/+ and CFTR-/- were sacrificed before and 6 h after inoculation with luminescent Pseudomonas aeruginosa into the tracheal carina. Tracheal mucosa and the bronchoalveolar lavage (BAL) fluid were collected to determine the level of mucin O-glycosylation, bacteria binding to mucins and the airways transcriptome. Disturbances in mucociliary transport were determined by ex-vivo imaging of luminescent Pseudomonas aeruginosa.Results: We provide evidence of an increased sialylation of CF airway mucins and impaired mucociliary transport that occur before the onset of inflammation. Hypersialylation of mucins was reproduced on tracheal explants from non CF animals treated with GlyH101, an inhibitor of CFTR channel activity, indicating a causal relationship between the absence of CFTR expression and the sialylation of mucins. This increased sialylation was correlated to an increased adherence of P. aeruginosa to mucins. In vivo infection of newborn CF piglets by live luminescent P. aeruginosa demonstrated an impairment of mucociliary transport of this bacterium, with no evidence of pre-existing inflammation.Conclusions: Our results document for the first time in a well-defined CF animal model modifications that affect the O-glycan chains of mucins. These alterations precede infection and inflammation of airway tissues, and provide a favorable context for microbial development in CF lung that hallmarks this disease.

Approximately 1 billion people worldwide suffer from zinc deficiency, with severe consequences for their well-being, such as critically impaired intestinal health. In addition to an extreme degeneration of the intestinal epithelium, the intestinal mucus is seriously disturbed in zinc-deficient (ZD) animals. The underlying cellular processes as well as the relevance of zinc for the mucin-producing goblet cells, however, remain unknown. To this end, this study examines the impact of zinc deficiency on the synthesis, production, and secretion of intestinal mucins as well as on the zinc homeostasis of goblet cells using the in vitro goblet cell model HT-29-MTX. Zinc deprivation reduced their cellular zinc content, changed expression of the intestinal zinc transporters ZIP-4, ZIP-5, and ZnT1 and increased their zinc absorption ability, outlining the regulatory mechanisms of zinc homeostasis in goblet cells. Synthesis and secretion of mucins were severely disturbed during zinc deficiency, affecting both MUC2 and MUC5AC mRNA expression with ongoing cell differentiation. A lack of zinc perturbed mucin synthesis predominantly on the post-translational level, as ZD cells produced shorter O-glycans and the main O-glycan pattern was shifted in favor of core-3-based mucins. The expression of glycosyltransferases that determine the formation of core 1-4 O-glycans was altered in zinc deficiency. In particular, B3GNT6 mRNA catalyzing core 3 formation was elevated and C2GNT1 and C2GNT3 elongating core 1 were downregulated in ZD cells. These novel insights into the molecular mechanisms impairing intestinal mucus stability during zinc deficiency demonstrate the essentiality of zinc for the formation and maintenance of this physical barrier.

Aberrant glycosylation is a featured characteristic of cancer and plays a role in cancer pathology; thus an understanding of the compositions and functions of glycans is critical for discovering diagnostic biomarkers and therapeutic targets for cancer. In this study, we used MALDI-TOF-MS analysis to determine the O-glycan profiles of prostate cancer cells metastasized to bone (PC-3), brain (DU145), lymph node (LNCaP), and vertebra (VCaP) in comparison to immortalized RWPE-1 cells derived from normal prostatic tissue. Prostate cancer (CaP) cells exhibited an elevation of simple/short O-glycans, with a reduction of complex O-glycans, increased O-glycan sialylation and decreased fucosylation. Core 1 sialylation was increased dramatically in all CaP cells, and especially in PC-3 cells. The expression of Neu5Acα2-3Galβ1-3GalNAc- (sialyl-3T antigen) which is the product of α2,3-sialyltransferase-I (ST3Gal-I) was substantially increased. We therefore focused on exploring the possible function of ST3Gal-I in PC-3 cells. ST3Gal-I silencing studies showed that ST3Gal-I was associated with PC-3 cell proliferation, migration and apoptosis. Further in vivo studies demonstrated that down regulation of ST3Gal-I reduced the tumor size in xenograft mouse model, indicating that sialyl-3T can serve as a biomarker for metastatic prostate cancer prognosis, and that ST3Gal-I could be a target for therapeutic intervention in cancer treatment.

Bacterial infections are frequently based on the binding of lectin-like adhesins to specific glycan determinants exposed on host cell receptors. These interactions confer species-specific recognition and tropism for particular host tissues and represent attractive antibacterial targets. However, the wide structural diversity of carbohydrates hampers the characterization of specific glycan determinants. Here, we characterized the receptor recognition of type IV pili (Tfp), a key adhesive factor present in numerous bacterial pathogens, using Neisseria meningitidis as a model organism. We found that meningococcal Tfp specifically recognize a triantennary sialylated poly-N-acetyllactosamine–containing N-glycan exposed on the human receptor CD147/Basigin, while fucosylated derivatives of this N-glycan impaired bacterial adhesion. Corroborating the inhibitory role of fucosylation on receptor recognition, adhesion of the meningococcus on nonhuman cells expressing human CD147 required prior defucosylation. These findings reveal the molecular basis of the selective receptor recognition by meningococcal Tfp and thereby, identify a potential antibacterial target.

Bacterial infections are frequently based on the binding of lectin-like adhesins to specific glycan determinants exposed on host cell receptors. These interactions confer species-specific recognition and tropism for particular host tissues and represent attractive antibacterial targets. However, the wide structural diversity of carbohydrates hampers the characterization of specific glycan determinants. Here, we characterized the receptor recognition of type IV pili (Tfp), a key adhesive factor present in numerous bacterial pathogens, using Neisseria meningitidis as a model organism. We found that meningococcal Tfp specifically recognize a triantennary sialylated poly- N -acetyllactosamine–containing N -glycan exposed on the human receptor CD147/Basigin, while fucosylated derivatives of this N -glycan impaired bacterial adhesion. Corroborating the inhibitory role of fucosylation on receptor recognition, adhesion of the meningococcus on nonhuman cells expressing human CD147 required prior defucosylation. These findings reveal the molecular basis of the selective receptor recognition by meningococcal Tfp and thereby, identify a potential antibacterial target.

The work presented by Audry et al. (M. Audry, C. Robbe-Masselot, J.-P. Barnier, B. Gachet, et al., mSphere 4:e00494-19, 2019, https://doi.org/10.1128/mSphere.00494-19) gives new insight into the interactions of Neisseria meningitidis and the human nasopharynx., The work presented by Audry et al. (M. Audry, C. Robbe-Masselot, J.-P. Barnier, B. Gachet, et al., mSphere 4:e00494-19, 2019, https://doi.org/10.1128/mSphere.00494-19) gives new insight into the interactions of Neisseria meningitidis and the human nasopharynx. Using an air interface tissue culture model of a polarized, mucus-secreting epithelium, Audry et al. demonstrate that N. meningitidis bacteria do not commonly invade epithelial cells. Rather, they are trapped in the mucus layer, where they are protected from dessication. In this model, meningicocci fail to elicit a pro-inflammatory immune response and show growth effects in response to another nasopharyngeal colonizer, Streptococcus mitis. These findings prompt new questions about pathobiont behaviors, the role of mucus in bacterium-host interactions, and modeling human infection.

Mucus is the first biological barrier encountered by particles and pathogenic bacteria at the surface of secretory epithelia. The viscoelasticity of mucus is governed in part by low energy interactions that are difficult to assess. The CYS domain is a good candidate to support low energy interactions between GFMs and/or mucus constituents. Our aim was to stiffen the mucus from HT29-MTX cell cocultures and the colon of mice through the delivery of a recombinant protein made of hydrophobic CYS domains and found in multiple copies in polymeric mucins. The ability of the delivery of a poly-CYS molecule to stiffen mucus gels was assessed by probing cellular motility and particle diffusion. We demonstrated that poly-CYS enrichment decreases mucus permeability and hinders displacement of pathogenic flagellated bacteria and spermatozoa. Particle tracking microrheology showed a decrease of mucus diffusivity. The empirical obstruction scaling model evidenced a decrease of mesh size for mouse mucus enriched with poly-CYS molecules. Our data bring evidence that enrichment with a protein made of CYS domains stiffens the mucin network to provide a more impermeable and protective mucus barrier than mucus without such enrichment.

Neisseria meningitidisis an inhabitant of the nasopharynx, from which it is transmitted from person to person or disseminates in the blood and becomes a harmful pathogen. In this work, we addressed the colonization of the nasopharyngeal niche by focusing on the interplay between meningococci and the mucus that lines the mucosa of the host. Using Calu-3 cells grown in air-interfaced culture, we studied the meningococcal colonization of the mucus and the host response. Our results suggested thatN. meningitidisbehaved like commensal bacteria in mucus, without interacting with human cells or actively transmigrating through the cell layer. As such, meningococci did not trigger a strong innate immune response from the Calu-3 cells. Finally, we have shown that this model is suitable for studying interaction ofN. meningitidiswith other bacteria living in the nasopharynx, and thatStreptococcus mitisbut notMoraxella catarrhaliscan promote meningococcal growth in this model.

International audience; Streptococcus gallolyticus is a commensal bacterium responsible for infectious endocarditis in the elderly, which has frequently been associated with colonic carcinoma. Whether this species is a cause or a consequence of colorectal cancer remains unknown. We recently demonstrated that S. gallolyticus Pil3 pilus is required for adhesion to colonic mucus and for colonization of mouse distal colon. We show here that Pil3 pilus binds equally well to human colonic mucins derived from HT29-MTX cells and to human stomach mucins from healthy donors. In addition, we have found that Pil3 also binds to human fibrinogen, which expands the repertoire of Pil3 host ligands.

Mucus is a major component of the intestinal barrier involved both in the protection of the host and the fitness of commensals of the gut. Streptococcus thermophilus is consumed world-wide in fermented dairy products and is also recognized as a probiotic, as its consumption is associated with improved lactose digestion. We determined the overall effect of S. thermophilus on the mucus by evaluating its ability to adhere, degrade, modify, or induce the production of mucus and/or mucins. Adhesion was analyzed in vitro using two types of mucins (from pig or human biopsies) and mucus-producing intestinal HT29-MTX cells. The induction of mucus was characterized in two different rodent models, in which S. thermophilus is the unique bacterial species in the digestive tract or transited as a sub-dominant bacterium through a complex microbiota. S. thermophilus LMD-9 and LMG18311 strains did not grow in sugars used to form mucins as the sole carbon source and displayed weak binding to mucus/mucins relative to the highly adhesive TIL448 Lactococcus lactis. The presence of S. thermophilus as the unique bacteria in the digestive tract of gnotobiotic rats led to accumulation of lactate and increased the number of Alcian-Blue positive goblet cells and the amount of the mucus-inducer KLF4 transcription factor. Lactate significantly increased KLF4 protein levels in HT29-MTX cells. Introduction of S. thermophilusvia transit as a sub-dominant bacterium (103 CFU/g feces) in a complex endogenous microbiota resulted in a slight increase in lactate levels in the digestive tract, no induction of overall mucus production, and moderate induction of sulfated mucin production. We thus show that although S. thermophilus is a poor mucus-adhesive bacterium, it can promote mucus pathway at least in part by producing lactate in the digestive tract. 9

Background Titanium dioxide (TiO2) particles are commonly used as a food additive (E171 in the EU) for its whitening and opacifying properties. However, the risk of gut barrier disruption is an increasing concern because of the presence of a nano-sized fraction. Food-grade E171 may interact with mucus, a gut barrier protagonist still poorly explored in food nanotoxicology. To test this hypothesis, a comprehensive approach was performed to evaluate in vitro and in vivo interactions between TiO2 and intestinal mucus, by comparing food-grade E171 with NM-105 (Aeroxyde P25) OECD reference nanomaterial. Results We tested E171-trapping properties of mucus in vitro using HT29-MTX intestinal epithelial cells. Time-lapse confocal laser scanning microscopy was performed without labeling to avoid modification of the particle surface. Near-UV irradiation of E171 TiO2 particles at 364 nm resulted in fluorescence emission in the visible range, with a maximum at 510 nm. The penetration of E171 TiO2 into the mucoid area of HT29-MTX cells was visualized in situ. One hour after exposure, TiO2 particles accumulated inside “patchy” regions 20 µm above the substratum. The structure of mucus produced by HT29-MTX cells was characterized by MUC5AC immunofluorescence staining. The mucus layer was thin and organized into regular “islands” located approximately 20 µm above the substratum. The region-specific trapping of food-grade TiO2 particles was attributed to this mucus patchy structure. We compared TiO2-mediated effects in vivo in rats after acute or sub-chronic oral daily administration of food-grade E171 and NM-105 at relevant exposure levels for humans. Cecal short-chain fatty acid profiles and gut mucin O-glycosylation patterns remained unchanged, irrespective of treatment. Conclusions Food-grade TiO2 is trapped by intestinal mucus in vitro but does not affect mucin O-glycosylation and short-chain fatty acid synthesis in vivo, suggesting the absence of a mucus barrier impairment under “healthy gut” conditions.

Background Titanium dioxide (TiO2) particles are commonly used as a food additive (E171 in the EU) for its whitening and opacifying properties. However, the risk of gut barrier disruption is an increasing concern because of the presence of a nano-sized fraction. Food-grade E171 may interact with mucus, a gut barrier protagonist still poorly explored in food nanotoxicology. To test this hypothesis, a comprehensive approach was performed to evaluate in vitro and in vivo interactions between TiO2 and intestinal mucus, by comparing food-grade E171 with NM-105 (Aeroxyde P25) OECD reference nanomaterial. Results We tested E171-trapping properties of mucus in vitro using HT29-MTX intestinal epithelial cells. Time-lapse confocal laser scanning microscopy was performed without labeling to avoid modification of the particle surface. Near-UV irradiation of E171 TiO2 particles at 364 nm resulted in fluorescence emission in the visible range, with a maximum at 510 nm. The penetration of E171 TiO2 into the mucoid area of HT29-MTX cells was visualized in situ. One hour after exposure, TiO2 particles accumulated inside “patchy” regions 20 µm above the substratum. The structure of mucus produced by HT29-MTX cells was characterized by MUC5AC immunofluorescence staining. The mucus layer was thin and organized into regular “islands” located approximately 20 µm above the substratum. The region-specific trapping of food-grade TiO2 particles was attributed to this mucus patchy structure. We compared TiO2-mediated effects in vivo in rats after acute or sub-chronic oral daily administration of food-grade E171 and NM-105 at relevant exposure levels for humans. Cecal short-chain fatty acid profiles and gut mucin O-glycosylation patterns remained unchanged, irrespective of treatment. Conclusions Food-grade TiO2 is trapped by intestinal mucus in vitro but does not affect mucin O-glycosylation and short-chain fatty acid synthesis in vivo, suggesting the absence of a mucus barrier impairment under “healthy gut” conditions. Electronic supplementary material The online version of this article (10.1186/s12951-018-0379-5) contains supplementary material, which is available to authorized users.

Helicobacter pylori binds to the gastric mucin, MUC5AC, and to trefoil factor, TFF1, which has been shown to interact with gastric mucin. We examined the interactions of TFF1 and H. pylori with purified gastrointestinal mucins from different animal species and from humans printed on a microarray platform to investigate whether TFF1 may play a role in locating H. pylori in gastric mucus. TFF1 bound almost exclusively to human gastric mucins and did not interact with human colonic mucins. There was a strong correlation between binding of TFF1 and H. pylori to human gastric mucins, and between binding of both TFF1 and H. pylori to gastric mucins with that of Griffonia simplicifolia lectin-II, which is specific for terminal non-reducing α- or β-linked N-acetyl-d-glucosamine. These results suggest that TFF1 may help to locate H. pylori in a discrete layer of gastric mucus and hence restrain their interactions with epithelial cells.

International audience; Streptococcus gallolyticus is an increasing cause of bacteremia and infective endocarditis in the elderly. Several epidemiological studies have associated the presence of this bacterium with colorectal cancer. We have studied the interaction of S. gallolyticus with human colonic cells. S. gallolyticus strain UCN34, adhered better to mucus-producing cells such as HT-29-MTX than to the parental HT-29 cells. Attachment to colonic mucus is dependent on the pil3 pilus operon, which is heterogeneously expressed in the wild-type UCN34 population. We constructed a pil3 deletion mutant in a Pil3 overexpressing variant (Pil3+) and were able to demonstrate the role of Pil3 pilus in binding to colonic mucus. Importantly, we showed that pil3 deletion mutant was unable to colonize mice colon as compared to the isogenic Pil3+ variant. Our findings establish for the first time a murine model of intestinal colonization by S. gallolyticus.

Helicobacter pylori is a Gram-negative bacterium that colonizes the mucus niche of the gastric mucosa and infects more than half of the world's human population. Chronic infection may cause gastritis, duodenal ulcer, intestinal metaplasia or gastric cancer. In the stomach, H. pylori interacts with O-glycans of gastric mucins but the mechanism by which the bacteria succeed in altering the mucosa remains mainly unknown. To better understand the physiopathology of the infection, inhibitory adhesion assays were performed with various O-glycans expressed by human gastric mucins, and topographic expression of gastric mucins MUC5AC and MUC6 was analyzed for healthy uninfected individuals, for infected asymptomatic individuals and for patients infected by H. pylori and having the incomplete type of intestinal metaplasia. The glycosylation of the gastric mucosa of asymptomatic individuals infected by H. pylori was determined and compared with the glycosylation pattern found for patients with the incomplete type of intestinal metaplasia. Results show that H. pylori manages to modulate host's glycosylation during the course of infection in order to create a favorable niche, whereas asymptomatic infected individuals seem to counteract further steps of infection development by adapting their mucus glycosylation.

The gastrointestinal mucosal surface is the primary interface between internal host tissues and the vast microbiota. Mucins, key components of mucus, are high-molecular-weight glycoproteins characterized by the presence of many O-linked oligosaccharides to the core polypeptide. They play many biological functions, helping to maintain cellular homeostasis and to establish symbiotic relationships with complex microbiota. Mucin O-glycans exhibit a huge variety of peripheral sequences implicated in the binding of bacteria to the mucosal tissues, thereby playing a key role in the selection of specific species and in the tissue tropism displayed by commensal and pathogenic bacteria. Bacteria have evolved numerous strategies to colonize host mucosae, and among these are modulation of expression of cell surface adhesins which allow bacteria to bind to mucins. However, despite well structurally characterized adhesins and lectins, information on the nature and structure of oligosaccharides recognized by bacteria is still disparate. This review summarizes the current knowledge on the structure of epithelial mucin O-glycans and the interaction between host and commensal or pathogenic bacteria mediated by mucins.

Poster of distinctionPoster of distinction; International audience; Targeted epithelial disruption impacts colonic mucus and microbiota in mice. Digestive Disease Week

Human gastric mucin MUC5AC is secreted in the colonic mucus of cancer patients and is a specific marker of precancerous lesions called aberrant crypt foci. Using MUC5AC as a specific marker can improve sensitivity in the detection of early colorectal cancer. Here we demonstrated that the accumulation of MUC5AC in xenograft and mouse stomach can be detected by magnetic resonance imaging (MRI). We used ultrasmall particles of iron oxide (USPIOs) conjugated with disulfide constrained heptapeptide that were identified using a screening phage display. To accomplish this, we employed positive selection of the phage display library on MUC5AC purified from fresh human colonic adenomas in combination with negative selection of the phage library on purified human MUC2, which is predominantly found in normal colorectal tissues. This conjugate was tested on human colorectal cancer cell lines that were either able or unable to secrete MUC5AC, both in vitro and in vivo. MUC5AC-USPIO contrast agent and USPIOs alone were not detected in cell lines unable to secrete MUC5AC. A combination of MRI and microscopy studies was performed to detect a specific accumulation of the contrast agent in vivo. Thus, the MUC5AC contrast agent enabled non-invasive detection of precancerous lesions and colorectal cancer, highlighting its potential use in diagnostics, in the early detection of colorectal cancer recurrences after treatment and in mechanistic studies implicating MUC5AC. Copyright © 2016 John Wiley & Sons, Ltd.

Helicobacter pylori binds to the gastric mucin, MUC5AC, and to trefoil factor, TFF1, which has been shown to interact with gastric mucin. We examined the interactions of TFF1 and H. pylori with purified gastrointestinal mucins from different animal species and from humans printed on a microarray platform to investigate whether TFF1 may play a role in locating H. pylori in gastric mucus. TFF1 bound almost exclusively to human gastric mucins and did not interact with human colonic mucins. There was a strong correlation between binding of TFF1 and H. pylori to human gastric mucins, and between binding of both TFF1 and H. pylori to gastric mucins with that of Griffonia simplicifolia lectin-II, which is specific for terminal non-reducing α- or β-linked N-acetyl-d-glucosamine. These results suggest that TFF1 may help to locate H. pylori in a discrete layer of gastric mucus and hence restrain their interactions with epithelial cells.

The gastrointestinal tract is lined by a thick and complex layer of mucus that protects the mucosal epithelium from biochemical and mechanical aggressions. This mucus barrier confers protection against pathogens but also serves as a binding site that supports a sheltered niche of microbial adherence. The carcinogenic bacteria Helicobacter pylori colonize the stomach through binding to host glycans present in the glycocalyx of epithelial cells and extracellular mucus. The secreted MUC5AC mucin is the main component of the gastric mucus layer, and BabA-mediated binding of H. pylori to MUC5AC confers increased risk for overt disease. In this study we unraveled the O-glycosylation profile of Muc5ac from glycoengineered mice models lacking the FUT2 enzyme and therefore mimicking a non-secretor human phenotype. Our results demonstrated that the FUT2 determines the O-glycosylation pattern of Muc5ac, with Fut2 knock-out leading to a marked decrease in a1,2-fucosylated structures and increased expression of the terminal type 1 glycan structure Lewis-a. Importantly, for the first time, we structurally validated the expression of Lewis-a in murine gastric mucosa. Finally, we demonstrated that loss of mucin FUT2-mediated fucosylation impairs gastric mucosal binding of H. pylori BabA adhesin, which is a recognized feature of pathogenicity. The authors thank Nuno Mendes for excellent technical support at the animal facility of IPATIMUP, University of Porto; Dr. Jacques Bara from the U-673 INSERM, CNRS, Paris, France for providing the PM7, 45M1 and 7LE antibodies; Dr Ingemar Carlstedt, University of Lund, Sweden for providing LUM6-3 and LUM5-1 antibodies and Dr. Ola Söderberg from the Uppsala Universitet, Sweden for support with the PLA experiments. This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and the projects with the references FCOMP-01-0124-FEDER 028188 and FCOMP-01-0124-FEDER041276 (EXPL/CTM-BIO/0762/2013, PTDC/BBB-EBI/0786/2012 and PTDC/BBB-EBI/0567/2014). The authors acknowledge the support by Gastric Glyco Explorer Initial Training Network (Seventh Framework Programme GastricGlycoExplorer project, grant number 316929). AM received an individual grant (SFRH/BPD/75871/2011) from FCT, POPH (Programa Operacional Potencial Humano) and FSE (Fundo Social Europeu) and acknowledges EMBO for a Short-Term Fellowship (EMBO ASTF 330-212). The resources provided by the Consortium for Functional Glycomics were funded by NIGMS-GM62116. TB is supported by grants from Vetenskapsrådet/VR, Cancerfonden, and the J.C. Kempe and Seth M. Kempe Memorial Foundation and this work was in part performed within the Umeå Centre for Microbial Research (UCMR), and the Biochemical Imaging Center Umeå (BICU).

Human gastric mucin MUC5AC is secreted in the colonic mucus of cancer patients and is a specific marker of precancerous lesions called aberrant crypt foci. Using MUC5AC as a specific marker can improve sensitivity in the detection of early colorectal cancer. Here we demonstrated that the accumulation of MUC5AC in xenograft and mouse stomach can be detected by magnetic resonance imaging (MRI). We used ultrasmall particles of iron oxide (USPIOs) conjugated with disulfide constrained heptapeptide that were identified using a screening phage display. To accomplish this, we employed positive selection of the phage display library on MUC5AC purified from fresh human colonic adenomas in combination with negative selection of the phage library on purified human MUC2, which is predominantly found in normal colorectal tissues. This conjugate was tested on human colorectal cancer cell lines that were either able or unable to secrete MUC5AC, both in vitro and in vivo. MUC5AC-USPIO contrast agent and USPIOs alone were not detected in cell lines unable to secrete MUC5AC. A combination of MRI and microscopy studies was performed to detect a specific accumulation of the contrast agent in vivo. Thus, the MUC5AC contrast agent enabled non-invasive detection of precancerous lesions and colorectal cancer, highlighting its potential use in diagnostics, in the early detection of colorectal cancer recurrences after treatment and in mechanistic studies implicating MUC5AC. Copyright © 2016 John WileySons, Ltd.

International audience; The present study aimed at detecting the exogenously applied probiotic Lactobacillus farciminis in rats, after exposure to IBS-like chronic stress, based on 4-day Water Avoidance Stress (WAS). The presence of L. farciminis in both ileal and colonic mucosal tissues was demonstrated by FISH and qPCR, with ileum as the preferential niche, as for the SFB population. A different spatial distribution of the probiotic was observed: in the ileum, bacteria were organized in micro-colonies more or less close to the epithelium whereas, in the colon, they were mainly visualized far away from the epithelium. When rats were submitted to WAS, the L. farciminis population substantially decreased in both intestinal regions, due to a stress-induced increase in colonic motility and defecation, rather than a modification of bacterial binding to the intestinal mucin Muc2.