Airway environment drives the selection of quorum sensing mutants and promote Staphylococcus aureus chronic lifestyle.

Staphylococcus aureus is a predominant cause of chronic lung infections. While the airway environment is rich in highly sialylated mucins, the interaction of S. aureus with sialic acid is poorly characterized. Using S. aureus USA300 as well as clinical isolates, we demonstrate that quorum-sensing dysfunction, a hallmark of S. aureus adaptation, correlates with a greater ability to consume free sialic acid, providing a growth advantage in an air-liquid interface model and in vivo. Furthermore, RNA-seq experiment reveals that free sialic acid triggers transcriptional reprogramming promoting S. aureus chronic lifestyle. To support the clinical relevance of our results, we show the co-occurrence of S. aureus, sialidase-producing microbiota and free sialic acid in the airway of patients with cystic fibrosis. Our findings suggest a dual role for sialic acid in S. aureus airway infection, triggering virulence reprogramming and driving S. aureus adaptive strategies through the selection of quorum-sensing dysfunctional strains.

Global proteomic identifies multiple cancer-related signaling pathways altered by a gut pathobiont associated with colorectal cancer.

In this work, we investigated the oncogenic role of Streptococcus gallolyticus subsp. gallolyticus (SGG), a gut bacterium associated with colorectal cancer (CRC). We showed that SGG UCN34 accelerates colon tumor development in a chemically induced CRC murine model. Full proteome and phosphoproteome analysis of murine colons chronically colonized by SGG UCN34 revealed that 164 proteins and 725 phosphorylation sites were differentially regulated. Ingenuity Pathway Analysis (IPA) indicates a pro-tumoral shift specifically induced by SGG UCN34, as ~ 90% of proteins and phosphoproteins identified were associated with digestive cancer. Comprehensive analysis of the altered phosphoproteins using ROMA software revealed up-regulation of several cancer hallmark pathways such as MAPK, mTOR and integrin/ILK/actin, affecting epithelial and stromal colonic cells. Importantly, an independent analysis of protein arrays of human colon tumors colonized with SGG showed up-regulation of PI3K/Akt/mTOR and MAPK pathways, providing clinical relevance to our findings. To test SGG's capacity to induce pre-cancerous transformation of the murine colonic epithelium, we grew ex vivo organoids which revealed unusual structures with compact morphology. Taken together, our results demonstrate the oncogenic role of SGG UCN34 in a murine model of CRC associated with activation of multiple cancer-related signaling pathways.

Human Fecal Mucin Glycosylation as a New Biomarker in Inflammatory Bowel Diseases.

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.

Evidence of early increased sialylation of airway mucins and defective mucociliary clearance in CFTR-deficient piglets.

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.

Zinc Deficiency Disturbs Mucin Expression, O-Glycosylation and Secretion by Intestinal Goblet Cells.

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.

The expression and functional analysis of the sialyl-T antigen in prostate cancer.

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 Neu5Acα2-3Galß1-3GalNAc- (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.

Receptor recognition by meningococcal type IV pili relies on a specific complex N-glycan.

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.

Airway Mucus Restricts Neisseria meningitidis Away from Nasopharyngeal Epithelial Cells and Protects the Mucosa from Inflammation.

Neisseria meningitidis is an inhabitant of the nasopharynx, from which it is transmitted from person to person or disseminates in blood and becomes a harmful pathogen. In this work, we addressed colonization of the nasopharyngeal niche by focusing on the interplay between meningococci and the airway mucus that lines the mucosa of the host. Using Calu-3 cells grown in air interface culture (cells grown with the apical domain facing air), we studied meningococcal colonization of the mucus and the host response. Our results suggested that N. meningitidis behaved like commensal bacteria in mucus, without interacting with human cells or actively transmigrating through the cell layer. As a result, type IV pili do not play a role in this model, and 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 of N. meningitidis with other bacteria living in the nasopharynx and that Streptococcus mitis, but not Moraxella catarrhalis, can promote meningococcal growth in this model.IMPORTANCEN. meningitidis is transmitted from person to person by aerosol droplets produced by breathing, talking, or coughing or by direct contact with a contaminated fluid. The natural reservoir of N. meningitidis is the human nasopharynx mucosa, located at the back of the nose and above the oropharynx. The means by which meningococci cross the nasopharyngeal wall is still under debate, due to the lack of a convenient and relevant model mimicking the nasopharyngeal niche. Here, we took advantage of Calu-3 cells grown in air interface culture to study how meningococci colonize the nasopharyngeal niche. We report that the airway mucus is both a niche for meningococcal growth and a protective barrier against N. meningitidis infection. As such, N. meningitidis behaves like commensal bacteria and is unlikely to induce infection without an external trigger.

Mucin CYS domain stiffens the mucus gel hindering bacteria and spermatozoa.

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.

Sialic acid mediated mechanical activation of ß2 adrenergic receptors by bacterial pili.

Meningococcus utilizes ß-arrestin selective activation of endothelial cell ß2 adrenergic receptor (ß2AR) to cause meningitis in humans. Molecular mechanisms of receptor activation by the pathogen and of its species selectivity remained elusive. We report that ß2AR activation requires two asparagine-branched glycan chains with terminally exposed N-acetyl-neuraminic acid (sialic acid, Neu5Ac) residues located at a specific distance in its N-terminus, while being independent of surrounding amino-acid residues. Meningococcus triggers receptor signaling by exerting direct and hemodynamic-promoted traction forces on ß2AR glycans. Similar activation is recapitulated with beads coated with Neu5Ac-binding lectins, submitted to mechanical stimulation. This previously unknown glycan-dependent mode of allosteric mechanical activation of a G protein-coupled receptor contributes to meningococcal species selectivity, since Neu5Ac is only abundant in humans due to the loss of CMAH, the enzyme converting Neu5Ac into N-glycolyl-neuraminic acid in other mammals. It represents an additional mechanism of evolutionary adaptation of a pathogen to its host.

Characterization of Mucus-Related Properties of Streptococcus thermophilus: From Adhesion to Induction.

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.

Food-grade TiO2 is trapped by intestinal mucus in vitro but does not impair mucin O-glycosylation and short-chain fatty acid synthesis in vivo: implications for gut barrier protection.

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.

Binding of Helicobacter pylori to Human Gastric Mucins Correlates with Binding of TFF1.

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 ß-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.

A Sensitive and Rapid Method to Determin the Adhesion Capacity of Probiotics and Pathogenic Microorganisms to Human Gastrointestinal Mucins.

Mucus is the habitat for the microorganisms, bacteria and yeast that form the commensal flora. Mucins, the main macromolecules of mucus, and more specifically, the glycans that cover them, play essential roles in microbial gastrointestinal colonization. Probiotics and pathogens must also colonize mucus to have lasting positive or deleterious effects. The question of which mucin-harboured glycan motifs favour the adhesion of specific microorganisms remains very poorly studied. In the current study, a simple test based on the detection of fluorescent-labeled microorganisms raised against microgram amounts of mucins spotted on nitrocellulose was developed. The adhesion of various probiotic, commensal and pathogenic microorganisms was evaluated on a panel of human purified gastrointestinal mucins and compared with that of commercially available pig gastric mucins (PGM) and of mucins secreted by the colonic cancer cell line HT29-MTX. The latter two proved to be very poor indicators of adhesion capacity on intestinal mucins. Our results show that the nature of the sialylated cores of O-glycans, determined by MALDI MS-MS analysis, potentially enables sialic acid residues to modulate the adhesion of microorganisms either positively or negatively. Other identified factors affecting the adhesion propensity were O-glycan core types and the presence of blood group motifs. This test should help to select probiotics with enhanced adhesion capabilities as well as deciphering the role of specific mucin glycotopes on microbial adhesion.

How do they stick together? Bacterial adhesins implicated in the binding of bacteria to the human gastrointestinal mucins.

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.

The Pil3 pilus of Streptococcus gallolyticus binds to intestinal mucins and to fibrinogen.

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.

Muc5ac gastric mucin glycosylation is shaped by FUT2 activity and functionally impacts Helicobacter pylori binding.

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 α1,2-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 opgC gene is required for OPGs succinylation and is osmoregulated through RcsCDB and EnvZ/OmpR in the phytopathogen Dickeya dadantii.

Osmoregulated periplasmic glucans (OPGs) are a family of periplasmic oligosaccharides found in the envelope of most Proteobacteria. They are required for virulence of zoo- and phyto-pathogens. The glucose backbone of OPGs is substituted by various kinds of molecules depending on the species, O-succinyl residues being the most widely distributed. In our model, Dickeya dadantii, a phytopathogenic bacteria causing soft rot disease in a wide range of plant species, the backbone of OPGs is substituted by O-succinyl residues in media of high osmolarity and by O-acetyl residues whatever the osmolarity. The opgC gene encoding a transmembrane protein required for the succinylation of the OPGs in D. dadantii was found after an in silico search of a gene encoding a protein with the main characteristics recovered in the two previously characterized OpgC of E. coli and R. sphaeroides, i.e. 10 transmembrane segments and one acyl-transferase domain. Characterization of the opgC gene revealed that high osmolarity expression of the succinyl transferase is controlled by both the EnvZ-OmpR and RcsCDB phosphorelay systems. The loss of O-succinyl residue did not affect the virulence of D. dadantii, suggesting that only the glucose backbone of OPGs is required for virulence.

Early detection of colonic dysplasia by magnetic resonance molecular imaging with a contrast agent raised against the colon cancer marker MUC5AC.

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.