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Intestinal mucins play a crucial role in the mucosal barrier, serving as the body’s initial defense against microorganisms. However, how the host regulates the secretion and glycosylation of these mucins in response to bacterial invasion remains unclear. Our study demonstrates that when exposed to Streptococcus gallolyticus (SGG), a gut pathobiont, the host mucosa promptly adjusts the behavior of specialized goblet cells (GCs) located in the middle of the crypts. A subset of these cells undergoes a remodeling, becoming intercrypt goblet cells (icGCs), which do not detach from the surface but instead migrate along intercrypt spaces while secreting a mucus impermeable to bacterial pathogens. Significantly, a non-piliated SGG mutant unable to bind to mucus fails to induce icGCs, allowing its translocation through the mucosa and submucosa. Interestingly, a closely related nonpathogenic bacterium, SGM, able to bind to mucus, also triggers the differentiation of GCs into icGCs. This discovery opens new avenues for treating patients with a “leaky gut” as observed in intestinal diseases such as inflammatory bowel diseases and metabolic disorders, but also patients with a history of repeated antibiotic use. Utilizing mucus-adherent probiotics to induce icGCs represents a promising strategy for reinforcing the mucosal barrier.

Meningococcus utilizes β-arrestin selective activation of endothelial cell β2 adrenergic receptor (β2AR) to cause meningitis in humans. Here authors report that Meningococcus triggers β2AR signaling by exerting forces on β2AR glycans that terminally expose N-acetyl-neuraminic acid (sialic acid, Neu5Ac) residues.

Abstract 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.

ABSTRACT 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. IMPORTANCE N. 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.

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.

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. [ABSTRACT FROM AUTHOR]

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.

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.

Abstract 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.

Our studies provide direct evidence that O-glycosylation pathways play a role in the regulation of cell growth through apoptosis and proliferation pathways. A series of small molecular weight analogs of the GalNAc-α-1-O-serine/threonine structure based on 1-benzyl-2-acetamido-2-deoxy-α-O-d-galactopyranoside have been synthesized and tested in the human colorectal cancer cell lines PC/AA/C1/SB10C and HCA7/C29. Three inhibitors, 1-benzyl-2-acetamido-2-deoxy-α-O-d-galactopyranoside, and the corresponding 2-azido- and C-glycoside analogs were screened in these colorectal cancer cell lines at 0.5 mM and showed induction of apoptosis and downregulation of proliferation. Treatment of both cell lines with inhibitors led to changes in glycosylation detected with peanut lectin. The inhibition of glycosyltransferase activity in cell homogenates from human colorectal mucosal cells and cultured cell lines could be shown. The competitive action of the inhibitors resulted in the intracellular formation of 28 aryl-glycan products which were identified by MALDI and electrospray mass spectroscopy. The structures showed a differential pattern for each of the inhibitors in both cell lines. Gene array analysis of the glycogenes illustrated a pattern of glycosyltransferases that matched the glycan structures found in glycoproteins and aryl-glycans formed in the PC/AA/C1/SB10C cells; however, there was no action of the three inhibitors on glycogene transcript levels. The inhibitors act at both intermediary metabolic and genomic levels, resulting in altered protein glycosylation and aryl-glycan formation. These events may play a part in growth arrest. [ABSTRACT FROM AUTHOR]