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

Abstract

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.

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

“…Besides, 12% long O -glycans based on the core 2 type (NeuAcα2-3Galβ1-3(NeuAcα2-3Galβ1-4GlcNAcβ1-6)GalNAc; m / z 1705) were found. Together with a small percentage of sialylated Tn antigens (NeuAcα2-6GalNAc), 79.8% of glycans produced by this cell line are sialylated, confirming previous findings [ 42 , 43 ]. Secreted mucins are indeed highly acidic, as already demonstrated with AB staining.…”

“…To investigate whether the lack of zinc alters mucin O -glycosylation, O -glycan pattern of mucins secreted by HT-29-MTX was analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) ( Figure 6 ; structures of detected O -glycans are summarized in Supplementary Table S2 ). Post-confluent zinc-sufficient HT-29-MTX cells ( Figure 6 A) mainly secreted mucins based on Thomsen-Friedenreich (TF) antigens (Galβ1-3GalNAc), a core 1 type mucin, similar to a recent study with differentiating HT-29-MTX [ 42 ]. A total of 55.4% of the detected O -glycans in supernatants of CTR cells were sialylated (NeuAcα2-3Galβ1-3GalNAc or Galβ1-3(NeuAcα2-6)GalNAc; m / z 895) and 10.1% disialylated TF antigens (NeuAcα2-3Galβ1-3(NeuAcα2-6)GalNAc; m / z 1256).…”

“…Twenty-four hours before collecting secreted mucins, medium was changed to DMEM w/o phenol red and 0% FCS. Supernatants were purified and oligosaccharides were released from mucins by alkaline borohydride treatment as described [ 42 ]. After the permethylation of oligosaccharides, the O -glycosylation of mucins was analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry in the positive ion mode [ 42 ].…”

Improved Gastric Acid Resistance and Adhesive Colonization of Probiotics by Mucoadhesive and Intestinal Targeted Konjac Glucomannan Microspheres

“…Furthermore, the number of WCFS1 remained on the OKGM or sOKGM films after the incubation and elution was counted. [38] The retention rates of probiotics on OKGM or sOKGM film were 83.0% and 97.2%, respectively (Figures S5 and 4F, Supporting Information), suggesting a stronger interaction between WCFS1 with sOKGM polymers, consistent with ITC results. The enhanced adhesion of WCFS1 to sOKGM film was further confirmed by the higher fluorescent intensity of Syto 9 stained WCFS1 remained on sOKGM film than OKGM film ( Figure 4G).…”

Special Issue: Gut Bacteria-Mucus Interaction

“…Four papers specifically address bacterial adhesion to mucus and mucin glycans using different methodologies including atomic force microscopy (AFM) and optical tweezers (OT) [3], carbohydrate or mucin microarrays [4], and in vitro adhesion plate assays [5] or slot-blot analysis [6] using fluorescent-labelled microorganisms. These articles, focusing on probiotic species such as Lactobacillus acidophilus [5] or pathogens species such as Helicobacter pylori [4] highlight how the origin, chemical structure and glycosylation pattern of different mucins as determined by MALDI MS-MS analysis may affect recognition by pathogenic or commensal bacteria [6]. This adhesion is often driven by several mechanisms such as cell-surface glycans or proteins that act in parallel or in a consecutive manner [3].…”