Mucin CYS domain stiffens the mucus gel hindering bacteria and spermatozoa

Bastien Demouveaux,Tetsuharu Narita,Valérie Gouyer,Jean-Luc Desseyn,Catherine Robbe-Masselot,Frédéric Gottrand

doi:10.1038/s41598-019-53547-x

Abstract

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.

Highlights

Mucus is the first biological barrier encountered by particles and pathogenic bacteria at the surface of secretory epithelia
The aim of this study was to probe the potential of a molecule made of 12 consecutive identical copies of the CYS domain in stiffening mucus from HT29-MTX cells (MTX) secreting mainly the respiratory Gel-forming mucins (GFMs) MUC5AC (9 CYS domains), and mucus from scrapped mouse colonic mucosa for which Muc[2] is the major, if not the only one, GFM
Recombinant CYSx12 production was checked in the MTX:MTX-rCYSx12 cocultures using immunofluorescence and Western blotting as described in the Online Resource 1 section with an antibody that recognizes a short peptide of the CYS domains Nos. 2, 3, and 5 of MUC5B27

Summary

Introduction

Mucus is the first biological barrier encountered by particles and pathogenic bacteria at the surface of secretory epithelia. The CYS domain is a good candidate to support low energy interactions between GFMs and/or mucus constituents. There are five human GFMs named MUC2, MUC5AC, MUC5B, MUC6, and MUC19, which form the mucus protein scaffold These mucins are conserved in mice (Muc[2], Muc5ac, Muc5b, Muc[6] and Muc[19]). The CYS domain intersects the central part of GFMs resulting in an alternation between hydrophilic highly O-glycosylated regions and hydrophobic CYS domains This supports a possible role in mucus architecture by non-covalent self-associations and www.nature.com/scientificreports interactions with other molecules of the mucus content such as lipids[5,15,16,19,21,22,23]. We suggested that the CYS domain regulates mucin trafficking through the presence of C-mannose[24]

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