Poster Session I - A28 MUCUS FROM A HYPERSECRETORY MUTANT GOBLET-LIKE CELL INCREASES ENTAMOEBA HISTOLYTICA VIRULENCE AND CYTOTOXICITY

TL;DR

This study shows that altered mucus from hypersecretory goblet cells increases the virulence and cytotoxicity of the parasite Entamoeba histolytica.

Contribution

The study reveals how changes in MUC2 mucus glycosylation impact the host defense against Entamoeba histolytica.

Findings

Hypersecretory MUC2 mucus is more adherent to Entamoeba histolytica but fails to protect epithelial cells.

Mutant mucus is easily ingested by the parasite, leading to increased expression of virulence genes.

Altered mucus glycans compromise barrier function and promote parasite-induced cytotoxicity.

Abstract

MUC2 mucin is produced by colonic goblet cells and forms a mucus bilayer that provides a physical barrier between potential pathogens in the lumen and the underlying epithelial cells. MUC2 is heavily glycosylated with a variety of glycans that imparts its functional properties. Mucus is the first line of innate host defense in the gut that colonic pathogens such as the parasite Entamoeba histolytica (Eh) degrades to cause amoebic colitis. As modifications in MUC2 mucus structure/function is a predisposing factor in Eh disease pathogenesis, we investigated how alterations in MUC2 glycans affected epithelial barrier functions against Eh.

The specific aims are:

1. To quantify hypersecretory mucus barrier protection in response to Eh

2. To quantify Eh virulence factor expression in response to hypersecretory MUC2 mucin

Two goblet cells lines were used; WT LS174T cells and a hypersecretory Mut LS174T clone selected using an unbiased screen. Whole genome sequencing revealed that Mut cells had mutations that upregulated the expression of TP53, AGR2 and MEK/MAPK that enhanced MUC2 production with an altered glycomics profile. MUC2 secretion in WT and Mut cells was quantified by 3H-metabolically labeled mucus secretion in response to Eh. Integrity of the mucus layer was enumerated by Eh adherence to and cytotoxicity of cells measured by LDH release. Eh degradation of mucus was quantified using mucin specific antibodies against MUC2 protein and glycans by immunoblotting. Eh mucophagy of WT or Mut mucus was quantified by confocal microscopy and imaging flow cytometry. Following mucophagy, modulation of Eh virulence genes was measured by RT-PCR and enzymatic activity quantified using enzyme specific substrates.

Mut cells constitutively produced and secreted significantly more MUC2 mucin in response to Eh as compared to WT cells. Even though Mut mucus was more adherent to Eh than WT mucus it was not protective resulting in increased cell cytotoxicity. Mut mucus was dispersed and easily disrupted and ingested (mucophagy) by Eh as compared to dense packed polymeric WT mucus. The higher ingestion of Mut mucus by Eh significantly upregulated parasite virulence genes with increased enzymatic activity of secreted proteinases that degraded Mut mucus in a time- and dose-dependent manner.

This study highlights the importance of mucus glycans in conferring structural and functional integrity of the mucus gel that provide barrier protective properties to the colonic epithelium. Understanding both quality and quantity of mucus may be an important predisposing factor against pathogen invasion and in GI disorders.

CIHR