<i>Giardia duodenalis</i> Alters Intestinal Mucin Transcription and Disrupts the Mucus Layer in a Cysteine Protease‐dependent Manner

Objective Giardia duodenalis is a diarrheagenic protozoan parasite that infects humans and animals across the world. Giardiasis is responsible for both acute and chronic illnesses, including post‐infectious irritable bowel syndrome (PI‐IBS) and extra‐intestinal complications. The mechanisms by which these occur remain unclear. Gastrointestinal mucus is mostly composed of MUC5AC mucin in the stomach and MUC2 in the intestine. We hypothesized that Giardia was able to overcome the protective functions of mucus to facilitate the onset and propagation of disease. Our aim was to assess whether and how Giardia proteases may impair the protective function of mucus. Methods Muc2−/− (KO‐inf) and C57BL/6 (WT‐inf) mice were gavaged with Giardia trophozoites. Seven days post‐infection, tissues were collected for histology, qPCR, and analysis of bacterial translocation. Human colonic biopsies from healthy donors were exposed to Giardia trophozoites, and amounts of intracellular mucins were quantified. In vitro , Giardia trophozoites were incubated with a human mucus‐producing cell line, LS174T, to assess intracellular mucin content and MUC2 transcription. Trophozoites were incubated with or without E64 inhibitor to assess the role of its cysteine proteases. Lastly, Giardia 's secreted products were incubated with purified human MUC2 to assess proteolysis via western blot. Results KO‐inf mice had a higher parasitic load compared to WT‐inf mice and lost weight over the period of infection. Compared to non‐infected mice, WT‐inf mice exhibited novel transcription of Muc5ac and decreased transcription of Muc2 in the jejunum. Both Muc2 and Muc5ac mRNA were increased in the colon. Muc5ac transcription was not induced in KO‐inf mice. WT‐inf mice had larger colonic mucus granules but a thinner mucus layer as demonstrated by staining with periodic‐acid Schiff/Alcian blue, wheat germ agglutinin, and fluorescent in situ hybridization for the location of commensal bacteria. Infected mice had bacterial translocation into the liver and spleen. Giardia spent media degraded human MUC2 in vitro . This was attenuated by E64. Human colonic biopsies exposed to Giardia had less intracellular mucin content. Giardia reduced intracellular mucin content in LS174T cells after 20, 40, and 60 minutes post‐infection, whereas it increased mucin content by 3 hours post‐infection. These effects were cysteine protease‐dependent and involved cellular protein kinase C. In vitro , Giardia increased MUC2 transcription after 1 hour post‐infection in a cysteine protease‐dependent manner. Conclusions Muc2 protects against Giardia accumulation and induced weight loss. Giardia disrupts this mucosal protective barrier by direct proteolysis of the mucus layer and by altering mucin gene transcription‐specifically an increase of Muc5ac and Muc2 in the colon, and a decrease of Muc2 in the jejunum. The resultant loss of the mucus barrier was associated with increased bacterial translocation. Giardia ‐induced alterations in mucin transcription, in addition to mucin degradation and depletion, were attenuated by a cysteine protease inhibitor, indicating a potential target to reduce Giardia ‐induced pathology. Future research is needed to determine how these effects may contribute to acute and chronic disease. Support or Funding Information Host‐Parasite Interactions NSERC CREATE and the Canadian Association of Gastroenterology

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