CO‐INFECTION WITH GIARDIA DUODENALIS PROTECTS THE HOST AGAINST ENTEROPATHOGENIC ESCHERICHIA COLI VIA NLRP3 INFLAMMASOME‐DEPENDENT ANTI‐MICROBIAL PEPTIDE PRODUCTION
Authors: Manko Anna , Motta Jean‐Paul , Cotton James , Oyeyemi Ayodele , Vallance Bruce , Beck Paul , Wallace John , Buret Andre 
Published: November 2023

BackgroundInfectious diarrheal disease represents a critical concern for child health in developing countries, and often occurs in the context of polymicrobial infections. Mechanisms whereby concurrent infections may alter clinical disease outcome remain obscure. Recent findings indicate that giardiasis could protect against pediatric diarrhea, but the mechanisms are unknown. We hypothesized that Giardia was able to change the pathogenic outcome of bacterial enteritis, either directly or indirectly.AimTo define the mechanisms by which cysteine protease(s) released by Giardia activate the NLPR3 inflammasome pathway to increase antimicrobial peptide (AMP) production during co‐infection with enteropathogenic Escherichia coli (EPEC).MethodsMice (wild type or NLRP3−/−) were infected with G. muris and/or Citrobacter rodentium to model co‐infections with Giardia and EPEC in humans. AMP production, bacterial pathogen burdens, and colonic disease activity were assessed. Human enterocytes (Caco‐2) were pretreated or not with glyburide (NLRP3 inhibitor; 100 mm, 30 min), and infected with Giardia duodenalis trophozoites (with or without pre‐treatment with cathepsin B‐like inhibitors) and EPEC separately or in combination. Human β‐defensin‐2 (HBD‐2), mouse β‐defensin‐3 (MBD‐3) and trefoil‐factor 3 (TFF3) protein and mRNA expression were assessed by immunofluorescent (IF) staining and by RT‐qPCR, respectively. Colonic Caspase‐1 and ‐11 protein levels were assessed by Western blot. Direct anti‐bacterial effects of Giardia were assessed in vitro.ResultsInfection with G. muris increased colonic β‐defensin and TFF3 expression, inhibited bacterial colonization, and reduced disease activity in co‐infected animals. These effects were lost in NLRP3 −/− mice. HBD‐2 and TFF3 IF staining intensity was highest in co‐infected epithelial cells. HBD‐2 and TFF3 mRNA levels were highest in co‐infected enterocytes, and the effect was lost upon glyburide treatment. Caspase‐1 and ‐11 protein levels were highest in co‐infected mice when compared to animals given C. rodentium alone. Pre‐treatment of Giardia trophozoites with a selective cathepsin B‐like inhibitor abolished the effects of G. intestinalis. Moreover, G. intestinalis directly inhibited EPEC growth in vitro, in a cathepsin B‐like‐dependent manner.ConclusionsCo‐infection with Giardia during EPEC infection activates the NLRP3 inflammasome, and increases AMP production in human enterocytes. Giardia cathepsin B‐like proteases contribute to this AMP‐mediated protective effect, which is partially NLRP3‐dependent. Giardia cathepsin‐like proteases have direct anti‐bacterial properties. Our data suggest a novel role for the inflammasome in the production of AMP, and reveals its protective effects using models of co‐infection with Giardia.Support or Funding InformationFunding for this research was provided by a discovery grant from the Natural Sciences and Engineering Research Council of Canada, and by a NSERC CREATE grant on Host‐Parasite Interactions.