Amebiasis is caused by the protozoan parasite Entamoeba histolytica (Eh), a potentially fatal disease occurring mainly in developing countries. How Eh interacts with innate host factors in the gut is poorly understood. Eh resides and feed in/on the outer colonic mucus layer and thus share an ecological niche with indigenous microbiota. As gut microbiota regulates innate immune responses, in this study we characterized the cooperative roles that microbiota and the mucus layer play in Eh-induced pro-inflammatory responses in the colon. To study this, we used antibiotics treated and non-treated specific pathogen free Muc2-/- and Muc2+/+ littermates and germ-free mice inoculated with Eh in colonic loops as a short infection model. In antibiotic treated Muc2-/- and Muc2+/+ littermates, Eh elicited robust mucus and water secretions, enhanced pro-inflammatory cytokines and chemokine expression with elevated MPO activity and higher pathology scores as compared to the modest response observed in non-antibiotic treated littermates. Host responses were microbiota specific as mucus secretion and pro-inflammatory responses were attenuated following homologous fecal microbial transplants in antibiotic-treated Muc2+/+ quantified by secretion of 3H-glucosamine newly synthesized mucin, Muc2 mucin immunostaining and immunohistochemistry. Eh-elicited pro-inflammatory responses and suppressed goblet cell transcription factor Math1 as revealed by in vivo imaging of Eh-colonic loops in Math1GFP mice, and in vitro using Eh-stimulated LS174T human colonic goblet cells. Eh in colonic loops increased bacterial translocation of bioluminescent E. coli and indigenous bacteria quantified by FISH and quantitative PCR. In germ-free animals, Eh-induced mucus/water secretory responses, but acute pro-inflammatory responses and MPO activity were severely impaired, allowing the parasite to bind to and disrupt mucosal epithelial cells. These findings have identified key roles for intestinal microbiota and mucus in regulating innate host defenses against Eh, and implicate dysbiosis as a risk factor for amebiasis that leads to exacerbated immune responses to cause life-threatening disease.

Background & Aims Alterations in intestinal MUC2 mucin and microbial diversity are closely linked with important intestinal pathologies; however, their impact on each other and on intestinal pathogenesis has been vaguely characterized. Therefore, it was of interest in this study to delineate distinct and cooperative function of commensal microbiota and the Muc2 mucus barrier in maintaining intestinal epithelial barrier function. Methods Muc2 mucin deficient ( Muc2 –/– ) and sufficient ( Muc2 +/+ ) littermates were used as a model for assessing the role of Muc2. To quantify the role of the microbiota in disease pathogenesis, Muc2 +/+ and Muc2 –/– littermates were treated with a cocktail of antibiotics that reduced indigenous bacteria, and then fecal transplanted with littermate stool and susceptibility to dextran sulphate sodium (DSS) quantified. Results Although, Muc2 +/+ and Muc2 –/– littermates share similar phyla distribution as evidenced by 16S sequencing they maintain their distinctive gastrointestinal phenotypes. Basally, Muc2 –/– showed low-grade colonic inflammation with high populations of inflammatory and tolerogenic immune cells that became comparable to Muc2 +/+ littermates following antibiotic treatment. Antibiotics treatment rendered Muc2 +/+ but not Muc2 –/– littermates highly susceptibility to DSS-induced colitis that was ILC3 dependent. Muc2 –/– microbiota was colitogenic to Muc2 +/+ as it worsened DSS-induced colitis. Microbiota dependent inflammation was confirmed by bone-marrow chimera studies, as Muc2 –/– receiving Muc2 +/+ bone marrow showed no difference in their susceptibility toward DSS induced colitis. Muc2 –/– microbiota exhibited presence of characteristic OTUs of specific bacterial populations that were transferrable to Muc2 +/+ littermates. Conclusions These results highlight a distinct role for Muc2 mucin in maintenance of healthy microbiota critical in shaping innate host defenses to promote intestinal homeostasis.

Entamoeba histolytica (Eh) is a protozoan parasite of humans that colonizes the outer colonic mucus layer. Under conditions not fully understood, Eh breaches innate host defenses and invades the intestinal mucosa-causing amebic colitis and liver abscess. In asymptomatic infection, Eh interacts with and feeds on resident microbiota that forms biofilms on the outer colonic mucus layer. Despite the close association between Eh and commensal microbiota, we still lack basic knowledge on whether microbiota and/or their metabolites influence Eh virulence traits critical in disease pathogenesis. In the pathogenesis of intestinal amebiasis, Eh overcomes the protective mucus layer using a combination of mucinase/glycosidase and potent mucus secretagogue activity. In this addendum, we discuss the interconnected role of a healthy mucus barrier and the role commensal microbiota play in shaping innate host defense against Eh-induced proinflammatory and secretory responses critical in disease pathogenesis.

Epidemiological studies suggest frequent association of enteropathogenic bacteria with Entamoeba histolytica during symptomatic infection. In this study, we sought to determine if the interaction with enteropathogenic (EPEC) or nonpathogenic Escherichia coli (strain DH5α) could modify the virulence of E. histolytica to cause disease in animal models of amebiasis. In vitro studies showed a 2-fold increase in CaCo2 monolayer destruction when E. histolytica interacted with EPEC but not with E. coli DH5α for 2.5 h. This was associated with increased E. histolytica proteolytic activity as revealed by zymogram analysis and degradation of the E. histolytica CP-A1/5 (EhCP-A1/5) peptide substrate Z-Arg-Arg-pNC and EhCP4 substrate Z-Val-Val-Arg-AMC. Additionally, E. histolytica-EPEC interaction increased EhCP-A1, -A2, -A4, and -A5, Hgl, Apa, and Cox-1 mRNA expression. Despite the marked upregulation of E. histolytica virulence factors, nonsignificant macroscopic differences in amebic liver abscess development were observed at early stages in hamsters inoculated with either E. histolytica-EPEC or E. histolytica-E. coli DH5α. Histopathology of livers of E. histolytica-EPEC-inoculated animals revealed foci of acute inflammation 3 h postinoculation that progressively increased, producing large inflammatory reactions, ischemia, and necrosis with high expression of il-1β, ifn-γ, and tnf-α proinflammatory cytokine genes compared with that in livers of E. histolytica-E. coli DH5α-inoculated animals. In closed colonic loops from mice, intense inflammation was observed with E. histolytica-EPEC manifested by downregulation of Math1 mRNA with a corresponding increase in the expression of Muc2 mucin and proinflammatory cytokine genes il-6, il-12, and mcp-1. These results demonstrate that E. histolytica/EPEC interaction enhanced the expression and production of key molecules associated with E. histolytica virulence, critical in pathogenesis and progression of disease.