Microbiota-derived indole differentially shapes Entamoeba histolytica physiology and promotes host-compatible colonization

Entamoeba histolytica is a pathogenic amoeba that colonizes the human large intestine and causes amoebiasis. In its natural gut environment, the parasite is exposed to microbiota-derived metabolites, including indole, a tryptophan-derived compound present at millimolar concentrations, whereas laboratory cultures contain negligible levels. We previously showed that bacterial metabolites such as queuine and oxaloacetate modulate parasite stress responses and virulence. Here, we investigated how acute versus long-term exposure to indole shapes E. histolytica physiology and host interactions. Trophozoites were gradually adapted to indole over two months. Proteomic profiling compared untreated (WT), acutely indole-exposed (WT + I), and indole-adapted (ADI) trophozoites. Cytoskeletal organization, motility, oxidative stress responses, and colonization capacity were assessed using imaging, functional assays, and a mouse cecum infection model. Host inflammatory responses were evaluated by measuring CXCL1 and lipocalin expression. Acute indole exposure inhibited parasite growth (IC₅₀ = 1.2 mM) and increased cytopathic activity. In contrast, ADI trophozoites displayed reduced cell size, increased F-actin formation, enhanced migration in vitro, and lower cytopathic activity. ADI trophozoites also showed improved survival following oxidative challenge, consistent with enrichment of oxidoreductases and chaperone-related proteins. In vivo, ADI trophozoites colonized the cecum more efficiently than WT or WT + I parasites. Infection with WT + I trophozoites induced higher CXCL1 and lipocalin expression than ADI trophozoites, indicating a stronger host inflammatory response to acutely indole-exposed parasites. These findings identify indole as an ecological cue that initially challenges E. histolytica but, upon sustained exposure, promotes adaptive reprogramming toward enhanced stress tolerance and improved host-compatible colonization.