Amoebiasis is an illness induced by the non-flagellated protozoan Entamoeba histolytica(Stanley, 2003; WHO/PAHO/UNESCO, 1997)ranking among the top fifteen causes of diarrhea in children up to two years of age. In certain regions of the world, the prevalence of this disease can reach 40%. However, due to its neglected status, accurate estimates defining the actual epidemiological parameters are lacking(Shirley et al., 2019).

Entamoeba disparwas recognized as a species in 1997(WHO/PAHO/UNESCO, 1997). Among the species of the genus Entamoeba, E. disparis the most prevalent (49.4%), followed by E. histolytica (32.3%) (dos Santos Zanetti et al., 2021).Despite being considered a non-invasive species, E. dispar has been detected in samples from patients with amoebic liver abscess(Ximénez et al., 2010), dysenteric colitis (Graffeo et al., 2014), non-dysenteric colitis(Costa et al., 2000)and polyxenic cultures were able to induce colitis(Prado et al., 2020) and amoebic liver abscess (ALA) in experimental animals(Costa et al., 2010).

Proteins present on the surface of trophozoites are associated with lesion development in the host(Blazquez et al., 2007; Espinosa-Cantellano and Martínez-Palomo, 2000; Ximénez et al., 2017). Although E. histolytica and E. dispar share 6,134 genes, the expression of surface proteins differs between the species, with the former expressing a higher number of proteins (Wilson et al., 2019). One of these proteins is the galactose and N-acetyl-D-galactosamine-binding lectin (Gal/GalNAc), which has fewer heavy subunit sequences in E. dispar compared to E. histolytica. It is utilized for the adhesion of trophozoites to the mucin layer and to the epithelial cells of the large intestine, inactivates complement proteins, and possesses lytic capacity, resulting in host cell death (Haque et al., 2003; Petri et al., 2002; Wilson et al., 2019).The Gal/GalNAc genes synthesizes a heterodimer composed of a light 35kDa, an intermediate 150kDa and a heavy 170kDa subunit, and it is associated with the production of invasive amoebiasis, similar to the cysteine proteinases gene (Wilson et al., 2019).

Cysteine proteinases (CPs) assist trophozoites in penetrating the intestinal mucosa by degrading mucoprotein 2 (MUC-2), a glycoprotein secreted by goblet cells that functions as a mechanical barrier of the villi. Additionally, CPs degrade elastin, fibrinogen, laminin, and collagen, facilitating the parasite's motility in the extracellular matrix. CPs also break down proteins such as the anaphylotoxins C3a and C5a, resulting in a reduction in the inflammatory response(Que and Reed, 1997; Reed et al., 1995; Seydel* et al., 1998).

Amoebapores are pore-forming proteins of 4-5 kDa and have three isoforms, A (APA), B (APB) and C (APC) (Leippe and Müller-Eberhard, 1994). They are structurally and functionally similar to NK-lysin, a polypeptide produced by natural killer cells and CD8+ T lymphocytes, capable of exertingcytolytic activity (BRUHN et al., 2003). Compared to NK-lysin, amoebapores have five times higher pore-forming capacity, attributed to their more hydrophobic structure(BRUHN et al., 2003).Additionally, they can induce lysis in various host cells, such as enterocytes and hepatocytes (Leippe et al., 1994).

Studies have demonstrated that bacteria from the host microbiota can interact with parasites, and this interaction could influence the parasite-host relationship(Yurchenko and Lukeš, 2018). In fact, an experimental study involving monoxenic and polyxenicE. disparstrains found that the polyxenic culture, where bacteria from the indigenous microbiota of the patient from which it was isolated are also present, was able to induce cytopathic effects and produce experimental lesions in hamsters, likeE. histolytica(Costa et al., 2006). These observations reinforce the idea that bacteria from the gut microbiota and even exogenous bacteria could influence E. dispar trophozoites. The association between microorganisms can be perceived as a community of species experiencing different selection pressures. An organism within a host can be mutualistic (both benefiting from the relationship), parasitic (one-sided benefits, where the parasite spoliates the host), or commensal (one-sided benefits, but without spoliation of the host)(Dheilly et al., 2019).

EnteropathogenicEscherichia coli (EPEC) is a Gram-negative bacterium that causes diarrhea(Clarke et al., 2003; Deborah Chen and Frankel, 2005; Pires et al., 2015).As EPEC can be isolated from various sources, including processed and raw foods, domestic and wild animals, and natural environments such as water and soil, host infection can occur easily(Souza et al., 2016). Experimental models of co-infection involving EPEC with other pathogens, aimed at understanding the consequences of this association, have sparked the interest of researchers(Fernández-López et al., 2019; Galván-Moroyoqui et al., 2008; Gorelik et al., 2019; Pakpinyo et al., 2003).Studies have indicated that symbiosis between bacteria and amoebas can modulate phenotypes, virulence properties and the pathogenicity of amoebas(Bhattacharya et al., 1992; Costa et al., 2006; Oliveira et al., 2021; Shibayama; et al., 2007). However, the interaction process of Entamoeba sp. with other microorganisms is not yet fully understood, including its consequences. Our research group recently discovered that the interaction between E. dispar and the enterobacterium Salmonella typhimuriumincreased the gene expression of the amoebic virulence factors such as amoebapore A, cysteine proteinase-5 and Gal/GalNAc-binding lectin. Additionally, an increased intensity of ulcerative amoebic colitis was observed, characterized by the production of larger zones of erosion and ulceration of the intestinal wall, heightened intensity of the inflammatory infiltrate, accompanied by an increased expression of the pro-inflammatory mediators cyclooxygenase 2, IL-1, and TNF-α(Oliveira et al., 2021).

There are still numerous unanswered questions regarding the symbiosis between bacteria and amoebae and its potential impact on the virulence and pathogenicity of trophozoites in amoebiasis, particularly in the context of the experimental lesions produced by E. dispar. In this study, we assessed the influence of Enteropathogenic Escherichia coli (EPEC) on ACFN and ADO strains of E. dispar by examining the gene expression of amoebic virulence factors both in vitro and in an experimental model of amoebic liver abscess. Our findings reveal that E. dispar strains exhibit distinct responses to interaction with EPEC. We observed differing behavior in the gene expression of virulence factors and, consequently, in the ability to induce lesions when EPEC interacts with ACFN and ADO strains of E. dispar.