Acetylcholine Upregulates Entamoeba histolytica Virulence Factors, Enhancing Parasite Pathogenicity in Experimental Liver Amebiasis

Abstract

Entamoeba histolytica is an invasive enteric protozoan, whose infections are associated to high morbidity and mortality rates. However, only less than 10% of infected patients develop invasive amebiasis. The ability of E. histolytica to adapt to the intestinal microenvironment could be determinant in triggering pathogenic behavior. Indeed, during chronic inflammation, the vagus nerve limits the immune response through the anti-inflammatory reflex, which includes acetylcholine (ACh) as one of the predominant neurotransmitters at the infection site. Consequently, the response of E. histolytica trophozoites to ACh could be implicated in the establishment of invasive disease. The aim of this study was to evaluate the effect of ACh on E. histolytica virulence. Methods include binding detection of ACh to plasma membrane, quantification of the relative expression of virulence factors by RT-PCR and western blot, evaluation of the effect of ACh in different cellular processes related to E. histolytica pathogenesis, and assessment of the capability of E. histolytica to migrate and form hepatic abscesses in hamsters. Results demonstrated that E. histolytica trophozoites bind ACh on their membrane and show a clear increase of the expression of virulence factors, that were upregulated upon stimulation with the neurotransmitter. ACh treatment increased the expression of L220, Gal/GalNAc lectin heavy subunit (170 kDa), amebapore C, cysteine proteinase 2 (ehcp-a2), and cysteine proteinase 5 (ehcp-a5). Moreover, erythrophagocytosis, cytotoxicity, and actin cytoskeleton remodeling were augmented after ACh treatment. Likewise, by assessing the formation of amebic liver abscess, we found that stimulated trophozoites to develop greater hamster hepatic lesions with multiple granulomas. In conclusion, ACh enhanced parasite pathogenicity by upregulating diverse virulence factors, thereby contributing to disease severity, and could be linked to the establishment of invasive amebiasis.

The Gal/GalNac lectin as a possible acetylcholine receptor in Entamoeba histolytica

“…The relative expression of genes coding for the intermediate and heavy subunit of the Gal/GalNAc lectin (150 and 170 kDa) ( Table 1 ) important participants in amoebic pathogenesis was evaluated; expression levels were normalized to the α-tubulin gene. It has been reported that the relative expression of these subunits increases after interaction with exogenous ACh ( Medina-Rosales et al., 2021 ). Furthermore, trophozoites inhibited at the Gal/GalNAc lectin binding sites with N-acetylgalactosamine and interacted with ACh did not express the 170 kDa and 150 kDa genes compared to the control; instead, trophozoites interacted directly with ACh did show an increased relative expression of the Gal/GalNAc lectin genes (150 and 170 kDa) ( Figure 5 ).…”

“…Therefore the presence of ACh at the site of infection can affect the elimination of the parasite from the body. In a previous study, our research group determined that the interaction between ACh and E. histolytica causes an increase in its virulence factors such as adhesion, synthesis, and secretion of amebapore C, and cysteine proteinase 2 ( ehcp-a2 ) and cysteine proteinase 5 ( ehcp-a5 ) and a significant increase in amebic liver abscess in hamsters ( Medina-Rosales et al, 2021 ).…”

“…Acetylcholine (ACh) has also been reported as a modulator of E. histolytica virulence; it is the main neurotransmitter in the colon and, during the inflammatory process, is released by the enteric autonomic nervous system to regulate the immune response, studies carried out with trophozoites stimulated with exogenous ACh evidenced a significant increase in virulence mechanisms such the adhesion, synthesis and secretion of C amebapores, cysteine proteinase 2 ( ehcp-a2 ) and cysteine proteinase 5 ( ehcp-a5 ); moreover, in the model of amoebic liver abscess in hamsters, an excellent progression of lesions was shown ( Medina-Rosales et al., 2021 ).…”

Amebic liver abscess by Entamoeba histolytica

“…E. histolytica is an anaerobic parasitic invasive enteric protozoan, and infections of E. histolytica correlate to high mortality and morbidity rates[ 11 , 12 ]. Each year, this protozoan causes 40000-100000 deaths, ranking only behind malaria in patient mortality[ 13 - 15 ].…”

“…Each year, this protozoan causes 40000-100000 deaths, ranking only behind malaria in patient mortality[ 13 - 15 ]. According to a previous report, invasive amebiasis develops in fewer than one-tenth of patient infections[ 11 ]. The geographic distribution of amebiasis has worldwide amplitude and a high rate of incidence, and it remains a public health concern in low- and middle-income developing countries in the tropics, particularly in environments that are crowded and lacking in adequate sanitation and clean water due to the oral-fecal route of pathogen transmission (including ingestion of food or water that contains cysts from this protozoan)[ 6 , 16 - 19 ].…”