Use and endocytosis of iron-containing proteins by Entamoeba histolytica trophozoites

Iron is essential for nearly all organisms; in mammals, it is part of proteins such as haemoglobin, and it is captured by transferrin and lactoferrin. Transferrin is present in serum, and lactoferrin is secreted by the mucosa and by neutrophils at infection sites, as a host iron-withholding response, sequestering iron away from invading microorganisms. Additionally, all cells contain ferritin, which sequesters iron when its intracellular levels are increased, detoxifying and preventing damage. Liver ferritin contains 50% of iron corporal reserves. During evolution, pathogens have evolved diverse strategies to obtain iron from their hosts in order to survive. The protozoan Entamoeba histolytica invades the intestinal mucosa, causing dysentery, and the trophozoites often travel to the liver producing hepatic abscesses; thus, intestine and liver proteins could be important iron supplies for E. histolytica. We found that E. histolytica trophozoites can grow in both ferrous and ferric iron, and that they can use haemoglobin, holo-transferrin, holo-lactoferrin, and ferritin as in vitro iron sources. These proteins supported the amoeba growth throughout consecutive passages, similarly to ferric citrate. By confocal microscopy and immunoblotting, iron-binding proteins were observed specifically bound to the amoeba surface, and they were endocytosed, trafficked through the endosomal/lysosomal route, and degraded by neutral and acidic cysteine-proteases. Transferrin and ferritin were mainly internalized through clathrin-coated vesicles, and holo-lactoferrin was mainly internalized by caveola-like structures. In contrast, apo-lactoferrin bound to membrane lipids and cholesterol, inducing cell death. The results suggest that in vivo trophozoites secrete products that can destroy enterocytes, erythrocytes, and hepatocytes, releasing transferrin, haemoglobin, ferritin, and other iron-containing proteins, which, together with lactoferrin derived from neutrophils and acinar cells, could be used as abundant iron supplies by amoebas.