Knockdown of Five Genes Encoding Uncharacterized Proteins Inhibits Entamoeba histolytica Phagocytosis of Dead Host Cells

Abstract

Entamoeba histolytica is the protozoan parasite that causes invasive amebiasis, which is endemic to many developing countries and characterized by dysentery and liver abscesses. The virulence of E. histolytica correlates with the degree of host cell engulfment, or phagocytosis, and E. histolytica phagocytosis alters amebic gene expression in a feed-forward manner that results in an increased phagocytic ability. Here, we used a streamlined RNA interference screen to silence the expression of 15 genes whose expression was upregulated in phagocytic E. histolytica trophozoites to determine whether these genes actually function in the phagocytic process. When five of these genes were silenced, amebic strains with significant decreases in the ability to phagocytose apoptotic host cells were produced. Phagocytosis of live host cells, however, was largely unchanged, and the defects were surprisingly specific for phagocytosis. Two of the five encoded proteins, which we named E. histolytica ILWEQ (EhILWEQ) and E. histolytica BAR (EhBAR), were chosen for localization via SNAP tag labeling and localized to the site of partially formed phagosomes. Therefore, both EhILWEQ and EhBAR appear to contribute to E. histolytica virulence through their function in phagocytosis, and the large proportion (5/15 [33%]) of gene-silenced strains with a reduced ability to phagocytose host cells validates the previously published microarray data set demonstrating feed-forward control of E. histolytica phagocytosis. Finally, although only limited conclusions can be drawn from studies using the virulence-deficient G3 Entamoeba strain, the relative specificity of the defects induced for phagocytosis of apoptotic cells but not healthy cells suggests that cell killing may play a ratelimiting role in the process of Entamoeba histolytica host cell engulfment. Entamoeba histolytica infection, which causes dysentery and amebic liver abscesses, is an enormous public health problem in developing countries (1, 2). In areas where the infection is highly endemic, such as the Mirpur region of Dhaka, Bangladesh, over 50% of children may become infected by 5 years of age (3), yet only about 10% of all infections become symptomatic (4, 5). Isolated strains of E. histolytica have been shown to have different levels of virulence both in vivo and in vitro (6-8), which may in part determine whether or not a particular infection becomes invasive.Multiple mechanisms contribute to the ability of E. histolytica to destroy the host intestinal mucosa and cause disease (9). Following excystation within the small intestinal lumen, trophozoites attach to host mucous and epithelial cells in the colon, largely through the multisubunit amebic GalNAc lectin (10, 11). Trophozoites also secrete multiple cysteine proteases, which degrade mucin and the extracellular matrix (12-14), and they kill resident host cells through a contact-dependent process that remains poorly understood (15-17). Host cells appear to suffer from a disruption of the cell membrane with rapid changes in int...

AGC family kinase 1 participates in trogocytosis but not in phagocytosis in Entamoeba histolytica

“…Our observations indicate that EhAGCK1 is recruited after EhAGCK2, which is similar to the sequential recruitment of PKCε and PKCα during FcγR-mediated phagocytosis 36 . EhAGCK1 may play a role in downstream signalling necessary for formation of initiation complexes, which may include membrane-remodelling molecules such as BAR domain-containing proteins that have been shown to be associated with phagocytosis and virulence 37 . In contrast, in case of phagocytosis of pre-killed CHO cells, only EhAGCK2 is recruited to the phagocytic cups (Fig.…”

Coactosin Phosphorylation Controls Entamoeba histolytica Cell Membrane Protrusions and Cell Motility

“…7B ). Furthermore, since cell killing appears to be the rate-limiting step in rapid phagocytosis of viable cells ( 28 ), we assayed phagocytosis of apoptotic cells and again found no significant difference ( Fig. 7C ).…”

“…Given the known roles of actin polymerization in amoebic adherence and phagocytosis and that EhCoactosin localizes within phagocytic cups and its overexpression reduced phagocytosis by ∼50% in a prior report ( 20 ), it is somewhat surprising that adherence and phagocytosis were not significantly affected by Coac-A and Coac-D overexpression. Furthermore, although cell killing was not directly assayed, we note that cell killing is the rate-limiting step in phagocytosis ( 28 ); thus, the absence of an effect on phagocytosis of viable cells suggests that cell killing is also unaffected by expression of these protein constructs. As noted above, a limitation of this experimental system is the ongoing expression of the genomic copy of the EhCoactosin gene, which might cause false-negative results.…”

The actin cytoskeleton orchestra in Entamoeba histolytica

“…Thus, presence of these proteins in amoeba is an interesting avenue. A study reported that silencing of EhBAR (C4M128) impacted phagocytosis by the amoebic trophozoites, 101 further strengthening the implications of this domain in actin regulation.…”

“…ENTH domain containing proteins are functionally involved in actin regulation 119 . C4M9B6 was shown to be an important protein during phagocytosis 101 . C4LYB3 (1623 aa.)…”