Summary paragraph Entamoeba histolytica is the causative agent of amoebiasis, a potentially fatal diarrheal disease in the developing world. The parasite was named “histolytica” for its ability to destroy host tissues, which is most likely driven by direct killing of human cells. The mechanism of human cell killing has been unclear, though the accepted model was that the parasites use secreted toxic effectors to kill cells prior to ingestion1. Here we report the surprising discovery that amoebae kill by biting off and ingesting distinct pieces of living human cells, resulting in intracellular calcium elevation and eventual cell death. After cell killing, amoebae detach and cease ingestion. Ingestion of bites is required for cell killing, and also contributes to invasion of intestinal tissue. The internalization of bites of living human cells is reminiscent of trogocytosis (Greek trogo–, nibble) observed between immune cells2–6, but amoebic trogocytosis differs since it results in death. The ingestion of live cell material and the rejection of corpses illuminate a stark contrast to the established model of dead cell clearance in multicellular organisms7. These findings change the paradigm for tissue destruction in amoebiasis and suggest an ancient origin of trogocytosis as a form of intercellular exchange.
Phagocytosis is a process whereby particles are taken in by cells through mechanisms super ficially similar to those for endocytosis. It serves a wide range of functions, from providing nutrition in unicellular organisms to initiation of both innate and adaptive immunity in vertebrates. In the protozoan parasite Entamoeba histolytica, it has an essential role in survival and pathogenesis. In this study, we show that EhC2PK, a C2 domain containing protein kinase, and the Ca 2 + and actin binding protein, EhCaBP1, are involved in the initiation of phagocytosis in E. histolytica. Conditional suppression of EhC2PK expression and overexpression of a mutant form reveals its role in the initiation of phagocytic cups. EhC2PK binds phosphatidylserine in the presence of Ca 2 + and thereby recruits EhCaBP1 and actin to the membrane. Identification of these proteins in phagocytosis is an important step in amoebic biology and these molecules could be the important targets for developing novel therapies against amoebiasis.
The protozoan parasite Entamoeba histolytica is the aetiologic agent of amoebiasis, an endemic infection in developing countries with considerable morbidity and mortality. Recently, trogocytosis has been recognized as the key step in amoebic cytolysis and invasion, a paradigm shift in understanding pathogenicity of this organism. Here we report that AGC family kinase 1 is specifically involved in trogocytosis of live human cells and does not participate in phagocytosis of dead cells. Live imaging reveals localization of this kinase in the long and thin tunnels formed during trogocytosis but not in the trogosomes (endosomes formed after trogocytosis). Silencing of the specific gene leads to a defect in CHO cell destruction and trogocytosis while other endocytic processes remain unaffected. The results suggest that the trogocytic pathway is likely to be different from phagocytosis though many of the steps and molecules involved may be common.
Entamoeba histolytica is a protist parasite that is the causative agent of amoebiasis, and is a highly motile organism. The motility is essential for its survival and pathogenesis, and a dynamic actin cytoskeleton is required for this process. EhCoactosin, an actin-binding protein of the ADF/cofilin family, participates in actin dynamics, and here we report our studies of this protein using both structural and functional approaches. The X-ray crystal structure of EhCoactosin resembles that of human coactosin-like protein, with major differences in the distribution of surface charges and the orientation of terminal regions. According to in vitro binding assays, full-length EhCoactosin binds both F- and G-actin. Instead of acting to depolymerize or severe F-actin, EhCoactosin directly stabilizes the polymer. When EhCoactosin was visualized in E. histolytica cells using either confocal imaging or total internal reflectance microscopy, it was found to colocalize with F-actin at phagocytic cups. Over-expression of this protein stabilized F-actin and inhibited the phagocytic process. EhCoactosin appears to be an unusual type of coactosin involved in E. histolytica actin dynamics.
Background: Novel protein kinase EhC2PK in E. histolytica is involved in initiation of erythrophagocytosis. Results: The autophosphorylation at Ser 428 position occurs through a trans-reaction and is important for stimulation of kinase activity. Conclusion: Phosphorylation at Ser428 is critical in initiation of erythrophagocytosis. Significance: It provides description of a molecular mechanism that shows importance of trans-autophosphorylation of EhC2PK in E. histolytica erythrophagocytosis.
Amebiasis is a neglected tropical disease which is caused by the protozoan parasite Entamoeba histolytica. This disease is one of the leading causes of diarrhea globally, affecting largely impoverished residents in developing countries. Amebiasis also remains one of the top causes of gastrointestinal diseases in returning international travellers. Despite having many side effects, metronidazole remains the drug of choice as an amebicidal tissue-active agent. However, emergence of metronidazole resistance in pathogens having similar anaerobic metabolism and also in laboratory strains of E. histolytica has necessitated the identification and development of new drug targets and therapeutic strategies against the parasite. Recent research in the field of amebiasis has led to a better understanding of the parasite’s metabolic and cellular pathways and hence has been useful in identifying new drug targets. On the other hand, new molecules effective against amebiasis have been mined by modifying available compounds, thereby increasing their potency and efficacy and also by repurposing existing approved drugs. This review aims at compiling and examining up to date information on promising drug targets and drug molecules for the treatment of amebiasis.
Motility and phagocytosis are the two important processes that are intricately linked to survival and virulence potential of the protist parasite Entamoeba histolytica. These processes primarily rely on actin-dependent pathways, and regulation of these pathways is critical for understanding the pathology of E. histolytica. Generally, phosphoinositides dynamics have not been explored in amoebic actin dynamics and particularly during phagocytosis in E. histolytica. We have explored the roles of PtdIns(4,5)P 2 as well as the enzyme that produces this metabolite, EhPIPKI during phagocytosis. Immunofluorescence and live cell images showed enrichment of EhPIPKI in different stages of phagocytosis from initiation till the cups progressed towards closure. However, the enzyme was absent after phagosomes are pinched off from the membrane. Overexpression of a dominant negative mutant revealed a reduction in the formation of phagocytic cups and inhibition in the rate of engulfment of erythrocytes. Moreover, EhPIPKI binds directly to F and G-actin unlike PIPKs from other organisms. PtdIns(4,5)P 2 , the product of the enzyme, also followed a similar distribution pattern during phagocytosis as determined by a GFP-tagged PH-domain from PLCδ, which specifically binds PtdIns(4,5)P 2 in trophozoites. In summary, EhPIPKI regulates initiation of phagocytosis by regulating actin dynamics.
For the protist parasite Entamoeba histolytica, endocytic processes, such as phagocytosis, are essential for its survival in the human gut. The actin cytoskeleton is involved in the formation of pseudopods and phagosomal vesicles by incorporating a number of actin‐binding and modulating proteins along with actin in a temporal manner. The actin dynamics, which comprises polymerization, branching, and depolymerization is very tightly regulated and takes place directionally at the sites of initiation of phagocytosis. Formin and profilin are two actin‐binding proteins that are known to regulate actin cytoskeleton dynamics and thereby, endocytic processes. In this article, we report the participation of formin and profilin in E. histolytica phagocytosis and propose that these two proteins interact with each other and their sequential recruitment at the site is required for the successful completion of phagocytosis. The evidence is based on detailed microscopic, live imaging, interaction studies, and expression downregulation. The cells downregulated for expression of formin show absence of profilin at the site of phagocytosis, whereas downregulation of profilin does not affect formin localization.
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