Abstract
Phagocytosis is required for proliferation and pathogenesis of Entamoeba histolytica and erythrophagocytosis is considered to be a marker of invasive amoebiasis. Ca2+ has been found to play a central role in the process of phagocytosis. However, the molecular mechanisms and the signalling mediated by Ca2+ still remain largely unknown. Here we show that Calmodulin-like calcium binding protein EhCaBP3 of E. histolytica is directly involved in disease pathomechanism by its capacity to participate in cytoskeleton dynamics and scission machinery during erythrophagocytosis. Using imaging techniques EhCaBP3 was found in phagocytic cups and newly formed phagosomes along with actin and myosin IB. In vitro studies confirmed that EhCaBP3 directly binds actin, and affected both its polymerization and bundling activity. Moreover, it also binds myosin 1B in the presence of Ca2+. In cells where EhCaBP3 expression was down regulated by antisense RNA, the level of RBC uptake was reduced, myosin IB was found to be absent at the site of pseudopod cup closure and the time taken for phagocytosis increased, suggesting that EhCaBP3 along with myosin 1B mediate the closure of phagocytic cups. Experiments with EhCaBP3 mutant defective in Ca2+ -binding showed that Ca2+ binding is required for phagosome formation. Liposome binding assay revealed that EhCaBP3 recruitment and enrichment to membrane is independent of any cellular protein as it binds directly to phosphatidylserine. Taken together, our results suggest a novel pathway mediating phagocytosis in E. histolytica, and an unusual mechanism of modulation of cytoskeleton dynamics by two calcium binding proteins, EhCaBP1 and EhCaBP3 with mostly non-overlapping functions.
Highlights
A variety of cell types, such as macrophages and neutrophils and many unicellular eukaryotes have the ability to engulf particles of size greater than 0.5 mm through a process called phagocytosis
Our results demonstrate the complex role of Ca2+ binding proteins, EhCaBP1 and EhCaBP3 in regulation of phagocytosis in the protist parasite E. histolytica and the novel mechanisms of manipulating actin dynamics at multiple levels
Since multiple CaBPs are likely to be involved in different steps of phagocytosis, the subcellular localization of EhCaBP3 was checked during red blood cells (RBCs) uptake by immunostaining with specific anti-EhCaBP3 antibody
Summary
A variety of cell types, such as macrophages and neutrophils and many unicellular eukaryotes have the ability to engulf particles of size greater than 0.5 mm through a process called phagocytosis. In the former this process has evolved as one of the critical elements of host defence, while in the latter it serves as a mode of nutrition. It is believed that this disassembly of the F-actin rim is necessary for phagosome maturation, as it may act as a barrier for phagosome-vesicle fusion [8,9,10,11]. Spatial and temporal regulation of actin dynamics is the key to controlling phagocytosis
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