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Abstract
Phagocytosis plays a key role in nutrient uptake and virulence of the protist parasite Entamoeba histolytica. Phagosomes have been characterized by proteomics, and their maturation in the cells has been studied. However, there is so far not much understanding about initiation of phagocytosis and formation of phagosomes at the molecular level. Our group has been studying initiation of phagocytosis and formation of phagosomes in E. histolytica, and have described some of the molecules that play key roles in the process. Here we show the involvement of EhAK1, an alpha kinase and a SH3 domain containing protein in the pathway that leads to formation of phagosomes using red blood cell as ligand particle. A number of approaches, such as proteomics, biochemical, confocal imaging using specific antibodies or GFP tagged molecules, expression down regulation by antisense RNA, over expression of wild type and mutant proteins, were used to understand the role of EhAK1 in phagocytosis. EhAK1 was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. It is recruited to the phagosomes through interaction with the calcium binding protein EhCaBP1. A reduction in phagocytosis was observed when EhAK1 was down regulated by antisense RNA, or by over expression of the kinase dead mutant. G-actin was identified as one of the major substrates of EhAK1. Phosphorylated actin preferentially accumulated at the phagocytic cups and over expression of a phosphorylation defective actin led to defects in phagocytosis. In conclusion, we describe an important component of the pathway that is initiated on attachment of red blood cells to E. histolytica cells. The main function of EhAK1 is to couple signalling events initiated after accumulation of EhC2PK to actin dynamics.
Highlights
Phagocytosis is an essential process both in unicellular organisms which use this process to obtain their food [1], and multicellular organisms, where it plays a central role in the innate immune system [2]
Our results indicate that erythrophagocytosis is initiated by the recruitment of EhAK1 with the help of EhCaBP1 at the RBC attachment site, which is followed by recruitment of actin
Our previous work has shown the importance of EhCaBP1 in RBC phagocytosis
Summary
Phagocytosis is an essential process both in unicellular organisms which use this process to obtain their food [1], and multicellular organisms, where it plays a central role in the innate immune system [2]. A number of cell surface molecules, such as Gal/GalNAc lectin [8], TMK96 [9], TMK39 [10], SREHP [11] and EhROM1 [12] have been shown to be involved in adherence to other cells It is not yet clear if these molecules are amoebic receptors during phagocytosis of different particles, such as RBC, bacteria and apoptotic human cells [13]. Analysis of the phagosome proteome has revealed involvement of a large number of proteins in phagosome formation and subsequent maturation [14,16,17,18,19] Some of these, such as actin [20], Arp proteins, actin binding proteins, PI3 kinase [21], P21 activated protein kinase (PAK) [22], and different GTPases are already known to be part of phagocytic and signalling pathways [23,24]. Previous studies from our laboratory have shown that calcium binding protein (EhCaBP1) is involved in the initiation of Author Summary
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