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.
Entamoeba histolytica is one of the major causes of morbidity and mortality in developing countries. Phagocytosis plays an important role in both survival and virulence, and has been used as one of the virulence markers. E. histolytica displays a high rate of phagocytosis and offers a unique system to understand the mechanism of this important biological process seen in many eukaryotic cells. However, the molecular mechanism of the process is still largely unknown in E. histolytica, though this pathway has been characterized in many systems. We have been studying this pathway using red blood cells, and have identified a number of molecules that are involved during initiation. Here, we demonstrate that an alpha kinase like atypical kinase EhAK1 is an important component of the pathway that regulates erythrophagocytosis. We provide evidence that EhAK1 is recruited to the phagocytic cups through EhCaBP1. We also show that over expression of kinase defective mutant, or down regulation of the gene using antisense RNA, led to defects in phagocytosis. Actin appears to be one of the substrates of EhAK1 and phosphorylation of actin is required for phagocytosis. Our results suggest that E. histolytica has evolved a novel pathway to carry out phagocytosis.