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      A Novel Alpha Kinase EhAK1 Phosphorylates Actin and Regulates Phagocytosis in Entamoeba histolytica

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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.

          Author Summary

          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.

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          Most cited references 59

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          Shaping cups into phagosomes and macropinosomes.

           J. Swanson (2008)
          The ingestion of particles or cells by phagocytosis and of fluids by macropinocytosis requires the formation of large endocytic vacuolar compartments inside cells by the organized movements of membranes and the actin cytoskeleton. Fc-receptor-mediated phagocytosis is guided by the zipper-like progression of local, receptor-initiated responses that conform to particle geometry. By contrast, macropinosomes and some phagosomes form with little or no guidance from receptors. The common organizing structure is a cup-shaped invagination of the plasma membrane that becomes the phagosome or macropinosome. Recent studies, focusing on the physical properties of forming cups, indicate that a feedback mechanism regulates the signal transduction of phagocytosis and macropinocytosis.
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            A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba.

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              Phagocytosis and the actin cytoskeleton.

              The process of engulfing a foreign particle - phagocytosis - is of fundamental importance for a wide diversity of organisms. From simple unicellular organisms that use phagocytosis to obtain their next meal, to complex metazoans in which phagocytic cells represent an essential branch of the immune system, evolution has armed cells with a fantastic repertoire of molecules that serve to bring about this complex event. Regardless of the organism or specific molecules concerned, however, all phagocytic processes are driven by a finely controlled rearrangement of the actin cytoskeleton. A variety of signals can converge to locally reorganise the actin cytoskeleton at a phagosome, and there are significant similarities and differences between different organisms and between different engulfment processes within the same organism. Recent advances have demonstrated the complexity of phagocytic signalling, such as the involvement of phosphoinostide lipids and multicomponent signalling complexes in transducing signals from phagocytic receptors to the cytoskeleton. Similarly, a wide diversity of 'effector molecules' are now implicated in actin-remodelling downstream of these receptors.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                October 2014
                9 October 2014
                : 10
                : 10
                Affiliations
                [1 ]School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
                [2 ]School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India
                University of Virginia Health System, United States of America
                Author notes

                This study was funded in part by a grant from BNP Paribas. This does not alter our adherence to all PLOS Pathogens policies on sharing data and material.

                Conceived and designed the experiments: MSM SB AB. Performed the experiments: MSM. Analyzed the data: MSM SB AB. Contributed reagents/materials/analysis tools: SB AB. Wrote the paper: MSM SB AB.

                Article
                PPATHOGENS-D-14-01213
                10.1371/journal.ppat.1004411
                4192601
                25299184

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 22
                Funding
                MSM acknowledges Council of Scientific & Industrial Research (CSIR), India for fellowship. AB thanks Department of Science and Technology for JC Bose Fellowship, funding from Department of Biotechnology and a donation from BNP Paribas. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Cell Biology
                Microbiology
                Molecular Biology
                Parasitology
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

                Infectious disease & Microbiology

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