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A pinboard by
Ravi Bharadwaj

Research Associate, JNU

PINBOARD SUMMARY

Deciphering the molecular mechanism for phagocytosis and motility in Entamoeba histolytica

I have been working on understanding the molecular mechanism of pathogenicity of protozoan parasite Entamoeba histolytica. Amoebic infection causes amoebiasis and advance stages of infection may lead to liver, lung or brain abscesses. It infects 15- 20% of the world population and greatly contributes to significant cause of morbidity and mortality in developing countries including India. E. histolytica possesses a highly dynamic and actin-rich cytoskeleton that participates in many pathogenesis-related processes like pseudopod extension, blebbing regulated by extracellular matrix interactions involving rapid actin remodeling, help in motility of trophozoites on intestinal mucosa and phagocytosis, also associated with cytoskeleton actin remodeling. Rho GTPases are known as key regulators of the actin cytoskeleton and consequently influence the shape and movement of the cells. Our study is focused majorly on Rho GTPases, specifically to understand the role of EhRho1 in virulent behaviour of E. histolytica. We have shown that blebs are important to provide cell motility for parasite during host tissue invasion and EhRho1 plays an important role in regulates blebbing by modulating PIP2 level in plasma membrane through PI 3 kinases.Interestingly, alteration in lipid composition affects the interaction between plasma membrane and cortical actin which result in nucleation of bleb. We have also found that EhRho1 regulates phagocytosis by modulating actin dynamics through actin nucleating protein, EhFormin1. EhRho1 binds directly with EhFormin1 and promote actin polymerization by releasing it from intramolecular inhibitory state. EhRho1 translocate to phagocytic cups and influence EhFormin1 localization there to enhance the actin polymerization for extension of phagocytic arms. This has been shown by several lines of experiments. overexpression of dominant negative mutant or lowering the expression of EhRho1 in trophozoites cause decrease the cell motility and delocalization of EhFormin1 from phagocytic cups which results in impairment of phagocytic processes and decrease F-actin content. The overall results state that EhRho1 regulates two major virulent processes in parasite, cell motility and phagocytosis by regulating lipid and actin dynamics. E. histolytica being an early branching eukaryote, deciphering molecular mechanism of cell motility or phagocytosis in this organism will help us to understand the evolution of pathways and molecules in different system.

7 ITEMS PINNED

A novel alpha kinase EhAK1 phosphorylates actin and regulates phagocytosis in Entamoeba histolytica.

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.

Pub.: 10 Oct '14, Pinned: 10 Oct '17

Autophosphorylation at Thr279 of Entamoeba histolytica atypical kinase EhAK1 is required for activity and regulation of erythrophagocytosis.

Abstract: Phagocytosis plays a key role in survival and pathogenicity of Entamoeba histolytica. We have recently demonstrated that an atypical kinase EhAK1 is involved in phagocytosis in this parasite. It is recruited to the phagocytic cups through interaction with EhCaBP1. EhAK1 manipulates actin dynamics by multiple mechanisms including phosphorylation of G-actin. Biochemical analysis showed that EhAK1 is a serine/threonine kinase with broad ion specificity and undergoes multiple trans-autophosphorylation. Three autophosphorylation sites were identified by mass spectrometry. Out of these Thr279 appears to be involved in both autophosphorylation as well as substrate phosphorylation. Over expression of the mutant Thr279A inhibited erythrophagocytosis showing dominant negative phenotype. Multiple alignments of different kinases including alpha kinases displayed conserved binding sites that are thought to be important for function of the protein. Mutation studies demonstrated the importance of some of these binding sites in kinase activity. Binding studies with fluorescent-ATP analogs supported our prediction regarding ATP binding site based on sequence alignment. In conclusion, EhAK1 has multiple regulatory features and enrichment of EhAK1 at the site of phagocytosis stimulates trans-autophosphorylation reaction that increases kinase activity resulting in enhanced actin dynamics and phagocytosis. Some of the properties of EhAK1 are similar to that seen in alpha kinases.

Pub.: 08 Jan '16, Pinned: 10 Oct '17

Molecular Basis of Pathogenesis in Amoebiasis

Abstract: Abstract Amoebiasis is one of the major public health problems in developing countries. In spite of the availability of an effective drug and absence of overt drug resistance, the disease is still prevalent among large population and spread over a number of countries. It is caused by the protist parasite Entamoeba histolytica that essentially infects humans, though other species that infect a few animals have been reported. A number of molecular techniques have recently been developed. These have helped in understanding biological processes in E. histolytica and in the identification of key molecules that are involved in amoebic virulence and invasion. Moreover, developments in the area of disease and invasion models have allowed understanding of these processes at molecular level and circumvented lack of a good animal model of amoebiasis. All these knowledge will help us to design better therapeutics and allow us to control this important disease.AbstractAmoebiasis is one of the major public health problems in developing countries. In spite of the availability of an effective drug and absence of overt drug resistance, the disease is still prevalent among large population and spread over a number of countries. It is caused by the protist parasite Entamoeba histolytica that essentially infects humans, though other species that infect a few animals have been reported. A number of molecular techniques have recently been developed. These have helped in understanding biological processes in E. histolytica and in the identification of key molecules that are involved in amoebic virulence and invasion. Moreover, developments in the area of disease and invasion models have allowed understanding of these processes at molecular level and circumvented lack of a good animal model of amoebiasis. All these knowledge will help us to design better therapeutics and allow us to control this important disease.Entamoeba histolyticaE. histolytica

Pub.: 28 Sep '15, Pinned: 10 Oct '17