Research Associate, AIIMS
EhRho1 is an possible target for development of new therapeutic drugs for treatment of amoebiasis
Entamoeba histolytica, a protozoan parasite causes amoebic dysentery or amoebiasis by invading colonic mucosa of host tissue. Phagocytosis and motility of parasite have often been associated with virulence behavior of E. histolytica and are essential factors responsible for pathogenesis. During tissue invasion, E. histolytica cells display high degree of blebbing due to high friction surfaces of gut epithelial cell lining which tend to employ blebbing as a mode of migration over pseudopods. After invasion, parasites may reach to liver, brain or lung and cause abscess in the tissues. EhRho1 is a small GTPase that resides in the plasma membrane and enriches in the membrane, forming blebs. We have shown a novel mechanism for regulation of bleb formation in E. histolytica by EhRho1 through PI3 kinase pathway. On the other side, our results show that EhRho1 regulates actin dynamics and localizes in the phagocytic cups during erythrophagocytosis and remain associated with the newly formed phagosome. Binding of activated EhRho1 (GTP bound) with Rho bind domain of EhFormin1 releases it from intermolecular autoinhibitory state and recruits to phagocytic cups. The overall results concluded that EhRho1 regulates bleb based motility of parasite by regulating PIP2 level in plasma membrane through PI3 kinases pathway. Activation of EhFormin1 is required for progressions of phagocytic processes through the EhRho1 pathway in E. histolytica.
Abstract: Entamoeba histolytica transmembrane kinases (EhTMKs) can be grouped into six distinct families on the basis of motifs and sequences. Analysis of the E. histolytica genome revealed the presence of 35 EhTMKB1 members on the basis of sequence identity (>or=95%). Only six homologs were full length containing an extracellular domain, a transmembrane segment and an intracellular kinase domain. Reverse transcription followed by polymerase chain reaction (RT-PCR) of the kinase domain was used to generate a library of expressed sequences. Sequencing of randomly picked clones from this library revealed that about 95% of the clones were identical with a single member, EhTMKB1-9, in proliferating cells. On serum starvation, the relative number of EhTMKB1-9 derived sequences decreased with concomitant increase in the sequences derived from another member, EhTMKB1-18. The change in their relative expression was quantified by real time PCR. Northern analysis and RNase protection assay were used to study the temporal nature of EhTMKB1-9 expression after serum replenishment of starved cells. The results showed that the expression of EhTMKB1-9 was sinusoidal. Specific transcriptional induction of EhTMKB1-9 upon serum replenishment was further confirmed by reporter gene (luciferase) expression and the upstream sequence responsible for serum responsiveness was identified. EhTMKB1-9 is one of the first examples of an inducible gene in Entamoeba. The protein encoded by this member was functionally characterized. The recombinant kinase domain of EhTMKB1-9 displayed protein kinase activity. It is likely to have dual specificity as judged from its sensitivity to different kinase inhibitors. Immuno-localization showed EhTMKB1-9 to be a surface protein which decreased on serum starvation and got relocalized on serum replenishment. Cell lines expressing either EhTMKB1-9 without kinase domain, or EhTMKB1-9 antisense RNA, showed decreased cellular proliferation and target cell killing. Our results suggest that E. histolytica TMKs of B1 family are functional kinases likely to be involved in serum response and cellular proliferation.
Pub.: 10 Jun '10, Pinned: 29 Sep '17
Abstract: The parasite Entamoeba histolytica is the etiological agent of amoebiasis and phagocytosis plays a key role in virulence of this organism. Signaling pathways involved in activation of cytoskeletal dynamics required for phagocytosis remain to be elucidated. Phagocytosis is initiated with sequential recruitment of EhC2PK, EhCaBP1, EhCaBP3 and an atypical kinase EhAK1 after particle attachment. Here we show that EhARPC1, an essential subunit of the actin branching complex Arp 2/3 is recruited to the phagocytic initiation sites by EhAK1. Imaging, expression knockdown of different molecules and pull down experiments suggest that EhARPC1 interacts with EhAK1 and that it is required during initiation of phagocytosis and phagosome formation. Moreover, recruitment of EhARPC2 at the phagocytosis initiation by EhAK1 is also observed, indicating that the Arp 2/3 complex is recruited. In conclusion, these results suggests a novel mechanism of recruitment of Arp 2/3 complex during phagocytosis in E. histolytica.
Pub.: 10 Dec '15, Pinned: 29 Sep '17
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: 25 Sep '17
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: 25 Sep '17
Abstract: The protozoan parasite Entamoeba histolytica causes amoebiasis, a major public health problem in developing countries. Motility of E. histolytica is important for its pathogenesis. Blebbing is an essential process contributing to cellular motility in many systems. In mammalian cells, formation of plasma membrane blebs is regulated by Rho-GTPases through its effectors, such as ROCK, mDia1 and acto-myosin proteins. In the present study, we have illuminated the role of EhRho1 in bleb formation and motility of E. histolytica. EhRho1 was found at the site of bleb formation in plasma membrane of trophozoites. Overexpression of mutant EhRho1 defective for GTP-binding or down-regulating EhRho1 by anti-sense RNA resulted in reduced blebbing and motility. Moreover, serum-starvation reduced blebbing that was restored on serum-replenishment. Lysophosphatidic acid (LPA) treatment induced bleb formation, while wortmannin inhibited the process. In all these cases, concentration of GTP-EhRho1 (active) and Phosphatidylinositol 4,5-bisphosphate (PIP2) inversely correlated with the level of plasma membrane blebbing. Our study suggests the role of EhRho1 in blebbing and bleb-based motility through PI3 kinase pathway in E. histolytica.
Pub.: 10 May '17, Pinned: 25 Sep '17
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