EhRho1 regulates plasma membrane blebbing through PI3 kinase inEntamoeba histolytica

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 Rho kinase, mDia1, and acto-myosin proteins. In this 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 Guanosine triphosphate (GTP)-binding or down-regulating EhRho1 by antisense RNA resulted in reduced blebbing and motility. Moreover, serum-starvation reduced blebbing that was restored on serum-replenishment. Lysophosphatidic acid treatment induced bleb formation, whereas 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.

EhRho1 regulates phagocytosis by modulating actin dynamics through EhFormin1 and EhProfilin1 inEntamoeba histolytica

“…The killing of CHO cells was determined by counting after methylene blue staining box" that aids in binding to the plasma membrane and targeting protein complexes after activation (Bharadwaj et al, 2017a). The C-terminal region of EhRho1 has a membrane insertion region "CAAX FIGURE 9 Cell adhesion and killing by EhRho1 transfectants.…”

“…Lipid kinases are the core regulators to maintain lipid diversity in different membrane compartments. Increased PIP2 has been correlated with decrease in membrane flexibility that may have a role in phagocytosis and EhRho1 downregulation (Bharadwaj et al, 2017a). The main products of PI3 kinase are PIP3 and PI3P, results of phosphorylation at 3 position of the inositol head group of PIP2.…”

“…Initially, immunostaining was carried out to determine the localisation of EhRho1 during RBC uptake at different time intervals, using specific antibody generated against the recombinant protein (Bharadwaj, Arya, Shahid mansuri, Bhattacharya, & Bhattacharya, 2017b). Initially, immunostaining was carried out to determine the localisation of EhRho1 during RBC uptake at different time intervals, using specific antibody generated against the recombinant protein (Bharadwaj, Arya, Shahid mansuri, Bhattacharya, & Bhattacharya, 2017b).…”

“…Interestingly, E. histolytica expresses (or encodes) more than 19 Rho domain containing GTPases as compared to 18 found in human (Bharadwaj, Arya, Shahid mansuri, Bhattacharya, & Bhattacharya, 2017a;Bosch, Wittchen, Qiu, Burridge, & Siderovski, 2011). Interestingly, E. histolytica expresses (or encodes) more than 19 Rho domain containing GTPases as compared to 18 found in human (Bharadwaj, Arya, Shahid mansuri, Bhattacharya, & Bhattacharya, 2017a;Bosch, Wittchen, Qiu, Burridge, & Siderovski, 2011).…”

“…Though EhRho1 shows 47% sequence identity and 70% structural similarity with its human homologue, HsRhoA (Bharadwaj et al, 2017a), it is distinctly different as it lacks the signature "Rho insert" domain that separates Rho proteins from Ras superfamily. Moreover, EhRho1 is not a substrate for Clostridium botulinum C3 exoenzyme, a critical feature of mammalian Rho GTPases, but is glucosylated by Clostridum difficile toxin B and Clostridum novyi α-toxin (Lohia & Samuelson, 1993;Majumder, Schmidt, Lohia, & Aktories, 2006).…”