EhC2B, a C2 domain-containing protein, promotes erythrophagocytosis in Entamoeba histolytica via actin nucleation

Abstract

Remodelling of the actin cytoskeleton in response to external stimuli is obligatory for many cellular processes in the amoebic cell. A rapid and local rearrangement of the actin cytoskeleton is required for the development of the cellular protrusions during phagocytosis, trogocytosis, migration, and invasion. Here, we demonstrated that EhC2B, a C2 domain-containing protein, is an actin modulator. EhC2B was first identified as an effector of EhRab21 from E. histolytica. In vitro interaction studies including GST pull-down, fluorescence-based assay and ITC also corroborated with our observation. In the amoebic trophozoites, EhC2B accumulates at the pseudopods and the tips of phagocytic cups. FRAP based studies confirmed the recruitment and dynamics of EhC2B at the phagocytic cup. Moreover, we have shown the role of EhC2B in erythrophagocytosis. It is well known that calcium-dependent signal transduction is essential for the cytoskeletal dynamics during phagocytosis in the amoebic parasite. Using liposome pelleting assay, we demonstrated that EhC2B preferentially binds to the phosphatidylserine in the presence of calcium. The EhC2B mutants defective in calcium or lipid-binding failed to localise beneath the plasma membrane. The cells overexpressing these mutants have also shown a significant reduction in erythrophagocytosis. The role of EhC2B in erythrophagocytosis and pseudopod formation was also validated by siRNA-based gene knockdown approach. Finally, with the help of in vitro nucleation assay using fluorescence spectroscopy and total internal reflection fluorescence microscopy, we have established that EhC2B is an actin nucleator. Collectively, based on the results from the study, we propose that EhC2B acts like a molecular bridge which promotes membrane deformation via its actin nucleation activity during the progression of the phagocytic cup in a calcium-dependent manner.

EhRho6‐mediated actin degradation in Entamoeba histolytica is associated with compromised pathogenicity

“…To understand the role of the substrate EhRho6 in the pathogen, we steered our investigation to amoebic trophozoites. Stable amoebic transfectants expressing HA‐Rho6WT under cysteine synthase promoter were generated (Figure S2a) to attain a near endogenous level of expression (Nakada‐Tsukui et al, 2009; Tripathi et al, 2020; Verma et al, 2015). To assess the nucleotide dependency, stable cell lines expressing constitutively active (CA) and dominant‐negative (DN) mutants of EhRho6 were also prepared (Table S3).…”

“…E. histolytica trophozoites cell lines were generated through transfection by electroporation (Emmanuel et al, 2015; Tripathi et al, 2020), selected, and maintained at 6 μg ml −1 G418 (Sigma–Aldrich; Cat No. A1720) at 35.5°C.…”

MaSln1, a Conserved Histidine Protein Kinase, Contributes to Conidiation Pattern Shift Independent of the MAPK Pathway in Metarhizium acridum

“…MAD1 , a gene for an adhesin protein (MAC_00987) which plays important roles in conidia formation and cell cycle in M. anisopliae ( 46 ), was upregulated in Δ MaSln1 . In addition, many genes related to actin formation were also differentially expressed, such as a downregulated gene for a C2-structural protein (MAC_00302) which can act as a molecular bridge by inhibiting the nucleation activity of actin, thus promoting cell membrane flow ( 47 ). A gene encoding the long-chain reactive protein PIL-1 (MAC_03800), an important cytoskeleton component in fission yeast, was also significantly downregulated ( 48 ).…”

Analysis of Expression Profiles of mRNA and lncRNA in Oviduct During Estrus Phase of Sheep (Ovis Aries) with Two Fecundity (FecB Gene) Genotypes

“…It's worth noting that 4 mRNAs: C2CD5 (C2 calcium-dependent domain containing 5), EPB41L1(erythrocyte membrane protein band 4.1-like 1), EML4(echinoderm microtubule associated protein like 4), and VIRMA (vir like m6A methyltransferase associated) and LNC_006270 were found DE n all comparisons. Several researches have been conducted to assess the in uence of these genes on metabolic [31][32][33] and energy balance [34][35][36], however, little is known about the association between these candidates and reproduction, thus, we hypothesis that these genes may indirectly affect embryo development in sheep oviduct via metabolic progress.…”