An atypical EhGEF regulates phagocytosis in Entamoeba histolytica through EhRho1

Abstract

The parasite Entamoeba histolytica is the etiological agent of amoebiasis, a major cause of morbidity and mortality due to parasitic diseases in developing countries. Phagocytosis is an essential mode of obtaining nutrition and has been associated with the virulence behaviour of E. histolytica. Signalling pathways involved in activation of cytoskeletal dynamics required for phagocytosis remains to be elucidated in this parasite. 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 showed the involvement of non-Dbl Rho Guanine Nucleotide Exchange Factor, EhGEF in regulation of amoebic phagocytosis by regulating activation of EhRho1. EhGEF was found in the phagocytic cups during the progression of cups, until closure of phagosomes, but not in the phagosomes themselves. Our observation from imaging, pull down experiments and down regulating expression of different molecules suggest that EhGEF interacts with EhRho1 and it is required during initiation of phagocytosis and phagosome formation. Also, biophysical, and computational analysis reveals that EhGEF mediates GTP exchange on EhRho1 via an unconventional pathway. In conclusion, we describe a non-Dbl EhGEF of EhRho1 which is involved in endocytic processes of E. histolytica.

PI Kinase-EhGEF2-EhRho5 axis contributes to LPA stimulated macropinocytosis in Entamoeba histolytica

“…GST-RBD and GST-PBD have been frequently used as bait to look for the presence of the activated Rho-GTP population in numerous studies [ 19 , 51 ] including some in E . histolytica [ 52 , 53 ]. We cloned EhRho5 in bacterial expression vector pET28a+ with His tag.…”

“…Rho GTPases interact with the effectors only in their GTP bound form and this property has been thoroughly exploited in the field to identify activated pool of the GTPases using effector pulldown assay [ 32 , 53 ] The effector binding by Rho GTPases is generally attributed to Switch I region also known as the effector binding region (YVPTVFDNY; EhRho5) and the ‘Rho insert’ region (EAMIRKLADENQK; EhRho5) [ 76 ]. Among these, the switch I region is in close vicinity to the nucleotide moiety and responsible for sensing GTP/GDP through interaction with the Mg 2+ ion via a conserved threonine (Thr42; EhRho5).…”

Pathogenicity and virulence of Entamoeba histolytica , the agent of amoebiasis

“…Uptake and display of mammalian membrane proteins on the surface of the amoeba protect the parasite from lysis by inhibiting the complement cascade, indicating that trogocytosis contributes to amoebic defence strategies for immune evasion [ 181 , 182 ]. Trogocytosis and phagocytosis occur by different mechanisms, although many of the early steps and molecules involved may be common [ 183 , 184 ].…”

Endocytosis in anaerobic parasitic protists

“…Concerning the well-known phagocytic activity of E. histolytica , Bhararadwaj and colleagues 99 identified the involvement of non-Dbl Rho guanine exchange factor (EhGEF) in regulating this activity. Using immunofluorescence microscopy, they show that EhGEF is localised in the phagocytic cup even during the progression of the cups and the closure of the phagosome, but not in the formed phagosome.…”