An atypical EhGEF regulates phagocytosis in Entamoeba histolytica through EhRho1.

Ravi Bharadwaj,Tomoyoshi Nozaki,Krishna K Inampudi,Somlata Somlata,Azhar Ahmad,Tushar Kushwaha,William A Petri

doi:10.1371/journal.ppat.1010030

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.

Highlights

Entamoeba histolytica is the causative agent of amoebic dysentery or amoebiasis, a major public health problem throughout the world, in developing countries and is one of the major causes of morbidity and mortality [1,2]
E. histolytica is causative agent of amoebiasis in humans, which is of major concern in children, as repeated infection leads to stunted physical and mental growth
The molecules, which participate in these endocytic processes may prove to be good therapeutic targets, as endocytic processes are necessary for parasite growth, survival, and pathogenesis in host system

Summary

Introduction

Entamoeba histolytica is the causative agent of amoebic dysentery or amoebiasis, a major public health problem throughout the world, in developing countries and is one of the major causes of morbidity and mortality [1,2]. A number of cell surface molecules, such as Gal/GalNAc lectin [11], TMK96 [12], TMK39 [13], SREHP [14,15] and EhROM1[16] have been shown to be involved in adherence to other cells It is not yet clear if these molecules are amoebic receptors during phagocytosis of prey, such as RBC, bacteria and apoptotic human cells [17,18]. Analysis of the phagosome proteome has revealed involvement of a large number of proteins in phagosome formation and subsequent maturation [19,20,21,22] Some of these, such as actin, Arp proteins, actin binding proteins, PI3 kinase, activated protein kinase (PAK), and Rho GTPases are already known to be part of phagocytic and signalling pathways [23,24,25,26,27]. E. histolytica genome codes for large number of small GTPases belonging to Rho, Ras and Rac families and it has been suggested that the function and regulation of these molecules are intimately related to pathogenic mechanisms of E. histolytica [23,28,29]

Methods

Results

Discussion

Conclusion