An atypical EhGEF regulates phagocytosis in Entamoeba histolytica through EhRho1.

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. Author summary: 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. Parasite display variety of endocytic processes, like phagocytosis, trogocytosis and micropinocytosis to name a few. The molecular mechanism of these processes is still largely unknown. 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. Here in this this report, we have focussed on an unconventional EhGEF, which was identified as EhRho1 interacting protein by mass spectrometry. Our microscopy experiments show EhGEF participates in phagocytosis and trogocytosis and is highly dynamic in nature. The molecule also plays a crucial role in activation and recruitment of EhRho1 to the site of phagocytosis, and downregulation of EhGEF mimics phenotype similar to overexpression of activity dead mutant (T34N) of EhRho1. Our finding reveals EhGEF to play important role in actin dependent endocytic processes through EhRho1 and thereby, regulating actin dynamics during the process through EhFormin1 and EhProfilin1. [ABSTRACT FROM AUTHOR]