Identification and functional analysis of a stress-responsive MAPK15 in Entamoeba invadens

Abstract

E. histolytica, a protozoan parasite is the causative agent of amoebiasis in human beings. It exists in two different forms - the motile trophozoite form which undergoes encystation under starvation conditions to form the non-motile, osmotically resistant cyst form. Cellular stresses stimulate several signaling cascades which assist the parasite in counter-attacking such conditions thereby, promoting cell survival. To study the stress-associated pathways activated during encystation, we have used Entamoeba invadens, a reptilian parasite as a model organism because of its ability to undergo encystation under in vitro conditions. In this study, we have identified a stress-responsive MAPK which gets upregulated under different stress conditions, including encystation. Sequence analysis and phylogenetic classification show that the MAPK belongs to the atypical MAPK15 family (henceforth, named EiMAPK15), which does not require an upstream MAPKK for its phosphorylation and activation. The in vitro kinase activity of recombinant EiMAPK15 exhibits its auto-phosphorylation ability. Immunolocalization studies reveal that the protein is mainly cytosolic under normal growing conditions but gets translocated into the nucleus under stress conditions. Knockdown of EiMAPK15 using double-stranded RNA was found to reduce the expression of other encystation-specific genes which in turn, resulted in the decline of the overall encystation efficiency of the cells. Overall, the present work has laid the platform for further characterization of this important MAPK gene in Entamoeba invadens.

Keywords: Auto-phosphorylation; Cellular stress; Double-stranded RNA; Encystation; MAPK15.