Stress-responsive AMP Kinase like protein regulates encystation of Entamoeba invadens.

ABSTRACT

Starvation is always accompanied by an increase in the ratio of AMP/ATP followed by activation of AMPK. It is one of the sensors for cellular energy status and is highly conserved across various species. Its role in the stage differentiation process of protozoan species like Giardia, Plasmodium, Trypanosome, and Toxoplasma has been reported. Since Entamoeba undergoes encystation in glucose-starved conditions; it intrigued us to investigate the existence and role of AMPK during the differentiation of trophozoites to the cyst. By employing in silico approaches, we have identified an AMPK homologue which is denominated here as EiAMPK (AMPK-like protein in Entamoeba invadens). Sequence and structural analysis indicate that EiAMPK is sequentially and structurally similar to the AMPK alpha subunit of other organisms. The recombinant form of EiAMPK was functionally active and in accordance, its activity was inhibited by an AMPK-specific inhibitor (eg. Compound C). The increased expression of EiAMPK during different stresses indicated that EiAMPK is a stress-responsive gene. To further investigate, whether EiAMPK has any role in encystation, we employed RNAi-mediated gene silencing that demonstrated its active involvement in encystation. It is known that Entamoeba maintains a flow of glucose from the glycolytic pathway to chitin synthesis for cyst wall formation during encystation. It is conceivable that EiAMPK might have a command over such glucose metabolism. As anticipated, the chitin synthesis was found greatly inhibited in both EiAMPK knockdown and Compound C treated cells, indicating that EiAMPK regulates the cyst wall chitin synthesis.