Functional roles of AMP-activated protein kinase (AMPK) complexes containing TcAMPKα1 or TcAMPKα2 in energy homeostasis regulation and cell culture progression in Trypanosoma cruzi

Abstract

The AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme involved in maintaining energy homeostasis in response to different stresses in many organisms. During the transition between the mammalian host and the insect vector, Trypanosoma cruzi, the causative agent of Chagas disease, faces different types of environmental fluctuations, all of which prompt the parasite to remodel its metabolism. Recently, it was shown that Trypanosoma brucei AMPK is involved in the differentiation from the bloodstream slender to stumpy stage and in surface protein expression changes in response to nutritional stress. This underscores the relevance of AMPK for parasite life cycle progression.We identified four candidate genes for the AMPK subunits of T. cruzi (alpha1 and alpha2 catalytic subunits, beta and gamma regulatory subunits). The beta and gamma subunits are largely conserved in their domain structure relative to the mammalian orthologs. However, the alpha subunits show significant sequence and structure differences from the human counterparts. The presence of these subunits in T. cruzi epimastigotes was confirmed by RT-PCR, Western blot using a phospho-AMPKa specific antibody, mass spectrometry and by kinase activity assays using the specific AMPK substrate SAMS. TcAMPKa1 over-expressing epimastigotes showed a lower growth rate in basal culture conditions compared to the control. On the other hand, alpha2 over-expression had the opposite effect. Additionally, we observed upregulation of AMPK activity under epimastigote starvation, and that dorsomorphin, a specific AMPK inhibitor, also inhibits T. cruzi AMPK. Moreover, each of these subunits could complement S. cerevisiae conditional mutants lacking the respective subunit of the AMPK ortholog SNF1. Finally, starving assays with AMPKa over-expressing parasites also showed a possible role of AMPK in autophagy. Overall, our results show for the first time, the presence of a functional AMPK orthologue in Trypanosoma cruzi.