TcAMPK: identification and characterization of a cellular energy homeostasis hub regulator in Trypanosoma cruzi

PATRICIO GENTA; STERNLIEB T; ALONSO GD; SCHOIJET AC

Mar del Plata

Congreso; XXXI Reunión de la Sociedad Argentina de Protozoología (Sap). 13-16 de noviembre de 2019; 2019

TheAMP-activated protein kinase (AMPK) is a heterotrimeric enzyme involved inmaintaining energy homeostasis in response to different stresses in manyorganisms. During the transition between the mammalian host and the insectvector, Trypanosoma cruzi, the causative agent of Chagas disease, faces different types of environmentalfluctuations, all of which prompt the parasite to remodel its metabolism toadapt, survive and differentiate into the next stages of its life cycle.Recently, it was shown that Trypanosomabrucei AMPK is involved in the differentiation from the bloodstream slenderto stumpy stage and in surface protein expression changes in response tonutritional stress. This underscores the relevance of AMPK for parasite lifecycle progression.Weidentified four candidate genes for the AMPK subunits of T. cruzi (α1 and α2 catalytic subunits, β and γ regulatorysubunits). The β and γ subunits are largely conserved in their domain structurerelative to the mammalian orthologs. However, the alpha subunits showsignificant sequence and structure differences from the human counterparts. Thepresence of these subunits in T. cruziepimastigotes was confirmed by RT-PCR, Western blot using a phospho-AMPKαspecific antibody, mass spectrometry and by kinase activity assays using the specificAMPK substrate SAMS. TcAMPKα1 over-expressing epimastigotes showed a lowergrowth rate in basal culture conditions compared to the control, while α2over-expression had the opposite effect. We found there is upregulation of AMPKactivity under epimastigote starvation, and that dorsomorphin, a specific AMPK inhibitor,also inhibits T. cruzi AMPK. Each of these subunits could complement the?glucose dependent? phenotype of S.cerevisiae conditional mutants lacking the respective subunit of the AMPKortholog SNF1. Starving assays with AMPKα over-expressing parasites also showeda possible role of AMPK in autophagy. Our results show, for the first time, thepresence of a functional AMPK orthologue in Trypanosomacruzi.