Abstract
ABSTRACTProtein phosphorylation is involved in several key biological roles in the complex life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease, and protein kinases are potential drug targets. Here, we report that the AGC essential kinase 1 (TcAEK1) exhibits a cytosolic localization and a higher level of expression in the replicative stages of the parasite. A CRISPR/Cas9 editing technique was used to generate ATP analog-sensitive TcAEK1 gatekeeper residue mutants that were selectively and acutely inhibited by bumped kinase inhibitors (BKIs). Analysis of a single allele deletion cell line (TcAEK1-SKO), and gatekeeper mutants upon treatment with inhibitor, showed that epimastigote forms exhibited a severe defect in cytokinesis. Moreover, we also demonstrated that TcAEK1 is essential for epimastigote proliferation, trypomastigote host cell invasion, and amastigote replication. We suggest that TcAEK1 is a pleiotropic player involved in cytokinesis regulation in T. cruzi and thus validate TcAEK1 as a drug target for further exploration. The gene editing strategy we applied to construct the ATP analog-sensitive enzyme could be appropriate for the study of other proteins of the T. cruzi kinome.IMPORTANCE Chagas disease affects 6 to 7 million people in the Americas, and its treatment has been limited to drugs with relatively high toxicity and low efficacy in the chronic phase of the infection. New validated targets are needed to combat this disease. In this work, we report the chemical and genetic validation of the protein kinase AEK1, which is essential for cytokinesis and infectivity, using a novel gene editing strategy.
Highlights
Protein phosphorylation is involved in several key biological roles in the complex life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease, and protein kinases are potential drug targets
Putative phosphorylation sites, which are conserved in AGC kinase proteins to regulate their function, are predicted in TcAEK1: the first is located in the kinase domain at the “activation loop” (S216, corresponding to HsAKT3 T305), and the second is located in the AGC kinase C-terminal domain (S382, corresponding to HsAKT3 S472) (Fig. 1A)
We found that parasites hemizygous for AEK1 and those in which AEK1 was inhibited through a chemical genetic approach showed similar phenotypes
Summary
Protein phosphorylation is involved in several key biological roles in the complex life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease, and protein kinases are potential drug targets. IMPORTANCE Chagas disease affects 6 to 7 million people in the Americas, and its treatment has been limited to drugs with relatively high toxicity and low efficacy in the chronic phase of the infection. Trypanosoma cruzi, the causative agent of Chagas disease, is a parasitic protist that belongs to the order Kinetoplastida and exhibits a complex life cycle. This parasite has four major developmental stages, epimastigotes and metacyclic trypomastigotes in the insect vector and cell-derived trypomastigotes and amastigotes in the mammalian host, with two replicative stages: the epimastigote and amastigote forms. Protein kinases constitute an attractive class of molecular targets for drug discovery for the treatment of T. cruzi infection
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