Category: publications

Trypanosoma cruzi, the agent of Chagas disease, accumulates polyphosphate (polyP) and Ca²⁺ inside acidocalcisomes. The alkalinization of this organelle stimulates polyP hydrolysis and Ca²⁺ release. Here, we report that histidine ammonia lyase (HAL), an enzyme that catalyzes histidine deamination with production of ammonia (NH₃) and urocanate, is responsible for acidocalcisome alkalinization. Histidine addition to live parasites expressing HAL fused to the pH-sensitive emission biosensor green fluorescent protein (GFP) variant pHluorin induced alkalinization of acidocalcisomes. PolyP decreased HAL activity of epimastigote lysates or the recombinant protein but did not cause its polyphosphorylation, as determined by the lack of HAL electrophoretic shift on NuPAGE gels using both in vitro and in vivo conditions. We demonstrate that HAL binds strongly to polyP and localizes to the acidocalcisomes and cytosol of the parasite. Four lysine residues localized in the HAL C-terminal region are instrumental for its polyP binding, its inhibition by polyP, its function inside acidocalcisomes, and parasite survival under starvation conditions. Expression of HAL in yeast deficient in polyP degradation decreased cell fitness. This effect was enhanced by histidine and decreased when the lysine-rich C-terminal region was deleted. In conclusion, this study highlights a mechanism for stimulation of acidocalcisome alkalinization linked to amino acid metabolism.

IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and is characterized by the presence of acidocalcisomes, organelles rich in phosphate and calcium. Release of these molecules, which are necessary for growth and cell signaling, is induced by alkalinization, but a physiological mechanism for acidocalcisome alkalinization was unknown. In this work, we demonstrate that a histidine ammonia lyase localizes to acidocalcisomes and is responsible for their alkalinization.

Brian S Mantilla, Cristina Azevedo, Paul W Denny, Adolfo Saiardi, Roberto Docampo. mBio. 2021 Nov 2;e0198121. doi: 10.1128/mBio.01981-21.

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. 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.

Miguel A Chiurillo, Bryan C Jensen, Roberto Docampo. Microbiol Spectr. 2021 Sep 29;e0073821. doi: 10.1128/Spectrum.00738-21.