Abstract
ABSTRACTTrypanosoma 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.
Highlights
IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and is characterized by the presence of acidocalcisomes, organelles rich in phosphate and calcium
We identified a C-terminal lysine-rich region in histidine ammonia lyase (HAL) whose deletion inhibits epimastigote growth in starvation medium supplemented with histidine as an energy source and which reverses its toxicity when expressed in yeast
Alkalinization of acidocalcisomes is important for polyP hydrolysis and Ca21 release from these organelles [6], but a physiological mechanism for this process had not been identified until now
Summary
IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and is characterized by the presence of acidocalcisomes, organelles rich in phosphate and calcium. Trypanosoma cruzi, the etiologic agent of Chagas disease, is characterized by the presence of acidocalcisomes, lysosome-related organelles containing large amounts of polyphosphate (polyP) bound to organic cations, such as basic amino acids and polyamines, and inorganic cations like calcium, magnesium, potassium, sodium, and zinc [1]. These organelles are acidified by two proton pumps, a vacuolar H1-ATPase (V-ATPase) [2] and a vacuolar H1 pyrophosphatase (VP1) [3], and possess a P-type Ca21-ATPase for Ca21 uptake [4] and an inositol 1,4,5-trisphosphate receptor (IP3R) for Ca21 release [5]. This enzyme is developmentally regulated and highly expressed in epimastigotes [11]
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