Assessing the calcium binding properties of the kinetoplastid-specific protein TcCAL1

Abstract

Trypanosoma cruzi, the causative agent of Chagas disease, survives by invading different organisms and facing diverse environments. The parasite successfully completes its life cycle by differentiating into various developmental stages adapted to proliferate alternately in the extra- or intracellular space. Calcium (Ca²⁺) plays important roles during mammalian host cell invasion and the differentiation from epimastigotes into infective metacyclic trypomastigotes, a process called metacyclogenesis. Although it is well established that the intracellular Ca²⁺ concentration [Ca²⁺]i increases during these events, the mechanisms that generate and decode this signal are not yet fully elucidated. Recently, we identified an hypothetical protein with putative Ca²⁺-binding domains named TcCAL1 to study its potential relevance in the parasite´s biology. TcCAL1 lacks homologs in mammalian genomes, contains two predicted EF-hand domains for Ca²⁺ binding, and is expressed throughout the entire T. cruzi life cycle. We found that the overexpression of TcCAL1 facilitates both the adhesion and internalization of T. cruzi into in vitro-cultured mammalian cells, thereby increasing the parasite´s virulence. We also demonstrated that TcCAL1 overexpression impairs metacyclogenesis in a dose-dependent manner. In this study, we investigated whether this phenotype is mediated by the Ca²⁺-binding properties of TcCAL1. To this end, we first employed different approaches to assay the association of Ca²⁺ using recombinant TcCAL1x6His protein produced in bacteria (rTcCAL1). We found that rTcCAL1 associates with Ca²⁺, emitting a dose-dependent signal when incubated in the presence of the fluorescent probe Quin-2. By contrast, studies using circular dichroism and nuclear magnetic resonance could neither confirm nor refute that rTcCAL1 binds Ca²⁺, suggesting that the wild-type version of the protein may be needed to demonstrate ion association by these methodologies#. We then measured the intracellular Ca²⁺ concentration [Ca²⁺]i in epimastigotes overexpressing TcCAL1 using the ratiometric indicator Fura-2AM and a FlexStation3 robotic multi-plate spectrofluorometer. These experiments showed that, in response to specific stimuli such as mild digitonin treatment, the increase in [Ca²⁺]i is significantly higher in control parasites compared to those overexpressing TcCAL1. This suggests that transgenic cultures exhibit more efficient buffering capacity against a Ca²⁺ influx into the cytoplasm due to the overexpression of TcCAL1 and its ion-binding property. Overall, these results encourage us to move forward with more studies aimed at elucidating the role of this kinetoplastid-specific protein in the metacyclogenesis of T. cruzi, as well on the invasion into the mammalian hosts.