Cellular mechano-transduction is important for detecting environmental changes and Piezo channels, which were discovered in 2010, have crucial roles in several processes in mammalian cells, such as vascular development and function, pulmonary respiration, and sensory transduction. Their conservation in unicellular organisms and their function in Ca2+ signaling is an untapped area of research. Ca2+ is a potent signal for controlling a variety of downstream effectors that stimulate specific biological responses. We and others discovered years ago that contact with host cells triggers an increase in cytosolic Ca2+ of the infective trypomastigote stage of Trypanosoma cruzi, the etiologic agent of Chagas disease. Preventing this Ca2+ increase with intracellular Ca2+ chelators prevented host cell invasion. A role for Ca2+ in T. cruzi replication and differentiation was also proposed. The mechanism involved in the Ca2+ increase upon contact of trypomastigotes with host cells was never identified but its contact-dependence suggests a mechano-transduction process. Stimulation of Piezo channels preferentially leads to Ca2+ and in some cases Na+ and Ca2+ entry into cells. Two paralogs, TcPiezo1 and TcPiezo2, are present in T. cruzi. Using CRISPR/Cas9 techniques we have C-terminally tagged TcPiezo1 and localized it to the T. cruzi plasma membrane and an intracellular spot. We also obtained TcPiezo1-KO mutants, which are deficient in host cell invasion and intracellular replication. We propose to study the role of both Piezo channels in Ca2+ signaling during T. cruzi host cell invasion and replication. Our hypothesis is that T. cruzi Piezo channels will function at the plasma membrane as Ca2+ entry channels that will activate a signaling cascade culminating in specific parasite functions like activation of invasion and differentiation. With the aim of studying cytosolic Ca2+ concentration fluctuations in the infective stages of T. cruzi we propose to use genetically encoded Ca2+ indicators (GECIs), in addition to the traditional chemical indicators. GECIs are powerful tools that allow the noninvasive imaging of defined cells and compartments. The use of these new tools will facilitate direct real-time observation of Ca2+ changes during T. cruzi host cell invasion and replication, the investigation of the role of Piezo channels in the generation of these changes, and the study of other potential Ca2+ roles in future work. We propose to generate T. cruzi different stages expressing GECIs targeted to their cytosol and to their mitochondria to study the dynamics of Ca2+ in live parasites and explore the requirement for Ca2+ signaling during host cell invasion by trypomastigotes and during replication and differentiation of amastigotes, and the role of Piezo channels in these processes. T. cruzi Piezo channels will likely have lineage specific characteristics, which are different from those of mammalian cells Piezo channels, and their study could lead to new insights into the T. cruzi biology and the potential identification of new targets.
Approximately 7-8 million people are infected with Trypanosoma cruzi. The goal of this application is to study the role of Piezo channels in calcium ion (Ca2+) signaling during T. cruzi host cell invasion and replication. Our hypothesis is that Piezo channels functions have lineage specific characteristics, which are different from those of mammalian cells and that our studies will lead to new insights into the T. cruzi biology and the potential identification of new targets.
