Statistical treatment of the data.

Trypanosoma cruzi, the causative agent of Chagas disease, is a parasitic protist that affects millions of people worldwide. Currently there are no fully effective drugs or vaccines available. Contact of T. cruzi infective forms with their host cells or with the extracellular matrix increases their intracellular Ca2+ concentration suggesting a mechano-transduction process. We report here that T. cruzi possesses two distinct mechanosensitive Piezo channels, named TcPiezo1 and TcPiezo2, with different subcellular localizations but similarly essential for normal proliferation, differentiation, and infectivity. While TcPiezo1 localizes to the plasma membrane, TcPiezo2 localizes to the lysosomes. Downregulation of TcPiezo1 expression by a novel ligand-regulated hammerhead ribozyme (HHR) significantly inhibited Ca2+ entry in cells expressing a genetically encoded Ca2+ indicator while downregulation of TcPiezo2 expression inhibited Ca2+ release from lysosomes, which are now identified as novel acidic Ca2+ stores in trypanosomes. The channels are activated by contact with extracellular matrix and by hypoosmotic stress. The results establish the essentiality of Piezo channels for the life cycle and Ca2+ homeostasis of T. cruzi and a novel lysosomal localization for a Piezo channel in eukaryotes.