Fig 1.
Conditional knockout of TcPiezo1 and subcellular localization.
(A) Schematic representation of CRISPR/Cas9 mediated endogenous C-terminal tagging of TcPiezo1 with an aptazyme cassette Shark1 or Shark3 (composed of 3 × Ty tag, ribozyme, GAPDH 3’UTR, and Bsd gene) for conditional knockout (CKO) of TcPiezo1. (B) Ty1-tagged TcPiezo1 Tet-OFF colocalization with T. cruzi plasma membrane marker H+-ATPase (TcHAf or α-HAf) in the plasma membrane of epimastigote (Pearson’s correlation coefficient (PCC): 0.8750), trypomastigote (PCC: 0.6021) and amastigote (PCC: 0.9240), as detected by immunofluorescence analyses with antibodies against Ty1. Yellow in merged images indicates colocalization. DIC, differential interference contrast microscopy. DAPI staining in blue. Scale bars 5 and 10 µm, as indicated. (C) Growth of TcPiezo1 Tet-OFF epimastigotes in the absence (black line, − Tet) or presence (red line, + Tet) of 5 μg/ml tetracycline for the indicated number of days. Western blot analyses of TcPiezo1 Tet-OFF epimastigotes grown in the absence (0) or presence (2–6) of tetracycline. Total lysates (30 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis before transfer to a nitrocellulose membrane and then stained with antibodies against Ty1 (top). One band of ~ 290 kDa was detected in epimastigote homogenates. Membranes were stripped and re-incubated with antibody against Alpha-tubulin as a loading control (bottom, Tub). (D) Percentage (%) of metacyclic trypomastigotes in TcPiezo1 Tet-OFF epimastigote cultures after incubation in TAU 3AAG medium in the absence (-Tet) or presence (+Tet) of 5 µ g/ml tetracycline after 96 h. Differentiation of epimastigotes to metacyclic trypomastigotes was quantified by staining with DAPI to distinguish the position of the kinetoplast by fluorescence microscopy. (E) Effect of non-induced (-Tet) and induced (+Tet) TcPiezo1 Tet-OFF on trypomastigote infection of Vero cells after 4 h. (F) Effect of non-induced (-Tet) and induced (+Tet) TcPiezo1 Tet-OFF on amastigote replication after 72 h. In panels C, D, E, F, values are means ± s.d. (n = 3). One-way ANOVA with multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig 2.
Downregulation of TcPiezo1 expression decreases Ca2+ entry in T. cruzi.
Intracellular Ca2+ concentrations of 5 × 107 TcPiezo1 Tet-OFF epimastigotes (A-C) or 2 x 107 trypomastigotes (D) expressing jGCaMP7s were measured by jGCaMP7s signal fluorescence in arbitrary units of fluorescence (AU). An increase in jGCaMP7s fluorescence indicates increasing cytosolic Ca2+. (A) Addition of 1.8 mM Ca2+ in Tet-induced TcPiezo1 cells (+Tet) elicited a lower increase in intracellular Ca2+ than in non-induced cells (-Tet). (B) Downregulation of TcPiezo1 expression showed a significant decrease of intracellular Ca2+ (+Tet). Addition of 10 µ M nifedipine (the TcCav inhibitor) slightly reduced intracellular Ca2+ in non-induced cells (-Tet + Nif) while it had an additive effect in Tet-induced cells (+Tet + Nif). (C) Addition of 10 µ M GsMTx4 showed a significant reduction of intracellular Ca2+ in non-induced cells (-Tet + GsM) comparable to downregulation of TcPiezo1 (+Tet) and had an additive effect in Tet-induced cells (+Tet + GsM). (D) Extracellular matrix (ECM) triggered TcPiezo1-mediated Ca2+ entry in tissue culture-derived trypomastigotes. Addition of 40 µg ECM to non-induced (-Tet) trypomastigotes significantly increased intracellular Ca2+, compared to Tet-induced cells (+Tet). In panels A-D, addition of buffer A with glucose (BAG), instead of 1.8 mM Ca2+, was used as a control baseline labeled with B. Values are means ± s.d. (n = 3). One-way ANOVA with multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig 3.
Subcellular localization and conditional knockout (CKO) of TcPiezo2.
(A) Endogenous C-terminally smV5-tagged TcPiezo2 colocalized with T. cruzi cruzipain (TcCZP) to the reservosomes/lysosomes of epimastigotes (PCC: 0.7294), trypomastigotes (PCC: 0.7443) and amastigotes (PCC: 0.8524). Yellow in merged images indicates colocalization. DIC, differential interference contrast microscopy. DAPI staining in blue. Scale bars 5 and 10 µm, as indicated. (B) Growth of TcPiezo2 Tet-OFF epimastigotes in the absence (black line, − Tet) or presence (red line, + Tet) of 5 μg/ml tetracycline for the indicated number of days. Western blot analyses of TcPiezo2 Tet-OFF epimastigotes grown in the absence (0) or presence (2–6) of tetracycline. Total lysates (30 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis before transfer to a nitrocellulose membrane and then stained with antibodies against Ty1 (top). One band of ~ 280 kDa was detected in epimastigote homogenates. Membranes were stripped and re-incubated with antibody against Alpha-tubulin as a loading control (bottom, Tub). (C) Percentage of metacyclic trypomastigotes in TcPiezo2 Tet-OFF epimastigote cultures after incubation in TAU 3AAG medium in the absence (-Tet) and presence (+Tet) of 5 µ g/ml tetracycline after 96 h. (D) Effect of non-induced (-Tet) and induced (+Tet) TcPiezo2 Tet-OFF on trypomastigote infection of Vero cells after 4 h. (E) Effect of non-induced (-Tet) and induced (+Tet) TcPiezo2 Tet-OFF on amastigote replication after 72 h. In panels B, C, D, E, values are means ± s.d. (n = 3). One-way ANOVA with multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001).
Fig 4.
Effects of TcPiezo2 expression downregulation on intracellular Ca2+ stores in T. cruzi.
Intracellular Ca2+ concentrations of 5 × 107 TcPiezo2 Tet-OFF epimastigotes expressing jGCaMP7s were measured by jGCaMP7s signal fluorescence in AU. (A) TcPiezo2 mediated lysosomal Ca2+ release. Addition of 70 µ M Glycyl-L-phenylalanine 2-naphthylamide (GPN) for activation of lysosomal Ca2+ release in Tet-induced TcPiezo2 cells (+Tet) elicited less increase in intracellular Ca2+ than in non-induced cells (-Tet). (B) Addition of 30 µ M cyclopiazonic acid (CPA) for activation of ER Ca2+ release demonstrated no significant difference between Tet-induced (+Tet) and non-induced (-Tet) cells. (C) Addition of 1.8 mM Ca2+ showed no significant difference in intracellular Ca2+ between Tet-induced (+Tet) and non-induced (-Tet) cells. (D) Addition of 1 µ M ionomycin in Tet-induced cells (+Tet) elicited less increase of intracellular Ca2+ than in non-induced cells (-Tet). 100 µ M EGTA was added to remove extracellular Ca2+ in A-D. Addition of DMSO or BAG was used as control (baseline) labeled with B. Values are means ± s.d. (n = 3). One-way ANOVA with multiple comparisons (*P < 0.05, **P < 0.01).
Fig 5.
Effects of activators and inhibitors on TcPiezo channels.
Intracellular Ca2+ changes of 5 × 107 TcPiezo Tet-OFF epimastigotes expressing jGCaMP7s were measured by jGCaMP7s signal fluorescence in AU. (A) Yoda1 activated TcPiezo channels. Addition of 20 µ M or 40 µ M Yoda1 to TcPiezo1 Tet-OFF cells elicited two distinct peaks of Ca2+ increase, indicating that TcPiezo1 and TcPiezo2 were independently activated. (B) Addition of 20 or 40 µ M Dooku1 to the cells elicited two different peaks of Ca2+ rise, also suggesting that the two TcPiezo channels were successively activated. (C) Two Ca2+ spikes were generated in Tet-induced (+Tet) and non-induced (-Tet) TcPiezo1 Tet-OFF cells, respectively, by the additions of 1.8 mM Ca2+ and 40 µ M Dooku1. (D) Changes in jGCaMP7s fluorescence (first peak and second peak in Fig 5C) in Tet-induced (+Tet) and non-induced (-Tet) cells. Downregulation of TcPiezo1 expression significantly decreased Dooku1-evoked TcPiezo1-mediated Ca2+ entry (left bar graph) but had no effect on Dooku1-evoked TcPiezo2-mediated Ca2+ release (right bar graph). (E) 100 µ M EGTA was incubated with TcPiezo2 Tet-OFF cells to remove extracellular Ca2+, abolishing TcPiezo1-mediated Ca2+ entry. 70 µ M GPN was added to activate Ca2+ release from lysosomes. Downregulation of TcPiezo2 expression (+Tet) showed a significant decrease of lysosomal Ca2+ release as in Fig 4A. Addition of 10 µ M GsMTx4 showed a significant reduction of intracellular Ca2+ in non-induced cells (-Tet + GsM) like downregulation of TcPiezo2 (+Tet) and had an additive effect on TcPiezo2-mediated Ca2+ release in Tet-induced cells (+Tet + GsM). (F) Changes in jGCaMP7s fluorescence (left bar graph) and the rates of fluorescence increase (right bar graph) upon GsM inhibition in Tet-induced (+Tet ± GsM) and non-induced (-Tet ± GsM) cells in Fig 5E. (G) 100 µ M EGTA and 70 µ M GPN were added to TcPiezo2 Tet-OFF cells (±Tet), as indicated. Addition of 20 µ M Yoda1 (+Yoda1) dramatically stimulated TcPiezo2-mediated Ca2+ release from lysosomes. (H) Changes in fluorescence (left bar graph) and the rates of fluorescence increase (right bar graph) upon Yoda1 activation in Tet-induced (+Tet±Yoda1) and non-induced (-Te±Yoda1) cells in Fig 5G. Addition of DMSO, instead of Yoda1/Dooku1/GsM, was used as control (baseline) labeled with B. In panels D, F, H, values are means ± s.d. (n = 3). One-way ANOVA with multiple comparisons (**P < 0.01, ***P < 0.001).
Fig 6.
TcPiezo-CKO responses to hypoosmotic stress.
5 × 107 TcPiezo-CKO epimastigotes expressing jGCaMP7s in 600 µl isosmotic buffer (300 mOsm) was submitted to hypoosmotic stress (150 mOsm) by adding 600 µl H2O at 50 s. Upon hypoosmotic stress, the cells swelled and the intracellular Ca2+ rose (control, C or -Tet). No intracellular Ca2+ was increased by adding 600 µl isosmotic buffer (baselines, B). (A) Non-induced TcPiezo1 Tet-OFF intracellular Ca2+ responses to the addition of 100 µ M EGTA, 1 µ M Xestospongin C (XestC), or 4 µ M ruthenium red (RR). The addition of EGTA did not affect intracellular Ca2+ rise, but the addition of XestC or RR did. (B) Tet-induced TcPiezo1 Tet-OFF cells (+Tet) had slightly lower Ca2+ rise than non-induced cells (-Tet). (C) Non-induced TcPiezo2 Tet-OFF (-Tet) intracellular Ca2+ responses to the addition of 1 µ M XestC or 4 µ M RR. The addition of XestC or RR significantly affected intracellular Ca2+ rise. (D) Tet-induced TcPiezo2 Tet-OFF cells (+Tet) had a significant decrease of intracellular Ca2+ rise, compared to non-induced cells (-Tet). 100 µ M EGTA was added to remove extracellular Ca2+, abolishing Ca2+ entry, as indicated. Values are means ± s.d. (n = 3). One-way ANOVA with multiple comparisons (*P < 0.05, **P < 0.01, *** P < 0.001).
Fig 7.
Scheme of TcPiezo-mediated Ca2+ entry and intracellular Ca2+ release in T. cruzi. TcPiezo 1 mediates Ca2+ entry through the plasma membrane (PM), which is activated by Yoda1, Dooku1 and extracellular matrix (ECM), and inhibited by spider mechanotoxin 4 (GsMTx4) and ruthenium red (RR), respectively.
TcPiezo 2 mediates Ca2+ release from the lysosomes, which is activated by Yoda1and Dooku1, and blocked by GsMTx4, respectively. Intracellular Ca2+ changes are detected by using the cytosolic expressing jGCaMP7s. Downregulation of TcPiezo1 or TcPiezo2 by a novel conditional expression system using hammerhead ribozymes (HHR)-Shark1 and Shark3 establishes the essentiality of Piezo channels for the life cycle of T. cruzi.