Ca²⁺ entry at the plasma membrane and uptake by acidic stores is regulated by the activity of the V‐H⁺‐ATPase in Toxoplasma gondii

Abstract

Ca2+ is a universal intracellular signal that regulates many cellular functions. In Toxoplasma gondii, the controlled influx of extracellular and intracellular Ca2+ into the cytosol initiates a signaling cascade that promotes pathogenic processes like tissue destruction and dissemination. In this work, we studied the role of proton transport in cytosolic Ca2+ homeostasis and the initiation of Ca2+ signaling. We used a T. gondii mutant of the V‐H+‐ATPase, a pump previously shown to transport protons to the extracellular medium, and to control intracellular pH and membrane potential and we show that proton gradients are important for maintaining resting cytosolic Ca2+ at physiological levels and for Ca2+ influx. Proton transport was also important for Ca2+ storage by acidic stores and, unexpectedly, the endoplasmic reticulum. Proton transport impacted the amount of polyphosphate (polyP), a phosphate polymer that binds Ca2+ and concentrates in acidocalcisomes. This was supported by the co‐localization of the vacuolar transporter chaperone 4 (VTC4), the catalytic subunit of the VTC complex that synthesizes polyP, with the V‐ATPase in acidocalcisomes. Our work shows that proton transport regulates plasma membrane Ca2+ transport and control acidocalcisome polyP and Ca2+ content, impacting Ca2+ signaling and downstream stimulation of motility and egress in T. gondii.

Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress

“…These differences would impact Ca 2+ storage pools, affecting cytosolic Ca 2+ and signaling. For example, our findings indicate that bradyzoites show reduced responses to ionomycin and thapsigargin, which release Ca 2+ primarily from the ER, and in response to NH 4 Cl, which releases Ca 2+ from acidocalcisomes and likely other acidic stores ( Moreno and Zhong, 1996 ; Stasic et al, 2021 ). Consistent with these dampened responses, bradyzoites showed significantly reduced expression of the Ca 2+ -ATPases TgSERCA ( Nagamune et al, 2007 ) and TgA1 ( Luo et al, 2004 ), which are involved in transporting cytosolic Ca 2+ into the ER and acidocalcisome, respectively.…”

Toxoplasma bradyzoites exhibit physiological plasticity of calcium and energy stores controlling motility and egress

“…Based on the above findings, it is likely that bradyzoites possess different mechanisms to control Ca 2+ homeostasis, including differences in expression of calcium channels, calcium pumps and calcium storage pools relative to tachyzoites. For example, our findings indicate that bradyzoites show reduced responses to ionomycin and thapsigargin, which release Ca 2+ from the ER, and in response to NH 4 Cl, which releases Ca 2+ from acidocalcisomes and likely other acidic stores [35,55]. Consistent with these dampened responses, bradyzoites showed significantly reduced expression of the Ca 2+ -ATPases TgSERCA [34] and TgA1 [36], which are involved in transporting cytosolic Ca 2+ into the ER and acidocalcisome, respectively.…”

The Toxoplasma plant‐like vacuolar compartment (PLVAC)

“…Two genes with homology to the yeast VTC proteins ScVtc2p (VTC2) and ScVtc4p (VTC4) were identified in T . gondii and we have shown that the VTC4 subunit colocalizes with the V‐H + ‐ATPase to acidocalcisomes and the PLVAC (Stasic et al, 2021). As polyP is negatively charged, it is most likely involved in chelating positively charged ions like Ca 2+ , Zn 2+ , and others.…”