The role of potassium and host calcium signaling in Toxoplasma gondii egress

Highlights

• Toxoplasma gondii replicates inside host cells and takes up Ca2+ from the host cytosol.

• T. gondii uses extracellular Ca2+ which contributes to reach a threshold needed for egress.

• Two peaks of Ca2+ precede parasite egress. Intracellular and extracellular stores contribute.

• It is possible to patch infected cells to deliver defined Ca2+ concentrations to trigger egress.

• Reduction of the surrounding potassium concentration modulates the rate of egress.

Abstract

Toxoplasma gondii is an obligate intracellular parasite and replicates inside a parasitophorous vacuole (PV) within the host cell. The membrane of the PV (PVM) contains pores that permits for equilibration of ions and small molecules between the host cytosol and the PV lumen. Ca²⁺ signaling is universal and both T. gondii and its mammalian host cell utilize Ca²⁺ signals to stimulate diverse cellular functions. Egress of T. gondii from host cells is an essential step for the infection cycle of T. gondii, and a cytosolic Ca²⁺ increase initiates a Ca²⁺ signaling cascade that culminates in the stimulation of motility and egress. In this work we demonstrate that intracellular T. gondii tachyzoites are able to take up Ca²⁺ from the host cytoplasm during host cell signaling events. Both intracellular and extracellular Ca²⁺ sources are important in reaching a threshold of parasite cytosolic Ca²⁺ needed for successful egress. Two peaks of Ca²⁺ were observed in egressing single parasites with the second peak resulting from Ca²⁺ entry. We patched infected host cells to allow the delivery of precise concentrations of Ca²⁺ for the stimulation of motility and egress. Using this approach of patching infected host cells, allowed us to determine that increasing the host cytosolic Ca²⁺ to a specific concentration can trigger egress, which is further accelerated by diminishing the concentration of potassium (K⁺).

Introduction

Toxoplasma gondii is an obligate intracellular parasite that infects approximately one third of the world's population. T. gondii replicates inside cells and causes disease by engaging in multiple rounds of a lytic cycle, which consists of invasion of host cells, replication inside a parasitophorous vacuole (PV), egress resulting in lysis of the host cell, and invasion of a new host cell [1,2]. Several key steps of the lytic cycle of T. gondii: motility, attachment, invasion, and egress, are regulated by fluctuations in its cytosolic Ca²⁺ concentration ([Ca²⁺]_c) [3,4].

Ca²⁺ signaling is universal and plays important roles in the regulation of a large number of cellular functions [5]. The [Ca²⁺]_c is highly regulated, because prolonged high cytosolic Ca²⁺ levels are toxic and may result in cell death. A variety of Ca²⁺ pumps, channels, and transporters, located at the plasma membrane (PM) and intracellular organelles (endoplasmic reticulum (ER), acidic stores, and mitochondria) are involved in regulating cytosolic Ca²⁺ [6].

In T. gondii, the controlled influx of extracellular and intracellular Ca²⁺ into the parasite cytosol initiates a cascade of signaling pathways that promotes progression through the biological steps of the parasite lytic cycle. Motile parasites loaded with fluorescent Ca²⁺ indicators, as well as expressing Genetically Encoded Calcium Indicators (GECIs) exhibit Ca²⁺ oscillations [7,8]. Previous studies have shown that a rise in the cytosolic Ca²⁺ activates the motility machinery leading to egress. Blocking these cytosolic Ca²⁺ fluxes with BAPTA-AM (membrane permeable cytosolic Ca²⁺ chelator), blocks motility, conoid extrusion (apical tip of the parasite necessary for attachment), invasion, and host cell egress [9].

Measurements of T. gondii [Ca²⁺]_c (using Fura2-AM) showed influx of Ca²⁺ from the extracellular environment, which did not operate as store-operated calcium entry (SOCE) as shown with experiments testing surrogate ions like Mn [10]. This result was supported by the lack of components of the SOCE pathway, STIM and ORAI from the T. gondii genome [11]. We showed that influx of Ca²⁺, as well as invasion-linked traits [10] and egress [8] were inhibited by nifedipine a Voltage Operated Calcium Chanel blocker (VOCC), supporting the function of this type of channel in Ca²⁺ influx.

Active egress of T. gondii from host cells requires rupture of the parasitophorous vacuole membrane (PVM) and the host cell membrane [12]. Egress is essential for the dissemination of the infection, and it has been known for several years that Ca²⁺ ionophores can trigger egress [13]. The final and conclusive evidence of a cytosolic Ca²⁺ increase preceding egress was obtained by expressing GECIs in the cytosol of T. gondii tachyzoites [8]. Secretion of the Perforin-Like Protein 1 (TgPLP1) from micronemes (specialized secretory organelles involved in egress, motility, and invasion by tachyzoites), assists in the permeabilization of the PVM and host cell membrane [14]. Both secretion of the microneme protein TgPLP1 and initiation of motility during egress are stimulated by an increase in cytosolic Ca²⁺. It has been proposed that the trigger for this cytosolic Ca²⁺ increase is the rupture of the host plasma membrane and the ensuing reduction in the concentration of the surrounding potassium [K⁺]. It was proposed that low [K⁺] would activate a phospholipase C activity in T. gondii that, in turn, would cause an increase in [Ca²⁺]_c in the parasite via Ca²⁺ release from intracellular stores [15].

As an obligate intracellular parasite, T. gondii resides and replicates within the PV that functions as a molecular sieve and passively permits the exchange of small molecules; thus, the surrounding milieu of intracellular parasites is likely in equilibrium with the host cell cytoplasm [16]. Therefore, intracellular parasites would be exposed to the same fluctuations of the host cytosolic ionic composition. The host cytosolic Ca²⁺ is highly regulated and the resting [Ca²⁺]_c is maintained at ∼70−100 nM, which is similar to the [Ca²⁺]_c of replicating parasites. Ca²⁺ efflux from intracellular stores of the parasite must be the first step of the Ca²⁺ signaling pathway leading to activation of egress, so maintaining these stores replenished with Ca²⁺ is fundamental for continuation of the lytic cycle.

In this work, we investigated how T. gondii manages to replenish its intracellular Ca²⁺ stores during its intracellular replication and how the decrease of the surrounding K⁺ concentration impacts parasite egress. Using a variety of pharmacological tools, fluorescence microscopy, and a new approach using patched infected host cells, we show that Ca²⁺ signaling of the host cell influences parasite cytosolic Ca²⁺ and contributes to parasite egress. Rupture of the host cell during egress facilitates Ca²⁺ influx. The ensuing decrease in the K⁺ concentration modulates but does not trigger egress directly.