A novel dense granule protein, GRA41, regulates timing of egress and calcium sensitivity in Toxoplasma gondii (original) (raw)
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The Journal of biological chemistry, 2015
Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole (PV) where it communicates with the host cell cytosol through the PV membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca2+ oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, critical steps of the lytic cycle. Extracellular Ca2+ enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors and measured Ca2+ changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca2+ oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasite...
The Role of Host Calcium Signaling in Toxoplasma gondii egress
Toxoplasma gondii, an obligate intracellular parasite, is capable of invading virtually any nucleated cell. Active invasion by the parasite is a precise process and intracellular parasites reside and replicate inside a parasitophorous vacuole (PV). The membrane of the PV functions as a molecular sieve allowing for its ionic milieu to be in equilibrium with the host cytosol. The parasite would thus be exposed to the ionic fluctuations of the host cytoplasm, such as increases in host cytosolic Ca 2+ during Ca 2+ signaling events. Ca 2+ is a universal signaling molecule and both the host cell and T. gondii will utilize Ca 2+ signaling to stimulate diverse cellular functions. Using T. gondii tachyzoites and host cells expressing genetically encoded Ca 2+ indicators, we demonstrate that host Ca 2+ signaling impacts intracellular Ca 2+ levels of the parasite. We propose that Ca 2+ influx derived from host Ca 2+ signaling into the parasite is utilized to maintain the intracellular Ca 2+ stores replenished as the parasite replicates within the low Ca 2+ environment of the host cell. Intracellular Ca 2+ store(s) release is needed to reach the initiation spike in Ca 2+ that meets the threshold of Ca 2+ needed to initiate the tightly coordinated process of egress. Post activation of the signal and subsequent microneme secretion, would cause breakdown of the host cell and allow for extracellular Ca 2+ influx, causing a second, larger spike in Ca 2+ and activation of the glideosome, the main motility machinery. To directly deliver precise concentrations of ions needed for egress, we used patch-clamped infected host cells and monitored parasite egress. In doing so, we discovered an alternative role for K + in timing parasite egress. This is the first study using wholecell patch clamp to study the role of ions such as K + and Ca 2+ in T. gondii egress.
Calcium entry in Toxoplasma gondii and its enhancing effect of invasion-linked traits
The Journal of biological chemistry, 2014
During invasion and egress from their host cells, Apicomplexan parasites face sharp changes in the surrounding calcium ion (Ca(2+)) concentration. Our work with Toxoplasma gondii provides evidence for Ca(2+) influx from the extracellular milieu leading to cytosolic Ca(2+) increase and enhancement of virulence traits, such as gliding motility, conoid extrusion, microneme secretion, and host cell invasion. Assays of Mn(2+) and Ba(2+) uptake do not support a canonical store-regulated Ca(2+) entry mechanism. Ca(2+) entry was blocked by the L-type Ca(2+) channel inhibitor nifedipine and stimulated by the increase in cytosolic Ca(2+) and by the specific L-type Ca(2+) channel agonist Bay K-8644. Our results demonstrate that Ca(2+) entry is critical for parasite virulence. We propose a regulated Ca(2+) entry mechanism activated by cytosolic Ca(2+) that has an enhancing effect on invasion-linked traits.
Calcium-mediated protein secretion potentiates motility in Toxoplasma gondii
Journal of Cell Science, 2004
Apicomplexans such as Toxoplasma gondii actively invade host cells using a unique parasite-dependent mechanism termed gliding motility. Calcium-mediated protein secretion by the parasite has been implicated in this process, but the precise role of calcium signaling in motility remains unclear. Here we used calmidazolium as a tool to stimulate intracellular calcium fluxes and found that this drug led to enhanced motility by T. gondii. Treatment with calmidazolium increased the duration of gliding and resulted in trails that were twice as long as those formed by control parasites. Calmidazolium also increased microneme secretion by T. gondii, and studies with a deletion mutant of the accessory protein m2AP specifically implicated that adhesin MIC2 was important for gliding. The effects of calmidazolium on gliding and secretion were due to increased release of calcium from intracellular stores and calcium influx from the extracellular milieu. In addition, we demonstrate that calmidazolium-stimulated increases in intracellular calcium were highly dynamic, and that rapid fluxes in calcium levels were associated with parasite motility. Our studies suggest that oscillations in intracellular calcium levels may regulate microneme secretion and control gliding motility in T. gondii.
PLOS Pathogens
Fundamental processes that govern the lytic cycle of the intracellular parasite Toxoplasma gondii are regulated by several signalling pathways. However, how these pathways are connected remains largely unknown. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels. We show that both egress inducers trigger indistinguishable signalling responses and provide evidence for a positive feedback loop linking calcium and cyclic nucleotide signalling. Using WT and conditional knockout parasites of the non-essential calcium-dependent protein kinase 3 (CDPK3), which display a delay in calcium inonophore-mediated egress, we explore changes in phosphorylation and lipid signalling in sub-minute timecourses after inducing Ca2+ release. These studies indicate that cAMP and lipid metabolism are central to the feedback loop, which is partly dependent on CDPK3 and allows the parasite to respond faster to inducers of ...
Journal of Cell Biology, 2004
alcium is a critical mediator of many intracellular processes in eukaryotic cells. In the obligate intracellular parasite Toxoplasma gondii , for example, a rise in [Ca 2 ϩ ] is associated with significant morphological changes and rapid egress from host cells. To understand the mechanisms behind such dramatic effects, we isolated a mutant that is altered in its responses to the Ca 2 ϩ ionophore A23187 and found the affected gene encodes a homologue of Na ϩ /H ϩ exchangers (NHEs) located on the parasite's C plasma membrane. We show that in the absence of TgNHE1, Toxoplasma is resistant to ionophore-induced egress and extracellular death and amiloride-induced proton efflux inhibition. In addition, the mutant has increased levels of intracellular Ca 2 ϩ , which explains its decreased sensitivity to A23187. These results provide direct genetic evidence of a role for NHE1 in Ca 2 ϩ homeostasis and important insight into how this ubiquitous pathogen senses and responds to changes in its environment.
Calcium ionophore-induced egress of Toxoplasma gondii shortly after host cell invasion
Veterinary Parasitology, 2007
Calcium plays crucial roles in important events of Toxoplasma gondii life cycle, including motility, invasion and egress from the host cell. Calcium ionophore has been used to artificially trigger release of the parasites from infected cells. In this report we describe that calcium ionophore A21387 induced T. gondii egress from LLC-MK2 cells at times as early as 2 h after entry. Addition of kinase inhibitors as staurosporine, wortmanine and genistein to the incubation medium significantly reduced ionophore-induced egress. The same occurred when the actin inhibitor cytochalasin D was used. Parasites egressed 2 h post-infection from ionophoretreated cultures were unable of establishing infection in a new cell. S-VHS recording of egressing parasites showed that they assume an hourglass shape as they cross the plasma membrane, similar to the moving junction constriction observed during active invasion, and extrudes the conoid, similarly to what is also observed during invasion. Transmission and high resolution scanning electron microscopy revealed that the egressing tachyzoites are free from host cell derived membranes. These include plasma membrane and parasitophorous vacuole membranes as well as associated endoplasmic reticulum membranes. Taken together, these results indicate that although invasion and egress may share similar signaling pathways, as indicated by the effect of kinase and actin inhibitors, the tachyzoites move freely in the cytosol, a phenomenon very distinctive from invasion and that deserves attention. #
Journal of Biological Chemistry, 2019
Protozoan parasites of the phylum Apicomplexa actively move through tissue in order to initiate and perpetuate infection. The regulation of parasite motility relies on cyclic nucleotide-dependent kinases, but how these kinases are activated remains unknown. Here, using an array of biochemical and cell biology approaches, we show that the apicomplexan parasite Toxoplasma gondii expresses a large guanylate cyclase protein (TgGC), which contains several upstream ATPase transporter-like domains. We show that TgGC has a dynamic localization, being concentrated at the apical tip in extracellular parasites, which relocates to a more cytosolic distribution during intracellular replication. Conditional TgGC knockdown revealed that this protein is essential for acute-stage tachyzoite growth, as TgGC-deficient parasites were defective in motility, host cell attachment, invasion, and subsequent host cell egress. We show that TgGC is critical for a rapid rise in cytosolic [Ca 2+ ] and for secretion of microneme organelles upon stimulation with a cGMP agonist, but these deficiencies can be bypassed by direct activation of signaling by a Ca 2+ ionophore. Further, we found that TgGC is required for transducing changes in extracellular pH and [K + ] to activate cytosolic [Ca 2+ ] flux. Together, the results of our work implicate TgGC as a putative signal transducer that activates Ca 2+ signaling and motility in Toxoplasma.