Date Published: May 23, 2019
Publisher: Public Library of Science
Author(s): Hayley E. Bullen, Hugo Bisio, Dominique Soldati-Favre, Laura J Knoll.
To elicit effective invasion and egress from infected cells, obligate intracellular parasites of the phylum Apicomplexa rely on the timely and spatially controlled exocytosis of specialized secretory organelles termed the micronemes. The effector molecules and signaling events underpinning this process are intricate; however, recent advances within the field of Toxoplasma gondii research have facilitated a broader understanding as well as a more integrated view of this complex cascade of events and have unraveled the importance of phosphatidic acid (PA) as a lipid mediator at multiple steps in this process.
At any point during intracellular replication, deleterious environmental changes resulting in a loss of host-cell integrity can trigger T. gondii tachyzoite egress from infected cells via activation of microneme exocytosis and the actomyosin system. Extrinsic and intrinsic signals are likely to govern parasite egress from infected cells; however, the studies performed to date have implicated only specific extrinsic stimuli including low potassium (K+), low pH [1, 2], and serum albumin . During the intracellular cycle, T. gondii tachyzoites are surrounded by a parasitophorous vacuole membrane (PVM), which is permeable to small molecules, and changes in K+ or H+ levels are sensed by the parasite through unknown mechanisms to promote microneme secretion. Interestingly, exposure to an acidic environment can overcome a high potassium–induced block in microneme exocytosis, implying that K+ and pH are sensed by different receptors or that pH sensing is downstream of K+ detection . These extrinsic signals feed into a pathway within which phosphoinositide-phospholipase C (PI-PLC) forms a signaling node, hydrolyzing phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) to generate diacylglycerol (DAG) and inositol triphosphate (IP3) to produce PA and to mobilize calcium, respectively, to ultimately trigger microneme exocytosis . Further feeding this pathway are diverse signaling events carried out by cyclic nucleotides (Fig 1 and Table 1).
Cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are important second messengers with diverse roles in eukaryotic cells. Within the Apicomplexa, cGMP is a vital upstream mediator of signaling events leading presumably to PI-PLC activation and subsequent microneme secretion. Protein kinase G (PKG) senses fluctuations in cGMP levels, and its association with the parasite plasma membrane by acylation is a necessary modification to function in controlling motility and invasion . PKG critically participates in the mobilization of intracellular calcium, and its specific inhibition with either compound 1 (tri-substituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine) or, less selectively, compound 2 (4-[7-[(dimethylamino) methyl]-2-(4-fluorophenyl) imidazo[1,2-a] pyridin-3-yl] pyrimidin-2-amine) has been shown to impact microneme secretion and tachyzoite egress [6, 7].
Downstream of PKG, PKA, and PI-PLC activity is IP3 production and the ensuing release of calcium (Ca2+), a process previously reported to be sensitive to IP3 receptor inhibitors . Despite the strong pharmacological evidence for their existence, canonical genes encoding IP3 receptors cannot be identified. Apicomplexan parasites possess several putative Ca2+ stores, including the acidocalcisomes, the mitochondrion, the inner membrane complex (IMC), and most relevantly, the endoplasmic reticulum (ER). Treatment of parasites with the sarcoplasmic/ER Ca2+-ATPase (SERCA) inhibitor thapsigargin has been shown to prevent reentry of Ca2+ in the ER and to stimulate microneme exocytosis, presumably by blocking the influx of leaked Ca2+ from the ER . The recent advance in developing genetically encoded Ca2+ indicators has offered a powerful tool to visualize Ca2+ during egress and was exploited to identify compounds modulating Ca2+ signaling via a cell-based phenotypic screen for compounds that modulate Ca2+ signaling [13–15]. Additionally, Ca2+ is proposed to be mobilized in response to abscisic acid (ABA) via the production of cyclic ADP (cADP) ribose . Although T. gondii possesses ADP ribosyl cyclase and hydrolase  and a putative ABA-binding G-protein–coupled receptor (GPCR) receptor (G-protein–coupled receptor 89 [GPR89], TGGT1_286490), no pathway for the biosynthesis of ABA or ryanodine receptor could be found.
The production of IP3 by PI-PLC and concurrent Ca2+ mobilization preceding microneme secretion is also linked to the generation of DAG, which can be interconverted to PA through the action of DAG kinases (DGKs), and PA phosphatases (PAPs) . In mammalian cells, PA has been linked to exocytosis, and similarly, blocking PA production with either specific DGK inhibitors or through conditional depletion of the plasma membrane–associated DGK1 reduces PA production and blocks microneme secretion in T. gondii . Moreover, microneme secretion can be induced by treatment of T. gondii with the PAP inhibitor propranolol, implying that the ensuing buildup of PA facilitates exocytosis . Although the signaling pathway culminating in microneme release is slowly being unraveled, little is known about the actual fusion event at the parasite plasma membrane. Work delineating the importance of PA signaling at the parasite plasma membrane has, however, revealed the presence of a novel, PA-binding acylated pleckstrin homology domain–containing protein (APH) at the surface of the micronemes . Conditional depletion of the APH gene resulted in a selective block in microneme secretion, leading to impairments in parasite motility, invasion, and egress . APH is able to cluster multiple phosphate head-groups at the bilayer-binding surface , which is presumably critical to bringing membranes together during microneme exocytosis to induce SNAP receptor (SNARE)-mediated fusion . SNARES and C2 domain–containing proteins mediate vesicle–membrane fusion in diverse cell types in response to Ca2+ and membrane curvature. In both T. gondii and Plasmodium falciparum, a conserved C2 domain–containing protein termed DOC2.1 has been shown to play a role in microneme secretion, as well as the associated events of egress, motility, and invasion . Although DOC2.1 is yet to be localized, it has been proposed to mediate microneme release by assisting SNARE-like protein/complex formation in a Ca2+-dependent manner to prompt microneme–plasma membrane fusion  (Fig 1 and Table 1).
Motility and host-cell entry and egress involve a parasite-derived molecular motor termed the glideosome, components of which are located between the parasite plasma membrane and IMC, as well as specific parasite surface adhesins. An armadillo repeat–containing protein identified as glideosome-associated connector (GAC) was found to be crucial for motility, invasion, and egress without impacting microneme secretion . GAC not only acts as a connector between F-actin and the tail of the transmembrane microneme protein 2 (MIC2) adhesin but also possesses a PA-binding pleckstrin homology domain , which is likely involved in binding GAC to the parasite apex during motility following the DGK1-related up-regulation of PA. The dual binding of GAC to the MIC2 tail and PA presumably ensures higher affinity and selectivity for the secreted adhesin by combining two weak binding interactions, engendering strong binding. This finding not only presents a crucial link between the parasite surface adhesins and the underlying actomyosin network but also adds to the importance of PA signaling during invasion-related events not solely limited to microneme exocytosis.
Microneme exocytosis is the culmination of a complex series of events that are slowly being unraveled. Further investigations into the roles of CDPKs, PI-PLC regulation, cyclic nucleotides, and integration of potassium, pH, and PA sensing will be required to give a global overview of how tachyzoites govern natural and induced egress as well as invasion to perpetuate their pathogenicity.