Calcium controls a number of critical events including motility, secretion, cell invasion, and egress by protozoan parasites 1. Compared to animal 2 and plant cells 3 , the molecular mechanisms that govern calcium signaling in parasites are poorly understood. Here we demonstrate that the production of the phytohormone abscisic acid (ABA) controls calcium signaling within the apicomplexan parasite Toxoplasma gondii, an important human pathogen. In plants, ABA controls a number of important events including environmental stress responses, embryo development, and seed dormancy 4 ,5 . ABA induces production of the second-messenger cyclic ADP ribose (cADPR), which controls release of intracellular calcium stores in plants 6 . cADPR also controls intracellular calcium release in the protozoan parasite T. gondii 7,8 ; however, previous studies have not revealed the molecular basis of this pathway 9 . Addition of exogenous ABA induced formation of cADPR in T. gondii, stimulated calcium-dependent protein secretion, and induced parasite egress from the infected host cell in a density-dependent manner. Production of endogenous ABA within the parasite was confirmed by HPLC purification and GC-MS analysis. Selective disruption of ABA synthesis by the inhibitor fluridone (FLU) delayed egress and induced development of the slow-growing, dormant cyst stage of the parasite. Thus, ABA-mediated calcium signaling controls the decision between lytic and chronic stage growth, a developmental switch that is central in pathogenesis and transmission. The pathway for ABA production was likely acquired with an algal endosymbiont that was retained as a non-photosynthetic plastid known as the apicoplast. The plant-like nature of this pathway may be exploited therapeutically as shown by the ability of a specific inhibitor of ABA synthesis to prevent toxoplasmosis in the mouse model.Calcium-mediated secretion in T. gondii controls both motility and cell invasion and previous studies have demonstrated that these processes utilize the second messenger cADPR, yet the signals triggering this pathway remain unresolved 7,8 . In plants 6 , hydra 10 , and sponges 11 , ABA stimulates release of intracellular calcium through elevation of the cyclic nucleotide cADPR. Addition of exogenous ABA proved to be a potent agonist of secretion in T. gondii as shown by the release of the protein MIC2, a parasite adhesin that is discharged into the supernatant in response to increases in intracellular calcium (Fig. 1A). Induction of MIC2 secretion by ABA was highly specific to (±) -ABA and was not induced by (−) -ABA, the precursor β-carotene, or retinoic acid (Fig. 1B). Treatment with ABA lead to a dose-dependent increase in the second messenger cADPR in T. gondii, suggesting ABA may be a natural agonist for calcium signaling in parasites (Fig. 1C). Finally, chelation of intracellular calcium in the parasite blocked secretion induced by ABA, confirming that it acts through release of an intracellular calcium pool (Fig. 1D). Collectively these results ind...

Trypanosoma brucei, the protozoan parasite responsible for sleeping sickness, evades the immune response of mammalian hosts and digestion in the gut of the insect vector by means of its coat proteins tethered to the cell surface via glycosylphosphatidylinositol (GPI) anchors. To evaluate the importance of GPI for parasite survival, we cloned and disrupted a trypanosomal gene, TbGPI10, involved in biosynthesis of GPI. TbGPI10 encodes a protein of 558 amino acids having 25% and 23% sequence identity to human PIG-B and Saccharomyces cerevisiae Gpi10p, respectively. TbGPI10 restored biosynthesis of GPI in a mouse mutant cell line defective in mouse Pig-b gene. TbGPI10 also rescued the inviability of GPI10-disrupted S. cerevisiae, indicating that TbGPI10 is the orthologue of PIG-B͞GPI10 that is involved in the transfer of the third mannose to GPI. The bloodstream form of T. brucei could not lose TbGPI10; therefore, GPI synthesis is essential for growth of mammalian stage parasites. Procyclic form cells (insect stage parasites) lacking the surface coat proteins because of disruption of TbGPI10 are viable and grow slower than normal, provided that they are cultured in nonadherent flasks. In regular flasks, they adhered to the plastic surface and died. Infectivity to tsetse flies is partially impaired, particularly in the early stage. Therefore, parasitespecific inhibition of GPI biosynthesis should be an effective chemotherapy target against African trypanosomiasis.T rypanosoma brucei is a protozoan parasite invading humans and other mammals by transmission via tsetse flies. It causes sleeping sickness in humans and nagana disease in domestic animals living in the ''tsetse belt'' in central Africa. These are serious medical and agricultural problems for which safe and effective therapeutic and protective measures are highly desirable (1, 2).T. brucei has two distinct proliferative stages, a bloodstream stage living free in mammalian blood and an insect stage (or procyclic form) living in the midguts of tsetse flies. The cell surface of both stages of this unicellular parasite is covered by a large amount of glycosylphosphatidylinositol (GPI)-anchored proteins (3, 4): 10 7 variant surface glycoproteins per cell for the bloodstream form of the parasite and 3 ϫ 10 6 to 6 ϫ 10 6 procyclins (or procyclic acidic repetitive proteins) per cell of the procyclic form of the parasite (4-6), corresponding to 10% and 1-3%, respectively, of total proteins in these parasite stages (7,8). T. brucei evades the host's immune response by expressing structurally different forms of variant surface glycoproteins (4). Procyclins are thought to protect procyclic cells from digestion by the digestive enzymes in the fly (4, 6). In addition, T. brucei expresses a number of other GPI-anchored proteins, such as transferrin receptors in the bloodstream form (3, 4). Thus, the importance of GPI anchors for the survival and infection of T. brucei has been suggested, leading to the notion that the GPI biosynthesis pathway may be a good target for chemo...

The phylum Apicomplexa comprises a large group of early branching eukaryotes that includes a number of human and animal parasites. Calcium controls a number of vital processes in apicomplexans including protein secretion, motility, and differentiation. Despite the importance of calcium as a second messenger, very little is known about the systems that control homeostasis or that regulate calcium signaling in parasites. The recent completion of many apicomplexan genomes provides new opportunity to define calcium response pathways in this group of parasites in comparison to model organisms. Whole-genome comparison between the apicomplexans Plasmodium spp., Cryptosporidium spp., and Toxoplasma gondii revealed the presence of several P-Type Ca2+ transporting ATPases including a single endoplasmic reticulum (ER)-type sarcoplasmic-endoplasmic reticulum Ca2+ ATPase, several Golgi-like Ca2+ ATPases, and a single Ca2+/H+ exchanger. Only T. gondii showed evidence of plasma membrane-type Ca2+ ATPases or voltage-gated calcium channels. Despite pharmacological evidence for IP3 and ryanodine-mediated calcium release, animal-type calcium channels were not readily identified in parasites, indicating they are more similar to plants. Downstream of calcium release, a variety of EF-hand-containing proteins regulate calcium responses. Our analyses detected a single conserved calmodulin (CaM) homologue, 3 distinct centrin (CETN)-caltractin-like proteins, one of which is shared with ciliates, and a variety of deep-branching, CaM-CETN-like proteins. Apicomplexans were also found to contain a wide array of calcium-dependent protein kinases (CDPKs), which are commonly found in plants. Toxoplasma gondii contains more than 20 CDPK or CDPK-related kinases, which likely regulate a variety of responses including secretion, motility, and differentiation. Genomic and phylogenetic comparisons revealed that apicomplexans contain a variety of unusual calcium response pathways that are distinct from those seen in vertebrates. Notably, plant-like pathways for calcium release channels and calcium-dependent kinases are found in apicomplexans. The experimental flexibility of T. gondii should allow direct experimental manipulation of these pathways to validate their biological roles. The central importance of calcium in signaling and development, and the novel characteristics of many of these systems, indicates that parasite calcium pathways may be exploited as new therapeutic targets for intervention.

Toxoplasma virulence factor ROP18 targets endoplasmic reticulum–bound transcription factor ATF6β in the host cell, leading to the detrimental loss of ATF6β through proteasome-dependent degradation.

Intracellular calcium controls several crucial cellular events in apicomplexan parasites, including protein secretion, motility, and invasion into and egress from host cells. The plant compound thapsigargin inhibits the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA), resulting in elevated calcium and induction of protein secretion in Toxoplasma gondii. Artemisinins are natural products that show potent and selective activity against parasites, making them useful for the treatment of malaria. While the mechanism of action is uncertain, previous studies have suggested that artemisinin may inhibit SERCA, thus disrupting calcium homeostasis. We cloned the single-copy gene encoding SERCA in T. gondii (TgSERCA) and demonstrate that the protein localizes to the endoplasmic reticulum in the parasite. In extracellular parasites, TgSERCA partially relocalized to the apical pole, a highly active site for regulated secretion of micronemes. TgSERCA complemented a calcium ATPase-defective yeast mutant, and this activity was inhibited by either thapsigargin or artemisinin. Treatment of T. gondii with artemisinin triggered calcium-dependent secretion of microneme proteins, similar to the SERCA inhibitor thapsigargin. Artemisinin treatment also altered intracellular calcium in parasites by increasing the periodicity of calcium oscillations and inducing recurrent, strong calcium spikes, as imaged using Fluo-4 labeling. Collectively, these results demonstrate that artemisinin perturbs calcium homeostasis in T. gondii, supporting the idea that Ca 2؉ -ATPases are potential drug targets in parasites.

A picomplexan parasites rely on calcium-mediated signaling for a variety of vital functions including protein secretion, motility, cell invasion, and differentiation. These functions are controlled by a variety of specialized systems for uptake and release of calcium, which acts as a second messenger, and on the functions of calcium-dependent pro› teins. Defining these systems in parasites has been complicated by their evolutionary dis› tance from model organisms and practical concerns in working with small, and somewhat fastidious cells. Comparative genomic analyses of Toxoplasma gondii, Plasmodium spp. and Cryptosporidium spp. reveal several interesting adaptations for calcium-related processes in parasites. Apicomplexans contain several P-type Ca ^ ATPases including an ER-type reuptake mechanism (SERCA), which is the proposed target of artemisinin. All three organisms also contain several genes related to Golgi PMR-like calcium transporters, and a Ca^VH^ ex› changer, while plasma membrane-type (PMCA) Ca'^^ ATPases and voltage-dependent cal› cium channels are exclusively found in T gondii. Pharmacological evidence supports the presence of IP3 and ryanodine channels for calcium-mediated release. Collectively these sys› tems regulate calcium homeostasis and release calcium to act as a signal. Downstream re› sponses are controlled by a family of EF-hand containing calcium binding proteins includ› ing calmodulin, and an array of centrin and caltractin-like genes. Most surprising, apicomplexans contain a diversity of calcium-dependent protein kinases (CDPK), which are commonly found in plants. Toxoplasma contains more than 20 CDPK or CDPK-like pro› teases, while Plasmodium and Cryptosporidium have fewer than half this number. Several of these CDPKs have been shown to play vital roles in protein secretion, invasion, and differen› tiation, indicating that disruption of calcium-regulated pathways may provide a novel means for selective inhibition of parasites. Defining Calcium Regulation in an Early Branching EukaryoteApicomplexan parasites are most similar to ciliates and dinoflagellates and only distandy related to plants, fungi, and animals typically used as model organisms. Apicomplexans con› tain a remnant plastid derived from a secondary endosymbiont, called the apicoplast. A num› ber of plant-like metabolic systems are found in apicomplexans either due to retention of *Corresponding

cChronic infection with Toxoplasma gondii becomes established in tissues of the central nervous system, where parasites may directly or indirectly modulate neuronal function. Epidemiological studies have revealed that chronic infection in humans is a risk factor for developing mental diseases. However, the mechanisms underlying parasite-induced neuronal dysfunction in the brain remain unclear. Here, we examined memory associated with conditioned fear in mice and found that T. gondii infection impairs consolidation of conditioned fear memory. To examine the brain pathology induced by T. gondii infection, we analyzed the parasite load and histopathological changes. T. gondii infects all brain areas, yet the cortex exhibits more severe tissue damage than other regions. We measured neurotransmitter levels in the cortex and amygdala because these regions are involved in fear memory expression. The levels of dopamine metabolites but not those of dopamine were increased in the cortex of infected mice compared with those in the cortex of uninfected mice. In contrast, serotonin levels were decreased in the amygdala and norepinephrine levels were decreased in the cortex and amygdala of infected mice. The levels of cortical dopamine metabolites were associated with the time spent freezing in the fear-conditioning test. These results suggest that T. gondii infection affects fear memory through dysfunction of the cortex and amygdala. Our findings provide insight into the mechanisms underlying the neurological changes seen during T. gondii infection.T oxoplasma gondii is one of the most successful brain parasites, infecting approximately one-third of the human population (1). T. gondii can persist in brain and muscle throughout the host's life, and chronic infection is asymptomatic in immunocompetent humans (2). However, recent studies have suggested that T. gondii infection is a risk factor for developing mental diseases, such as schizophrenia and depression, as well as human behavior and personality changes and suicide (3, 4). Interestingly, T. gondii infection increases the risk of schizophrenia roughly 2.7 times, which is higher than that for genes associated with schizophrenia (5). Several studies have also suggested that rodents infected with T. gondii exhibit decreased avoidance behavior in response to cat odors, indicating manipulation of the host's behavior by T. gondii to facilitate the parasite's transmission and complete sexual replication in the definitive host (6-11).To date, research on the mechanism(s) underlying behavioral changes following T. gondii infection has been conducted primarily from two points of view. First, the relationship between parasite localization in the brain and behavioral changes has been investigated, with a previous study reporting that T. gondii has no obvious tropism in the brain (12-15). However, another study found that tissue cyst density in amygdalar areas (the medial and basolateral amygdala) is 2-fold higher than that in nonamygdalar areas (9), whereas the presence of tissue cy...

The African trypanosome Trypanosoma brucei, which causes sleeping sickness in humans and Nagana disease in livestock, is spread via blood-sucking Tsetse flies. In the fly's intestine, the trypanosomes survive digestive and trypanocidal environments, proliferate, and translocate into the salivary gland, where they become infectious to the next mammalian host. Here, we show that for successful survival in Tsetse flies, the trypanosomes use trans-sialidase to transfer sialic acids that they cannot synthesize from host's glycoconjugates to the glycosylphosphatidylinositols (GPIs), which are abundantly expressed on their surface. Trypanosomes lacking sialic acids due to a defective generation of GPI-anchored trans-sialidase could not survive in the intestine, but regained the ability to survive when sialylated by means of soluble trans-sialidase. Thus, surface sialic acids appear to protect the parasites from the digestive and trypanocidal environments in the midgut of Tsetse flies.