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.