Calcium-dependent signaling and kinases in apicomplexan parasites

Abstract

Calcium controls many critical events in the complex life cycles of apicomplexan parasites including protein secretion, motility, and development. Calcium levels are normally tightly regulated and rapid release of calcium into the cytosol activates a family of calcium-dependent protein kinases (CDPKs), which are normally characteristic of plants. CDPKs present in apicomplexans have acquired a number of unique domain structures likely reflecting their diverse functions. Calcium regulation in parasites is closely linked to signaling by cyclic nucleotides and their associated kinases. This Review summarizes the pivotal roles that calcium- and cyclic nucleotide-dependent kinases play in unique aspects of parasite biology.

Figure 1. Intracellular cycle of T. gondii

Tachyzoites display gliding motility on the substratum and along the surface of cells and use this unique form of actin-myosin based motility to actively invade into the host cell. Attachment is mediated by secretion of microneme proteins from the apex of the parasite marked by the conical cap known as the conoid. At the point of invasion, a tight constriction occurs between the host and parasite membranes, formed by a ring of proteins secreted from the rhoptries (depicted as a red ring). Intracellular replication leads to enlargement of the vacuole and accumulation of abscisic acid (ABA), which eventually triggers egress. Blocking production of ABA with the plant herbicide fluoridone (FLU) prevents egress and leads to development of semi-dormant cysts. Changes in cytosolic calcium have been shown to oscillate during gliding motility, control microneme secretion, and activate egress, as described in the text.

Figure 2. Calcium-response pathways controlling secretion and motility in T. gondii

The primary mobilizable store for calcium in T. gondii is the endoplasmic reticulum (ER), which is filled by the action of a Ca²⁺ ATPAse called SERCA. Stimulation of yet unidentified receptors (R) at the plasma membrane generates inositol triphosphate (IP₃ ) and diacyl glycerol (DAG) from phopsphatidyl inositol bisphosphate (PIP₂) through the activation of phospholipase C (PLC). Signaling, through a unique or common receptor, also results in activation of ADP ribose (ADPR) cyclase to produce cADPR. Phamacological and biochemical evidence supports the existence of calcium release channels that respond to cADPR (ryanodine receptors (R_YR) and through IP₃ receptors (IP₃R) (Lovett et al., 2002). Released calcium activates calcium-dependent protein kinases such as TgCDPK1, which has previously been implicated in microneme (MIC) secretion in T. gondii (Kieschnick et al., 2001). Independently, activation of guanylyl cyclase to generate cyclic GMP (cGMP) is important for activating protein kinase G (PKG), which also controls microneme secretion (Wiersma et al., 2004). Other studies in P. falciparum indicate that CDPKs may act on the myosin motor complex (Green et al., 2008), which is anchored in the inner membrane complex (IMC).

Figure 3. Phylogenetic analyses of calcium-dependent kinases in apicomplexans and plants

Comparison of calcium dependent protein kinases in higher plants (green), ciliates (light green), and apicomplexans. The canonical domain architecture is represented by plant CDPKs consisting of an N-terminal kinase domain followed by four EF hand domains (EF). Two groups of apicompelxan CDPKs fit this canonical profile (blue). One group has relatively short N-terminal regions (right side), while most members of the other have an extended N-terminus that is not homologous to other known domains (left side). Alternative domain structures are shown for other apicomplexan CDPKs including: a group with only three C-terminal EF hand domains (purple), a group with two or three N-terminal EF hand domains followed by a plekstrin homology (PH) and kinase domains (yellow), a group with one or more N-terminal EF hand domains, a kinase domain and three or four C-terminal EF hand domains (red). Representative models are shown, ( ) indicate variable number of domains. Gene names are based on previously published names or based on orthologous groupings defined here. Phylogenetic tree drawn using a distance matrix based on comparison of the kinase domains using Neighbor joining, BioNJ algorithym in PAUP* (Swofford, 1991), bootstrap values shown in filled circles (n=100). Taxa: Tg, T. gondii; Pf, P. falciparum; Cp, C. parvum; Tt, Tetrahymena thermophila; At, Arabidopsis thaliana; Os, Oryza sativa; Dc, Daucus carota; Cr, Chlamydomonas reinhardtii’, Pt,Paramecium tetraurelia; Li, Lilium longiflorum; Nt, Nicotiana tabacum. A complete listing of organisms, genes, and accession numbers is provided in the supplemental Table 1.

Figure 4. Schematic of Plasmodium life cycle

The life cycle of malaria is depicted in both the mosquito and vertebrate host. The cycle begins with injection of sporozoites into the dermis during initial probing by the mosquito. Sporozoites migrate in the skin, enter capillaries, and are carried to the liver where they cross the sinusoidal epithelium. Sporozoites migrate through cells and eventually enter hepatocytes where they develop and divide mitotically. Following rupture, liberated merozoites invade red cells and undergo successive round of intracellular replication, egress, and re-invasion. Over time, some parasites differentiate into gametocytes that circulate within red blood cells. When gametocytes are taken up by a feeding mosquito (the proboscis now enters directly into a capillary), they emerge in the insect midgut where they undergo a developmental transition, fuse to form a zygote, and develop into a motile stage called the ookinete. Migration of the ookinete across the midgut epithelium leads to encystation and development of sporozoites. Once liberated, sporozoites migrate to the salivary gland, penetrate to the lumen and await transmission. Calcium-dependent protein kinases (CDPKs) and cyclic nucleotide dependent protein kinases have been implicated in a number of steps involving motility and development as shown in the diagram and discussed further in the text. An emerging theme is that these two pathways intersect to control responses to calcium in a temporally and spatially complex manner. PKA, protein kinase A; PKB, protein kinase B; PKG, protein kinase G; PfPDE•, P. falciparum phosphodiesterase delta; PbGCβ, P. bergehi guanylyl cyclase beta ; PbMap2, P. berghei mitogen activated protein kinase 2; PbACα, P. berghei adenylyl cyclase alpha.