falciparumPHL dhfr-PfPKAr merozoites in IC buffer are treated with DiB indicating that PKA does not play a role in regulating Ca2+levels in merozoites

falciparumPHL dhfr-PfPKAr merozoites in IC buffer are treated with DiB indicating that PKA does not play a role in regulating Ca2+levels in merozoites. parasites invade and multiply within host erythrocytes. The process of erythrocyte invasion requires specific interactions between host receptors and parasite ligands. Many of the important parasite proteins that bind host receptors are localized in apical organelles called micronemes. Here, we demonstrate that cAMP serves as a key regulator that controls the timely secretion of microneme proteins during invasion. We show that exposure of merozoites to a low K+environment, as found in blood plasma, prospects to a rise in cytosolic cAMP levels due to XY101 activation of the cytoplasmic, bicarbonate-sensitive adenylyl cyclase (PfAC). A rise in cAMP activates protein kinase A (PKA), which regulates microneme secretion. In addition, cAMP triggers a rise in cytosolic Ca2+levels through the Epac pathway. Increases in both cAMP and Ca2+levels are essential for triggering microneme secretion. Identification of the different elements in the cAMP-dependent signaling pathways that regulate microneme secretion during invasion provides novel targets to block erythrocyte invasion, inhibit blood stage parasite growth and prevent malaria. == Introduction == All the clinical symptoms ofPlasmodium falciparummalaria are attributed to the blood stage of the parasite life cycle. The intra-erythrocytic stage of the life cycle is initiated when liberatedP. falciparummerozoites invade and multiply within host reddish blood cells. Following the development of mature schizonts, next generation merozoites egress from infected erythrocytes and invade uninfected erythrocytes to start a new cycle of contamination. Invasion of erythrocytes byP. falciparummerozoites is usually a complex multi-step process that is mediated by specific molecular interactions between reddish cell surface receptors and parasite protein ligands[1],[2]. A number of parasite ligands that mediate receptor binding during invasion reside in apical membrane-bound organelles known as micronemes and rhoptries[1],[2]. Timely secretion of these parasite ligands to the merozoite surface is critical for successful invasion[3],[4]. Microneme and rhoptry proteins are secreted from freeP. falciparummerozoites in a two-step process[5]. First, exposure of extracellular merozoites to a low [K+] environment common of blood plasma prospects to a rise in cytosolic Ca2+via a phospholipase C (PLC)-dependent pathway, which triggers translocation of microneme proteins such as 175 kD erythrocyte binding antigen (EBA175) and apical merozoite antigen-1 (PfAMA1) to the merozoite surface[5]. Subsequently, binding of EBA175 and its homologs to their erythrocyte MME receptors triggers secretion of rhoptry XY101 proteins such as PfRH2b, Clag3.1 and PfTRAMP[5],[6]. The pathways by which exposure ofP. falciparummerozoites to a low K+environment triggers a rise in cytosolic Ca2+and microneme secretion are not understood. Here, we demonstrate that another ubiquitous second messenger, namely, 3′-5′ cyclic adenosine monophosphate (cAMP), plays a central role in regulating cytosolic Ca2+levels and microneme secretion during merozoite invasion of reddish blood cells. We demonstrate that exposure of merozoites to a low K+environment as found in blood plasma activates the bicarbonate-sensitive cytoplasmic adenylyl cyclase (PfAC) leading to a rise in cytosolic cAMP levels and activation of protein kinase A (PKA), which regulates microneme secretion. In mammalian cells, the cAMP responsive PKA, which regulates diverse cellular processes in response to a rise in cytosolic cAMP levels, is composed of two catalytic subunits and two regulatory subunits[7]. Unlike mammalian cells,P. falciparumhas a single inhibitory regulatory subunit (PfPKAr) and a single catalytic subunit (PfPKAc)[8][12]. As the PfPKAr subunit is not predicted to dimerize, the holoenzyme is likely XY101 to be composed of a one-to-one ratio of PfPKArPfPKAc[12]. The PfPKAr subunit is usually predicted to have 2 cyclic nucleotide binding domains. When cAMP XY101 binds to one or both XY101 of these it provokes a conformational switch that engenders the dissociation of the PfPKArPfPKAc complex and activation of the released PfPKAc subunit that phosphorylates its specific substrates[8][12]. LikePlasmodium,Toxoplasmaalso encodes cAMP-dependent PKA and its inhibition prospects to a block in tachyzoite growth[13]. Increase in cytosolic cAMP levels that would activateToxoplasmaPKA also mediates the tachyzoite to bradyzoite developmental switch[14][16]. In addition to activating PKA, we demonstrate that cAMP activates the Epac pathway[17]inP. falciparummerozoites, which triggers a rise in cytosolic Ca2+leading to microneme release..