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Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress.

Collins CR, Hackett F, Strath M, Penzo M, Withers-Martinez C, Baker DA, Blackman MJ - PLoS Pathog. (2013)

Bottom Line: Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct merozoite organelles called micronemes.Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites.Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.

View Article: PubMed Central - PubMed

Affiliation: Division of Parasitology, MRC National Institute for Medical Research, Mill Hill, London, United Kingdom.

ABSTRACT
The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.

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Model of the role of PKG in egress.(A) PfSUB1 and PfAMA1 are stored in exonemes and micronemes respectively in developing intracellular merozoites. Rapid turnover of cGMP maintains PfPKG in an ‘off’ state. (B) Upon an endogenous or exogenously-supplied signal (which can be mimicked by zaprinast-mediated inhibition of one or more parasite PDEs), cGMP levels are raised above threshold levels, resulting in activation of PfPKG and phosphorylation of its substrates. This leads to discharge of both PfSUB1 and PfAMA1, perhaps via an increase in cytosolic Ca2+ released from intracellular stores. PfAMA1 translocates onto the merozoite surface, whereas PfSUB1 is released into the PV. PVM rupture rapidly ensues, as well as limited permeabilization of the RBC membrane. (C) The Ca2+ flux may activate CDPK5 for a downstream role in egress. Eventual rupture of the RBC membrane is mediated by at least one E64-sensitive cysteine protease, allowing egress. PfPKG may rapidly return to an inactive state upon lowering of cGMP levels, whilst PfPKG substrates may be dephosphorylated through the action of endogenous phosphatases.
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ppat-1003344-g007: Model of the role of PKG in egress.(A) PfSUB1 and PfAMA1 are stored in exonemes and micronemes respectively in developing intracellular merozoites. Rapid turnover of cGMP maintains PfPKG in an ‘off’ state. (B) Upon an endogenous or exogenously-supplied signal (which can be mimicked by zaprinast-mediated inhibition of one or more parasite PDEs), cGMP levels are raised above threshold levels, resulting in activation of PfPKG and phosphorylation of its substrates. This leads to discharge of both PfSUB1 and PfAMA1, perhaps via an increase in cytosolic Ca2+ released from intracellular stores. PfAMA1 translocates onto the merozoite surface, whereas PfSUB1 is released into the PV. PVM rupture rapidly ensues, as well as limited permeabilization of the RBC membrane. (C) The Ca2+ flux may activate CDPK5 for a downstream role in egress. Eventual rupture of the RBC membrane is mediated by at least one E64-sensitive cysteine protease, allowing egress. PfPKG may rapidly return to an inactive state upon lowering of cGMP levels, whilst PfPKG substrates may be dephosphorylated through the action of endogenous phosphatases.

Mentions: The second major conclusion of our study is that cGMP - through its capacity to activate PfPKG - is an endogenous regulator of malarial egress, and treatments that increase parasite cGMP levels induce premature egress in a PfPKG-dependent manner. Natural egress of P. falciparum schizonts is likely triggered by a signal that increases parasite cGMP levels. Zaprinast treatment presumably mimics that signal. This study did not extend to identifying the target of zaprinast in P. falciparum. However, the pfpdeα and pfpdeδ genes are non-essential in blood stages [20], [30], whilst the pfpdeβ gene is refractory to disruption (unpublished, D. Baker), indicating an indispensable role. PfPDEβ is therefore a good candidate for the target of zaprinast. Natural egress may be triggered by stimuli that down-regulate its activity, or by other signals that affect cGMP homeostasis (e.g. activation of guanylyl cyclase activity). Further work will be required to dissect this pathway, including the identification of PfPKG substrates. Toxoplasma egress involves mobilisation of Ca2+ from internal stores [24], [25]. Our findings that BAPTA-AM inhibits egress implies a similar calcium-dependence in Plasmodium. Indeed, during preparation of this manuscript, Agarwal et al. [31] reported that a sharp rise in intracellular Ca2+ is detectable in schizonts shortly before egress, and that treatment of schizonts with BAPTA-AM or a phospholipase C inhibitor (which likely inhibits Ca2+ release from intracellular stores) blocks egress, with a concomitant block in PfSUB1 discharge. A rise in intracellular Ca2+ just prior to egress of P. falciparum was also observed in a very recent study of Glushakova and colleagues [32]. The Ca2+ flux was unaffected by exogenous chelating agents in the culture medium, but was inhibited by BAPTA-AM, which also inhibited egress. In contrast, egress was accelerated by inhibitors of the parasite endoplasmic reticulum (ER) Ca2+-ATPase which pumps Ca2+ from the cytosol into the ER, as well as by the calcium ionophore A23187, further supporting the notion that the required Ca2+ is obtained from internal stores. Our present work showing that the BAPTA-AM-mediated egress block is overcome by zaprinast suggests that PfPKG acts to increase cytosolic Ca2+, or that it functions downstream or even independently of calcium in the regulatory pathway. The Ca2+ increase may be important for activation of CDPK5, the other Plasmodium kinase implicated in egress [7]. A simple model for the role of PKG in egress which may integrate our findings with these other strands of work is presented in Figure 7. Calcium signalling also regulates discharge of microneme proteins from extracellular merozoites [33], though how that is controlled independently of PfSUB1 discharge prior to egress is unknown.


Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress.

Collins CR, Hackett F, Strath M, Penzo M, Withers-Martinez C, Baker DA, Blackman MJ - PLoS Pathog. (2013)

Model of the role of PKG in egress.(A) PfSUB1 and PfAMA1 are stored in exonemes and micronemes respectively in developing intracellular merozoites. Rapid turnover of cGMP maintains PfPKG in an ‘off’ state. (B) Upon an endogenous or exogenously-supplied signal (which can be mimicked by zaprinast-mediated inhibition of one or more parasite PDEs), cGMP levels are raised above threshold levels, resulting in activation of PfPKG and phosphorylation of its substrates. This leads to discharge of both PfSUB1 and PfAMA1, perhaps via an increase in cytosolic Ca2+ released from intracellular stores. PfAMA1 translocates onto the merozoite surface, whereas PfSUB1 is released into the PV. PVM rupture rapidly ensues, as well as limited permeabilization of the RBC membrane. (C) The Ca2+ flux may activate CDPK5 for a downstream role in egress. Eventual rupture of the RBC membrane is mediated by at least one E64-sensitive cysteine protease, allowing egress. PfPKG may rapidly return to an inactive state upon lowering of cGMP levels, whilst PfPKG substrates may be dephosphorylated through the action of endogenous phosphatases.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649973&req=5

ppat-1003344-g007: Model of the role of PKG in egress.(A) PfSUB1 and PfAMA1 are stored in exonemes and micronemes respectively in developing intracellular merozoites. Rapid turnover of cGMP maintains PfPKG in an ‘off’ state. (B) Upon an endogenous or exogenously-supplied signal (which can be mimicked by zaprinast-mediated inhibition of one or more parasite PDEs), cGMP levels are raised above threshold levels, resulting in activation of PfPKG and phosphorylation of its substrates. This leads to discharge of both PfSUB1 and PfAMA1, perhaps via an increase in cytosolic Ca2+ released from intracellular stores. PfAMA1 translocates onto the merozoite surface, whereas PfSUB1 is released into the PV. PVM rupture rapidly ensues, as well as limited permeabilization of the RBC membrane. (C) The Ca2+ flux may activate CDPK5 for a downstream role in egress. Eventual rupture of the RBC membrane is mediated by at least one E64-sensitive cysteine protease, allowing egress. PfPKG may rapidly return to an inactive state upon lowering of cGMP levels, whilst PfPKG substrates may be dephosphorylated through the action of endogenous phosphatases.
Mentions: The second major conclusion of our study is that cGMP - through its capacity to activate PfPKG - is an endogenous regulator of malarial egress, and treatments that increase parasite cGMP levels induce premature egress in a PfPKG-dependent manner. Natural egress of P. falciparum schizonts is likely triggered by a signal that increases parasite cGMP levels. Zaprinast treatment presumably mimics that signal. This study did not extend to identifying the target of zaprinast in P. falciparum. However, the pfpdeα and pfpdeδ genes are non-essential in blood stages [20], [30], whilst the pfpdeβ gene is refractory to disruption (unpublished, D. Baker), indicating an indispensable role. PfPDEβ is therefore a good candidate for the target of zaprinast. Natural egress may be triggered by stimuli that down-regulate its activity, or by other signals that affect cGMP homeostasis (e.g. activation of guanylyl cyclase activity). Further work will be required to dissect this pathway, including the identification of PfPKG substrates. Toxoplasma egress involves mobilisation of Ca2+ from internal stores [24], [25]. Our findings that BAPTA-AM inhibits egress implies a similar calcium-dependence in Plasmodium. Indeed, during preparation of this manuscript, Agarwal et al. [31] reported that a sharp rise in intracellular Ca2+ is detectable in schizonts shortly before egress, and that treatment of schizonts with BAPTA-AM or a phospholipase C inhibitor (which likely inhibits Ca2+ release from intracellular stores) blocks egress, with a concomitant block in PfSUB1 discharge. A rise in intracellular Ca2+ just prior to egress of P. falciparum was also observed in a very recent study of Glushakova and colleagues [32]. The Ca2+ flux was unaffected by exogenous chelating agents in the culture medium, but was inhibited by BAPTA-AM, which also inhibited egress. In contrast, egress was accelerated by inhibitors of the parasite endoplasmic reticulum (ER) Ca2+-ATPase which pumps Ca2+ from the cytosol into the ER, as well as by the calcium ionophore A23187, further supporting the notion that the required Ca2+ is obtained from internal stores. Our present work showing that the BAPTA-AM-mediated egress block is overcome by zaprinast suggests that PfPKG acts to increase cytosolic Ca2+, or that it functions downstream or even independently of calcium in the regulatory pathway. The Ca2+ increase may be important for activation of CDPK5, the other Plasmodium kinase implicated in egress [7]. A simple model for the role of PKG in egress which may integrate our findings with these other strands of work is presented in Figure 7. Calcium signalling also regulates discharge of microneme proteins from extracellular merozoites [33], though how that is controlled independently of PfSUB1 discharge prior to egress is unknown.

Bottom Line: Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct merozoite organelles called micronemes.Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites.Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.

View Article: PubMed Central - PubMed

Affiliation: Division of Parasitology, MRC National Institute for Medical Research, Mill Hill, London, United Kingdom.

ABSTRACT
The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.

Show MeSH
Related in: MedlinePlus