Phosphodiesterase beta is the master regulator of cAMP signalling during malaria parasite invasion

Flueck, Christian; Drought, Laura; Jones, Andrew W.; Patel, Avnish; Perrin, Abigail J.; Walker, E. A.; Nofal, Stephanie D.; Snijders, Ambrosius P.; Blackman, Michael J.; Baker, David A.

doi:10.1371/journal.pbio.3000154

Cited by 48 publications

(78 citation statements)

References 59 publications

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“…Conditional ablation of PDEβ led to a 70% reduction in invasion and increased phosphorylation of over 230 parasite protein phosphosites, most of which contained a minimal PKA consensus motif (R/K, x, pS/pT), suggesting that the PDEβ knockout phenotype resulted from inappropriate hyper-activation of PKA due to uncontrolled cAMP levels. Of note, phosphorylation of AMA1 Ser 610 was up-regulated in the PDEβ null mutant, further supporting the notion that it is a PKA substrate [25]. In the present study, we have examined the role of cAMP signalling in P .…”

Section: Introductionsupporting

confidence: 80%

“…Consistent with this, motif analysis showed that the phosphorylation sites fit a PKA consensus and closely resemble those identified in a recent study of sites enriched in a P . falciparum PDEβ knockout in which PKA activity was enhanced due to raised cAMP levels [25] (S2C Fig and S2D Fig). A total of 77 sites that we identified as being significantly hypophosphorylated in both the ACβ- and PKA-deficient parasites were also quantified in the recent PDEβ-dependent phosphoproteome.…”

Section: Resultsmentioning

confidence: 99%

“…We recently reported that regulation of cAMP levels in asexual blood stage P . falciparum is governed by a dual-specific phosphodiesterase called phosphodiesterase beta (PDEβ) [25]. Conditional ablation of PDEβ led to a 70% reduction in invasion and increased phosphorylation of over 230 parasite protein phosphosites, most of which contained a minimal PKA consensus motif (R/K, x, pS/pT), suggesting that the PDEβ knockout phenotype resulted from inappropriate hyper-activation of PKA due to uncontrolled cAMP levels.…”

Section: Introductionmentioning

confidence: 99%

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Cyclic AMP signalling controls key components of malaria parasite host cell invasion machinery

et al. 2019

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Cyclic AMP (cAMP) is an important signalling molecule across evolution, but its role in malaria parasites is poorly understood. We have investigated the role of cAMP in asexual blood stage development of Plasmodium falciparum through conditional disruption of adenylyl cyclase beta (ACβ) and its downstream effector, cAMP-dependent protein kinase (PKA). We show that both production of cAMP and activity of PKA are critical for erythrocyte invasion, whilst key developmental steps that precede invasion still take place in the absence of cAMP-dependent signalling. We also show that another parasite protein with putative cyclic nucleotide binding sites, Plasmodium falciparum EPAC (PfEpac), does not play an essential role in blood stages. We identify and quantify numerous sites, phosphorylation of which is dependent on cAMP signalling, and we provide mechanistic insight as to how cAMP-dependent phosphorylation of the cytoplasmic domain of the essential invasion adhesin apical membrane antigen 1 (AMA1) regulates erythrocyte invasion.

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Section: Introductionsupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cyclic AMP signalling controls key components of malaria parasite host cell invasion machinery

et al. 2019

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“…[46,47] (data available in PlasmoDB) indicated that PF3D7_0507400 is transcribed in asexual blood stages, with peak expression in mature schizonts. To evaluate expression at the protein level we examined a recently obtained global proteomic dataset [48]. This clearly identified with high confidence two tryptic peptides of SUB1-ProM (corresponding to 17.6% coverage) in extracts of P. falciparum asexual blood stage schizonts (Supplementary Figure S3), confirming that the protein is expressed in these clinically relevant developmental stages of the parasite lifecycle.…”

Section: Sub1-prom Is Expressed In Asexual Blood Stages Of P Falciparummentioning

confidence: 74%

A malaria parasite subtilisin propeptide-like protein is a potent inhibitor of the egress protease SUB1

Tarr

Withers‐Martinez

Flynn

et al. 2020

Biochemical Journal

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Subtilisin-like serine peptidases (subtilases) play important roles in the life cycle of many organisms, including the protozoan parasites that are the causative agent of malaria, Plasmodium spp. As with other peptidases, subtilase proteolytic activity has to be tightly regulated in order to prevent potentially deleterious uncontrolled protein degradation. Maturation of most subtilases requires the presence of an N-terminal propeptide that facilitates folding of the catalytic domain. Following its proteolytic cleavage, the propeptide acts as a transient, tightly bound inhibitor until its eventual complete removal to generate active protease. Here we report the identification of a stand-alone malaria parasite propeptide-like protein, called SUB1-ProM, encoded by a conserved gene that lies in a highly syntenic locus adjacent to three of the four subtilisin-like genes in the Plasmodium genome. Template-based modelling and ab initio structure prediction showed that the SUB1-ProM core structure is most similar to the X-ray crystal structure of the propeptide of SUB1, an essential parasite subtilase that is discharged into the parasitophorous vacuole (PV) to trigger parasite release (egress) from infected host cells. Recombinant Plasmodium falciparum SUB1-ProM was found to be a fast-binding, potent inhibitor of P. falciparum SUB1, but not of the only other essential blood-stage parasite subtilase, SUB2, or of other proteases examined. Mass-spectrometry and immunofluorescence showed that SUB1-ProM is expressed in the PV of blood stage P. falciparum, where it may act as an endogenous inhibitor to regulate SUB1 activity in the parasite.

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“…Synchronized cultures are of great value in the study of many processes in the asexual cycle and the ability to synchronize egress and invasion to within minutes will allow the investigation of these rapid processes on different scales, allowing for a wide range of studies, including large-scale biochemical investigation of proteins involved, such as the proteolysis of MSP1 and AMA1 proteins [54-56]. Furthermore, as it makes timing of egress and invasion very predictable, this method will greatly aid the imaging of egress and invasion by video microscopy.…”

Section: Discussionmentioning

confidence: 99%

Use of a highly specific kinase inhibitor for rapid, simple and precise synchronization ofPlasmodium falciparumandPlasmodium knowlesiasexual stage parasites

Ressurreição

Thomas

Nofal

et al. 2020

Preprint

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During the course of the asexual erythrocytic stage of development, Plasmodium spp. parasites undergo a series of morphological changes and induce alterations in the host cell. At the end of this stage, the parasites exit the host cell, after which the progeny invade a new host cell. These processes are rapid and occur in a time-dependent manner. Of particular importance, egress and invasion of erythrocytes by the parasite are difficult to capture in an unsynchronized culture, or even a culture that has been synchronized to within hours. Therefore, precise synchronization of parasite cultures is of paramount importance for the investigation of these processes. Here we describe a method for synchronizing Plasmodium falciparum and Plasmodium knowlesi asexual blood stage parasites with ML10, a highly specific inhibitor of the cGMP-dependent protein kinase (PKG) that arrests parasite growth approximately 15 minutes prior to egress. This inhibitor allows parasite cultures to be synchronized to within minutes, with a simple wash step. Furthermore, we show that parasites remain viable for several hours after becoming arrested by the compound and that ML10 has advantages over the previously used PKG inhibitor Compound 2. Here, we demonstrate that ML10 is an invaluable tool for the study of Plasmodium spp. asexual blood stage biology and for the routine synchronization of P. falciparum and P. knowlesi cultures.

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Phosphodiesterase beta is the master regulator of cAMP signalling during malaria parasite invasion

Cited by 48 publications

References 59 publications

Cyclic AMP signalling controls key components of malaria parasite host cell invasion machinery

Cyclic AMP signalling controls key components of malaria parasite host cell invasion machinery

A malaria parasite subtilisin propeptide-like protein is a potent inhibitor of the egress protease SUB1

Use of a highly specific kinase inhibitor for rapid, simple and precise synchronization ofPlasmodium falciparumandPlasmodium knowlesiasexual stage parasites

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