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Regulation of the cGMP/Ca2+ signalling module in malaria parasites

English title Regulation of the cGMP/Ca2+ signalling module in malaria parasites
Applicant Brochet Mathieu
Number 155852
Funding scheme Temporary Backup Schemes
Research institution Dépt Microbiologie et Médecine Moléculaire Faculté de Médecine Université de Genève
Institution of higher education University of Geneva - GE
Main discipline Experimental Microbiology
Start/End 01.08.2015 - 31.07.2020
Approved amount 1'498'952.00
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All Disciplines (2)

Discipline
Experimental Microbiology
Molecular Biology

Keywords (6)

Development and transmission of malaria parasites; Intracellular signalling; Calcium; Secondary messengers; Chemical genetics; Anti-plasmodium

Lay Summary (French)

Lead
Le paludisme causé par les parasites de l’espèce Plasmodium représente un problème de santé publique majeur. Durant son cycle de vie le parasite est confronté à des environnements très variés tels que le sang et le foie chez l’homme ou l’intestin et les glandes salivaires chez le moustique. La perception des changements environnementaux par le parasite repose sur des molécules appelées messagers secondaires qui lui permettent d’adapter son comportement à son environnement.Chez Plasmodium le calcium est un messager secondaire crucial tout au long du cycle de vie du parasite dont on ne connait pas les mécanismes d’action. Le projet apporte sa contribution à ce domaine de recherche.
Lay summary

Contenu et objectifs du travail de recherche

Plasmodium est un parasite primitif éloigné des organismes modèles tels que la levure du boulanger couramment utilisée en laboratoire. Ceci rend son étude difficile et par exemple,  les protéines impliquées dans l’activation du calcium ne sont toujours pas connues.

Dans une première phase, nous identifierons ces protéines  grâce à des approches expérimentales récemment développées. Dans une seconde phase, nous déterminerons comment une fois activé, le calcium modifie le comportement du parasite pour s’adapter à son environnement. Enfin nous étudierons comment brouiller le message transmis par le calcium pour bloquer le développement du parasite.

 

Contexte scientifique et social du projet de recherche

Le projet relève de la recherche fondamentale. Les stratégies de contrôle du paludisme doivent être basées sur une connaissance fine de la biologie du parasite et de ses interactions avec  leurs hôtes et vecteurs. Le caractère primitif de l’organisme étudié permettra également de comprendre l’évolution des systèmes de communication intracellulaire impliqués dans la perception de l’environnement.

Direct link to Lay Summary Last update: 24.07.2015

Responsible applicant and co-applicants

Employees

Publications

Publication
A divergent cyclin/cyclin-dependent kinase complex controls the atypical replication of a malaria parasite during gametogony and transmission
Balestra Aurélia C, Zeeshan Mohammad, Rea Edward, Pasquarello Carla, Brusini Lorenzo, Mourier Tobias, Subudhi Amit Kumar, Klages Natacha, Arboit Patrizia, Pandey Rajan, Brady Declan, Vaughan Sue, Holder Anthony A, Pain Arnab, Ferguson David JP, Hainard Alexandre, Tewari Rita, Brochet Mathieu (2020), A divergent cyclin/cyclin-dependent kinase complex controls the atypical replication of a malaria parasite during gametogony and transmission, in eLife, 9, e56474.
Phosphatidic acid governs natural egress in Toxoplasma gondii via a guanylate cyclase receptor platform
Bisio Hugo, Lunghi Matteo, Brochet Mathieu, Soldati-Favre Dominique (2019), Phosphatidic acid governs natural egress in Toxoplasma gondii via a guanylate cyclase receptor platform, in Nature Microbiology, 4(3), 420-428.
Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites
Fang Hanwei, Gomes Ana Rita, Klages Natacha, Pino Paco, Maco Bohumil, Walker Eloise M., Zenonos Zenon A., Angrisano Fiona, Baum Jake, Doerig Christian, Baker David A., Billker Oliver, Brochet Mathieu (2018), Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites, in Nature Communications, 9(1), 4248-4248.
cGMP Signalling: Malarial Guanylyl Cyclase Leads the Way
Brochet Mathieu (2018), cGMP Signalling: Malarial Guanylyl Cyclase Leads the Way, in Current Biology, 28(17), R939-R941.
Sub-minute Phosphoregulation of Cell Cycle Systems during Plasmodium Gamete Formation
Invergo Brandon, Brochet Mathieu, Yu Lu, Choudhary Jyoti, Beltrao Pedro, Billker Oliver (2017), Sub-minute Phosphoregulation of Cell Cycle Systems during Plasmodium Gamete Formation, in Cell Reports, 21(7), 2017-2029.
Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii
Jia Yonggen, Marq Jean-Baptiste, Bisio Hugo, Jacot Damien, Mueller Christine, Yu Lu, Choudhary Jyoti, Brochet Mathieu, Soldati-Favre Dominique (2017), Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii, in EMBO journal, 36(21), 3250-3267.
A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress
Pino Paco, Caldelari Reto, Mukherjee Budhaditya, Vahokoski Juha, Kursula Inari, Blackman Michael, Heussler Volker, Brochet Mathieu, Soldati-Favre Dominique (2017), A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress, in Science, 358(6362), 522-528.
Invasion of hepatocytes by Plasmodium sporozoites requires cGMP-dependent protein kinase and calcium dependent protein kinase 4.
Govindasamy K, Jebiwott S, Jaijyan DK, Davidow A, Ojo KK, Van Voorhis WC, Brochet M, Billker O, Bhanot P (2016), Invasion of hepatocytes by Plasmodium sporozoites requires cGMP-dependent protein kinase and calcium dependent protein kinase 4., in Molecular Microbiology, 102(2), 349-363.
Calcium signalling in malaria parasites
Brochet Mathieu, Billker Oliver (2016), Calcium signalling in malaria parasites, in Molecular Microbiology, 100(3), 397-408.
Multiple short windows of calcium-dependent protein kinase 4 activity coordinate distinct cell cycle events during Plasmodium gametogenesis
Fang Hanwei, Klages Natacha, Baechler Bastien, Hillner Evelyn, Yu Lu, Pardo Mercedes, Choudhary Jyoti, Brochet Mathieu (2016), Multiple short windows of calcium-dependent protein kinase 4 activity coordinate distinct cell cycle events during Plasmodium gametogenesis, in Elife, 6, e26524.

Datasets

CDPK4 is a pleiotropic regulator controlling the atypical Plasmodium cell cycle during mosquito transmission

Author Choudhary, Jyoti
Publication date 24.05.2017
Persistent Identifier (PID) PXD005884
Repository PRIDE
Abstract
Transmission of malaria parasites from humans to the mosquito vector exclusively relies on the sexual reproduction of the parasite within the mosquito blood meal. Upon ingestion, Plasmodium male gametocyte undergoes explosive development: within 10 minutes, it completes three rounds of genome replication and endomitosis, assembles eight axonemes, and emerges from the host red blood cell. The plant-like calcium-dependent protein kinase 4 (CDPK4) was previously shown to be essential for male gametogenesis placing this kinase as an attractive drug target to block malaria transmission. Here we have identified and resolved three distinct molecular functions of CDPK4 during male gametogenesis. Activity of a myristoylated isoform is first required up to 20 seconds after activation to load the Mini-Chromosome Maintenance complex onto origins of replication. This role is partially mediated by a conserved protein belonging to the SAPS-domain family which is involved in the G1 to S-phase transition in eukaryotes. At the same time, activity of myristoylated CDPK4 is required to phosphorylate a Plasmodium-specific microtubule-associated protein necessary for mitotic spindle assembly. Finally, activity of a short non-myristoylated isoform of CDPK4 is essential seconds prior to completion of cytokinesis and the activation of male gamete motility. This late role has been linked to another Plasmodium-specific protein that is incorporated into axonemes during gametogenesis. This study reveals how a kinase of a protist parasite integrates and transduces multiple signals with a high spatiotemporal resolution to control both evolutionarily conserved and Plasmodium-specific biological processes.

Sub-minute Phosphoregulation of Cell Cycle Systems during Plasmodium Gamete Formation

Author Choudhary, Jyoti
Publication date 29.11.2017
Persistent Identifier (PID) PXD006266
Repository PRIDE
Abstract
As a first step in the transmission of malaria, Plasmodium parasites ingested by a mosquito must rapidly initiate a gametogenesis process prior to sexual reproduction. The phosphorylation-based signalling mechanisms underlying the initiation of this process remain largely unmapped. We have measured a high-resolution time course of phosphorylation in P. berghei gametocytes during the first minute of gametogenesis to elucidate the scope and dynamics of the response. The data reveals rapid phosphoregulation of microtubule motor proteins and DNA replication initiation proteins within the first 18 s and of DNA replication machinery by 60 s. The data also implicate several protein kinases and phosphatases in the gametogenesis signalling pathway. Through gene knock-out experiments, we verify that the protein kinases CDPK4 and SRPK1 have distinct influences over the phosphorylation of similar downstream targets. Together, the results show that key cell-cycle systems related to both replication and mitosis undergo simultaneous, rapid phosphoregulation.

Epistasis studies reveal redundancy among calcium-dependent protein kinases in motility and invasion of malaria parasites

Author Brochet, Mathieu
Publication date 17.09.2018
Persistent Identifier (PID) PXD011096
Repository PRIDE
Abstract
In malaria parasites, evolution of parasitism has been linked to functional optimisation. Despite this optimisation, most members of a calcium-dependent protein kinase (CDPK) family show genetic redundancy during erythrocytic proliferation. To identify relationships between phospho-signalling pathways, we here screen 294 genetic interactions among protein kinases in Plasmodium berghei. This reveals a synthetic negative interaction between a hypomorphic allele of the protein kinase G (PKG) and CDPK4 to control erythrocyte invasion which is conserved in P. falciparum. CDPK4 becomes critical when PKG-dependent calcium signals are attenuated to phosphorylate proteins important for the stability of the inner membrane complex, which serves as an anchor for the acto-myosin motor required for motility and invasion. Finally, we show that multiple kinases functionally complement CDPK4 during erythrocytic proliferation and transmission to the mosquito vector. This study reveals how CDPKs are wired within a stage-transcending signalling network to control motility and host cell invasion in malaria parasites.

A unique and divergent cyclin/cyclin-dependent kinase complex controls DNA replication during atypical cell-cycles of Plasmodium berghei gametogony

Author Hainard, Alexandre
Publication date 18.02.2020
Persistent Identifier (PID) PXD17283
Repository PRIDE
Abstract
Cell-cycle transitions are generally triggered by variations in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria parasites express ancestral CDKs and cyclins, whose functions and interdependency remain elusive. Here, we show that the unique Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical cell-cycle regulator of gametogony required for transmission to the mosquito. It is essential for DNA replication and phosphorylates canonical S/TPxK CDK motifs on components of the pre-replicative complex otherwise regulated by distinct kinases in other eukaryotes. Over a replicative cycle, CRK5 stably interacts with a single Haemosporidia-specific cyclin (SOC2) with no evidence of SOC2 degradation. Regulation of CRK5 activity relies instead on dynamic phosphorylation of a C-terminus extension that mediates its interaction with SOC2. Our results present evidence that during the atypical cell cycles of Plasmodium gametogony, a unique and divergent cyclin/CDK pair fulfils the functional space of multiple eukaryotic cell-cycle kinases to initiate DNA replication.

Cross-talk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii

Author Choudhary, Jyoti
Publication date 12.10.2017
Persistent Identifier (PID) PXD006045
Repository PRIDE
Abstract
Toxoplasma gondii encodes three Protein Kinase A catalytic (PKAc1-3) and one regulatory (PKAr) subunits to integrate cAMP-dependent signals. Here, we show that inactive PKAc1 is maintained at the parasite pellicle by interacting with dually acylated PKAr. Either a conditional knockdown of PKAr or the overexpression of PKAc1 results in a block in parasite division. In contrast, conditional expression of a dominant negative PKAr isoform unable to bind cAMP, triggers premature egress of parasites from infected cells. This untimely egress critically depends on parasite density and host cell acidification. A comparative phosphoproteome analysis reveals that PKA genetic inhibition significantly changed the phosphorylation profile of a putative cGMP-phosphodiesterase, PDE2. Consistently, the phenotype of PKA genetic inhibition is alleviated by chemical inhibition of the cGMP-dependent protein kinase G (PKG). A phosphodiesterase inhibitor is able to circumvent egress repression by PKA or pH neutralisation, indicating that environmental acidification and PKA signalling act as balancing regulators of cGMP degradation to control PKG-mediated egress. Collectively, these results reveal a cross-talk between PKA and PKG pathways to govern egress in T. gondii.

Phosphatidic acid governs natural egress in Toxoplasma gondii via a guanylate cyclase receptor platform

Author Brochet, Mathieu
Publication date 14.11.2018
Persistent Identifier (PID) PXD011692
Repository PRIDE
Abstract
Toxoplasma gondii is able to establish a life-long chronic infection in human and animals. Host cell entry and egress from infected cells are two key steps in the lytic cycle of this obligate intracellular parasite that ensures its survival and dissemination. Egress is a temporally orchestrated process underpinned by the regulated exocytosis of the apical secretory organelles, termed micronemes. At any point during intracellular replication, deleterious environmental changes resulting from a loss of host cell integrity can trigger egress via the activation of the cGMP-dependent protein kinase G (PKG). Remarkably, even in the absence of extrinsic alarming signals, the parasites naturally egress from infected cells in a coordinated manner after ca. 5 to 6 cycles of binary fission. Here we show that diacylglycerol kinase 2 (DGK2) is secreted into the parasitophorous vacuole where it produces phosphatidic (PA), which acts as intrinsic signal to elicit natural egress. PA activates a large guanylate cyclase (GC) receptor uniquely conserved in apicomplexans and composed of one P4-ATPase and two guanylate cyclase domains. Assembly of a functional GC at the parasite plasma membrane critically depends on the association with two proteins acting as cofactors. This study reveals the existence of a unique and versatile signaling platform able to respond to an intrinsic lipid mediator and other extrinsic alarming signals to control programmed and induced egress, respectively.

A divergent cyclin/cyclin-dependent kinase complex controls the atypical replication of Plasmodium berghei during gametogony and parasite transmission.

Author Bottrill, Andrew
Publication date 17.06.2020
Persistent Identifier (PID) PXD017622
Repository PRIDE
Abstract
Cell cycle transitions are generally triggered by variation in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. We also provide evidence for indirect regulation of the concomitant M-phase progression. During a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this unique cyclin/CDK pair fills the functional space of multiple eukaryotic cell-cycle kinases controlling DNA replication and M-phase progression.

A unique and divergent cyclin/cyclin-dependent kinase complex controls DNA replication during atypical cell-cycles of Plasmodium berghei gametogony

Author Alexandre, Hainard
Publication date 18.02.2020
Persistent Identifier (PID) PXD017283
Repository PRIDE
Abstract
Cell-cycle transitions are generally triggered by variations in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria parasites express ancestral CDKs and cyclins, whose functions and interdependency remain elusive. Here, we show that the unique Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical cell-cycle regulator of gametogony required for transmission to the mosquito. It is essential for DNA replication and phosphorylates canonical S/TPxK CDK motifs on components of the pre-replicative complex otherwise regulated by distinct kinases in other eukaryotes. Over a replicative cycle, CRK5 stably interacts with a single Haemosporidia-specific cyclin (SOC2) with no evidence of SOC2 degradation. Regulation of CRK5 activity relies instead on dynamic phosphorylation of a C-terminus extension that mediates its interaction with SOC2. Our results present evidence that during the atypical cell cycles of Plasmodium gametogony, a unique and divergent cyclin/CDK pair fulfils the functional space of multiple eukaryotic cell-cycle kinases to initiate DNA replication.

A divergent cyclin/cyclin-dependent kinase complex controls progression through the atypical replicative cycles during Plasmodium berghei gametogony

Author Subudhi, Amit Kumar
Publication date 17.06.2020
Persistent Identifier (PID) GSE144743
Repository GEO
Abstract
Cell-cycle transitions are generally triggered by variations in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have evolved unique cell-cycles with a repertoire of ancestral CDKs and cyclins whose functions and interdependency remain elusive. Here, we show that the divergent Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical cell-cycle regulator of gametogony required for transmission to the mosquito. It phosphorylates canonical CDK motifs on components of the pre-replicative complex and is essential for DNA replication. We also provide evidence for indirect regulation of the concomitant progression through M-phase. Over a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2) with no evidence of SOC2 cycling through transcription, translation nor degradation. Our results present evidence that during Plasmodium gametogony, a unique and divergent cyclin/CDK pair evolved to fulfil the functional space of multiple eukaryotic cell-cycle kinases controlling S-phase entry and progression through M-phase.

Collaboration

Group / person Country
Types of collaboration
Swiss Tropical and Public Health Institute Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
Université de Genève Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Exchange of personnel
Universtiy of Nottingham Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
The Crick Institute Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Rutgers - New Jersey Medical School United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Wellcome Trust sanger Institute Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Universitätsklinikum Heidelberg Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
BioMalPar XVI Biology and Pathology of the Malaria Parasite Talk given at a conference A divergent cyclin/cyclin-dependent kinase complex controls the atypical replication of Plasmodium berghei during gametogony and parasite transmission 18.05.2020 Heidelberg, Germany Brochet Mathieu; Balestra Aurélia;
BioMalPar XVI Biology and Pathology of the Malaria Parasite Talk given at a conference Malaria parasite PKG binds to and phosphorylates a putative transporter essential for critical calcium signals during erythrocytic proliferation and transmission to the mosquito 18.05.2020 Heidelberg, Germany Brochet Mathieu; Balestra Aurélia;
Molecular Parasitology Meeting Talk given at a conference A divergent cyclin/cyclin-dependent kinase complex controls progression through S-and M-phases during Plasmodium berghei gametogony 15.09.2019 Woodshole, United States of America Balestra Aurélia;
BioMalPar XV Biology and Pathology of the Malaria Parasite Poster Characterisation of an unusual cyclin/cyclin-dependent kinase complex governing the cell cycle of malaria parasites 28.05.2019 Heidelberg, Germany Balestra Aurélia; Brochet Mathieu;
Molecular advances and parasite strategies in host infection Poster Characterization of an unusual cyclin/cyclin-dependent kinase complex governing the cell cycle of malaria parasites 30.09.2018 Les Embiez, France Brochet Mathieu; Balestra Aurélia;
BioMalPar VIX: Biology and Pathology of the Malaria Parasite Talk given at a conference Genetic interactions reveal functional redundancy between calcium-dependent protein kinases 4 and 1 in motility and invasion of malaria parasites 23.05.2018 Heidelberg, Germany Fang Hanwei; Balestra Aurélia; Brochet Mathieu;
Annual Swiss Proteomics Meeting 2018 Talk given at a conference Sub-minute Phosphoregulation of Cell Cycle Systems during Plasmodium Gamete Formation 19.04.2018 Montreux, Switzerland Brochet Mathieu;
Swiss cytometry meeting Talk given at a conference Flow cytometry-based strategies to study malaria transmission 07.02.2018 Lausanne, Switzerland Brochet Mathieu;
2nd international parfrap conference Talk given at a conference CDPK4 is a pleiotropic regulator of Plasmodium gametogenesis 02.10.2016 Les Embiez, France Brochet Mathieu;
BioMalPar XII: Biology and Pathology of the Malaria Parasite Talk given at a conference CDPK4 is a pleiotropic regulator of Plasmodium gametogenesis 18.05.2016 Heidelberg, Germany Brochet Mathieu;


Awards

Title Year
European Molecular Biology Organisation Young Investigator Award 2019

Abstract

Malaria caused by Plasmodium parasites is a profound human health problem. To eliminate malaria, broadly acting medicines must be developed to target Plasmodium pathways essential across all life stages. The ability of Plasmodium parasites to perceive and respond to the multiple environments they face throughout their life cycle critically relies on signalling. Understanding signalling is of particular relevance to malaria because a portfolio of anti-malarial drug development projects is targeting parasite signalling pathways. However, due to the lack of robust genetic tools and molecular phenotyping, signalling in Plasmodium is poorly understood. By developing new chemical genetic tools and deep quantitative molecular phenotyping approaches, I recently showed that phosphoinositide metabolism links cGMP signalling to crucial Ca2+ signals at key decision points in the life cycle of malaria parasites. This is an important discovery as it defines the cGMP/Ca2+ signalling module as a key Plasmodium vulnerability across stages. I now propose a project at the interface of fundamental biology of malaria parasites and early-stage drug discovery aiming to i) characterise the molecular mechanisms by which cGMP signalling controls phosphoinositide and Ca2+ across stages and species, ii) to comprehensively identify and characterise all pathways regulating the cGMP/Ca2+ signalling module, and iii) to identify anti-plasmodial leads modulating cGMP/Ca2+ regulation and define their molecular targets for therapeutic and transmission-blocking interventions. This project will therefore provide new fundamental insights into a signalling pathway of universal importance in the Plasmodium life cycle to reveal novel targets for chemical intervention. As the cGMP/Ca2+ module connects diverse territories of the lipid metabolism with signalling, I expect this project to provide many unique starting points to further study the regulation of key virulence mechanisms of malaria parasites.
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