Egress and the parasite phosphatase PP1 in Plasmodium falciparum.

Project on egress and the parasite phosphatase PP1

Asexual proliferation of the malaria parasite Plasmodium falciparum in the red blood cells (RBC) follows a developmental program that alternates intraerythrocytic replication within a parasitophorous vacuole (PV) with dissemination to new host cells. Egress from erythrocytes is timely stimulated by a cGMP- and calcium-dependent pathway that activates the cGMP-dependent protein kinase PfPKG and the calcium-dependent kinase PfCDPK5 respectively. This in turn triggers the exocytosis of secretory organelles, namely exonemes and micronemes, enabling the release of key effectors required for the rupture of the surrounding PV and host membranes. Preceding PfPKG activation, early PV morphological modifications have been described, including PV poration and PV swelling, also known as the “rounding-up” step, but the molecular effectors involved are still unknown.


The egress scheme pathway in P. falciparum. Egress of the merozoites from the host red blood cell (RBC) requires the parasite to breach sequentially the surrounding membranes: the parasitophorous vacuole membrane (PVM) and the RBC membrane (RBCM). Following the end of schizogony (step 1), two morphological modifications of the PV take place, i.e. PVM poration and PV rounding-up (step 2). Then, an increase in cGMP concentration, likely regulated by the parasite phosphatase PP1, activates its downstream effector, the parasite kinase PKG that in turn leads to an increase in intracellular calcium concentration activating the kinase CDPK5 (step 3). These kinases are required to trigger the exocytosis of specific parasite organelles, namely the exonemes and micronemes, thereby releasing key effectors of egress. PP1, PKG and CDPK5 are necessary for PVM rupture (step 3). Finally, it has been observed that one single pore is formed and stabilized in the RBCM and that curling and buckling of this membrane allows the efficient release and dispersion of the merozoites in the extracellular milieu (steps 4 and 5). In addition to the pore formation, destabilization of the RBC cytoskeleton by parasite proteins is also required. Source from Collins et al. 2013, Brochet et al. 2014, Yeoh et al. 2007, Dvorin et al. 2010, Garg et al. 2013, Absalon et al. 2018, Abkarian et al. 2011, Das et al. 2015, Thomas et al. 2018, Hale et al. 2017, Glushakiva et al. 2018, Paul et al. 2020.

 

We have identified the protein phosphatase PP1 (PfPP1) as a master regulator of the egress signaling pathway6. A DiCre conditional mutant line for PfPP1 (PfPP1-iKO), fails to rupture the PV membrane (PVM) due to an incapacity to secrete exonemes and micronemes. A phosphoproteomic analysis of PfPP1 mutant revealed phosphoregulation of guanylate cyclase (GC), the enzyme responsible for cGMP synthesis, thereby placing PfPP1 upstream of the well characterized PfPKG in the egress signaling cascade.


Figure: PP1 is required for PVM rupture at the time of egress.
(A) IFA showing the depletion of the protein PP1-HA3 upon rapamycin treatment in the inducible PP1 knock-out line (PP1-iKO), based on the conditional excision of pp1 gene by a dimerizable Cre recombinase. (B) IFA showing that in control parasites (- rapamycin), PVM rupture takes place as shown by the detection of membrane whorls, in contrast to PP1-depleted parasites (+ rapamycin) that remain enclosed within the PVM.

 

As the mechanistic of the early steps of egress is poorly understood, our project aims at using PP1 to expand our knowledge of the egress pathway and to explore its contribution in the biology of Plasmodium sexual stages, especially during gametocytes egress. For this, we will combine cutting-edge technologies in cellular and molecular parasitology to precisely define the egress step controlled by PP1, to evaluate PP1 contribution in sexual stages development, to identify egress-specific targets of the phosphatase and to determine to which extent those targets represent new molecular effectors of the egress signaling pathway. The better comprehension of PP1 functions in Plasmodium biology and egress may open the avenue for future intervention strategy.                                                                   

Publications

Co-option of Plasmodium falciparum PP1 for egress from host erythrocytes.
Paul AS, Miliu A, Paulo JA, Goldberg JM, Bonilla AM, Berry L, Seveno M, Braun-Breton C, Kosber AL, Elsworth B, Arriola JSN, Lebrun M, Gygi SP, Lamarque MH, Duraisingh MT.Nat Commun. 2020 Jul 15;11(1):3532. doi: 10.1038/s41467-020-17306-1.PMID: 32669539 

Efficient invasion by Toxoplasma depends on the subversion of host protein networks.
Guérin A, Corrales RM, Parker ML, Lamarque MH, Jacot D, El Hajj H, Soldati-Favre D, Boulanger MJ, Lebrun M.Nat Microbiol. 2017 Oct;2(10):1358-1366. doi: 10.1038/s41564-017-0018-1. Epub 2017 Aug 28.PMID: 28848228

Shelph2, a bacterial-like phosphatase of the malaria parasite Plasmodium falciparum, is dispensable during asexual blood stage.
Miliu A, Lebrun M, Braun-Breton C, Lamarque MH.PLoS One. 2017 Oct 26;12(10):e0187073. doi: 10.1371/journal.pone.0187073. eCollection 2017.PMID: 29073264 

Plasticity and redundancy among AMA-RON pairs ensure host cell entry of Toxoplasma parasites.
Lamarque MH, Roques M, Kong-Hap M, Tonkin ML, Rugarabamu G, Marq JB, Penarete-Vargas DM, Boulanger MJ, Soldati-Favre D, Lebrun M.Nat Commun. 2014 Jun 17;5:4098. doi: 10.1038/ncomms5098.PMID: 24934579 

Structural and functional insights into the malaria parasite moving junction complex.
Vulliez-Le Normand B, Tonkin ML, Lamarque MH, Langer S, Hoos S, Roques M, Saul FA, Faber BW, Bentley GA, Boulanger MJ, Lebrun M.PLoS Pathog. 2012;8(6):e1002755. doi: 10.1371/journal.ppat.1002755. Epub 2012 Jun 21.PMID: 22737069 

Identification of a new rhoptry neck complex RON9/RON10 in the Apicomplexa parasite Toxoplasma gondii.
Lamarque MH, Papoin J, Finizio AL, Lentini G, Pfaff AW, Candolfi E, Dubremetz JF, Lebrun M.PLoS One. 2012;7(3):e32457. doi: 10.1371/journal.pone.0032457. Epub 2012 Mar 12.PMID: 22427839 

Host cell invasion by apicomplexan parasites: insights from the co-structure of AMA1 with a RON2 peptide.
Tonkin ML, Roques M, Lamarque MH, Pugnière M, Douguet D, Crawford J, Lebrun M, Boulanger MJ.Science. 2011 Jul 22;333(6041):463-7. doi: 10.1126/science.1204988.PMID: 21778402 

Identification of a new rhoptry neck complex RON9/RON10 in the Apicomplexa parasite Toxoplasma gondii.
Lamarque MH, Papoin J, Finizio AL, Lentini G, Pfaff AW, Candolfi E, Dubremetz JF, Lebrun M.PLoS One. 2012;7(3):e32457. doi: 10.1371/journal.pone.0032457. Epub 2012 Mar 12.PMID: 22427839 

The RON2-AMA1 interaction is a critical step in moving junction-dependent invasion by apicomplexan parasites.
Lamarque M, Besteiro S, Papoin J, Roques M, Vulliez-Le Normand B, Morlon-Guyot J, Dubremetz JF, Fauquenoy S, Tomavo S, Faber BW, Kocken CH, Thomas AW, Boulanger MJ, Bentley GA, Lebrun M. PLoS Pathog. 2011 Feb 10;7(2):e1001276. doi: 10.1371/journal.ppat.1001276. PMID: 21347343

Food vacuole proteome of the malarial parasite Plasmodium falciparum.
Lamarque M, Tastet C, Poncet J, Demettre E, Jouin P, Vial H, Dubremetz JF. Proteomics Clin Appl. 2008 Sep;2(9):1361-74. doi: 10.1002/prca.200700112. Epub 2008 Jul 21. PMID: 21136929

 

Group members

Theme leader


LAMARQUE  Mauld  
  
Assistant Professor (MC) University of Montpellier 
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