Evidence that the Malaria Parasite Plasmodium falciparum Putative Rhoptry Protein 2 Localizes to the Golgi Apparatus throughout the Erythrocytic Cycle

Hallée, Stéphanie; Richard, Dave

doi:10.1371/journal.pone.0138626

Cited by 10 publications

(11 citation statements)

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“…Our attempts at knockdown of PfFH levels by removal of trimethoprim from cultures of FH-RFA expressing strain, where FH is fused to EcDHFR degradation domain, did not result in lowering of protein levels. It has been shown that proteins targeted to organelles and possessing a signal sequence lack accessibility to proteasomal degradation machinery and hence the conditional degradation approach may be unsuitable for achieving knockdown of these protein levels (36). Therefore, the essentiality of FH was examined in P. berghei with BALB/c mice as host.…”

Section: Essentiality Of Fumarate Hydratase For Pmentioning

confidence: 99%

Biochemical characterization and essentiality of fumarate hydratase

Jayaraman¹,

Suryavanshi²,

Kalale

et al. 2018

Journal of Biological Chemistry

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Plasmodium falciparum (Pf), the causative agent of malaria, has an iron-sulfur cluster-containing class I fumarate hydratase (FH) that catalyzes the interconversion of fumarate to malate, a wellknown reaction in the tricarboxylic acid cycle. In humans, the same reaction is catalyzed by class II FH that has no sequence or structural homology with the class I enzyme from Plasmodium. Fumarate is generated in large quantities in the parasite as a byproduct of AMP synthesis and is converted to malate by FH and then used in the generation of the key metabolites oxaloacetate, aspartate, and pyruvate. Previous studies have identified the FH reaction as being essential to P. falciparum, but biochemical characterizations of PfFH that may provide leads for the development of specific inhibitors are lacking. Here, we report on the kinetic characterization of purified recombinant PfFH, functional complementation of fh deficiency in Escherichia coli, and mitochondrial localization in the parasite. We found that the substrate analog mercaptosuccinic acid is a potent PfFH inhibitor, with a K i value in the nanomolar range. The fh gene could not be knocked out in Plasmodium berghei when transfectants were introduced into BALB/c mice; however, fh knockout was successful when C57BL/6 mice were used as host, suggesting that the essentiality of the fh gene to the parasite was mouse strain dependent.Plasmodium falciparum (Pf), the causative agent of the most lethal form of malaria, during its intra-erythrocytic asexual stages, derives ATP primarily from glycolysis with low contribution from mitochondrial pathways (1, 2). The bulk of pyruvate formed is converted to lactic acid with a minor amount entering the tricarboxylic acid (TCA) cycle, the flux through which is upregulated in sexual stages (2). Key intermediates that anaplerotically feed into the TCA cycle are α-ketoglutarate derived from glutamate, oxaloacetate (OAA) from phosphoenolpyruvate, and fumarate from adenosine 5΄-monophosphate (AMP) synthesis. Synthesis of AMP in the parasite is solely from inosine-5΄-monophosphate (IMP) through a pathway involving the enzymes adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL). The net reaction of ADSS and ASL involves consumption of GTP and aspartate and, generation of GDP, P i and fumarate. In the rapidly dividing parasite with an AT-rich genome and high energy requirements, leading to a high demand for adenine pools, one would expect a high flux of fumarate generation. The parasite does not secrete fumarate but instead, the carbon derived from this metabolite can be traced in malate, OAA, aspartate, pyruvate (through PEP) and lactate (3). The metabolic significance of this fumarate anaplerosis is still obscure. In this context, fumarate hydratase (FH, fumarase) the key enzyme to metabolize fumarate becomes an Fumarate hydratase (fumarase, E.C. 4.2.1.2) catalyzes the reversible conversion of fumarate to malate. The stereospecific reaction involves the anti-addition of a water molecule across the carbon-carbon...

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Section: Essentiality Of Fumarate Hydratase For Pmentioning

confidence: 99%

Biochemical characterization and essentiality of fumarate hydratase

Jayaraman¹,

Suryavanshi²,

Kalale

et al. 2018

Journal of Biological Chemistry

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“…We had previously unsuccessfully tried to conditionally regulate the expression of GP1 by using the DHFR destabilisation domain (Muralidharan, Oksman, Iwamoto, Wandless, & Goldberg, 2011, Hallee & Richard, 2015 so we decided to switch to the glmS ribozyme system and generated a marker-free GP1-3HA-glmS tagged line by using the CRISPR-Cas9 technology ( Figure S5A; Ghorbal et al, 2014, Theriault & Richard, 2017. Proper integration of the 3HA-glmS cassette at the 3′ end of the GP1 gene was verified by Southern blot and PCR before and after parasite cloning ( Figure S5B-C).…”

Section: Knockdown Of Gp1 Affects In Vitro P Falciparum Asexual Stmentioning

confidence: 99%

“…Our previous work determined that the P. falciparum Putative Rhoptry Protein 2 (PRP2, PF3D7_1320000) resided in the Golgi and not in the rhoptries, which led us to rename it Golgi Protein 1 (GP1; Tufet‐Bayona et al, , Hallee & Richard, ). We show here that GP1 interacts with a unique and previously uncharacterised transmembrane protein (PF3D7_1123500), which we have named Golgi Protein 2 (GP2).…”

Section: Introductionmentioning

confidence: 99%

Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum

Hallée

Thériault

Gagnon

et al. 2018

Cellular Microbiology

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Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans-cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis-Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages.

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“…Fluorescence microscopy acquisition was performed as previously described (Hallee & Richard, ; Counihan et al, ) and using a GE Applied Precision Deltavision Elite microscope and an Olympus IX71 microscope with 100 × 1.4NA objective and with a sCMOS camera and deconvolved with the SoftWorx software. For immunofluorecence assays, parasites were fixed with cold MeOH (Dietz et al, ), cold 90% acetone/10% MeOH, or 4% paraformaldehyde (ProSciTech).…”

Section: Methodsmentioning

confidence: 99%

“…Fluorescence microscopy acquisition was performed as previously described (Hallee & Richard, 2015;Counihan et al, 2017)…”

Section: Microscopymentioning

confidence: 99%

The malaria parasite Plasmodium falciparum Sortilin is essential for merozoite formation and apical complex biogenesis

Hallée

Counihan

Matthews

et al. 2018

Cellular Microbiology

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The inner membrane complex and the apical secretory organelles are defining features of apicomplexan parasites. Despite their critical roles, the mechanisms behind the biogenesis of these structures in the malaria parasite Plasmodium falciparum are still poorly defined. We here show that decreasing expression of the P. falciparum homologue of the conserved endolysomal escorter Sortilin-VPS10 prevents the formation of the inner membrane complex and abrogates the generation of new merozoites. Moreover, protein trafficking to the rhoptries, the micronemes, and the dense granules is disrupted, which leads to the accumulation of apical complex proteins in the endoplasmic reticulum and the parasitophorous vacuole. We further show that protein export to the erythrocyte and transport through the constitutive secretory pathway are functional. Taken together, our results suggest that the malaria parasite P. falciparum Sortilin has potentially broader functions than most of its other eukaryotic counterparts.

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Evidence that the Malaria Parasite Plasmodium falciparum Putative Rhoptry Protein 2 Localizes to the Golgi Apparatus throughout the Erythrocytic Cycle

Cited by 10 publications

References 50 publications

Biochemical characterization and essentiality of fumarate hydratase

Biochemical characterization and essentiality of fumarate hydratase

Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum

The malaria parasite Plasmodium falciparum Sortilin is essential for merozoite formation and apical complex biogenesis

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