Pan-active imidazolopiperazine antimalarials target the Plasmodium falciparum intracellular secretory pathway.

A promising new compound class for treating human malaria is the imidazolopiperazines (IZP) class. IZP compounds KAF156 (Ganaplacide) and GNF179 are effective against Plasmodium symptomatic asexual blood-stage infections, and are able to prevent transmission and block infection in animal models. But despite the identification of resistance mechanisms in P. falciparum, the mode of action of IZPs remains unknown. To investigate, we here combine in vitro evolution and genome analysis in Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods in P. falciparum. Our findings reveal that IZP-resistant S. cerevisiae clones carry mutations in genes involved in Endoplasmic Reticulum (ER)-based lipid homeostasis and autophagy. In Plasmodium, IZPs inhibit protein trafficking, block the establishment of new permeation pathways, and cause ER expansion. Our data highlight a mechanism for blocking parasite development that is distinct from those of standard compounds used to treat malaria, and demonstrate the potential of IZPs for studying ER-dependent protein processing

Inhibition of Plasmepsin V activity demonstrates its essential role in protein export, PfEMP1 display, and survival of malaria parasites

The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of P. falciparum-infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target

Spatial Localisation of Actin Filaments across Developmental Stages of the Malaria Parasite

Actin dynamics have been implicated in a variety of developmental processes during the malaria parasite lifecycle. Parasite motility, in particular, is thought to critically depend on an actomyosin motor located in the outer pellicle of the parasite cell. Efforts to understand the diverse roles actin plays have, however, been hampered by an inability to detect microfilaments under native conditions. To visualise the spatial dynamics of actin we generated a parasite-specific actin antibody that shows preferential recognition of filamentous actin and applied this tool to different lifecycle stages (merozoites, sporozoites and ookinetes) of the human and mouse malaria parasite species Plasmodium falciparum and P. berghei along with tachyzoites from the related apicomplexan parasite Toxoplasma gondii. Actin filament distribution was found associated with three core compartments: the nuclear periphery, pellicular membranes of motile or invasive parasite forms and in a ring-like distribution at the tight junction during merozoite invasion of erythrocytes in both human and mouse malaria parasites. Localisation at the nuclear periphery is consistent with an emerging role of actin in facilitating parasite gene regulation. During invasion, we show that the actin ring at the parasite-host cell tight junction is dependent on dynamic filament turnover. Super-resolution imaging places this ring posterior to, and not concentric with, the junction marker rhoptry neck protein 4. This implies motor force relies on the engagement of dynamic microfilaments at zones of traction, though not necessarily directly through receptor-ligand interactions at sites of adhesion during invasion. Combined, these observations extend current understanding of the diverse roles actin plays in malaria parasite development and apicomplexan cell motility, in particular refining understanding on the linkage of the internal parasite gliding motor with the extra-cellular milieu

Plasmodium falciparum coronin organizes arrays of parallel actin filaments potentially guiding directional motility in invasive malaria parasites

Background: Gliding motility in Plasmodium parasites, the aetiological agents of malaria disease, is mediated by an actomyosin motor anchored in the outer pellicle of the motile cell. Effective motility is dependent on a parasite myosin motor and turnover of dynamic parasite actin filaments. To date, however, the basis for directional motility is not known. Whilst myosin is very likely orientated as a result of its anchorage within the parasite, how actin filaments are orientated to facilitate directional force generation remains unexplained. In addition, recent evidence has questioned the linkage between actin filaments and secreted surface antigens leaving the way by which motor force is transmitted to the extracellular milieu unknown. Malaria parasites possess a markedly reduced repertoire of actin regulators, among which few are predicted to interact with filamentous (F)-actin directly. One of these, PF3D7_1251200, shows strong homology to the coronin family of actin-filament binding proteins, herein referred to as PfCoronin. Methods: Here the N terminal beta propeller domain of PfCoronin (PfCor-N) was expressed to assess its ability to bind and bundle pre-formed actin filaments by sedimentation assay, total internal reflection fluorescence (TIRF) microscopy and confocal imaging as well as to explore its ability to bind phospholipids. In parallel a tagged PfCoronin line in Plasmodium falciparum was generated to determine the cellular localization of the protein during asexual parasite development and blood-stage merozoite invasion. Results: A combination of biochemical approaches demonstrated that the N-terminal beta-propeller domain of PfCoronin is capable of binding F-actin and facilitating formation of parallel filament bundles. In parasites, PfCoronin is expressed late in the asexual lifecycle and localizes to the pellicle region of invasive merozoites before and during erythrocyte entry. PfCoronin also associates strongly with membranes within the cell, likely mediated by interactions with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at the plasma membrane. Conclusions: These data suggest PfCoronin may fulfil a key role as the critical determinant of actin filament organization in the Plasmodium cell. This raises the possibility that macro-molecular organization of actin mediates directional motility in gliding parasites. Electronic supplementary material The online version of this article (doi:10.1186/s12936-015-0801-5) contains supplementary material, which is available to authorized users

The Metabolite Repair Enzyme Phosphoglycolate Phosphatase Regulates Central Carbon Metabolism and Fosmidomycin Sensitivity in Plasmodium falciparum

The malaria parasite has a voracious appetite, requiring large amounts of glucose and nutrients for its rapid growth and proliferation inside human red blood cells. The host cell is resource rich, but this is a double-edged sword; nutrient excess can lead to undesirable metabolic reactions and harmful by-products. Here, we demonstrate that the parasite possesses a metabolite repair enzyme (PGP) that suppresses harmful metabolic by-products (via substrate dephosphorylation) and allows the parasite to maintain central carbon metabolism. Loss of PGP leads to the accumulation of two damaged metabolites and causes a domino effect of metabolic dysregulation. Accumulation of one damaged metabolite inhibits an essential enzyme in the pentose phosphate pathway, leading to substrate accumulation and secondary inhibition of glycolysis. This work highlights how the parasite coordinates metabolic flux by eliminating harmful metabolic by-products to ensure rapid proliferation in its resource-rich niche.Members of the haloacid dehalogenase (HAD) family of metabolite phosphatases play an important role in regulating multiple pathways in Plasmodium falciparum central carbon metabolism. We show that the P. falciparum HAD protein, phosphoglycolate phosphatase (PGP), regulates glycolysis and pentose pathway flux in asexual blood stages via detoxifying the damaged metabolite 4-phosphoerythronate (4-PE). Disruption of the P. falciparumpgp gene caused accumulation of two previously uncharacterized metabolites, 2-phospholactate and 4-PE. 4-PE is a putative side product of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, and its accumulation inhibits the pentose phosphate pathway enzyme, 6-phosphogluconate dehydrogenase (6-PGD). Inhibition of 6-PGD by 4-PE leads to an unexpected feedback response that includes increased flux into the pentose phosphate pathway as a result of partial inhibition of upper glycolysis, with concomitant increased sensitivity to antimalarials that target pathways downstream of glycolysis. These results highlight the role of metabolite detoxification in regulating central carbon metabolism and drug sensitivity of the malaria parasite

<i>In silico</i> integrative genomic search strategy to identify <i>P. falciparum</i> invasins.

<p>(<b>A</b>) To compile a list of proteins that include invasins, <i>P. falciparum</i> genes with homologues in the tight junction forming <i>T. gondii</i>, <i>P. berghei, P. chabaudi, P. vivax, P. yoelii</i> and <i>P. knowlesi</i> were selected (blue). Orthologues found in the non-tight junction forming <i>C. parvum</i> and <i>C. hominus</i> (pink) were removed from the dataset. Transcriptomic and proteomic data from <i>P. berghei</i> and <i>P. gallinaceum</i> ookinetes (brown) was used to remove proteins involved in motility but not invasion (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046160#pone.0046160.s003" target="_blank">Table S1</a> and Supplemental Experimental Procedures for data sources). (<b>B</b>) The top 50 candidate invasins ranked according to <i>P. falciparum</i> asexual cycle maximum fold change in transcript expression and relative protein abundance in <i>P. falciparum</i> merozoite and sporozoite proteomes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046160#pone.0046160.s004" target="_blank">Table S2</a>). Accession numbers are from PlasmoDB version 8.2. Number of transmembrane domains (TM), presence of a signal peptide (SP) and expression maximum during intra-erythrocytic cycle are listed. Heat diagram demonstrate intra-erythrocytic expression levels, given across 48 hr lifecycle with red representing high relative and green low relative expression. Proteins tagged in this study with an HA epitope are highlighted in yellow, and proteins where tagging was attempted but unsuccessful are highlighted blue.</p

Spatial localisation of different rhoptry proteins before and during merozoite invasion.

<p>(<b>A</b>) IEM of free PfRON2-HA merozoites (pre-invasion) dual labeled with immunogold anti-HA (18 nm) and rhoptry bulb marker RAP1 (12 nm). Scale bar = 0.2 µm. (<b>B</b>) IEM of free PFF0645c-HA merozoites (pre-invasion) dual labeled with immunogold anti-HA (18 nm) and rhoptry bulb marker RAP1 (12 nm). Scale bar = 0.2 µm. (<b>C</b>) Widefield IFA of E64-treated schizonts (to prevent egress – see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046160#s4" target="_blank">Materials and Methods</a>) labeled with anti-PfRh2, anti-PfRON4 and DAPI. Scale bar = 5 µm. (<b>D–G</b>) Independent replicate imaging of merozoites from (<b>D</b>) PfRON2-HA, (<b>E</b>) PfASP-HA (two classes of distribution seen), (<b>F</b>) RAP1 and (<b>G</b>) PFF0645c-HA mid-way through invasion colabeled with anti-PfRON4 and DAPI.</p