Plasmodium falciparum parasites deploy RhopH2 into the host erythrocyte to obtain nutrients, grow and replicate

Plasmodium falciparum parasites, the causative agents of malaria, modify their host erythrocyte to render them permeable to supplementary nutrient uptake from the plasma and for removal of toxic waste. Here we investigate the contribution of the rhoptry protein RhopH2, in the formation of new permeability pathways (NPPs) in Plasmodium-infected erythrocytes. We show RhopH2 interacts with RhopH1, RhopH3, the erythrocyte cytoskeleton and exported proteins involved in host cell remodeling. Knockdown of RhopH2 expression in cycle one leads to a depletion of essential vitamins and cofactors and decreased de novo synthesis of pyrimidines in cycle two. There is also a significant impact on parasite growth, replication and transition into cycle three. The uptake of solutes that use NPPs to enter erythrocytes is also reduced upon RhopH2 knockdown. These findings provide direct genetic support for the contribution of the RhopH complex in NPP activity and highlight the importance of NPPs to parasite survival

Aryl amino acetamides prevent Plasmodium falciparum ring development via targeting the lipid-transfer protein PfSTART1.

With resistance to most antimalarials increasing, it is imperative that new drugs are developed. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited the ring-stage development of newly invaded merozoites. Here, we select parasites resistant to M-833 and identify mutations in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introducing PfSTART1 mutations into wildtype parasites reproduces resistance to M-833 as well as to more potent analogues. PfSTART1 binding to the analogues is validated using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assays. Imaging of invading merozoites shows the inhibitors prevent the development of ring-stage parasites potentially by inhibiting the expansion of the encasing parasitophorous vacuole membrane. The PfSTART1-targeting compounds also block transmission to mosquitoes and with multiple stages of the parasite's lifecycle being affected, PfSTART1 represents a drug target with a new mechanism of action

Table_1_PfATP4 inhibitors in the Medicines for Malaria Venture Malaria Box and Pathogen Box block the schizont-to-ring transition by inhibiting egress rather than invasion.xlsx

The cation efflux pump Plasmodium falciparum ATPase 4 (PfATP4) maintains Na+ homeostasis in malaria parasites and has been implicated in the mechanism of action of many structurally diverse antimalarial agents, including >7% of the antimalarial compounds in the Medicines for Malaria Venture’s ‘Malaria Box’ and ‘Pathogen Box’. Recent screens of the ‘Malaria Box’ and ‘Pathogen Box’ revealed that many PfATP4 inhibitors prevent parasites from exiting their host red blood cell (egress) or entering new host cells (invasion), suggesting that these compounds may have additional molecular targets involved in egress or invasion. Here, we demonstrate that five PfATP4 inhibitors reduce egress but not invasion. These compounds appear to inhibit egress by blocking the activation of protein kinase G, an enzyme that, once stimulated, rapidly activates parasite egress. We establish a direct link between egress and PfATP4 function by showing that the inhibition of egress is attenuated in a Na+-depleted environment and in parasites with a mutation in pfatp4. Finally, we show that PfATP4 inhibitors induce host cell lysis when administered prior to the completion of parasite replication. Since host cell lysis mimics egress but is not followed by invasion, this phenomenon likely explains why several PfATP4 inhibitors were previously classified as invasion inhibitors. Collectively, our results confirm that PfATP4-mediated Na+ efflux is critical to the regulation of parasite egress.</p

Presentation_1_PfATP4 inhibitors in the Medicines for Malaria Venture Malaria Box and Pathogen Box block the schizont-to-ring transition by inhibiting egress rather than invasion.pdf

The cation efflux pump Plasmodium falciparum ATPase 4 (PfATP4) maintains Na+ homeostasis in malaria parasites and has been implicated in the mechanism of action of many structurally diverse antimalarial agents, including >7% of the antimalarial compounds in the Medicines for Malaria Venture’s ‘Malaria Box’ and ‘Pathogen Box’. Recent screens of the ‘Malaria Box’ and ‘Pathogen Box’ revealed that many PfATP4 inhibitors prevent parasites from exiting their host red blood cell (egress) or entering new host cells (invasion), suggesting that these compounds may have additional molecular targets involved in egress or invasion. Here, we demonstrate that five PfATP4 inhibitors reduce egress but not invasion. These compounds appear to inhibit egress by blocking the activation of protein kinase G, an enzyme that, once stimulated, rapidly activates parasite egress. We establish a direct link between egress and PfATP4 function by showing that the inhibition of egress is attenuated in a Na+-depleted environment and in parasites with a mutation in pfatp4. Finally, we show that PfATP4 inhibitors induce host cell lysis when administered prior to the completion of parasite replication. Since host cell lysis mimics egress but is not followed by invasion, this phenomenon likely explains why several PfATP4 inhibitors were previously classified as invasion inhibitors. Collectively, our results confirm that PfATP4-mediated Na+ efflux is critical to the regulation of parasite egress.</p

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation

Resistant genomes pooled variant summary.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.</div

MMV291 analogues interfere with actin polymerisation in the presence of profilin in vitro.

PfACT1 (4 μM) under polymerizing conditions was quantified in the supernatant and pellet fractions in the presence of the MMV291 analogues (25 μM) or DMSO and upon addition of PfPFN (16 μM). (A) In the absence of PfPFN, 80 ± 4% of PfACT1 sedimented to the pellet fraction with the vehicle DMSO treatment. S-W936 decreased the amount of actin in the pellet to 68 ± 7%, while the remaining compounds had no significant effects on actin sedimentation. (B) Upon addition of PfPFN, actin sedimentation decreased to 21 ± 1% with DMSO treatment. All MMV291 analogues, S-MMV291, R-MMV291, S-W936, R-W936, S-W414, and S-W827, decreased the amount of actin in the pellet further to 11 ± 1%, 15 ± 2%, 8 ± 2%, 10 ± 4%, 9 ± 4%, and 5 ± 2%, respectively. Results are plotted as mean ± standard deviation of the relative amounts of actin in the pellet fraction. The data are based on at least 3 independent assays each performed in triplicate. Statistical significances were determined using an unpaired two-tailed t test, where ** P ≤ 0.01 and *** P ≤ 0.001, and **** ≤ 0.0001. No bar indicates not significant. Source data can be found in S1 Data.</p

A model of the comparison between mutation locations in human and <i>P</i>. <i>falciparum</i>.

An X-ray structure human profilin (gold) (PDB: 2PBD) and a homology model of human actin (green) (created by SWISS-MODEL [106] using O. cuniculus actin (PDB: 2PBD) [56] aligned with P. falciparum profilin (pink) (PDB: 2JKG) [36] and actin (blue) (PDB: 6I4E) [42] showing the similarity of the heterodimeric complex. The positions of the MMV291 P. falciparum mutations and the associated human amino acids are shown for comparison. (TIF)</p

MMV291 does not affect actin filaments in HeLa cells.

HeLa cells labelled with SiR-Actin imaged by lattice light-sheet microscopy upon stimulation with DMSO Control, 5 μM Latrunculin B, 200 nM CytD, 2.5 μM MMV291, 10 μM MMV291, and 20 μM MMV291 over a time course of 3 hours. Movies represent maximum intensity projections with the contrast scaled between 100–400 counts. Time is presented as HH:MM, and the scale bar represents 20 μm. (MP4)</p