Manipulating Eryptosis of Human Red Blood Cells: A Novel Antimalarial Strategy?

Malaria is a major global health burden, affecting over 200 million people worldwide. Resistance against all currently available antimalarial drugs is a growing threat, and represents a major and long-standing obstacle to malaria eradication. Like many intracellular pathogens, Plasmodium parasites manipulate host cell signaling pathways, in particular programmed cell death pathways. Interference with apoptotic pathways by malaria parasites is documented in the mosquito and human liver stages of infection, but little is known about this phenomenon in the erythrocytic stages. Although mature erythrocytes have lost all organelles, they display a form of programmed cell death termed eryptosis. Numerous features of eryptosis resemble those of nucleated cell apoptosis, including surface exposure of phosphatidylserine, cell shrinkage and membrane ruffling. Upon invasion, Plasmodium parasites induce significant stress to the host erythrocyte, while delaying the onset of eryptosis. Many eryptotic inducers appear to have a beneficial effect on the course of malaria infection in murine models, but major gaps remain in our understanding of the underlying molecular mechanisms. All currently available antimalarial drugs have parasite-encoded targets, which facilitates the emergence of resistance through selection of mutations that prevent drug-target binding. Identifying host cell factors that play a key role in parasite survival will provide new perspectives for host-directed anti-malarial chemotherapy. This review focuses on the interrelationship between Plasmodium falciparum and the eryptosis of its host erythrocyte. We summarize the current knowledge in this area, highlight the different schools of thoughts and existing gaps in knowledge, and discuss future perspectives for host-directed therapies in the context of antimalarial drug discovery

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

Red Blood Cell BCL-xL Is Required for Plasmodium falciparum Survival: Insights into Host-Directed Malaria Therapies

The development of antimalarial drug resistance is an ongoing problem threatening progress towards the elimination of malaria, and antimalarial treatments are urgently needed for drug-resistant malaria infections. Host-directed therapies (HDT) represent an attractive strategy for the development of new antimalarials with untapped targets and low propensity for resistance. In addition, drug repurposing in the context of HDT can lead to a substantial decrease in the time and resources required to develop novel antimalarials. Host BCL-xL is a target in anti-cancer therapy and is essential for the development of numerous intracellular pathogens. We hypothesised that red blood cell (RBC) BCL-xL is essential for Plasmodium development and tested this hypothesis using six BCL-xL inhibitors, including one FDA-approved compound. All BCL-xL inhibitors tested impaired proliferation of Plasmodium falciparum 3D7 parasites in vitro at low micromolar or sub-micromolar concentrations. Western blot analysis of infected cell fractions and immunofluorescence microscopy assays revealed that host BCL-xL is relocated from the RBC cytoplasm to the vicinity of the parasite upon infection. Further, immunoprecipitation of BCL-xL coupled with mass spectrometry analysis identified that BCL-xL forms unique molecular complexes with human μ-calpain in uninfected RBCs, and with human SHOC2 in infected RBCs. These results provide interesting perspectives for the development of host-directed antimalarial therapies and drug repurposing efforts

Live cell video of <i>Plasmodium falciparum</i> merozoites egressing from schizont in the presence of Cy.007 Fab at 400 μg/mL.

No merozoite invasions were observed and most extracellular merozoites did not change during the 10 minute imaging period. Video playback is 10x live imaging speed. (AVI)</p

Effects of PfRH5 and PfCyRPA antibodies upon the efficiency with which merozoites convert into ring or pseudo-ring stage parasites shown in S4 Fig.

Table A, Egress to start of spinning. Table B, Duration of spinning. Table C, End of spinning to first pseudopod visible. Table D, Pseudopod first visible to pre-ring formation. Table E, Pre-ring formation to complete ring formation. (XLSX)</p

Quantification of the effects of antibodies to PfRH5 and PfCyRPA upon the invasion of RBCs by <i>Plasmodium falciparum</i> 3D7 merozoites.

Video microscopy of several merozoite egress events was observed in the presence of antibodies with concentrations in μg/mL indicated in brackets. (A) The times from egress to first contact indicate were not significantly different indicating the imaging conditions were consistent. (B) The degree of deformation of merozoites on erythrocyte surfaces was quantified according to [20] in the presence of antibodies. R5.004-(22) caused significantly less deformation than the control or parasite antibodies using chi-squared analysis. (C-G) The timings of other invasion stage as indicated on the y-axes were measured using the antibody combinations names and concentrations (μg/mL) indicated below the x-axes. Antibody 9 is Cy.007 Fab-(400). Each event measured is represented by a symbol with bars indicating the median. Statistical analyses were performed using unpaired t tests in GraphPad Prism V 9.0. The asterisks indicate where parasite mAbs have altered the number of events significantly from the EBL 040 control with *p (TIF)</p

Parasite specific mAbs to PfRH5 and PfCyRPA inhibit <i>Plasmodium falciparum</i> invasion of human RBCs.

(A-F) Several live cell videos of P. falciparum merozoites egressing and attempting to invade erythrocytes in the presence of each of the antibodies (concentrations and combinations indicated) were analyzed. The number of successful events is presented for each parameter indicated by the y axis. Full antibody names and concentrations (μg/mL) are indicated below bottom graphs. Each event is represented by a symbol and bars indicate the median number of events (A,B,E) or the percentage of events (C,D,F). Statistical analyses were performed using unpaired t tests in GraphPad Prism V 9.0. The asterisks indicate where parasite mAbs have altered the number or percentage of events significantly from the EBL 040 control with *p<0.05, **p<0.01 and ***p<0.001.</p

Effects of PfRH5 and PfCyRPA antibodies upon parasite invasion efficiency shown in Fig 2.

Table A, Number of contacts per egress. Table B, Number of invasions per egress. Table C, % contacts that invade. Table D, % of invasions that regress. Table E, Productive invasions per egress. Table F, % of productive invasions per contact. (XLSX)</p

Live cell video of <i>Plasmodium falciparum</i> merozoites egressing from schizont in the presence of R5.004 IgG at 22 μg/mL.

There are no erythrocyte invasions observed and black arrows indicates selected extracellular merozoites that begin to differentiate into pseudo-rings about 3 minutes after egress. Video playback is 10x live imaging speed. (AVI)</p

Live cell video of <i>Plasmodium falciparum</i> merozoites egressing from schizont in the presence of R5.008 IgG at 40 μg/mL.

Successful invasions are indicated with white arrows and black arrows indicate selected extracellular merozoites that begin to differentiate into pseudo-rings beginning about 5 minutes and 40 seconds after egress. Video playback is 10x live imaging speed. (AVI)</p