Platelet factor 4 activity against P. falciparum and its translation to nonpeptidic mimics as antimalarials

Abstract

Plasmodium falciparum pathogenesis is affected by various cell types in the blood, including platelets, which can kill intraerythrocytic malaria parasites. Platelets could mediate these antimalarial effects through human defense peptides (HDPs), which exert antimicrobial effects by permeabilizing membranes. Therefore, we screened a panel of HDPs and determined that human platelet factor 4 (hPF4) kills malaria parasites inside erythrocytes by selectively lysing the parasite digestive vacuole (DV). PF4 rapidly accumulates only within infected erythrocytes and is required for parasite killing in infected erythrocyte-platelet cocultures. To exploit this antimalarial mechanism, we tested a library of small, nonpeptidic mimics of HDPs (smHDPs) and identified compounds that kill P. falciparum by rapidly lysing the parasite DV while sparing the erythrocyte plasma membrane. Lead smHDPs also reduced parasitemia in a murine malaria model. Thus, identifying host molecules that control parasite growth can further the development of related molecules with therapeutic potential.

Figure 1. hPF4 Acts as a HDP against P. falciparum via Lysis of the Parasite DV

(A) Screen of human HDPs found in blood for P. falciparum killing and hemolysis at 15 µM, including Regulated upon Activation, Normal T cell Expressed, and Secreted (RANTES); hPF4; Fibrinopeptide-A (FP-A); Fibrinopeptide-B (FP-B); Human Neutrophil Peptides 1 and 2 (HNP-1; HNP-2); and Cathelicidin (LL-37). Parasite survival was normalized to an untreated control. Artesunate (500 nM) and the HDP melittin (15 µM) were used as positive controls for parasite death without and with hemolysis, respectively. Data shown are means ± SEM. (B) Parasite-infected erythrocytes were incubated for 24 hr with 2.5 × 10⁸ platelets from either littermate WT, PF4 KO, or hPF4⁺ mice in the presence or absence of 5 µM AYP to induce platelet activation. Parasite survival was assayed by flow cytometry using SYTOX green as a measurement of DNA content and normalized to an untreated control. Recombinant hPF4 and mPF4 (10 µM) were used as positive controls. Data shown are means ± SEM. (C) Parasite-infected erythrocytes preincubated with rhodamine 123 (1 µM for parasite plasma membrane potential; 0.2 µM for mitochondrial potential) or 10 nM LysoTracker Red and then treated over a 4 hr time course with 10 µM PF4 or a 10 µM mixture of ionophores Monensin and Nigericin (Mon/Nig). Length bar is 10 µm in each figure. (D) hPF4 accumulates in the infected erythrocyte cytoplasm prior to lysis of the DV by immunofluorescence. Erythrocytes infected with parasites expressing plasmepsin-II-GFP (PM-II-GFP) were treated with 1 µM hPF4 over a 10 min time course. Within 1 min of PF4 incubation, PF4 staining is seen in the cytoplasm of infected erythrocytes until entering and inducing lysis of the parasite DV, as shown by GFP signal diffusion through the entire parasite cytoplasm. (Green, PM-IIGFP [DV]; red, hPF4 IFA; blue, parasite nuclei.) (E) Western blot analysis of erythrocyte fractions or parasite lysates shows early accumulation of hPF4 in infected erythrocytes, followed by persistence in the parasite. (F) ImageStream flow cytometry shows a dose-dependent increase in PM-II-GFP parasites with a fluorescence surface area of >2 µm², an indication of DV lysis, upon treatment with hPF4. Intact DVs have fluorescent surface areas <0.2 µm². Data shown are means ± SEM (*p < 0.05, **p < 0.01). (G) TEM images reveal dissolution of the DV membrane upon treatment with 10 µM PF4, as well as dispersal of hemozoin fragments throughout the cytoplasm (red arrowheads). Mock-treated controls showed complete integrity of the DV membrane (white arrowheads), encapsulating all hemozoin crystals. (N: parasite nucleus). See also Figure S1 and Movie S1.

Figure 2. The C-Terminal Amphipathic Helical Domain of PF4 Retains the HDP Activity against P. falciparum

(A) Projected helical wheel of C12 with each amino acid numbered. (B) Peptide derivatives of the PF4 C-terminal domain with lysine residues mutated to alanines to determine the necessity of positive charges for C12 antimalarial activity. Mutated residues are denoted by “A” followed by the residue number. IC₅₀s are means ± SEM (n = 3). (C) Dose-response curves for PF4, C12, and the series of C12 lysine mutants. Data shown are means ± SEM. (D) Parasite plasma membrane, mitochondrial, and DV potential were examined upon C12 treatment (see Figure 1B for details). (E) C12-treated PM-II-GFP parasites showed loss of DV integrity (green, PM-II-GFP [DV]; blue, parasite nuclei.) (F) C12-TAMRA treatment of PM-II-GFP parasites shows accumulation within infected erythrocytes and parasite bodies within 15 min of exposure, followed by DV lysis within 60 min of treatment. See also Figure S2.

Figure 3. Screen of smHDPs Reveals Potent Inhibitors of P. falciparum Growth

(A) Conceptual design of smHDPs from HDPs. Top: Amphipathic structure of magainin 2; cationic groups in blue, nonpolar groups in green, peptide backbone in yellow. Bottom: de novo designed smHDPs capture the facially amphipathic architecture and critical physicochemical properties needed to establish robust antimicrobial activity. (B) Screen of 920 smHDPs at 500 nM against P. falciparum. Parasite death was normalized to a chloroquine control (14.3% hit rate, Z′ = 0.720). (C) Lead smHDPs were screened against a panel of chloroquine-sensitive and chloroquine-resistant P. falciparum lines. Chloroquine was used as a positive control for parasite death. IC₅₀s are as mean ± SEM, n > 3 for each compound. Cytotoxicity (EC₅₀) determined against mouse 3T3 fibroblasts and human transformed liver HepG2 cells using an MTS viability assay. (D) Comparison of anti-P. falciparum (Pf; 3D7 strain) and antibacterial activities. Bacteria strains: E. coli 25922 (EC), S. aureus 27660 (SA), E. faecalis 29212 (EF), P. aeruginosa 10145 (PA), K. pneumoniae 13883 (KP). (E) Chemical structures of PMX1207 and PMX207. See also Figure S3 and Table S1.

Figure 4. smHDP Leads Kill P. falciparum via Parasite DV Lysis and Decrease Parasitemia in a Murine Malaria Model

(A) Parasite plasma membrane and mitochondrial potential was examined during treatment with PMX207 or PMX1207, with no discernible loss of fluorescence, unlike the positive controls. Compromise of DV integrity was monitored with Lyso Tracker Red, wherein loss of DV fluorescence was seen within 15 min of smHDP treatment, though no loss of parasite plasma membrane potential occurs. (B) Loss of DV integrity upon smHDP treatment of PM-II-GFP P. falciparum parasites (green, PM-II-GFP [DV]; blue, parasite nuclei.) (C) ImageStream flow cytometry shows a dose-dependent increase in PM-II-GFP parasites showing a fluorescence surface area of >2 µm², an indication of DV lysis, upon treatment with PMX207 or PMX1207. Intact DVs have fluorescent surface areas <0.2 µm². Data shown are means ± SEM (*p < 0.05, **p < 0.01). (D) TEM images reveal complete dissolution of the DV membrane upon PMX207 or PMX1207 treatment, with dispersal of hemozoin crystals (red arrowheads) throughout the cytoplasm and an accumulation of undigested hemoglobin-containing vesicles (yellow arrowheads). Mock-treated parasites retained a clear delineating DV membrane (white arrowheads), encapsulating all hemozoin crystals (N, parasite nucleus). (E) Parasitemias of Swiss Webster mice infected with P. yoelii 17XNL parasitized erythrocytes via i.v. injection and treated with vehicle (n = 5), 5 mg/kg PMX1207 (n = 7), or 20 mg/kg of PMX207 (n = 5). Parasitemia was assessed on days 4 and 6. Shown are the means ± SEM (*p < 0.05; ***p < 0.001). (F) Parasitemias of C57BL/6 mice infected with P. berghei ANKA parasitized erythrocytes via i.v. injection and treated with vehicle (n = 10), 5 mg/kg PMX1207 (n = 5), or 20 mg/kg of PMX207 (n = 5). Parasitemia was assessed on days 5 and 7. Shown are the means ± SEM (*p < 0.05; ***p < 0.001). (G) Survival curves for P. berghei ANKA infected mice. p = 0.0189 in the log rank Mantel-Cox test. See also Figure S4 and Movie S2.