Defense Peptides Engineered from Human Platelet Factor 4 Kill Plasmodium by Selective Membrane Disruption

Abstract

Malaria is a serious threat to human health and additional classes of antimalarial drugs are greatly needed. The human defense protein, platelet factor 4 (PF4), has intrinsic antiplasmodial activity but also undesirable chemokine properties. We engineered a peptide containing the isolated PF4 antiplasmodial domain, which through cyclization, retained the critical structure of the parent protein. The peptide, cPF4PD, killed cultured blood-stage Plasmodium falciparum with low micromolar potency by specific disruption of the parasite digestive vacuole. Its mechanism of action involved selective penetration and accumulation inside the intraerythrocytic parasite without damaging the host cell or parasite membranes; it did not accumulate in uninfected cells. This selective activity was accounted for by observations of the peptide's specific binding and penetration of membranes with exposed negatively charged phospholipid headgroups. Our findings highlight the tremendous potential of the cPF4PD scaffold for developing antimalarial peptide drugs with a distinct and selective mechanism of action.

Cyclic peptide scaffold with ability to stabilize and deliver a helical cell-impermeable cargo across membranes of cultured cancer cells

“…37 The ability of PF4 scaffold and grafted peptides to target and enter cancer cells to neutralize more negatively charged mitochondrial membranes represents an interesting parallel with cPF4PD's ability to target and enter RBCs infected with Plasmodium parasites to lyse the more negatively charged digestive vacuole membranes. 19 This crossover highlights the utility of selective membrane-active peptides for developing targeted therapies to a range of diseases.…”

“…The ability of the peptides to lyse neutral POPC or anionic POPC/POPS (4 : 1) membranes was investigated by measuring leakage of CF from large unilamellar vesicles (LUVs). 19,30 The dose response curves for peptide-induced membrane lysis are shown in Fig. 3B; the peptide concentration required to induce leakage for 50% of the vesicles (LC 50 ; final lipid concentration of 5 mM) and the relative selectivity for lysing LUVs composed of POPC/POPS (4 : 1) compared to POPC are shown in Table S1 (ESI †).…”

“…19 The direct entry of cPF4PD, and the larger PF4 protein, into the cytoplasm of malaria parasites inside RBCs suggests that endocytic pathways are not required for cell entry. 19 This entry mechanism is more desirable than endocytic mechanisms employed by other CPPs, for example the Tat peptide, that was also developed from a transduction domain of a larger protein. 12,20 The ability of PF4 and cPF4PD to enter infected RBCs, but not uninfected cells, is probably due to interaction between cationic residues within the amphipathic helices and anionic PS-headgroups on the outer leaflet of infected RBC membranes.…”

Immune role of platelets in malaria

“…AMPs interact with, penetrate and disrupt cell membranes containing exposed negatively charged phospholipids, which are absent in healthy mammalian cells but feature prominently in microbes and Plasmodium ‐infected cells . Indeed, peptides containing the AMP domain of PF4 show modest antiplasmodial activity , while a circularized dimer of the domain displays relatively potent activity and kills parasites without the requirement for red cell DARC expression (see below). PF4 and these PF4 AMP domain peptides were also shown to accumulate within the parasitized cell.…”

Dynamics of Gene Expression in Mice Infected with Different Genotypes of Toxoplasma gondii

“…3F to 3I). Since Pf4 was reported to be released from alpha-granules of activated platelets that could fight some parasite infections [47,48,49], the data imply that platelet may play an important role in host defense to the infections with different T. gondii genotype; however, the detailed mechanism needs to be further studied. The roles of three other down-regulated genes (Prg4, CXCL13 and Selp) in T. gondii infection also need to be identified in the future study.…”