From the Cover: Malaria parasite exit from the host erythrocyte: A two-step process requiring extraerythrocytic proteolysis

Abstract

Intraerythrocytic malaria parasites replicate by the process of schizogeny, during which time they copy their genetic material and package it into infective merozoites. These merozoites must then exit the host cell to invade new erythrocytes. To better characterize the events of merozoite escape, erythrocytes containing Plasmodium falciparum schizonts were cultured in the presence of the cysteine protease inhibitor, L-transepoxy-succinyl-leucylamido-(4-guanidino)butane (E64). This treatment resulted in the accumulation of extraerythrocytic merozoites locked within a thin, transparent membrane. Immunomicroscopy demonstrated that the single membrane surrounding the merozoites is not erythrocytic but rather is derived from the parasitophorous vacuolar membrane (PVM). Importantly, structures identical in appearance can be detected in untreated cultures at low frequency. Further studies revealed that (i) merozoites from the PVM-enclosed merozoite structures (PEMS) are invasive, viable, and capable of normal development; (ii) PEMS can be purified easily and efficiently; and (iii) when PEMS are added to uninfected red blood cells, released merozoites can establish a synchronous wave of infection. These observations suggest that L-transepoxy-succinyl-leucylamido-(4-guanidino)butane (E64) causes an accumulation of an intermediate normally present during the process of rupture. We propose a model for the process of rupture: merozoites enclosed within the PVM first exit from the host erythrocyte and then rapidly escape from the PVM by a proteolysis-dependent mechanism.M alaria afflicts an estimated 300 million to 500 million people and is responsible for the death of nearly 2 million children each year (1). Malaria is caused by several species of obligate intracellular protozoan parasites, the most deadly of which is Plasmodium falciparum. The malaria parasite completes its asexual life cycle in the mature human erythrocyte within a vacuole, delimited by the parasitophorous vacuole membrane (PVM). While in the red cell, the parasite undergoes three distinct stages of development: the ring, the trophozoite, and the schizont stage. During schizony, the parasite divides within the vacuole to form between 16 and 32 merozoites. Approximately 48 h after infection, the merozoites rupture from within the red cell to invade new cells and continue the cycle.In a 1986 study by Lyon and Haynes (2), purified P. falciparum schizonts that were cultured in the presence of small concentrations of leupeptin and chymostatin failed to properly rupture and, instead, formed ''protease inhibitor clusters of merozoites.'' A number of proteases are suspected to be involved in parasite exit, based on circumstantial evidence. The stage-specific expression of a cysteine protease (35,000-40,000 M r ) and a serine protease (75,000 M r ) found in mature schizonts and merozoites, respectively, implicates them in the process of rupture or invasion (3). The P. falciparum serine repeat antigen, known as SERA, P126, SERA-1, and SERP, is one of several homolo...

New antimalarial indolone-N-oxides, generating radical species, destabilize the host cell membrane at early stages of Plasmodium falciparum growth: role of band 3 tyrosine phosphorylation

“…Dephosphorylation and the variability in the patterns observed at schizont stage may be due to the combined effects of variable degrees of proteolysis [26] and to the energetic decay that characterizes the terminal parasite stages [27] (data not shown).…”

High Yield Purification of <em>Plasmodium falciparum</em> Merozoites For Use in Opsonizing Antibody Assays

“…Recently the cysteine protease inhibitor trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E64) has been used to generate viable merozoites. E64 prevents schizont rupture, generating membrane enclosed merozoites 14 , which can be disrupted by filtration to liberate viable merozoites 15,16 . This technique has lead to the spatial resolution of numerous proteins during erythrocyte invasion 15,[17][18][19] and has clarified the stage specific effect of several antimalarial drugs 16,20 .…”

Chemical biology approaches for the study of apicomplexan parasites

“…Treatment of intra-erythrocytic P. falciparum parasites with E-64 results in two distinct phenotypes. A swollen food-vacuole defect and an inhibition of host cell egress [11, 12]. The molecular basis of each phenotype has subsequently been determined; the swollen food vacuole defect arises from an inability of the parasite to digest hemoglobin imported to the food vacuole from the host cell for detoxification [13].…”

“…4: SAK1, a vinyl sulfone that inhibits PfDPAP3 and blocks P. falciparum merozoite egress [34]. 5: E64, an epoxy succinate that inhibits host calpain to block P. falciparum merozoite egress [15], and inhibits the activity of parasite digestive food vacuole falcipains [12]. …”