Plasmodium falciparum perforin like proteins (PfPLPs) are an important arsenal for the entry and exit of malaria parasites. These proteins bind and oligomerize on the membrane in calcium dependent manner and form an open pore. The calcium dependent pore forming activity of PLPs is usually conferred by their C2 like C-terminal domain. Here, we have tried to elucidate the calcium binding residues in the C-terminal domain of PfPLP1, a member of P. falciparum PLPs, playing a crucial role in calcium dependent egress of blood stage parasites. Through our in silico study, we have found that the C-terminal domain of all PfPLPs is rich in β-pleated sheets and is structurally similar to C2 domain of human perforin. Furthermore, homology search based on 3-D structure of PfPLP1 confirmed that it is structurally homologous to the calcium binding C2 domain of many proteins. On further elucidation of the calcium-binding pocket of the C2 like domain of PfPLP1 showed that it binds to two calcium molecules. The calcium-binding pocket could be a target of novel chemotherapeutics for studying functional role of PfPLPs in parasite biology as well as for limiting blood stage growth of malaria parasite. © 2015, Springer Science+Business Media Dordrecht.

Garg S.

Sharma V.

Ramu D.

Singh S.

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Shiv Nadar University

Systems and Synthetic Biology

The pore forming Plasmodium Perforin Like Proteins (PPLP), expressed in all stages of the parasite life cycle are critical for completion of the parasite life cycle. The high sequence similarity in the central Membrane Attack Complex/ Perforin (MACPF) domain among PLPs and their distinct functional overlaps define them as lucrative target for developing multi-stage antimalarial therapeutics. Herein, we evaluated the mechanism of Pan-active MACPF Domain (PMD), a centrally located and highly conserved region of PPLPs, and deciphered the inhibitory potential of specifically designed PMD inhibitors. The E. coli expressed rPMD interacts with erythrocyte membrane and form pores of ~10.5 nm height and ~24.3 nm diameter leading to hemoglobin release and dextran uptake. The treatment with PMD induced erythrocytes senescence which can be hypothesized to account for the physiological effect of disseminated PLPs in loss of circulating erythrocytes inducing malaria anemia. The anti-PMD inhibitors effectively blocked intraerythrocytic growth by suppressing invasion and egress processes and protected erythrocytes against rPMD induced senescence. Moreover, these inhibitors also blocked the hepatic stage and transmission stage parasite development suggesting multi-stage, transmission-blocking potential of these inhibitors. Concievably, our study has introduced a novel set of anti-PMD inhibitors with pan-inhibitory activity against all the PPLPs members which can be developed into potent cross-stage antimalarial therapeutics along with erythrocyte senescence protective potential to occlude PPLPs mediated anemia in severe malaria. © Copyright © 2020 Garg, Shivappagowdar, Hada, Ayana, Bathula, Sen, Kalia, Pati, Singh and Singh.

Frontiers in Cellular and Infection Microbiology

Plasmodium Perforin-Like Protein Pores on the Host Cell Membrane Contribute in Its Multistage Growth and Erythrocyte Senescence

Egress of malaria parasites from the host cell requires the concerted rupture of its enveloping membranes. Hence, we investigated the role of the plasmodial perforin-like protein PPLP2 in the egress of Plasmodium falciparum from erythrocytes. PPLP2 is expressed in blood stage schizonts and mature gametocytes. The protein localizes in vesicular structures, which in activated gametocytes discharge PPLP2 in a calcium-dependent manner. PPLP2 comprises a MACPF domain and recombinant PPLP2 has haemolytic activities towards erythrocytes. PPLP2-deficient [PPLP2(-)] merozoites show normal egress dynamics during the erythrocytic replication cycle, but activated PPLP2(-) gametocytes were unable to leave erythrocytes and stayed trapped within these cells. While the parasitophorous vacuole membrane ruptured normally, the activated PPLP2(-) gametocytes were unable to permeabilize the erythrocyte membrane and to release the erythrocyte cytoplasm. In consequence, transmission of PPLP2(-) parasites to the Anopheles vector was reduced. Pore-forming equinatoxin II rescued both PPLP2(-) gametocyte exflagellation and parasite transmission. The pore sealant Tetronic 90R4, on the other hand, caused trapping of activated wild-type gametocytes within the enveloping erythrocytes, thus mimicking the PPLP2(-) loss-of-function phenotype. We propose that the haemolytic activity of PPLP2 is essential for gametocyte egress due to permeabilization of the erythrocyte membrane and depletion of the erythrocyte cytoplasm. © 2014 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.

Cellular Microbiology

Perforin-like protein PPLP2 permeabilizes the red blood cell membrane during egress of Plasmodium falciparum gametocytes

Clinical malaria is associated with proliferation of blood-stage parasites. During the blood stage, Plasmodium parasites invade host red blood cells, multiply, egress and reinvade uninfected red blood cells to continue the life cycle. Here we demonstrate that calcium-dependent permeabilization of host red blood cells is critical for egress of Plasmodium falciparum merozoites. Although perforin-like proteins have been predicted to mediate membrane perforation during egress, the expression, activity and mechanism of action of these proteins have not been demonstrated. Here, we show that two perforin-like proteins, perforin-like protein 1 and perforin-like protein 2, are expressed in the blood stage. Perforin-like protein 1 localizes to the red blood cell membrane and parasitophorous vacuolar membrane in mature schizonts following its Ca2+ -dependent discharge from micronemes. Furthermore, perforin-like protein 1 shows Ca2+ -dependent permeabilization and membranolytic activities suggesting that it may be one of the effector proteins that mediate Ca2+ -dependent membrane perforation during egress. © 2013 Macmillan Publishers Limited. All rights reserved.