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.

Garg S.

Agarwal S.

Kumar S.

Shams Yazdani S.

Chitnis C.E.

Singh S.

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Shiv Nadar University

Nature Communications

Severe malaria caused by Plasmodium falciparum poses a major global health problem with high morbidity and mortality. P. falciparum harbors a family of pore-forming proteins (PFPs), known as perforin like proteins (PLPs), which are structurally equivalent to prokaryotic PFPs. These PLPs are secreted from the parasites and, they contribute to disease pathogenesis by interacting with host cells. The severe malaria pathogenesis is associated with the dysfunction of various barrier cells, including endothelial cells (EC). Several factors, including PLPs secreted by parasites, contribute to the host cell dysfunction. Herein, we have tested the hypothesis that PLPs mediate dysfunction of barrier cells and might have a role in disease pathogenesis. We analyzed various dysfunctions in barrier cells following rPLP2 exposure and demonstrate that it causes an increase in intracellular Ca2+ levels. Additionally, rPLP2 exposed barrier cells displayed features of cell death, including Annexin/PI positivity, depolarized the mitochondrial membrane potential, and ROS generation. We have further performed the time-lapse video microscopy of barrier cells and found that the treatment of rPLP2 triggers their membrane blebbing. The cytoplasmic localization of HMGB1, a marker of necrosis, further confirmed the necrotic type of cell death. This study highlights the role of parasite factor PLP in endothelial dysfunction and provides a rationale for the design of adjunct therapies against severe malaria.

Toxins

Pathogenic Pore Forming Proteins of Plasmodium Triggers the Necrosis of Endothelial Cells Attributed to Malaria Severity

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

Type-2 phosphatidic acid phosphatase (PAP2) a member of PAP2 superfamily mediates the conversion of phosphatidic acid (PA) to diacylglycerol (DAG) and thus plays a pivotal role in numerous cellular signaling processes in diverse organisms. An elevated level of intracellular PA is detrimental for the cell and induces cell death. In this study we identified and characterized a PAP2 homologue in Plasmodium falciparum, PfPAP2 and further elucidated its significance in regulation of PA homeostasis in parasite life cycle. PfPAP2 is expressed in the blood stage and harbors the canonical acid phosphatase domain (APD) with signature motifs. PfPAP2 catalyzes the dephosphorylation of PA to produce DAG and inorganic phosphate (Pi). Propranolol, a generic inhibitor of PAP2, inhibited the phosphatase activity of PfPAP2 by binding to the active site of APD domain as evident by in silico docking and confirmed by surface plasmon resonance (SPR) analysis. Inhibition of native PfPAP2 by propranolol led to rise in intracellular PA mediating disruption of intracellular PA homeostasis in parasites. The propranolol mediated inhibition of PfPAP2 directed early secretion of a micronemal Perforin like Protein, PfPLP1 leading to untimely permeabilization and host cell egress. The merozoites following premature egress were non-invasive and were attenuated to invade erythrocytes and cannot continue next cycle growth. This study demonstrates that disruption of PA homeostasis can cause growth retardation in malaria parasites, and thus its master regulator, PfPAP2, can serve as a very good molecular target for antimalarial chemotherapeutic interventions. © 2019, The Author(s).

Cell Death Discovery

Phosphatidic acid homeostasis regulated by a type-2 phosphatidic acid phosphatase represents a novel druggable target in malaria intervention

Lipid-based palmitoylation is a post-translation modification (PTM) which acts as a biological rheostat in life cycle progression of a deadly human malaria parasite, Plasmodium falciparum. P. falciparum palmitoylation is catalyzed by 12 putative palmitoyl acyl-transferase enzymes containing the conserved DHHC-CRD (DHHC motif within a cysteine-rich domain) which can serve as a druggable target. However, the paucity of high-throughput assays has impeded the design of drugs targeting palmitoylation. We have developed a novel strategy which involves engineering of Escherichia coli, a PTM-null system, to enforce ectopic expression of palmitoyl acyl-transferase in order to study Plasmodium-specific palmitoylation and screening of inhibitors. In this study, we have developed three synthetic E. coli strains expressing Plasmodium-specific DHHC proteins (PfDHHC7/8/9). These cells were used for validating acyl-transferase activity via acyl-biotin exchange (ABE) and clickable chemistry methods. E. coli proteome was found to be palmitoylated in PfDHHC-expressing clones, suggesting that plasmodium DHHC can catalyze palmitoylation of E. coli proteins. Upon treatment with generic inhibitor 2-bromopalmitate (2-BMP), a predominant reduction in palmitic acid incorporation is detected. Overall, these findings suggest that synthetic E. coli strains expressing PfDHHCs can enforce global palmitoylation in the E. coli proteome. Interestingly, this finding was corroborated by our in silico palmitoylome profiling, which revealed that out of the total E. coli proteome, 108 proteins were predicted to be palmitoylated as represented by the presence of three cysteine consensus motifs (cluster type I, II, III). In summary, our study reports a proof of concept for screening of chemotherapeutics targeting the palmitoylation machinery using a high-throughput screening platform. © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

FEBS Open Bio

Plasmodium palmitoylation machinery engineered in E. coli for high-throughput screening of palmitoyl acyl-transferase inhibitors

During the blood stage, Plasmodium spp. merozoites invade host red blood cells (RBCs), multiply, exit, and reinvade uninfected RBCs in a continuing cycle that is responsible for all the clinical symptoms associated with malaria. Entry into (invasion) and exit from (egress) RBCs are highly regulated processes that are mediated by an array of parasite proteins with specific functional roles. Many of these parasite proteins are stored in specialized apical secretory vesicles, and their timely release is critical for successful invasion and egress. For example, the discharge of parasite protein ligands to the apical surface of merozoites is required for interaction with host receptors to mediate invasion, and the timely discharge of proteases and pore-forming proteins helps in permeabilization and dismantling of limiting membranes during egress. This review focuses on our understanding of the signaling mechanisms that regulate apical organelle secretion during host cell invasion and egress by malaria parasites. The review also explores how understanding key signaling mechanisms in the parasite can open opportunities to develop novel strategies to target Plasmodium parasites and eliminate malaria. © 2017 Cold Spring Harbor Laboratory Press. All rights reserved.

Cold Spring Harbor Perspectives in Medicine

Molecular signaling involved in entry and exit of malaria parasites from host erythrocytes

Cellular Microbiology

Ca2+-mediated exocytosis of subtilisin-like protease 1: a key step in egress ofPlasmodium falciparummerozoites

Trends in Immunology

Protecting a serial killer: pathways for perforin trafficking and self-defence ensure sequential target cell death

Immunity

Protection from Endogenous Perforin: Glycans and the C Terminus Regulate Exocytic Trafficking in Cytotoxic Lymphocytes

Blood

A novel mechanism for egress of malarial parasites from red blood cells

Infection and Immunity

Global Identification of Multiple Substrates forPlasmodium falciparumSUB1, an Essential Malarial Processing Protease

Calcium-dependent permeabilization of erythrocytes by a perforin-like protein during egress of malaria parasites