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Drug-induced permeabilization of parasite's digestive vacuole is a key trigger of programmed cell death in Plasmodium falciparum.

ABSTRACT: Having previously characterized chloroquine (CQ)-induced programmed cell death (PCD) hallmarks in the malaria parasite Plasmodium falciparum and delineating a pathway linking these features, the roles of non-classical mediators were investigated in this paper. It was shown that the later stages of this pathway are Ca(2+)-dependent and transcriptionally regulated. Moreover, it was demonstrated for the first time that micromolar concentrations of CQ partially permeabilized the parasite's digestive vacuole (DV) membrane and that this important upstream event appears to precede mitochondrial dysfunction. This permeabilization of the DV occurred without rupture of the DV membrane and was reminiscent of lysosome-mediated cell death in mammalian cells. As such micromolar concentrations of CQ are found in the patient's plasma after initial CQ loading, this alludes to a clinically relevant antimalarial mechanism of the drug which has yet to be recognized. Furthermore, other 'non-antimalarial' lysosomotropic compounds were also shown to cause DV permeabilization, triggering PCD in both CQ-sensitive and -resistant parasites. These findings present new avenues for antimalarial developments, which induce DV destabilization to kill parasites.

SUBMITTER: Ch'ng JH

PROVIDER: S-EPMC3219093 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Oriented nucleation of hemozoin at the digestive vacuole membrane in Plasmodium falciparum.

Project description:Heme detoxification is a critical step in the life cycle of malaria-causing parasites, achieved by crystallization into physiologically insoluble hemozoin. The mode of nucleation has profound implications for understanding the mechanism of action of antimalarial drugs that inhibit hemozoin growth. Several lines of evidence point to involvement of acylglycerol lipids in the nucleation process. Hemozoin crystals have been reported to form within lipid nanospheres; alternatively, it has been found in vitro that they are nucleated at an acylglycerol lipid-water interface. We have applied cryogenic soft X-ray tomography and three-dimensional electron microscopy to address the location and orientation of hemozoin crystals within the digestive vacuole (DV), as a signature of their nucleation and growth processes. Cryogenic soft X-ray tomography in the "water window" is particularly advantageous because contrast generation is based inherently on atomic absorption. We find that hemozoin nucleation occurs at the DV inner membrane, with crystallization occurring in the aqueous rather than lipid phase. The crystal morphology indicates a common {100} orientation facing the membrane as expected of templated nucleation. This is consistent with conclusions reached by X-ray fluorescence and diffraction in a companion work. Uniform dark spheres observed in the parasite were identified as hemoglobin transport vesicles. Their analysis supports a model of hemozoin nucleation primarily in the DV. Modeling of the contrast at the DV membrane indicates a 4-nm thickness with patches about three times thicker, possibly implicated in the nucleation.

| S-EPMC3396484 | biostudies-literature

Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite's food vacuole and alter drug sensitivities.

Project description:Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs.

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On Programmed Cell Death in Plasmodium falciparum: Status Quo.

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Plasmodium falciparum Rab5B is an N-terminally myristoylated Rab GTPase that is targeted to the parasite's plasma and food vacuole membranes.

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A Plasmodium falciparum strain expressing GFP throughout the parasite's life-cycle.

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Role of Plasmodium falciparum digestive vacuole plasmepsins in the specificity and antimalarial mode of action of cysteine and aspartic protease inhibitors.

Project description:Hemoglobin (Hb) degradation is essential for the growth of the intraerythrocytic stages of malarial parasites. This process, which occurs inside an acidic digestive vacuole (DV), is thought to involve the action of four aspartic proteases, termed plasmepsins (PMs). These enzymes have received considerable attention as potential antimalarial drug targets. Leveraging the availability of a set of PM-knockout lines generated in Plasmodium falciparum, we report here that a wide range of previously characterized or novel aspartic protease inhibitors exert their antimalarial activities independently of their effect on the DV PMs. We also assayed compounds previously shown to inhibit cysteine proteases residing in the DV. The most striking observation was a ninefold increase in the potency of the calpain inhibitor N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) against parasites lacking all four DV PMs. Genetic ablation of PM III or PM IV also decreased the level of parasite resistance to the beta-hematin binding antimalarial chloroquine. On the basis of the findings of drug susceptibility and isobologram assays, as well as the findings of studies of the inhibition of Hb degradation, morphological analyses, and stage specificity, we conclude that the DV PMs and falcipain cysteine proteases act cooperatively in Hb hydrolysis. We also identify several aspartic protease inhibitors, designed to target DV PMs, which appear to act on alternative targets early in the intraerythrocytic life cycle. These include the potent diphenylurea compound GB-III-32, which was found to be fourfold less potent against a P. falciparum line overexpressing plasmepsin X than against the parental nontransformed parasite line. The identification of the mode of action of these inhibitors will be important for future antimalarial drug discovery efforts focusing on aspartic proteases.

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Protein targeting to the parasitophorous vacuole membrane of Plasmodium falciparum.

Project description:Red blood cell (RBC) invasion and parasitophorous vacuole (PV) formation by Plasmodium falciparum are critical for the development and pathogenesis of malaria, a continuing global health problem. Expansion of the PV membrane (PVM) during growth is orchestrated by the parasite. This is particularly important in mature RBCs, which lack internal organelles and no longer actively synthesize membranes. Pfs16, a 16-kDa integral PVM protein expressed by gametocytes, was chosen as a model for studying the trafficking of material from the parasite across the PV space to the PVM. The locations of Pfs16-green fluorescent protein (GFP) reporter proteins containing distinct regions of Pfs16 were tracked from RBC invasion to emergence. Inclusion of the 53 C-terminal amino acids (aa) of Pfs16 to a GFP reporter construct already containing the N-terminal secretory signal sequence was sufficient for targeting to and retention on the PVM. An amino acid motif identified in this region was also found in seven other known PVM proteins. Removal of the 11 C-terminal aa did not affect PVM targeting, but membrane retention was decreased. Additionally, during emergence from the PVM and RBC, native Pfs16 and the full-length Pfs16-GFP reporter protein were found to concentrate on the ends of the gametocyte. Capping was not observed in constructs lacking the amino acids between the N-terminal secretory signal sequence and the transmembrane domain, suggesting that this region, which is not required for PVM targeting, is involved in capping. This is the first report to define the amino acid domains required for targeting to the P. falciparum PVM.

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Turning up the heat: heat stress induces markers of programmed cell death in Plasmodium falciparum in vitro.

Project description:Malaria is characterised by cyclical febrile episodes that result from the rupture of mature schizont-infected erythrocytes releasing merozoites. In patients infected with Plasmodium falciparum, fever may reach peak temperatures as high as 41 °C. Febrile episodes typically have a deleterious effect on parasites and probably benefit the host by aiding parasite clearance; however, the parasite may also gain advantage from limiting its burden on the host and prolonging infection to ensure development and transmission of slow-maturing gametocytes. Programmed cell death (PCD) may provide the parasite with a mechanism of self-limitation, although the occurrence and phenotype of PCD in the erythrocytic stages remain controversial due to conflicting data. This study aimed to characterise the cell death phenotype of P. falciparum in response to in vitro heat stress. A variety of biochemical markers of PCD, including DNA fragmentation, mitochondrial dysregulation and phosphatidylserine externalisation, as well as morphological studies of Giemsa-stained thin smears and real-time microscopy were utilised to characterise the phenotype. Heat stress decreased P. falciparum growth and development in vitro. Late-stage parasites were more susceptible, although early stages were more affected than expected. Early-stage parasites exposed to 41 °C exhibited markers of an apoptosis-like PCD phenotype, including DNA fragmentation and mitochondrial depolarisation. Heat-stressed late-stage parasites showed no significant DNA fragmentation or mitochondrial dysregulation; however, cytoplasmic vacuolisation was suggestive of an autophagy-like form of PCD. Our results therefore showed that biochemical and morphological markers of PCD varied with intra-erythrocytic parasite development and that P. falciparum exhibited facets of both apoptosis- and autophagy-like phenotypes after exposure to febrile temperatures, which may reflect a unique PCD phenotype.

| S-EPMC3877572 | biostudies-literature

The neutral lipid composition present in the digestive vacuole of Plasmodium falciparum concentrates heme and mediates ?-hematin formation with an unusually low activation energy.

Project description:In eukaryotic cells, neutral lipids serve as major energy storage molecules; however, in Plasmodium falciparum, a parasite responsible for causing malaria in humans, neutral lipids may have other functions during the intraerythrocytic stage of the parasite life cycle. Specifically, experimental data suggest that neutral lipid structures behave as a catalyst for the crystallization of hemozoin, a detoxification byproduct of several blood-feeding organisms, including malaria parasites. Synthetic neutral lipid droplets (SNLDs) were produced by depositing a lipid blend solution comprised of mono- and diglycerides onto an aqueous surface. These lipid droplets are able to mediate the production of brown pigments that are morphologically and chemically identical to hemozoin. The partitioning of heme into these SNLDs was examined by employing Nile Red, a lipid specific dye. Soluble ferriprotoporphyrin IX was observed to spontaneously localize to the lipid droplets, partitioning in a pH-dependent manner with an estimated log P of 2.6. Interestingly, the pH profile of heme partitioning closely resembles that of ?-hematin formation. Differential scanning calorimetry and kinetic studies demonstrated that the SNLDs provide a unique environment that promotes hemozoin formation. SNLD-mediated formation of the malaria pigment displayed an activation energy barrier lower than those of individual lipid components. In particular, lipid droplets composed of diglycerides displayed activation barriers lower than those composed of monoglycerides. This difference was attributed to the greater fluidity of these lipids. In conjunction with the known pattern of lipid body proliferation, it is suggested that neutral lipid structures within the digestive vacuole not only are the location of in vivo hemozoin formation but are also essential for the survival of the parasite by functioning as a kinetically competent and site specific mediator for heme detoxification.

| S-EPMC2996888 | biostudies-literature

High-Content Screening of the Medicines for Malaria Venture Pathogen Box for Plasmodium falciparum Digestive Vacuole-Disrupting Molecules Reveals Valuable Starting Points for Drug Discovery.

Project description:Plasmodium falciparum infections leading to malaria have severe clinical manifestations and high mortality rates. Chloroquine (CQ), a former mainstay of malaria chemotherapy, has been rendered ineffective due to the emergence of widespread resistance. Recent studies, however, have unveiled a novel mode of action in which low-micromolar levels of CQ permeabilized the parasite's digestive vacuole (DV) membrane, leading to calcium efflux, mitochondrial depolarization, and DNA degradation. These phenotypes implicate the DV as an alternative target of CQ and suggest that DV disruption is an attractive target for exploitation by DV-disruptive antimalarials. In the current study, high-content screening of the Medicines for Malaria Venture (MMV) Pathogen Box (2015) was performed to select compounds which disrupt the DV membrane, as measured by the leakage of intravacuolar Ca2+ using the calcium probe Fluo-4 AM. The hits were further characterized by hemozoin biocrystallization inhibition assays and dose-response half-maximal (50%) inhibitory concentration (IC50) assays across resistant and sensitive strains. Three hits, MMV676380, MMV085071, and MMV687812, were shown to demonstrate a lack of CQ cross-resistance in parasite strains and field isolates. Through systematic analyses, MMV085071 emerged as the top hit due to its rapid parasiticidal effect, low-nanomolar IC50, and good efficacy in triggering DV disruption, mitochondrial degradation, and DNA fragmentation in P. falciparum These programmed cell death (PCD)-like phenotypes following permeabilization of the DV suggests that these compounds kill the parasite by a PCD-like mechanism. From the drug development perspective, MMV085071, which was identified to be a potent DV disruptor, offers a promising starting point for subsequent hit-to-lead generation and optimization through structure-activity relationships.

| S-EPMC5826141 | biostudies-literature

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