scholarly journals Cholesterol dynamics are essential for the growth and development of Plasmodium falciparum within the erythrocyte

2021 ◽  
Author(s):  
Avantika I. Ahiya ◽  
Suyash Bhatnagar ◽  
Joanne Morrisey ◽  
Josh R. Beck ◽  
Akhil B. Vaidya
Keyword(s):  
Infected Erythrocyte ◽  
De Novo ◽  
Cholesterol Content ◽  
Cholesterol Homeostasis ◽  
Parasite Development ◽  
Trophozoite Stage ◽  
Parasite Survival ◽  
Erythrocytic Cycle ◽  
Erythrocyte Plasma Membrane ◽  
Rapid Expulsion

AbstractPlasmodium spp. lack de novo cholesterol synthetic pathways and can only scavenge it from their host erythrocyte. Here we report that depletion of cholesterol from the erythrocyte plasma membrane by methyl-β-cyclodextrin (MBCD) has dramatic consequences. The removal of cholesterol results in invasion defects as well as inhibition of parasite development through the intra-erythrocytic cycle. These defects could be rescued by reconstitution with cholesterol and desmosterol but not with epicholesterol. By using live microscopy of fluorescently tagged trophozoite stage parasites, we detected rapid expulsion of the parasites from erythrocyte when exposed to MBCD for just 30 mins. Strikingly, the parasites transition from being intra-erythrocytic to extracellular within 10 seconds and do so without rupturing the erythrocyte membrane. These extruded parasites were still surrounded by the parasitophorous vacuolar membrane (PVM) and remained tethered to the erythrocyte. Electron microscopy revealed that although extracellular parasites retained their PVM, it was heavily compromised. Treatment with antimalarials that disrupt cholesterol homeostasis prior to MBCD exposure prevented the extrusion of trophozoites. These results reveal importance of cholesterol during the intra-erythrocytic development of P. falciparum and the dramatic consequences resulting from tampering with cholesterol content in the infected erythrocyte. These findings suggest dynamic nature of cholesterol within the infected erythrocyte that is critical for parasite survival.

scholarly journals Toxoplasma LIPIN is essential in channeling host lipid fluxes through membrane biogenesis and lipid storage

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sheena Dass ◽  
Serena Shunmugam ◽  
Laurence Berry ◽  
Christophe-Sebastien Arnold ◽  
Nicholas J. Katris ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Phosphatidic Acid ◽  
De Novo ◽  
Lipid Synthesis ◽  
Parasite Development ◽  
Membrane Biogenesis ◽  
Membrane Phospholipids ◽  
Phosphatidic Acid Phosphatase ◽  
Intracellular Parasites ◽  
Parasite Survival

AbstractApicomplexa are obligate intracellular parasites responsible for major human diseases. Their intracellular survival relies on intense lipid synthesis, which fuels membrane biogenesis. Parasite lipids are generated as an essential combination of fatty acids scavenged from the host and de novo synthesized within the parasite apicoplast. The molecular and metabolic mechanisms allowing regulation and channeling of these fatty acid fluxes for intracellular parasite survival are currently unknown. Here, we identify an essential phosphatidic acid phosphatase in Toxoplasma gondii, TgLIPIN, as the central metabolic nexus responsible for controlled lipid synthesis sustaining parasite development. Lipidomics reveal that TgLIPIN controls the synthesis of diacylglycerol and levels of phosphatidic acid that regulates the fine balance of lipids between storage and membrane biogenesis. Using fluxomic approaches, we uncover the first parasite host-scavenged lipidome and show that TgLIPIN prevents parasite death by ‘lipotoxicity’ through effective channeling of host-scavenged fatty acids to storage triacylglycerols and membrane phospholipids.

Altered cholesterol homeostasis in cultured and in vivo models of cystic fibrosis

2007 ◽  
Vol 292 (2) ◽  
pp. L476-L486 ◽  
Author(s):  
Nicole M. White ◽  
Dechen Jiang ◽  
James D. Burgess ◽  
Ilya R. Bederman ◽  
Stephen F. Previs ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Cholesterol Synthesis ◽  
De Novo ◽  
Cholesterol Content ◽  
Cholesterol Homeostasis ◽  
Lysosomal Storage ◽  
In Vivo Models ◽  
Cellular Processes ◽  
Nasal Tissue

Determining how the regulation of cellular processes is impacted in cystic fibrosis (CF) is fundamental to understanding disease pathology and to identifying new therapeutic targets. In this study, unesterified cholesterol accumulation is observed in lung and trachea sections obtained from CF patients compared with non-CF tissues, suggesting an inherent flaw in cholesterol processing. An alternate staining method utilizing a fluorescent cholesterol probe also indicates improper lysosomal storage of cholesterol in CF cells. Excess cholesterol is also manifested by a significant increase in plasma membrane cholesterol content in both cultured CF cells and in nasal tissue excised from cftr −/− mice. Impaired intracellular cholesterol movement is predicted to stimulate cholesterol synthesis, a hypothesis supported by the observation of increased de novo cholesterol synthesis in lung and liver of cftr −/− mice compared with controls. Furthermore, pharmacological inhibition of cholesterol transport is sufficient to cause CF-like elevation in cytokine production in wild-type cells in response to bacterial challenge but has no effect in CF cells. These data demonstrate via multiple methods in both cultured and in vivo models that cellular cholesterol homeostasis is inherently altered in CF. This perturbation of cholesterol homeostasis represents a potentially important process in CF pathogenesis.

scholarly journals Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress and invasion

PLoS Biology ◽  
2021 ◽  
Vol 19 (10) ◽  
pp. e3001408
Author(s):  
Anja C. Schlott ◽  
Ellen Knuepfer ◽  
Judith L. Green ◽  
Philip Hobson ◽  
Aaron J. Borg ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Infected Erythrocyte ◽  
Complex Function ◽  
Pleiotropic Effect ◽  
Substantial Effect ◽  
Parasite Development ◽  
Human Malaria Parasite ◽  
Erythrocyte Invasion ◽  
Erythrocytic Cycle ◽  
Parasite Egress

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of “pseudoschizonts,” which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.

scholarly journals Involvement of δ-aminolaevulinate synthase encoded by the parasite gene in de novo haem synthesis by Plasmodium falciparum

2002 ◽  
Vol 367 (2) ◽  
pp. 321-327 ◽  
Author(s):  
S. VARADHARAJAN ◽  
S. DHANASEKARAN ◽  
Z.Q. BONDAY ◽  
P.N. RANGARAJAN ◽  
G. PADMANABAN
Keyword(s):  
Plasmodium Falciparum ◽  
Enzyme Activity ◽  
Drug Target ◽  
Malaria Parasite ◽  
De Novo ◽  
Parasite Growth ◽  
Pcr Analysis ◽  
Trophozoite Stage ◽  
Parasite Survival ◽  
A Cell

The malaria parasite can synthesize haem de novo. In the present study, the expression of the parasite gene for Δ-aminolaevulinate synthase (PfALAS) has been studied by reverse transcriptase PCR analysis of the mRNA, protein expression using antibodies to the recombinant protein expressed in Escherichia coli and assay of ALAS enzyme activity in Plasmodium falciparum in culture. The gene is expressed through all stages of intra-erythrocytic parasite growth, with a small increase during the trophozoite stage. Antibodies to the erythrocyte ALAS do not cross-react with the parasite enzyme and vice versa. The recombinant enzyme activity is inhibited by ethanolamine and the latter inhibits haem synthesis in P. falciparum and growth in culture. The parasite ALAS is localized in the mitochondrion and its import into mitochondria in a cell-free import assay has been demonstrated. The import is blocked by haemin. On the basis of these results, the following conclusions are arrived at: PfALAS has distinct immunological identity and inhibitor specificity and is therefore a drug target. The malaria parasite synthesizes haem through the mitochondrion/cytosol partnership, and this assumes significance in light of the presence of apicoplasts in the parasite that may be capable of independent haem synthesis. The PfALAS gene is functional and vital for parasite haem synthesis and parasite survival.

scholarly journals Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress, and invasion

2020 ◽  
Author(s):  
Anja C. Schlott ◽  
Ellen Knuepfer ◽  
Judith L. Green ◽  
Philip Hobson ◽  
Aaron J. Borg ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Infected Erythrocyte ◽  
Complex Function ◽  
Pleiotropic Effect ◽  
Substantial Effect ◽  
Parasite Development ◽  
Human Malaria Parasite ◽  
Erythrocyte Invasion ◽  
Erythrocytic Cycle ◽  
Parasite Egress

ABSTRACTWe have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last ten to fifteen hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified sixteen NMT substrates for which myristoylation was significantly reduced by NMT inhibitor treatment, and of these, six proteins were substantially reduced in abundance. In a viability screen, we showed that for four of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least three mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of ‘pseudoschizonts’, which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.

scholarly journals Variability of cholesterol accessibility in human red blood cells measured using a bacterial cholesterol-binding toxin

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Rima S Chakrabarti ◽  
Sally A Ingham ◽  
Julia Kozlitina ◽  
Austin Gay ◽  
Jonathan C Cohen ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Phospholipid Composition ◽  
Cholesterol Content ◽  
Individual Variability ◽  
Cholesterol Homeostasis ◽  
Bacterial Toxin ◽  
Human Red Blood Cells ◽  
Cholesterol Levels ◽  
Cholesterol Binding

Cholesterol partitions into accessible and sequestered pools in cell membranes. Here, we describe a new assay using fluorescently-tagged anthrolysin O, a cholesterol-binding bacterial toxin, to measure accessible cholesterol in human red blood cells (RBCs). Accessible cholesterol levels were stable within individuals, but varied >10 fold among individuals. Significant variation was observed among ethnic groups (Blacks>Hispanics>Whites). Variation in accessibility of RBC cholesterol was unrelated to the cholesterol content of RBCs or plasma, but was associated with the phospholipid composition of the RBC membranes and with plasma triglyceride levels. Pronase treatment of RBCs only modestly altered cholesterol accessibility. Individuals on hemodialysis, who have an unexplained increase in atherosclerotic risk, had significantly higher RBC cholesterol accessibility. Our data indicate that RBC accessible cholesterol is a stable phenotype with significant inter-individual variability. Factors both intrinsic and extrinsic to the RBC contribute to variation in its accessibility. This assay provides a new tool to assess cholesterol homeostasis among tissues in humans.

Wnt5a Promotes Lysosomal Cholesterol Egress and Protects Against Atherosclerosis

2021 ◽  
Author(s):  
Sara Awan ◽  
Magalie Lambert ◽  
Ali Imtiaz ◽  
Fabien Alpy ◽  
Catherine Tomasetto ◽  
...  
Keyword(s):  
Dietary Cholesterol ◽  
Cell Types ◽  
Cholesterol Content ◽  
Cholesterol Homeostasis ◽  
Cholesterol Trafficking ◽  
Atherosclerotic Lesions ◽  
Crucial Component ◽  
Multiple Cell ◽  
Niemann Pick

Background: Impairment of cellular cholesterol trafficking is at the heart of atherosclerotic lesions formation. This involves egress of cholesterol from the lysosomes and two lysosomal proteins, the Niemann-Pick C1 (NPC1) and NPC2 that promotes cholesterol trafficking. However, movement of cholesterol out the lysosome and how disrupted cholesterol trafficking leads to atherosclerosis is unclear. As the Wnt ligand, Wnt5a inhibits the intracellular accumulation of cholesterol in multiple cell types, we tested whether Wnt5a interacts with the lysosomal cholesterol export machinery and studied its role in atherosclerotic lesions formation. Methods: We generated mice deleted for the Wnt5a gene in vascular smooth muscle cells (VSMCs). To establish whether Wnt5a also protects against cholesterol accumulation in human VSMCs, we used a CRISPR/Cas9 guided nuclease approach to generate human VSMCs knockout for Wnt5a. Results: We show that Wnt5a is a crucial component of the lysosomal cholesterol export machinery. By increasing lysosomal acid lipase expression, decreasing metabolic signaling by the mTORC1 kinase, and through binding to NPC1 and NPC2, Wnt5a senses changes in dietary cholesterol supply and promotes lysosomal cholesterol egress to the endoplasmic reticulum (ER). Consequently, loss of Wnt5a decoupled mTORC1 from variations in lysosomal sterol levels, disrupted lysosomal function, decreased cholesterol content in the ER, and promoted atherosclerosis. Conclusions: These results reveal an unexpected function of the Wnt5a pathway as essential for maintaining cholesterol homeostasis in vivo.

scholarly journals Cholesterol homeostasis in the vertebrate retina: Biology and pathobiology

2020 ◽  
pp. jlr.TR120000979 ◽  
Author(s):  
Sriganesh Ramachandra Rao ◽  
Steven J. Fliesler
Keyword(s):  
De Novo ◽  
Current Knowledge ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Neural Retina ◽  
Cholesterol Homeostasis ◽  
Sterol Synthesis ◽  
Topic Area ◽  
Sterol Transport ◽  
Age Related

Cholesterol is a quantitatively and biologically significant constituent of all mammalian cell membrane, including those that comprise the retina. Retinal cholesterol homeostasis entails the interplay between de novo synthesis, uptake, intra-retinal sterol transport, metabolism and efflux. Defects in these complex processes are associated with several congenital and age-related disorders of the visual system. Herein, we provide an overview of the following topics: a) cholesterol synthesis in the neural retina; b) lipoprotein uptake and intraretinal sterol transport in the neural retina and the retinal pigment epithelium (RPE); c) cholesterol efflux from the neural retina and the RPE; and d) biology and pathobiology of defects in sterol synthesis and sterol oxidation in the neural retina and the RPE. We focus, in particular, on studies involving animal models of monogenic disorders pertinent to the above topics, as well as in vitro models using biochemical, metabolic, and omic approaches. We also identify current knowledge gaps as well as opportunities in the field that beg further research in this topic area.

scholarly journals RORγ is a targetable master regulator of cholesterol biosynthesis in a cancer subtype

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Demin Cai ◽  
Junjian Wang ◽  
Bei Gao ◽  
Jin Li ◽  
Feng Wu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Regression ◽  
Cholesterol Biosynthesis ◽  
Cholesterol Content ◽  
Cholesterol Homeostasis ◽  
Synthesis Rate ◽  
Master Regulator ◽  
Cancer Subtype ◽  
Estrogen Receptor Positive ◽  
Chromatin Acetylation

Abstract Tumor subtype-specific metabolic reprogrammers could serve as targets of therapeutic intervention. Here we show that triple-negative breast cancer (TNBC) exhibits a hyper-activated cholesterol-biosynthesis program that is strongly linked to nuclear receptor RORγ, compared to estrogen receptor-positive breast cancer. Genetic and pharmacological inhibition of RORγ reduces tumor cholesterol content and synthesis rate while preserving host cholesterol homeostasis. We demonstrate that RORγ functions as an essential activator of the entire cholesterol-biosynthesis program, dominating SREBP2 via its binding to cholesterol-biosynthesis genes and its facilitation of the recruitment of SREBP2. RORγ inhibition disrupts its association with SREBP2 and reduces chromatin acetylation at cholesterol-biosynthesis gene loci. RORγ antagonists cause tumor regression in patient-derived xenografts and immune-intact models. Their combination with cholesterol-lowering statins elicits superior anti-tumor synergy selectively in TNBC. Together, our study uncovers a master regulator of the cholesterol-biosynthesis program and an attractive target for TNBC.

scholarly journals Calcium transport of Plasmodium chabaudi-infected erythrocytes.

1982 ◽  
Vol 93 (3) ◽  
pp. 680-684 ◽  
Author(s):  
K Tanabe ◽  
R B Mikkelsen ◽  
D F Wallach
Keyword(s):  
Infected Erythrocyte ◽  
Calcium Content ◽  
Transport Processes ◽  
Metabolic Inhibitors ◽  
Parasite Development ◽  
Ionophore A23187 ◽  
Plasmodium Chabaudi ◽  
Carbonyl Cyanide ◽  
Flux Measurements ◽  
Infected Cells

The calcium content and transport processes of Plasmodium chabaudi-infected rat erythrocytes were analyzed by atomic absorption spectroscopy and 45Ca2+ flux measurements. Infected erythrocytes, after fractionation on metrizamide gradients according to stage of parasite development, exhibited progressively increasing levels of Ca2+ with schizont and gametocytes containing 10- to 20-fold greater calcium levels than normal cells (0.54 +/- 0.25 nmol/10(8) cells). 45Ca2+ flux experiments showed both increased influx and decreased efflux in infected erythrocytes. Tris/NH4Cl lysis of normal erythrocytes preloaded with 45Ca2+ with the Ca2+ ionophore A23187 released less than 90% of cell calcium after incubation in ethyleneglycol bis(aminoethylether) N,N'-tetraacetic acid containing buffer, whereas lysis of the infected erythrocyte membrane resulted in release of 10-20% cell Ca2+, with the remaining portion associated with the isolated parasite fraction. This information together with the effects of various metabolic inhibitors indicates the presence of a parasite Ca2+ compartment in P. chabaudi-infected erythrocytes. Dicyclohexylcarbodiimide (DCCD) an inhibitor of proton ATPases of chloroplasts, bacteria, yeast, and mitochondria, and the proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), inhibited Ca2+ influx and stimulated efflux from infected cells. These results combined with evidence for a DCCD- and CCCP-sensitive membrane potential in P. chabaudi-infected cells (Mikkelsen et al., accompanying manuscript) suggest that Ca2+ transport of intraerythrocytic parasites is coupled to a proton-motive force across the Plasmodia plasma membrane.

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