scholarly journals Biosynthesis, export and processing of a 45 kDa protein detected in membrane clefts of erythrocytes infected with Plasmodium falciparum

1994 ◽  
Vol 302 (2) ◽  
pp. 487-496 ◽  
Author(s):  
A Das ◽  
H G Elmendorf ◽  
W I Li ◽  
K Haldar
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Brefeldin A ◽  
Soluble Form ◽  
Parasite Development ◽  
Parasite Protein ◽  
Parasite Plasmodium Falciparum ◽  
Membrane Bound

During its asexual life cycle, the human malaria parasite Plasmodium falciparum exports numerous proteins beyond its surface to its host erythrocyte. We have studied the biosynthesis, processing and export of a 45 kDa parasite protein resident in membrane clefts in the erythrocyte cytoplasm. Our results indicate that this cleft protein is made as a single tightly membrane-bound 45 kDa polypeptide in ring- and trophozoite-infected erythrocytes (0-36 h in the life cycle). Using ring/trophozoite parasites released from erythrocytes, the 45 kDa protein is shown to be efficiently transported to the cell surface. This export is specifically blocked by the drug brefeldin A, and at 15 and 20 degrees C. These results indicate that transport blocks seen in the Golgi of mammalian cells are conserved in P. falciparum. Further, the newly synthesized 45 kDa protein passes through parasite Golgi compartments before its export to clefts in the erythrocyte. In mid-to-late-ring-infected erythrocytes, a fraction of the newly synthesized 45 kDa protein is processed to a second membrane-bound phosphorylated 47 kDa protein. The t1/2 of this processing step is about 4 h, suggesting that it occurs subsequent to protein export from the parasite. Evidence is presented that, in later trophozoite stages (24-36 h), the exported 45 and 47 kDa proteins are partially converted into soluble molecules in the intraerythrocytic space. Taken together, the results indicate that the lower eukaryote P. falciparum modulates a classical secretory pathway to support membrane export beyond its plasma membrane to clefts in the erythrocyte. Subsequent to export, phosphorylation and/or conversion into a soluble form may regulate the interactions of the 45 kDa protein with the clefts during parasite development.

scholarly journals Plasmodium falciparum-infected erythrocytes utilize a synthetic truncated ceramide precursor for synthesis and secretion of truncated sphingomyelin

1995 ◽  
Vol 308 (1) ◽  
pp. 335-341 ◽  
Author(s):  
I Ansorge ◽  
D Jeckel ◽  
F Wieland ◽  
K Lingelbach
Keyword(s):  
Plasmodium Falciparum ◽  
Mammalian Cells ◽  
Membrane Fraction ◽  
Infected Erythrocyte ◽  
Brefeldin A ◽  
Parasite Development ◽  
Phospholipid Content ◽  
Unicellular Eukaryote ◽  
Fungal Metabolite ◽  
Sphingomyelin Synthase

Plasmodium falciparum is an intracellular parasite of human erythrocytes. Parasite development is accompanied by an increase of the phospholipid content of the infected erythrocyte, but it results in a selective decrease of sphingomyelin. We have studied sphingomyelin biosynthesis in infected erythrocytes using as substrate a synthetic radiolabelled ceramide precursor, truncated in both hydrophobic chains. Lysates of infected, unlike those of non-infected, erythrocytes contained sphingomyelin synthase activity, which therefore is of parasite origin. The enzyme activity was associated with a membrane fraction. In contrast to mammalian cells, the parasite did not synthesize detectable levels of glycosphingolipids. In intact infected erythrocytes the ceramide precursor was converted into a correspondingly truncated soluble sphingomyelin which was released into the medium at 37 degrees C. Release of truncated sphingomyelin was inhibited by low temperature (15 degrees C) but not by the fungal metabolite brefeldin A which, however, arrests protein export from the parasite. While membranes of mammalian cells, including the plasma membrane of non-infected erythrocytes, are impermeable to truncated sphingomyelin, the membrane of infected erythrocytes allowed passage of the molecule in both directions. The results obtained with the unicellular eukaryote used here as an experimental model are discussed in comparison with sphingomyelin synthesis and transport in mammalian cells.

scholarly journals Variable oxygen environments and DNMT2 determine the DNA cytosine epigenetic landscape of Plasmodium falciparum

2021 ◽  
Author(s):  
Artur Scherf ◽  
Elie Hammam ◽  
Samia Miled ◽  
Frederic Bonhomme ◽  
Benoit Arcangioli ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Mammalian Cells ◽  
Dna Methyltransferase ◽  
Cytosine Methylation ◽  
Parasite Development ◽  
Epigenetic Landscape ◽  
Human Malaria Parasite ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum ◽  
Oxidized Products

DNA cytosine methylation and its oxidized products are important epigenetic modifications in mammalian cells. Although 5-methylcytosine (5mC) was detected in the human malaria parasite Plasmodium falciparum, the presence of oxidized 5mC forms remain to be characterized.Here we establish a protocol to explore nuclease-based DNA digestion for the extremely AT-rich genome of P. falciparum (>80% A+T) for quantitative LC-MS/MS analysis of 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). We demonstrate the presence of 5hmC, 5fC and 5caC cytosine modifications in a DNMT2-only organism and observe striking ratio changes between 5mC and 5hmC during the 48-hour blood stage parasite development. Parasite-infected red blood cells cultured in different physiological oxygen concentrations revealed a shift in the cytosine modifications distribution towards the oxidized 5hmC and 5caC forms. In the absence of the canonical C5-DNA methyltransferase (DNMT1 and DNMT3A/B) in P. falciparum, we show that all cytosine modifications depend on the presence of DNMT2. We conclude that DNMT2 and oxygen levels are critical determinants that shape the dynamic cytosine epigenetic landscape in this human pathogen.

Actomyosin motor in the merozoite of the malaria parasite, Plasmodium falciparum: implications for red cell invasion

1998 ◽  
Vol 111 (13) ◽  
pp. 1831-1839 ◽  
Author(s):  
J.C. Pinder ◽  
R.E. Fowler ◽  
A.R. Dluzewski ◽  
L.H. Bannister ◽  
F.M. Lavin ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Plasma Membrane ◽  
Toxoplasma Gondii ◽  
Malaria Parasite ◽  
Cellular Protein ◽  
Red Cell ◽  
Parasite Protein ◽  
Ionic Detergent ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum

The genome of the malaria parasite, Plasmodium falciparum, contains a myosin gene sequence, which bears a close homology to one of the myosin genes found in another apicomplexan parasite, Toxoplasma gondii. A polyclonal antibody was generated against an expressed polypeptide of molecular mass 27,000, based on part of the deduced sequence of this myosin. The antibody reacted with the cognate antigen and with a component of the total parasite protein on immunoblots, but not with vertebrate striated or smooth muscle myosins. It did, however, recognise two components in the cellular protein of Toxoplasma gondii. The antibody was used to investigate stage-specificity of expression of the myosin (here designated Pf-myo1) in P. falciparum. The results showed that the protein is synthesised in mature schizonts and is present in merozoites, but vanishes after the parasite enters the red cell. Pf-myo1 was found to be largely, though not entirely, associated with the particulate parasite cell fraction and is thus presumably mainly membrane bound. It was not solubilised by media that would be expected to dissociate actomyosin or myosin filaments, or by non-ionic detergent. Immunofluorescence revealed that in the merozoite and mature schizont Pf-myo1 is predominantly located around the periphery of the cell. Immuno-gold electron microscopy also showed the presence of the myosin around almost the entire parasite periphery, and especially in the region surrounding the apical prominence. Labelling was concentrated under the plasma membrane but was not seen in the apical prominence itself. This suggests that Pf-myo1 is associated with the plasma membrane or with the outer membrane of the subplasmalemmal cisterna, which forms a lining to the plasma membrane, with a gap at the apical prominence. The results lead to a conjectural model of the invasion mechanism.

scholarly journals Brefeldin A inhibits transport of the glycophorin-binding protein from Plasmodium falciparum into the host erythrocyte

1994 ◽  
Vol 300 (3) ◽  
pp. 821-826 ◽  
Author(s):  
J Benting ◽  
D Mattei ◽  
K Lingelbach
Keyword(s):  
Plasmodium Falciparum ◽  
Golgi Apparatus ◽  
Host Cell ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Binding Protein ◽  
Brefeldin A ◽  
Cell Cytoplasm ◽  
Parasite Cell ◽  
Host Cell Cytoplasm

Plasmodium falciparum, a protozoan parasite of the human erythrocyte, causes the most severe form of malaria. During its intraerythrocytic development, the parasite synthesizes proteins which are exported into the host cell. The compartments involved in the secretory pathway of P. falciparum are still poorly characterized. A Golgi apparatus has not been identified, owing to the lack of specific protein markers and Golgi-specific post-translational modifications in the parasite. The fungal metabolite brefeldin A (BFA) is known to inhibit protein secretion in higher eukaryotes by disrupting the integrity of the Golgi apparatus. We have used the parasite-encoded glycophorin-binding protein (GBP), a soluble protein found in the host cell cytoplasm, as a marker to investigate the effects of BFA on protein secretion in the intracellular parasite. In the presence of BFA, GBP was not transported into the erythrocyte, but remained inside the parasite cell. The effect caused by BFA was reversible, and the protein could be chased into the host cell cytoplasm within 30 min. Transport of GBP from the BFA-sensitive site into the host cell did not require protein synthesis. Similar observations were made when infected erythrocytes were incubated at 15 degrees C. Incubation at 20 degrees C resulted in a reduction rather than a complete block of protein export. The relevance of our findings to the identification of compartments involved in protein secretion from the parasite cell is discussed.

scholarly journals A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

1995 ◽  
Vol 6 (2) ◽  
pp. 135-150 ◽  
Author(s):  
N T Ktistakis ◽  
C Y Kao ◽  
R H Wang ◽  
M G Roth
Keyword(s):  
Chinese Hamster Ovary ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Secretory Activity ◽  
Specific Marker ◽  
Ovary Cells ◽  
Fluorescent Lipid

The use of reporter proteins to study the regulation of secretion has often been complicated by posttranslational processing events that influence the secretion of certain proteins, but are not part of the cellular mechanisms that specifically regulate secretion. This has been a particular limitation for the isolation of mammalian secretion mutants, which has typically been a slow process. To provide a reporter of secretory activity independent of protein processing events, cells were labeled with the fluorescent lipid analogue C5-DMB-ceramide (ceramide coupled to the fluorophore boron dipyrromethene difluoride) and its secretion was followed by fluorescence microscopy and fluorescence-activated cell sorting. Brefeldin A, which severely inhibits secretion in Chinese hamster ovary cells, blocked secretion of C5-DMB-ceramide. At high temperature, export of C5-DMB-ceramide was inhibited in HRP-1 cells, which have a conditional defect in secretion. Using C5-DMB-ceramide as a reporter of secretory activity, several different pulse-chase protocols were designed that selected mutant Chinese hamster ovary cells that were resistant to the drug brefeldin A and others that were defective in the transport of glycoproteins to the cell surface. Mutant cells of either type were identified in a mutagenized population at a frequency of 10(-6). Thus, the fluorescent lipid C5-DMB-ceramide can be used as a specific marker of secretory activity, providing an efficient, general approach for isolating mammalian cells with defects in the secretory pathway.

scholarly journals The Exported Chaperone PfHsp70x Is Dispensable for the Plasmodium falciparum Intraerythrocytic Life Cycle

mSphere ◽  
2017 ◽  
Vol 2 (5) ◽  
Author(s):  
David W. Cobb ◽  
Anat Florentin ◽  
Manuel A. Fierro ◽  
Michelle Krakowiak ◽  
Julie M. Moore ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Life Cycle ◽  
Host Cell ◽  
Protein Trafficking ◽  
Human Disease ◽  
Clinical Manifestations ◽  
Parasite Protein ◽  
Content Type ◽  
Parasite Survival ◽  
Outstanding Question

ABSTRACT Half of the world’s population lives at risk for malaria. The intraerythrocytic life cycle of Plasmodium spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite’s ability to survive within the erythrocyte is needed to combat the deadliest agent of malaria, P. falciparum. An outstanding question in the field is how P. falciparum undertakes the essential process of trafficking its proteins within the host cell. In most organisms, chaperones such as Hsp70 are employed in protein trafficking. Of the Plasmodium species causing human disease, the chaperone PfHsp70x is unique to P. falciparum, and it is the only parasite protein of its kind exported to the host (S. Külzer et al., Cell Microbiol 14:1784–1795, 2012). This has placed PfHsp70x as an ideal target to inhibit protein trafficking and kill the parasite. However, we show that PfHsp70x is not required for export of parasite effectors and it is not essential for parasite survival inside the RBC. Export of parasite proteins into the host erythrocyte is essential for survival of Plasmodium falciparum during its asexual life cycle. While several studies described key factors within the parasite that are involved in protein export, the mechanisms employed to traffic exported proteins within the host cell are currently unknown. Members of the Hsp70 family of chaperones, together with their Hsp40 cochaperones, facilitate protein trafficking in other organisms, and are thus likely used by P. falciparum in the trafficking of its exported proteins. A large group of Hsp40 proteins is encoded by the parasite and exported to the host cell, but only one Hsp70, P. falciparum Hsp70x (PfHsp70x), is exported with them. PfHsp70x is absent in most Plasmodium species and is found only in P. falciparum and closely related species that infect apes. Herein, we have utilized clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing in P. falciparum to investigate the essentiality of PfHsp70x. We show that parasitic growth was unaffected by knockdown of PfHsp70x using both the dihydrofolate reductase (DHFR)-based destabilization domain and the glmS ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of P. falciparum to proceed through its intraerythrocytic life cycle. The effect of PfHsp70x knockdown/knockout on the export of proteins to the host red blood cell (RBC), including the critical virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), was tested, and we found that this process was unaffected. These data show that although PfHsp70x is the sole exported Hsp70, it is not essential for the asexual development of P. falciparum. IMPORTANCE Half of the world’s population lives at risk for malaria. The intraerythrocytic life cycle of Plasmodium spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite’s ability to survive within the erythrocyte is needed to combat the deadliest agent of malaria, P. falciparum. An outstanding question in the field is how P. falciparum undertakes the essential process of trafficking its proteins within the host cell. In most organisms, chaperones such as Hsp70 are employed in protein trafficking. Of the Plasmodium species causing human disease, the chaperone PfHsp70x is unique to P. falciparum, and it is the only parasite protein of its kind exported to the host (S. Külzer et al., Cell Microbiol 14:1784–1795, 2012). This has placed PfHsp70x as an ideal target to inhibit protein trafficking and kill the parasite. However, we show that PfHsp70x is not required for export of parasite effectors and it is not essential for parasite survival inside the RBC.

scholarly journals Exploring the lower thermal limits for development of the human malaria parasite, Plasmodium falciparum

2019 ◽  
Vol 15 (6) ◽  
pp. 20190275 ◽  
Author(s):  
Jessica L. Waite ◽  
Eunho Suh ◽  
Penelope A. Lynch ◽  
Matthew B. Thomas
Keyword(s):  
Plasmodium Falciparum ◽  
Future Climate Change ◽  
Parasite Development ◽  
Temperature Dependent ◽  
Human Malaria Parasite ◽  
Thermal Limits ◽  
Degree Day ◽  
Extrinsic Incubation Period ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum

The rate of malaria transmission is strongly determined by parasite development time in the mosquito, known as the extrinsic incubation period (EIP), since the quicker parasites develop, the greater the chance that the vector will survive long enough for the parasite to complete development and be transmitted. EIP is known to be temperature-dependent but this relationship is surprisingly poorly characterized. There is a single degree-day model for EIP of Plasmodium falciparum that derives from a limited number of poorly controlled studies conducted almost a century ago. Here, we show that the established degree-day model greatly underestimates the rate of development of P. falciparum in both Anopheles stephensi and An. gambiae mosquitoes at temperatures in the range of 17–20°C. We also show that realistic daily temperature fluctuation further speeds parasite development. These novel results challenge one of the longest standing models in malaria biology and have potentially important implications for understanding the impacts of future climate change.

scholarly journals Scanning electron microscope-analysis of the protrusions (knobs) present on the surface of Plasmodium falciparum-infected erythrocytes.

1983 ◽  
Vol 97 (3) ◽  
pp. 795-802 ◽  
Author(s):  
J Gruenberg ◽  
D R Allred ◽  
I W Sherman
Keyword(s):  
Plasmodium Falciparum ◽  
Cell Surface ◽  
Parasite Development ◽  
Red Cell ◽  
Schizont Stage ◽  
Dynamic Changes ◽  
Trophozoite Stage ◽  
Parasite Plasmodium Falciparum ◽  
Surface Patterns ◽  
Scanning Electron

The nature of the surface deformations of erythrocytes infected with the human malaria parasite Plasmodium falciparum was analyzed using scanning electron microscopy at two stages of the 48-h parasite maturation cycle. Infected cells bearing trophozoite-stage parasites (24-36 h) had small protrusions (knobs), with diameters varying from 160 to 110 nm, and a density ranging from 10 to 35 knobs X micron-2. When parasites were fully mature (schizont stage, 40-44 h), knob size decreased (100-70 nm), whereas density increased (45-70 knobs X micron-2). Size and density of the knobs varied inversely, suggesting that knob production (a) occurred throughout intraerythrocytic parasite development from trophozoite to schizont and (b) was related to dynamic changes of the erythrocyte membrane. Variation in the distribution of the knobs over the red cell surface was observed during parasite maturation. At the early trophozoite stage of parasite development, knobs appeared to be formed in particular domains of the cell surface. As the density of knobs increased and they covered the entire cell surface, their lateral distribution was dispersive (more-than-random); this was particularly evident at the schizont stage. Regional surface patterns of knobs (rows, circles) were seen throughout parasite development. The nature of the dynamic changes that occurred at the red cell surface during knob formation, as well as the nonrandom distribution of knobs, suggested that the red cell cytoskeleton may have played a key role in knob formation and patterning.

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