scholarly journals Evaluation of pH during cytostomal endocytosis and vacuolar catabolism of haemoglobin in Plasmodium falciparum

2007 ◽  
Vol 407 (3) ◽  
pp. 343-354 ◽  
Author(s):  
Nectarios Klonis ◽  
Olivia Tan ◽  
Katherine Jackson ◽  
Daniel Goldberg ◽  
Michael Klemba ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Fluorescent Protein ◽  
Ph Value ◽  
Ph Dependence ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum ◽  
Intracellular Compartments ◽  
Green Fluorescent ◽  
Haem Detoxification ◽  
Endocytic Vesicles

The DV (digestive vacuole) of the malaria parasite, Plasmodium falciparum, is the site of Hb (haemoglobin) digestion and haem detoxification and, as a consequence, the site of action of CQ (chloroquine) and related antimalarials. However, the precise pH of the DV and the endocytic vesicles that feed it has proved difficult to ascertain. We have developed new methods using EGFP [enhanced GFP (green fluorescent protein)] to measure the pH of intracellular compartments. We have generated a series of transfectants in CQ-sensitive and -resistant parasite strains expressing GFP chimaeras of the DV haemoglobinase, plasmepsin II. Using a quantitative flow cytometric assay, the DV pH was determined to be 5.4–5.5. No differences were detected between CQ-sensitive and -resistant strains. We have also developed a method that relies on the pH dependence of GFP photobleaching kinetics to estimate the pH of the DV compartment. This method gives a pH estimate consistent with the intensity-based measurement. Accumulation of the pH-sensitive probe, LysoSensor Blue, in the DV confirms the acidity of this compartment and shows that the cytostomal vesicles are not measurably acidic, indicating that they are unlikely to be the site of Hb digestion or the site of CQ accumulation. We show that a GFP probe located outside the DV reports a pH value close to neutral. The transfectants and methods that we have developed represent useful tools for investigating the pH of GFP-containing compartments and should be of general use in other systems.

scholarly journals Trafficking of plasmepsin II to the food vacuole of the malaria parasite Plasmodium falciparum

2004 ◽  
Vol 164 (1) ◽  
pp. 47-56 ◽  
Author(s):  
Michael Klemba ◽  
Wandy Beatty ◽  
Ilya Gluzman ◽  
Daniel E. Goldberg
Keyword(s):  
Plasmodium Falciparum ◽  
Fluorescent Protein ◽  
Green Fluorescent Protein Fluorescence ◽  
Food Vacuole ◽  
Protein Fluorescence ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum ◽  
Plasmepsin Ii ◽  
Green Fluorescent ◽  
Secretory System

fA amily of aspartic proteases, the plasmepsins (PMs), plays a key role in the degradation of hemoglobin in the Plasmodium falciparum food vacuole. To study the trafficking of proPM II, we have modified the chromosomal PM II gene in P. falciparum to encode a proPM II–GFP chimera. By taking advantage of green fluorescent protein fluorescence in live parasites, the ultrastructural resolution of immunoelectron microscopy, and inhibitors of trafficking and PM maturation, we have investigated the biosynthetic path leading to mature PM II in the food vacuole. Our data support a model whereby proPM II is transported through the secretory system to cytostomal vacuoles and then is carried along with its substrate hemoglobin to the food vacuole where it is proteolytically processed to mature PM II.

scholarly journals Trafficking of the major virulence factor to the surface of transfected P falciparum–infected erythrocytes

Blood ◽  
2005 ◽  
Vol 105 (10) ◽  
pp. 4078-4087 ◽  
Author(s):  
Ellen Knuepfer ◽  
Melanie Rug ◽  
Nectarios Klonis ◽  
Leann Tilley ◽  
Alan F. Cowman
Keyword(s):  
Fluorescent Protein ◽  
Parasitophorous Vacuole ◽  
Human Red Blood Cells ◽  
Rbc Membrane ◽  
Parasite Plasmodium ◽  
Virulence Protein ◽  
Parasite Plasmodium Falciparum ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Falciparum Erythrocyte Membrane Protein

Abstract After invading human red blood cells (RBCs) the malaria parasite Plasmodium falciparum remodels the host cell by trafficking proteins to the RBC compartment. The virulence protein P falciparum erythrocyte membrane protein 1 (PfEMP1) is responsible for cytoadherence of infected cells to host endothelial receptors. This protein is exported across the parasite plasma membrane and parasitophorous vacuole membrane and inserted into the RBC membrane. We have used green fluorescent protein chimeras and fluorescence photobleaching experiments to follow PfEMP1 export through the infected RBC. Our data show that a knob-associated histidine-rich protein (KAHRP) N-terminal protein export element appended to the PfEMP1 transmembrane and C-terminal domains was sufficient for efficient trafficking of protein domains to the outside of the P falciparum–infected RBC. The physical state of the exported proteins suggests trafficking as a complex rather than in vesicles and supports the hypothesis that endogenous PfEMP1 is trafficked in a similar manner. This study identifies the sequences required for expression of proteins to the outside of the P falciparum–infected RBC membrane.

Roles of 1-Cys peroxiredoxin in haem detoxification in the human malaria parasite Plasmodium falciparum

FEBS Journal ◽  
2005 ◽  
Vol 272 (7) ◽  
pp. 1784-1791 ◽  
Author(s):  
Shin-ichiro Kawazu ◽  
Nozomu Ikenoue ◽  
Hitoshi Takemae ◽  
Kanako Komaki-Yasuda ◽  
Shigeyuki Kano
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Parasite ◽  
Human Malaria Parasite ◽  
Human Malaria ◽  
Parasite Plasmodium ◽  
Parasite Plasmodium Falciparum ◽  
Haem Detoxification

scholarly journals Visualization of retrovirus uptake and delivery into acidic endosomes

2011 ◽  
Vol 434 (3) ◽  
pp. 559-569 ◽  
Author(s):  
Kosuke Miyauchi ◽  
Mariana Marin ◽  
Gregory B. Melikyan
Keyword(s):  
Fluorescent Protein ◽  
Permissive Cell ◽  
Independent Manner ◽  
Protein Variant ◽  
Time Resolved ◽  
Enveloped Viruses ◽  
Intracellular Compartments ◽  
Green Fluorescent ◽  
Endocytic Vesicles ◽  
Acidic Compartments

Diverse enveloped viruses enter cells by endocytosis and fusion with intracellular compartments. Recent evidence suggests that HIV also infects permissive cell lines by fusing with endosomes in a pH-independent manner. This finding highlights the importance of time-resolved monitoring of viral uptake. In the present study, we designed an imaging-based assay to measure endocytosis in real-time through probing the virus' accessibility to external solutions. Exposure of viruses bearing a pH-sensitive GFP (green fluorescent protein) variant on their surface to solutions of different acidity altered the fluorescence of surface-accessible particles, but not internalized viruses. By sequentially applying acidic and alkaline buffers with or without ammonium chloride, we were able to quantify the fractions of internalized and non-internalized virions, as well as the fraction of detached particles, over time. The exact time of single-virus internalization was assessed from the point when a particle ceased to respond to a perfusion with alternating acidic and alkaline buffers. We found that, surprisingly, HIV pseudoparticles entered acidic compartments shortly after internalization. These results suggest that the virus might be sorted to a quickly maturing pool of endocytic vesicles and thus be trafficked to fusion-permissive sites near the cell nucleus.

Sign in / Sign up

Export Citation Format

Share Document