scholarly journals Parasitophorous vacuole poration precedes its rupture and rapid host erythrocyte cytoskeleton collapse inPlasmodium falciparumegress

2017 ◽  
Vol 114 (13) ◽  
pp. 3439-3444 ◽  
Author(s):  
Victoria L. Hale ◽  
Jean M. Watermeyer ◽  
Fiona Hackett ◽  
Gema Vizcay-Barrena ◽  
Christiaan van Ooij ◽  
...  
Keyword(s):  
Time Window ◽  
Electron Tomography ◽  
Shape Change ◽  
Parasitophorous Vacuole ◽  
Current Model ◽  
Initial Step ◽  
Cysteine Proteases ◽  
Erythrocyte Membranes ◽  
Membrane Perforation ◽  
Erythrocyte Cytoskeleton

In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential rupture of the vacuole and erythrocyte membranes. The current model is that PKG, a malarial cGMP-dependent protein kinase, triggers egress, activating malarial proteases and other effectors. Using selective inhibitors of either PKG or cysteine proteases to separately inhibit the sequential steps in membrane perforation, combined with video microscopy, electron tomography, electron energy loss spectroscopy, and soft X-ray tomography of mature intracellularPlasmodium falciparumparasites, we resolve intermediate steps in egress. We show that the parasitophorous vacuole membrane (PVM) is permeabilized 10–30 min before its PKG-triggered breakdown into multilayered vesicles. Just before PVM breakdown, the host red cell undergoes an abrupt, dramatic shape change due to the sudden breakdown of the erythrocyte cytoskeleton, before permeabilization and eventual rupture of the erythrocyte membrane to release the parasites. In contrast to the previous view of PKG-triggered initiation of egress and a gradual dismantling of the host erythrocyte cytoskeleton over the course of schizont development, our findings identify an initial step in egress and show that host cell cytoskeleton breakdown is restricted to a narrow time window within the final stages of egress.

scholarly journals Cellular electron tomography of the apical complex in the apicomplexan parasite Eimeria tenella shows a highly organised gateway for regulated secretion.

2021 ◽  
Author(s):  
Alana Burrell ◽  
Virginia Marugan-Hernandez ◽  
Flavia Moreira-Leite ◽  
David J P Ferguson ◽  
Fiona M Tomley ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Electron Tomography ◽  
Parasitophorous Vacuole ◽  
Three Dimensional ◽  
Eimeria Tenella ◽  
Structural Explanation ◽  
Apicomplexan Parasites ◽  
Host Cytoplasm ◽  
Fibre Number ◽  
Apical Complex

The apical complex of apicomplexan parasites is essential for host cell invasion and intracellular survival and as the site of regulated exocytosis from specialised secretory organelles called rhoptries and micronemes. Despite its importance, there is little data on the three-dimensional organisation and quantification of these organelles within the apical complex or how they are trafficked to this specialised region of plasma membrane for exocytosis. In coccidian apicomplexans there is an additional tubulin-containing hollow barrel structure, the conoid, which provides a structural gateway for this specialised secretion. Using a combination of cellular electron tomography and serial block face-scanning electron microscopy (SBF-SEM) we have reconstructed the entire apical end of Eimeria tenella sporozoites. We discovered that conoid fibre number varied, but there was a fixed spacing between fibres, leading to conoids of different sizes. Associated apical structures varied in size to accommodate a larger or smaller conoid diameter. However, the number of subpellicular microtubules on the apical polar ring surrounding the conoid did not vary, suggesting a control of apical complex size. We quantified the number and location of rhoptries and micronemes within cells and show a highly organised gateway for trafficking and docking of rhoptries, micronemes and vesicles within the conoid around a set of intra-conoidal microtubules. Finally, we provide ultrastructural evidence for fusion of rhoptries directly through the parasite plasma membrane early in infection and the presence of a pore in the parasitophorous vacuole membrane, providing a structural explanation for how rhoptry proteins (ROPs) may be trafficked between the parasite and the host cytoplasm

Dibucaine-Induced Modification of Sodium Transport in Toad Skin and of Model Membrane Structures

2001 ◽  
Vol 56 (7-8) ◽  
pp. 614-622 ◽  
Author(s):  
Mario Suwalsky ◽  
Carlos Schneider ◽  
Fernando Villena ◽  
Beryl Norris ◽  
Hernan Cárdenas ◽  
...  
Keyword(s):  
Human Erythrocyte ◽  
Shape Change ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Erythrocyte Membranes ◽  
Membrane Structures ◽  
Toad Skin ◽  
Large Unilamellar Vesicles ◽  
Human Erythrocyte Membranes ◽  
Membrane Models

The interaction of the local anesthetic dibucaine with the isolated toad skin and membrane models is described. The latter consisted of human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), large unilamellar vesicles (LUV) of dimyristoylphosphati-dylcholine (DMPC) and phospholipid multilayers built-up of DMPC and dimyristoylphos-phatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Results indicate a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of dibucaine in toad skin, which may be interpreted as reflecting inhibition of the active transport of ions. This finding might be explained on the basis of the results ob­ tained from fluorescence spectroscopy and X-ray diffraction studies on membrane models. In fact, dibucaine induced structural perturbations in IUM, DMPC LUV and phospholipid multilayers. Scanning electron microscopy revealed that dibucaine induced erythrocyte stomatocytosis. According to the bilayer couple hypothesis an echinocytic type of shape change would have been expected given the preferential interaction of dibucaine with DMPC. Although it is still premature to define the molecular mechanism of action of dibucaine, the experimental results confirm the important role played by the phospholipid bilayers in the association of the anesthetic with cell membranes

scholarly journals Inhibition of Malaria Parasite Development by a Cyclic Peptide That Targets the Vital Parasite Protein SERA5

2008 ◽  
Vol 76 (9) ◽  
pp. 4332-4344 ◽  
Author(s):  
W. Douglas Fairlie ◽  
Tim P. Spurck ◽  
Joanne E. McCoubrie ◽  
Paul R. Gilson ◽  
Susanne K. Miller ◽  
...  
Keyword(s):  
Late Stage ◽  
Biochemical Characterization ◽  
Cyclic Peptide ◽  
Parasitophorous Vacuole ◽  
Cysteine Proteases ◽  
Catalytic Triad ◽  
Parasite Development ◽  
Gene Encoding ◽  
Parasite Protein ◽  
Different Strains

ABSTRACT The serine repeat antigen (SERA) proteins of the malaria parasites Plasmodium spp. contain a putative enzyme domain similar to that of papain family cysteine proteases. In Plasmodium falciparum parasites, more than half of the SERA family proteins, including the most abundantly expressed form, SERA5, have a cysteine-to-serine substitution within the putative catalytic triad of the active site. Although SERA5 is required for blood-stage parasite survival, the occurrence of a noncanonical catalytic triad casts doubt on the importance of the enzyme domain in this function. We used phage display to identify a small (14-residue) disulfide-bonded cyclic peptide (SBP1) that targets the enzyme domain of SERA5. Biochemical characterization of the interaction shows that it is dependent on the conformation of both the peptide and protein. Addition of this peptide to parasite cultures compromised development of late-stage parasites compared to that of control parasites or those incubated with equivalent amounts of the carboxymethylated peptide. This effect was similar in two different strains of P. falciparum as well as in a transgenic strain where the gene encoding the related serine-type parasitophorous vacuole protein SERA4 was deleted. In compromised parasites, the SBP1 peptide crosses both the erythrocyte and parasitophorous vacuole membranes and accumulates within the parasitophorous vacuole. In addition, both SBP1 and SERA5 were identified in the parasite cytosol, indicating that the plasma membrane of the parasite was compromised as a result of SBP1 treatment. These data implicate an important role for SERA5 in the regulation of the intraerythrocytic development of late-stage parasites and as a target for drug development.

scholarly journals Cathepsin Cs Are Key for the Intracellular Survival of the Protozoan Parasite, Toxoplasma gondii

2006 ◽  
Vol 282 (7) ◽  
pp. 4994-5003 ◽  
Author(s):  
Xuchu Que ◽  
Juan C. Engel ◽  
David Ferguson ◽  
Annette Wunderlich ◽  
Stanislas Tomavo ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Cathepsin B ◽  
Parasitophorous Vacuole ◽  
Cathepsin L ◽  
Protozoan Parasite ◽  
Cysteine Proteases ◽  
Intracellular Survival ◽  
Chemotherapeutic Agents ◽  
Cathepsin C

Cysteine proteases play key roles in apicomplexan invasion, organellar biogenesis, and intracellular survival. We have now characterized five genes encoding papain family cathepsins from Toxoplasma gondii, including three cathepsin Cs, one cathepsin B, and one cathepsin L. Unlike endopeptidases cathepsin B and L, T. gondii cathepsin Cs are exopeptidases and remove dipeptides from unblocked N-terminal substrates of proteins or peptides. TgCPC1 was the most highly expressed cathepsin mRNA in tachyzoites (by real-time PCR), but three cathepsins, TgCPC1, TgCPC2, and TgCPB, were undetectable in in vivo bradyzoites. The specific cathepsin C inhibitor, Gly-Phe-dimethylketone, selectively inhibited the TgCPCs activity, reducing parasite intracellular growth and proliferation. The targeted disruption of TgCPC1 does not affect the invasion and growth of tachyzoites as TgCPC2 is then up-regulated and may substitute for TgCPC1. TgCPC1 and TgCPC2 localize to constitutive secretory vesicles of tachyzoites, the dense granules. T. gondii cathepsin Cs are required for peptide degradation in the parasitophorous vacuole as the degradation of the marker protein, Escherichia coli β-lactamase, secreted into the parasitophorous vacuole of transgenic tachyzoites was completely inhibited by the cathepsin C inhibitor. Cathepsin C inhibitors also limited the in vivo infection of T. gondii in the chick embryo model of toxoplasmosis. Thus, cathepsin Cs are critical to T. gondii growth and differentiation, and their unique specificities could be exploited to develop novel chemotherapeutic agents.

Amoeboid shape change and contact guidance: T-lymphocyte crawling through fibrillar collagen is independent of matrix remodeling by MMPs and other proteases

Blood ◽  
2003 ◽  
Vol 102 (9) ◽  
pp. 3262-3269 ◽  
Author(s):  
Katarina Wolf ◽  
Regina Müller ◽  
Stefan Borgmann ◽  
Eva.-B. Bröcker ◽  
Peter Friedl
Keyword(s):  
T Cell ◽  
Shape Change ◽  
Cathepsin L ◽  
Fluorescein Isothiocyanate ◽  
Cysteine Proteases ◽  
Dynamic Imaging ◽  
Basement Membranes ◽  
Contact Guidance ◽  
Matrix Remodeling ◽  
3 Dimensional

Abstract The passage of leukocytes through basement membranes involves proteolytic degradation of extracellular matrix (ECM) components executed by focalized proteolysis. We have investigated whether the migration of leukocytes through 3-dimensional collagenous tissue scaffolds requires similar ECM breakdown. Human T blasts and SupT1 lymphoma cells expressed mRNA of MMP-9, MT1-MMP, MT4-MMP, cathepsin L, uPA, and uPAR as well as ADAM-9, -10, -11, -15, and -17. Upon long-term migration within 3-dimensional collagen matrices, however, no in situ collagenolysis was obtained by sensitive fluorescein isothiocyanate–collagen fragmentation analysis and confocal fluorescence/backscatter microscopy. Consistent with nonproteolytic migration, T-cell crawling and path generation were not impaired by protease inhibitor cocktail targeting MMPs, serine proteases, cysteine proteases, and cathepsins. Dynamic imaging of cell-ECM interactions showed T-cell migration as an amoeba-like process driven by adaptive morphology, crawling along collagen fibrils (contact guidance) and squeezing through pre-existing matrix gaps by vigorous shape change. The concept of nonproteolytic amoeboid migration was confirmed for multicomponent collagen lattices containing hyaluronan and chondroitin sulfate and for other migrating leukocytes including CD8+ T blasts, monocyte-derived dendritic cells, and U937 monocytic cells. Together, amoeboid shape change and contact guidance provide constitutive protease-independent mechanisms for leukocyte trafficking through interstitial tissues that are insensitive toward pharmacologic protease inhibitors.

scholarly journals Maturation and substrate processing topography of the Plasmodium falciparum invasion/egress protease plasmepsin X

2022 ◽  
Author(s):  
Daniel Goldberg ◽  
Sumit Mukherjee ◽  
Eashan Sharma
Keyword(s):  
Catalytic Domain ◽  
Parasitophorous Vacuole ◽  
Aspartic Protease ◽  
Effector Proteins ◽  
Erythrocyte Membranes ◽  
Common Precursor ◽  
Quadruple Mutant ◽  
Substrate Processing ◽  
Mutant Forms

Abstract During the intravascular stage of infection, the malaria parasite Plasmodium invades a host erythrocyte, multiplies within a parasitophorous vacuole (PV) and exits upon rupture of the PV and erythrocyte membranes in a process known as egress. Both egress and invasion are controlled by effector proteins discharged from specialized secretory organelles. The aspartic protease plasmepsin X (PM X) regulates activity for many of these effectors, but it is unclear how PM X accesses its diverse substrates that reside in different organelles. PM X also processes itself to generate different isoforms that remain present in terminal schizonts. The function of these different forms is not understood. We have mapped the autoprocessing cleavage sites and constructed parasites with cleavage site mutations. Surprisingly, all the cleavage mutant forms of PM X, including a quadruple mutant that remained full-length, retained in vitro activity, were trafficked normally in the parasites, and supported parasite growth and normal egress and invasion. Further analysis showed that the N-terminal half of the prodomain stays bound to the catalytic domain even after processing and is required for proper folding and intracellular trafficking of PM X. We find that this enzyme cleaves microneme and exoneme substrates before discharge, possibly in a common precursor organelle, while the rhoptry substrates that are dependent on PM X activity are cleaved after exoneme discharge into the PV. The data give insight into the temporal, spatial and biochemical control of this unusual but important aspartic protease.

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