Subpellicular microtubules associate with an intramembranous particle lattice in the protozoan parasite Toxoplasma gondii

1997 ◽  
Vol 110 (1) ◽  
pp. 35-42 ◽  
Author(s):  
N.S. Morrissette ◽  
J.M. Murray ◽  
D.S. Roos
Keyword(s):  
Toxoplasma Gondii ◽  
Fourier Analysis ◽  
Host Cell ◽  
Morphological Changes ◽  
Shape Change ◽  
Freeze Fracture ◽  
Protozoan Parasite ◽  
Two Dimensional ◽  
Intramembranous Particle ◽  
Analysis Techniques

Application of Fourier analysis techniques to images of isolated, frozen-hydrated subpellicular microtubules from the protozoan parasite Toxoplasma gondii demonstrates a distinctive 32 nm periodicity along the length of the microtubules. A 32 nm longitudinal repeat is also observed in the double rows of intramembranous particles seen in freeze-fracture images of the parasite's pellicle; these rows are thought to overlie the subpellicular microtubules. Remarkably, the 32 nm intramembranous particle periodicity is carried over laterally to the single rows of particles that lie between the microtubule-associated double rows. This creates a two-dimensional particle lattice, with the second dimension at an angle of approximately 75 degrees to the longitudinal rows (depending on position along the length of the parasite). Drugs that disrupt known cytoskeletal components fail to destroy the integrity of the particle lattice. This intramembranous particle organization suggests the existence of multiple cytoskeletal filaments of unknown identity. Filaments associated with the particle lattice provide a possible mechanism for motility and shape change in Toxoplasma: distortion of the lattice may mediate the twirling motility seen upon host-cell lysis, and morphological changes observed during invasion.

Association of host cell endoplasmic reticulum and mitochondria with the Toxoplasma gondii parasitophorous vacuole membrane: a high affinity interaction

1997 ◽  
Vol 110 (17) ◽  
pp. 2117-2128 ◽  
Author(s):  
A.P. Sinai ◽  
P. Webster ◽  
K.A. Joiner
Keyword(s):  
Endoplasmic Reticulum ◽  
Toxoplasma Gondii ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Protozoan Parasite ◽  
Host Cells ◽  
Parasitophorous Vacuole Membrane ◽  
Vacuole Membrane ◽  
Protein Protein Interaction ◽  
High Affinity

The parasitophorous vacuole membrane (PVM) of the obligate intracellular protozoan parasite Toxoplasma gondii forms tight associations with host mitochondria and the endoplasmic reticulum (ER). We have used a combination of morphometric and biochemical approaches to characterize this unique phenomenon, which we term PVM-organelle association. The PVM is separated from associated mitochondria and ER by a mean distance of 12 and 18 nm, respectively. The establishment of PVM-organelle association is dependent on active parasite entry, but does not require parasite viability for its maintenance. Association is not a consequence of spatial constraints imposed on the growing vacuole. Morphometric analysis indicates that the extent of mitochondrial association with the PVM stays constant as the vacuole enlarges, whereas the extent of ER association decreases. Disruption of host cell microtubules partially blocks the establishment but not the maintenance of PVM-mitochondrial association, and has no significant effect on PVM-ER association. PVM-organelle association is maintained following disruption of infected host cells, as assessed by electron microscopy and by sub-cellular fractionation showing co-migration of fixed PVM and organelle markers. Taken together, the data suggest that a high affinity, potentially protein-protein interaction between parasite and organelle components is responsible for PVM-organelle association.

scholarly journals The Protozoan Parasite Toxoplasma gondii Selectively Reprograms the Host Cell Translatome

2018 ◽  
Vol 86 (9) ◽  
Author(s):  
Louis-Philippe Leroux ◽  
Julie Lorent ◽  
Tyson E. Graber ◽  
Visnu Chaparro ◽  
Laia Masvidal ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Activation ◽  
Immune Cell ◽  
Mrna Translation ◽  
Protozoan Parasite ◽  
Translational Efficiency ◽  
Type I ◽  
Content Type ◽  
Infection Inhibition

ABSTRACT The intracellular parasite Toxoplasma gondii promotes infection by targeting multiple host cell processes; however, whether it modulates mRNA translation is currently unknown. Here, we show that infection of primary murine macrophages with type I or II T. gondii strains causes a profound perturbation of the host cell translatome. Notably, translation of transcripts encoding proteins involved in metabolic activity and components of the translation machinery was activated upon infection. In contrast, the translational efficiency of mRNAs related to immune cell activation and cytoskeleton/cytoplasm organization was largely suppressed. Mechanistically, T. gondii bolstered mechanistic target of rapamycin (mTOR) signaling to selectively activate the translation of mTOR-sensitive mRNAs, including those with a 5′-terminal oligopyrimidine (5′ TOP) motif and those encoding mitochondrion-related proteins. Consistent with parasite modulation of host mTOR-sensitive translation to promote infection, inhibition of mTOR activity suppressed T. gondii replication. Thus, selective reprogramming of host mRNA translation represents an important subversion strategy during T. gondii infection.

scholarly journals Host Cell Invasion by Toxoplasma gondii Is Temporally Regulated by the Host Microtubule Cytoskeleton

2010 ◽  
Vol 9 (11) ◽  
pp. 1680-1689 ◽  
Author(s):  
Kristin R. Sweeney ◽  
Naomi S. Morrissette ◽  
Stephanie LaChapelle ◽  
Ira J. Blader
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Viral Infections ◽  
Protozoan Parasite ◽  
Host Cells ◽  
Cell Tropism ◽  
Microtubule Cytoskeleton ◽  
Parasite Invasion ◽  
Content Type ◽  
Microtubule Disruption

ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite that invades and replicates within most nucleated cells of warm-blooded animals. The basis for this wide host cell tropism is unknown but could be because parasites invade host cells using distinct pathways and/or repertoires of host factors. Using synchronized parasite invasion assays, we found that host microtubule disruption significantly reduces parasite invasion into host cells early after stimulating parasite invasion but not at later time points. Host microtubules are specifically associated with the moving junction, which is the site of contact between the host cell and the invading parasite. Host microtubules are specifically associated with the moving junction of those parasites invading early after stimulating invasion but not with those invading later. Disruption of host microtubules has no effect on parasite contact, attachment, motility, or rate of penetration. Rather, host microtubules hasten the time before parasites commence invasion. This effect on parasite invasion is distinct from the role that host microtubules play in bacterial and viral infections, where they function to traffic the pathogen or pathogen-derived material from the host cell's periphery to its interior. These data indicate that the host microtubule cytoskeleton is a structure used by Toxoplasma to rapidly infect its host cell and highlight a novel function for host microtubules in microbial pathogenesis.

scholarly journals Microneme Proteins 1 and 4 From Toxoplasma gondii Induce IL-10 Production by Macrophages Through TLR4 Endocytosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Rafael Ricci-Azevedo ◽  
Flavia Costa Mendonça-Natividade ◽  
Ana Carolina Santana ◽  
Juliana Alcoforado Diniz ◽  
Maria Cristina Roque-Barreira
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Inflammatory Cytokine ◽  
Early Stage ◽  
Protozoan Parasite ◽  
Cell Responses ◽  
Tnf Α ◽  
Host Inflammatory Response ◽  
Anti Inflammatory Cytokine ◽  
Microneme Proteins

The protozoan parasite Toxoplasma gondii modulates host cell responses to favor its success in the early stage of infections by secreting proteins from its apical organelles. Some of these proteins, including microneme proteins (MICs) 1 and 4, trigger pro-inflammatory host cell responses. The lectins MIC1 and MIC4 interact with N-linked glycans on TLR2 and TLR4, activating NF-κB and producing IL-12, TNF-α, and IL-6. Interestingly, MIC1 and MIC4 also trigger secretion of the anti-inflammatory cytokine IL-10 through mechanisms as yet unknown. Herein, we show that the ability of these MICs to induce macrophages to produce IL-10 depends on TLR4 internalization from the cell surface. Macrophages subjected to blockade of endocytosis by Dynasore continued to release TNF-α, but failed to produce IL-10, in response to MIC1 or MIC4 exposure. Similarly, IL-10 was not produced by Dynasore-conditioned T. gondii-infected macrophages. Furthermore, MIC1- or MIC4-stimulated macrophages gained transient tolerance to LPS. We report a previously undiscovered mechanism by which well-defined T. gondii components inhibit a host inflammatory response.

scholarly journals New Method for the Orthogonal Labeling and Purification of Toxoplasma gondii Proteins While Inside the Host Cell

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Gregory M. Wier ◽  
Erica M. McGreevy ◽  
Mark J. Brown ◽  
Jon P. Boyle
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Click Reaction ◽  
Severe Disease ◽  
Protozoan Parasite ◽  
Trna Synthetase ◽  
Content Type ◽  
Fluorescent Tags

ABSTRACTToxoplasma gondiiis an obligate intracellular protozoan parasite that is capable of causing severe disease in immunocompromised humans. How T. gondii is able to modulate the host cell to support itself is still poorly understood. Knowledge pertaining to the host-parasite interaction could be bolstered by developing a system to specifically label parasite proteins while the parasite grows inside the host cell. For this purpose, we have created a strain of T. gondii that expresses a mutant Escherichia coli methionyl-tRNA synthetase (MetRSNLL) that allows methionine tRNA to be loaded with the azide-containing methionine analog azidonorleucine (Anl). Anl-containing proteins are susceptible to a copper-catalyzed “click” reaction to attach affinity tags for purification or fluorescent tags for visualization. The MetRSNLL-Anl system labels nascent T. gondii proteins in an orthogonal fashion, labeling proteins only in MetRSNLL-expressing parasites. This system should be useful for nonradioactive pulse-chase studies and purification of nascently translated proteins. Although this approach allows labeling of a diverse array of parasite proteins, secreted parasite proteins appear to be only minimally labeled in MetRSNLL-expressing T. gondii. The minimal labeling of secreted proteins is likely a consequence of the selective charging of the initiator tRNA (and not the elongator methionine tRNA) by the heterologously expressed bacterial MetRS.IMPORTANCEStudying how T. gondii modifies the host cell to permit its survival is complicated by the complex protein environment of the host cell. The approach presented in this article provides the first method for specific labeling of T. gondii proteins while the parasite grows inside the host cell. We show that this approach is useful for pulse-chase labeling of parasite proteins duringin vitrogrowth. It should also be applicable duringin vivoinfections and in other apicomplexan parasites, including Plasmodium spp.

scholarly journals TgTKL1 Is a Unique Plant-Like Nuclear Kinase That Plays an Essential Role in Acute Toxoplasmosis

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Joseph M. Varberg ◽  
Isabelle Coppens ◽  
Gustavo Arrizabalaga ◽  
Rajshekhar Y. Gaji
Keyword(s):  
Toxoplasma Gondii ◽  
Tyrosine Kinase ◽  
Host Cell ◽  
Essential Role ◽  
Cell Attachment ◽  
Protozoan Parasite ◽  
Lytic Cycle ◽  
Host Cell Attachment ◽  
Acute Toxoplasmosis ◽  
Insight Into

ABSTRACT In the protozoan parasite Toxoplasma gondii , protein kinases have been shown to play key roles in regulating parasite motility, invasion, replication, egress, and survival within the host. The tyrosine kinase-like (TKL) family of proteins are an unexplored set of kinases in Toxoplasma . Of the eight annotated TKLs in the Toxoplasma genome, a recent genome-wide loss-of-function screen showed that six are important for tachyzoite fitness. By utilizing an endogenous tagging approach, we showed that these six T. gondii TKLs (TgTKLs) localize to various subcellular compartments, including the nucleus, the cytosol, the inner membrane complex, and the Golgi apparatus. To gain insight into the function of TKLs in Toxoplasma , we first characterized TgTKL1, which contains the plant-like enhanced disease resistance 1 (EDR1) domain and localizes to the nucleus. TgTKL1 knockout parasites displayed significant defects in progression through the lytic cycle; we show that the defects were due to specific impairment of host cell attachment. Transcriptomics analysis identified over 200 genes of diverse functions that were differentially expressed in TgTKL1 knockout parasites. Importantly, numerous genes implicated in host cell attachment and invasion were among those most significantly downregulated, resulting in defects in microneme secretion and processing. Significantly, all of the mice inoculated intraperitoneally with TgTKL1 knockout parasites survived the infection, suggesting that TgTKL1 plays an essential role in acute toxoplasmosis. Together, these findings suggest that TgTKL1 mediates a signaling pathway that regulates the expression of multiple factors required for parasite virulence, underscoring the potential of this kinase as a novel therapeutic target. IMPORTANCE Toxoplasma gondii is a protozoan parasite that can cause chronic and life-threatening disease in mammals; new drugs are greatly needed for treatment. One attractive group of drug targets consists of parasite kinases containing unique features that distinguish them from host proteins. In this report, we identify and characterize a previously unstudied kinase, TgTKL1, that localizes to the nucleus and contains a domain architecture unique to plants and protozoa. By disrupting TgTKL1, we showed that this kinase is required for the proper expression of hundreds of genes, including many that are required for the parasite to gain entry into the host cell. Specifically, parasites lacking TgTKL1 have defects in host cell attachment, resulting in impaired growth in vitro and a complete loss of virulence in mice. This report provides insight into the importance of the parasite tyrosine kinase-like kinases and establishes TgTKL1 as a novel and essential virulence factor in Toxoplasma .

Low Voltage Field Emission Scanning Electron Microscopy Provides Structural Evidence For Actin-Sized Filaments In Toxoplasma Gondii

1999 ◽  
Vol 5 (S2) ◽  
pp. 1130-1131
Author(s):  
Heide Schatten ◽  
David Sibley ◽  
Hans Ris
Keyword(s):  
Plasma Membrane ◽  
Toxoplasma Gondii ◽  
Host Cell ◽  
Field Emission ◽  
Cell Invasion ◽  
Low Voltage ◽  
Protozoan Parasite ◽  
Host Cell Invasion ◽  
Obligate Intracellular ◽  
Triton X

The protozoan parasite Toxoplasma gondiiis an obligate intracellular parasite that exhibits gliding and twisting motility during cell locomotion and host cell invasion. By using molecular and genetic approaches it has been determined that actin and myosin are localized beneath the parasite plasma membrane and produce the force for motility and active penetration during host cell invasion. However, structural evidence for actin fibers beneath the plasma membrane is still missing. Recently Chavez et al. demonstrated actin-like filaments in isolated cytoskeletal complexes. Our aproach has been to remove the cell membrane with 0.15% Triton X-100 in cytoskeleton preserving buffer, followed by imaging with low voltage field emission SEM. As seen in Fig. I, we could demonstrate the subpellicle actin network in parasites invading a host cell (arrow). Fig. 2 shows a similar subsurface network of actin filaments in a parasite gliding on glass.

scholarly journals Ethanol and acetaldehyde elevate intracellular [Ca2+] and stimulate microneme discharge in Toxoplasma gondii

1999 ◽  
Vol 342 (2) ◽  
pp. 379-386 ◽  
Author(s):  
Vern B. CARRUTHERS ◽  
Silvia N. J. MORENO ◽  
David L. SIBLEY
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Mammalian Cells ◽  
Model Organism ◽  
Protozoan Parasite ◽  
Host Cells ◽  
Sequential Addition ◽  
Dose Dependent ◽  
Related Compounds ◽  
Convenient Model

One of the first steps in host-cell invasion by the protozoan parasite Toxoplasma gondii occurs when the parasite attaches by its apical end to the target host cell. The contents of apical secretory organelles called micronemes have recently been implicated in parasite apical attachment to host cells. Micronemes are regulated secretory vesicles that discharge in response to elevated parasite intracellular Ca2+ levels ([Ca2+]i). In the present study we found that ethanol and related compounds produced a dose-dependent stimulation of microneme secretion. In addition, using fluorescence spectroscopy on tachyzoites loaded with the Ca2+-sensitive fluorescent dye fura-2, we demonstrated that ethanol stimulated microneme secretion by elevating parasite [Ca2+]i. Furthermore, sequential addition experiments with ethanol and other Ca2+-mobilizing drugs showed that ethanol probably elevated parasite [Ca2+]i by mobilizing Ca2+ from a thapsigargin-insensitive compartment of neutral pH. Earlier studies have shown that ethanol also elevates [Ca2+]i in mammalian cells. Thus, because it is genetically tractable, T. gondii might be a convenient model organism for studying the Ca2+-elevating effects of alcohol in higher eukaryotes.

scholarly journals Identification and Characterization of an Escorter for Two Secretory Adhesins in Toxoplasma gondii

2001 ◽  
Vol 152 (3) ◽  
pp. 563-578 ◽  
Author(s):  
Matthias Reiss ◽  
Nicola Viebig ◽  
Susan Brecht ◽  
Marie-Noelle Fourmaux ◽  
Martine Soete ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Secretory Pathway ◽  
Protozoan Parasite ◽  
Host Cells ◽  
Plausible Mechanism ◽  
Identification And Characterization ◽  
Intracellular Protozoan Parasite ◽  
Cargo Molecule

The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. Micronemes are apical secretory organelles releasing their contents upon contact with host cells. We have identified a transmembrane micronemal protein MIC6, which functions as an escorter for the accurate targeting of two soluble proteins MIC1 and MIC4 to the micronemes. Disruption of MIC1, MIC4, and MIC6 genes allowed us to precisely dissect their contribution in sorting processes. We have mapped domains on these proteins that determine complex formation and targeting to the organelle. MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. In contrast, MIC1 is linked to a quality control system and is absolutely required for the complex to leave the early compartments of the secretory pathway. MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act. We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.

scholarly journals Evidence that the cADPR signalling pathway controls calcium-mediated microneme secretion in Toxoplasma gondii

2005 ◽  
Vol 389 (2) ◽  
pp. 269-277 ◽  
Author(s):  
Eduardo N. Chini ◽  
Kisaburo Nagamune ◽  
Dawn M. Wetzel ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Calcium Release ◽  
Protozoan Parasite ◽  
Second Messenger ◽  
Ruthenium Red ◽  
Host Cell Invasion ◽  
Protozoan Parasites ◽  
Calcium Release Channels ◽  
Cyclic Adp Ribose

The protozoan parasite Toxoplasma gondii relies on calcium-mediated exocytosis to secrete adhesins on to its surface where they can engage host cell receptors. Increases in intracellular calcium occur in response to Ins(1,4,5)P3 and caffeine, an agonist of ryanodine-responsive calcium-release channels. We examined lysates and microsomes of T. gondii and detected evidence of cADPR (cyclic ADP ribose) cyclase and hydrolase activities, the two enzymes that control the second messenger cADPR, which causes calcium release from RyR (ryanodine receptor). We also detected endogenous levels of cADPR in extracts of T. gondii. Furthermore, T. gondii microsomes that were loaded with 45Ca2+ released calcium when treated with cADPR, and the RyR antagonists 8-bromo-cADPR and Ruthenium Red blocked this response. Although T. gondii microsomes also responded to Ins(1,4,5)P3, the inhibition profiles of these calcium-release channels were mutually exclusive. The RyR antagonists 8-bromo-cADPR and dantrolene inhibited protein secretion and motility in live parasites. These results indicate that RyR calcium-release channels that respond to the second-messenger cADPR play an important role in regulating intracellular Ca2+, and hence host cell invasion, in protozoan parasites.

Sign in / Sign up

Export Citation Format

Share Document