Besnoitia besnoiti and Toxoplasma gondii: two apicomplexan strategies to manipulate the host cell centrosome and Golgi apparatus

Parasitology ◽  
2014 ◽  
Vol 141 (11) ◽  
pp. 1436-1454 ◽  
Author(s):  
RITA CARDOSO ◽  
SOFIA NOLASCO ◽  
JOÃO GONÇALVES ◽  
HELDER C. CORTES ◽  
ALEXANDRE LEITÃO ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Golgi Apparatus ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Besnoitia Besnoiti ◽  
Microtubule Cytoskeleton ◽  
Cytoskeleton Organization ◽  
Evolutionary Paths ◽  
The Impact

SUMMARYBesnoitia besnoiti and Toxoplasma gondii are two closely related parasites that interact with the host cell microtubule cytoskeleton during host cell invasion. Here we studied the relationship between the ability of these parasites to invade and to recruit the host cell centrosome and the Golgi apparatus. We observed that T. gondii recruits the host cell centrosome towards the parasitophorous vacuole (PV), whereas B. besnoiti does not. Notably, both parasites recruit the host Golgi apparatus to the PV but its organization is affected in different ways. We also investigated the impact of depleting and over-expressing the host centrosomal protein TBCCD1, involved in centrosome positioning and Golgi apparatus integrity, on the ability of these parasites to invade and replicate. Toxoplasma gondii replication rate decreases in cells over-expressing TBCCD1 but not in TBCCD1-depleted cells; while for B. besnoiti no differences were found. However, B. besnoiti promotes a reorganization of the Golgi ribbon previously fragmented by TBCCD1 depletion. These results suggest that successful establishment of PVs in the host cell requires modulation of the Golgi apparatus which probably involves modifications in microtubule cytoskeleton organization and dynamics. These differences in how T. gondii and B. besnoiti interact with their host cells may indicate different evolutionary paths.

scholarly journals MYR1-Dependent Effectors Are the Major Drivers of a Host Cell’s Early Response to Toxoplasma , Including Counteracting MYR1-Independent Effects

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Adit Naor ◽  
Michael W. Panas ◽  
Nicole Marino ◽  
Michael J. Coffey ◽  
Christopher J. Tonkin ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Expression Profiles ◽  
Parasitophorous Vacuole ◽  
Effector Proteins ◽  
Gene Set Enrichment Analysis ◽  
Host Cells ◽  
Content Type ◽  
Cell Functions ◽  
The Impact

ABSTRACT The obligate intracellular parasite Toxoplasma gondii controls its host cell from within the parasitophorous vacuole (PV) by using a number of diverse effector proteins, a subset of which require the aspartyl protease 5 enzyme (ASP5) and/or the recently discovered MYR1 protein to cross the PV membrane. To examine the impact these effectors have in the context of the entirety of the host response to Toxoplasma , we used RNA-Seq to analyze the transcriptome expression profiles of human foreskin fibroblasts infected with wild-type RH (RH-WT), RHΔ myr1 , and RHΔ asp5 tachyzoites. Interestingly, the majority of the differentially regulated genes responding to Toxoplasma infection are MYR1 dependent. A subset of MYR1 responses were ASP5 independent, and MYR1 function did not require ASP5 cleavage, suggesting the export of some effectors requires only MYR1. Gene set enrichment analysis of MYR1-dependent host responses suggests an upregulation of E2F transcription factors and the cell cycle and a downregulation related to interferon signaling, among numerous others. Most surprisingly, “hidden” responses arising in RHΔ myr1 - but not RH-WT-infected host cells indicate counterbalancing actions of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1 dependent provide new insight into the parasite’s ability to co-opt host cell functions. IMPORTANCE Toxoplasma gondii is unique in its ability to successfully invade and replicate in a broad range of host species and cells within those hosts. The complex interplay of effector proteins exported by Toxoplasma is key to its success in co-opting the host cell to create a favorable replicative niche. Here we show that a majority of the transcriptomic effects in tachyzoite-infected cells depend on the activity of a novel translocation system involving MYR1 and that the effectors delivered by this system are part of an intricate interplay of activators and suppressors. Removal of all MYR1-dependent effectors reveals previously unknown activities that are masked or hidden by the action of these proteins.

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 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.

Secretion by Toxoplasma gondii of an antigen that appears to become associated with the parasitophorous vacuole membrane upon invasion of the host cell

1987 ◽  
Vol 88 (2) ◽  
pp. 231-239
Author(s):  
I. Kimata ◽  
K. Tanabe
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Anterior Part ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Immunoperoxidase Staining ◽  
Cell Plasma ◽  
Sds Page ◽  
Infected Cells ◽  
Triton X

Monoclonal antibodies against Toxoplasma gondii were prepared to characterize antigens of the parasite. Immunoperoxidase staining of parasites fixed with paraformaldehyde and glutaraldehyde (PFAGA) followed by Triton X-100 treatment showed that the antibody of clone I-63 recognized an antigen located in the anterior part of the parasite. When analysed by SDS-PAGE and immunoblotting, the antigen migrated in a 66 × 10(3) Mr region. The parasite antigen diminished greatly in parasites after invasion of host cells, but reappeared around a time when intracellular T. gondii multiplied. Immunodetection on PFAGA-fixed T. gondii-infected cells, whose membranes were permeabilized by freeze-thawing in the presence of 5% glycerol, demonstrated that, immediately after parasite invasion, the I-63 antibody-reactive antigen appeared to become associated with the parasitophorous vacuole (PV) membrane, that had been formed mainly by invagination of the host-cell plasma membrane so as to surround an invading parasite. The antigen remained associated with the PV membrane for some time, but disappeared later when the PV increased in size after the parasites had multiplied several times. These results were strengthened by immunoelectron microscopic observations: the antigen that had been localized at the anterior part of the parasite before invasion appeared in an area of the host cell cytoplasm around the tips of penetrating parasites and, thereafter, extended throughout the surface of the PV membrane when parasites completed invasion. Thus, it appears that the I-63-reactive antigen is secreted by T. gondii upon invasion of the host cell and becomes associated with the PV membrane shortly after invasion.

scholarly journals A Novel Secreted Protein, MYR1, Is Central to Toxoplasma ’s Manipulation of Host Cells

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Magdalena Franco ◽  
Michael W. Panas ◽  
Nicole D. Marino ◽  
Mei-Chong Wendy Lee ◽  
Kerry R. Buchholz ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Biology ◽  
Parasitophorous Vacuole ◽  
Critical Role ◽  
Host Cells ◽  
Secreted Protein ◽  
Parasitophorous Vacuole Membrane ◽  
Vacuole Membrane ◽  
Novel Protein

ABSTRACT The intracellular protozoan Toxoplasma gondii dramatically reprograms the transcriptome of host cells it infects, including substantially up-regulating the host oncogene c -myc . By applying a flow cytometry-based selection to infected mouse cells expressing green fluorescent protein fused to c-Myc (c-Myc–GFP), we isolated mutant tachyzoites defective in this host c-Myc up-regulation. Whole-genome sequencing of three such mutants led to the identification of MYR1 ( My c r egulation 1 ; TGGT1_254470 ) as essential for c-Myc induction. MYR1 is a secreted protein that requires TgASP5 to be cleaved into two stable portions, both of which are ultimately found within the parasitophorous vacuole and at the parasitophorous vacuole membrane. Deletion of MYR1 revealed that in addition to its requirement for c-Myc up-regulation, the MYR1 protein is needed for the ability of Toxoplasma tachyzoites to modulate several other important host pathways, including those mediated by the dense granule effectors GRA16 and GRA24. This result, combined with its location at the parasitophorous vacuole membrane, suggested that MYR1 might be a component of the machinery that translocates Toxoplasma effectors from the parasitophorous vacuole into the host cytosol. Support for this possibility was obtained by showing that transit of GRA24 to the host nucleus is indeed MYR1-dependent. As predicted by this pleiotropic phenotype, parasites deficient in MYR1 were found to be severely attenuated in a mouse model of infection. We conclude, therefore, that MYR1 is a novel protein that plays a critical role in how Toxoplasma delivers effector proteins to the infected host cell and that this is crucial to virulence. IMPORTANCE Toxoplasma gondii is an important human pathogen and a model for the study of intracellular parasitism. Infection of the host cell with Toxoplasma tachyzoites involves the introduction of protein effectors, including many that are initially secreted into the parasitophorous vacuole but must ultimately translocate to the host cell cytosol to function. The work reported here identified a novel protein that is required for this translocation. These results give new insight into a very unusual cell biology process as well as providing a potential handle on a pathway that is necessary for virulence and, therefore, a new potential target for chemotherapy.

scholarly journals Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular Parasite Toxoplasma gondii

2020 ◽  
Author(s):  
Suchita Rastogi ◽  
Yuan Xue ◽  
Stephen R. Quake ◽  
John C. Boothroyd
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Biology ◽  
Parasitophorous Vacuole ◽  
Dense Granule ◽  
Effector Proteins ◽  
Transcriptomic Analysis ◽  
Host Cells ◽  
Intracellular Parasite ◽  
I Cells

ABSTRACTThe intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultra-pure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single cell transcriptomic analysis at 1-3 hours post-infection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the host’s earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent pro-inflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs, (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggests that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes.IMPORTANCEThis work performs the first transcriptomic analysis of U-I cells, captures the earliest stage of a host cell’s interaction with Toxoplasma gondii, and dissects the effects of individual classes of parasite effectors on host cell biology.

scholarly journals The Toxoplasma gondii Dense Granule Protein GRA7 Is Phosphorylated upon Invasion and Forms an Unexpected Association with the Rhoptry Proteins ROP2 and ROP4

2008 ◽  
Vol 76 (12) ◽  
pp. 5853-5861 ◽  
Author(s):  
Joe Dan Dunn ◽  
Sandeep Ravindran ◽  
Seon-Kyeong Kim ◽  
John C. Boothroyd
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Opportunistic Pathogen ◽  
Dense Granule ◽  
Host Cells ◽  
Obligate Intracellular ◽  
Dense Granules ◽  
Obligate Intracellular Parasite ◽  
Granule Protein

ABSTRACT The obligate intracellular parasite Toxoplasma gondii infects warm-blooded animals throughout the world and is an opportunistic pathogen of humans. As it invades a host cell, Toxoplasma forms a novel organelle, the parasitophorous vacuole, in which it resides during its intracellular development. The parasite modifies the parasitophorous vacuole and its host cell with numerous proteins delivered from rhoptries and dense granules, which are secretory organelles unique to the phylum Apicomplexa. For the majority of these proteins, little is known other than their localization. Here we show that the dense granule protein GRA7 is phosphorylated but only in the presence of host cells. Within 10 min of invasion, GRA7 is present in strand-like structures in the host cytosol that contain rhoptry proteins. GRA7 strands also contain GRA1 and GRA3. Independently of its phosphorylation state, GRA7 associates with the rhoptry proteins ROP2 and ROP4 in infected host cells. This is the first report of interactions between proteins secreted from rhoptries and dense granules.

scholarly journals A Glycosylphosphatidylinositol-Anchored Carbonic Anhydrase-Related Protein of Toxoplasma gondii Is Important for Rhoptry Biogenesis and Virulence

mSphere ◽  
2017 ◽  
Vol 2 (3) ◽  
Author(s):  
Nathan M. Chasen ◽  
Beejan Asady ◽  
Leandro Lemgruber ◽  
Rossiane C. Vommaro ◽  
Jessica C. Kissinger ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Carbonic Anhydrase ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Lytic Cycle ◽  
Host Cells ◽  
Intracellular Pathogen ◽  
Related Protein ◽  
Content Type ◽  
Initial Interaction

ABSTRACT Toxoplasma gondii is an intracellular pathogen that infects humans and animals. The pathogenesis of T. gondii is linked to its lytic cycle, which starts when tachyzoites invade host cells and secrete proteins from specialized organelles. Once inside the host cell, the parasite creates a parasitophorous vacuole (PV) where it divides. Rhoptries are specialized secretory organelles that contain proteins, many of which are secreted during invasion. These proteins have important roles not only during the initial interaction between parasite and host but also in the formation of the PV and in the modification of the host cell. We report here the identification of a new T. gondii carbonic anhydrase-related protein (TgCA_RP), which localizes to rhoptries of mature tachyzoites. TgCA_RP is important for the morphology of rhoptries and for invasion and growth of parasites. TgCA_RP is also critical for parasite virulence. We propose that TgCA_RP plays a role in the biogenesis of rhoptries. Carbonic anhydrase-related proteins (CARPs) have previously been described as catalytically inactive proteins closely related to α-carbonic anhydrases (α-CAs). These CARPs are found in animals (both vertebrates and invertebrates) and viruses as either independent proteins or domains of other proteins. We report here the identification of a new CARP (TgCA_RP) in the unicellular organism Toxoplasma gondii that is related to the recently described η-class CA found in Plasmodium falciparum. TgCA_RP is posttranslationally modified at its C terminus with a glycosylphosphatidylinositol anchor that is important for its localization in intracellular tachyzoites. The protein localizes throughout the rhoptry bulbs of mature tachyzoites and to the outer membrane of nascent rhoptries in dividing tachyzoites, as demonstrated by immunofluorescence and immunoelectron microscopy using specific antibodies. T. gondii mutant tachyzoites lacking TgCA_RP display a growth and invasion phenotype in vitro and have atypical rhoptry morphology. The mutants also exhibit reduced virulence in a mouse model. Our results show that TgCA_RP plays an important role in the biogenesis of rhoptries. IMPORTANCE Toxoplasma gondii is an intracellular pathogen that infects humans and animals. The pathogenesis of T. gondii is linked to its lytic cycle, which starts when tachyzoites invade host cells and secrete proteins from specialized organelles. Once inside the host cell, the parasite creates a parasitophorous vacuole (PV) where it divides. Rhoptries are specialized secretory organelles that contain proteins, many of which are secreted during invasion. These proteins have important roles not only during the initial interaction between parasite and host but also in the formation of the PV and in the modification of the host cell. We report here the identification of a new T. gondii carbonic anhydrase-related protein (TgCA_RP), which localizes to rhoptries of mature tachyzoites. TgCA_RP is important for the morphology of rhoptries and for invasion and growth of parasites. TgCA_RP is also critical for parasite virulence. We propose that TgCA_RP plays a role in the biogenesis of rhoptries.

Three-Dimensional Imaging of Toxoplasma gondii–Host Cell Interactions within the Parasitophorous Vacuole

2004 ◽  
Vol 10 (5) ◽  
pp. 580-585 ◽  
Author(s):  
Heide Schatten ◽  
Hans Ris
Keyword(s):  
Toxoplasma Gondii ◽  
Cell Membrane ◽  
Host Cell ◽  
Parasitophorous Vacuole ◽  
Three Dimensional ◽  
Host Cells ◽  
Preparation Technique ◽  
Host Cell Membrane ◽  
Three Dimensional Imaging ◽  
Structural Interactions

The protozoan parasite Toxoplasma gondii is a representative of apicomplexan parasites that invades host cells through an unconventional motility mechanism. During host cell invasion it forms a specialized membrane-surrounded compartment that is called the parasitophorous vacuole. The interactions between the host cell and parasite membranes are complex and recent studies have revealed in more detail that both the host cell and the parasite membrane contribute to the formation of the parasitophorous vacuole. By using our a new specimen preparation technique that allows three-dimensional imaging of thick-sectioned internal cell structures with high-resolution, low-voltage field emission scanning electron microscopy, we were able to visualize continuous structural interactions of the host cell membrane with the parasite within the parasitophorous vacuole. Fibrous and tubular material extends from the host cell membrane and is connected to parasite membrane components. Shorter protrusions are also elaborated from the parasite. Several of these shorter fine protrusions connect to the fibrous material of the host cell membrane. The elaborate network may be used for modifications of the parasitophorous vacuole membrane that will allow utilization of nutrients from the host cell by the parisite while it is being protected from host cell attacks. The structural interactions between parasite and host cells undergo time-dependent changes, and a fission pore is the most prominent structure left connecting the parasite with the host cell. The fission pore is anchored in the host cell by thick structural components of unknown nature. The new information gained with this technique includes structural details of fibrous and tubular material that is continuous between the parasite and host cell and can be imaged in three dimensions. We present this technique as a tool to investigate more fully the complex structural interactions of the host cell and the parasite residing in the parasitophorous vacuole.

scholarly journals The Toxoplasma gondii Rhoptry Protein ROP4 Is Secreted into the Parasitophorous Vacuole and Becomes Phosphorylated in Infected Cells

2004 ◽  
Vol 3 (5) ◽  
pp. 1320-1330 ◽  
Author(s):  
Kimberly L. Carey ◽  
Artemio M. Jongco ◽  
Kami Kim ◽  
Gary E. Ward
Keyword(s):  
Toxoplasma Gondii ◽  
Infected Cell ◽  
Host Cell ◽  
Transmembrane Protein ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Cell Protein ◽  
Type I ◽  
Membrane Function ◽  
Vacuole Membrane

ABSTRACT Many intracellular pathogens are separated from the cytosol of their host cells by a vacuole membrane. This membrane serves as a critical interface between the pathogen and the host cell, across which nutrients are imported, wastes are excreted, and communication between the two cells takes place. Very little is known about the vacuole membrane proteins mediating these processes in any host-pathogen interaction. During a screen for monoclonal antibodies against novel surface or secreted proteins of Toxoplasma gondii, we identified ROP4, a previously uncharacterized member of the ROP2 family of proteins. We report here on the sequence, posttranslational processing, and subcellular localization of ROP4, a type I transmembrane protein. Mature, processed ROP4 is localized to the rhoptries, secretory organelles at the apical end of the parasite, and is secreted from the parasite during host cell invasion. Released ROP4 associates with the vacuole membrane and becomes phosphorylated in the infected cell. Similar results are seen with ROP2. Further analysis of ROP4 showed it to be phosphorylated on multiple sites, a subset of which result from the action of either host cell protein kinase(s) or parasite kinase(s) activated by host cell factors. The localization and posttranslational modification of ROP4 and other members of the ROP2 family of proteins within the infected cell make them well situated to play important roles in vacuole membrane function.

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