Toxoplasma gondii exploits the host ESCRT machinery for parasite uptake of host cytosolic proteins

Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole (PV), the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV-1 virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV-1 Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of TgGRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PV membrane (PVM). These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission.

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Toxoplasma gondii subverts the host ESCRT machinery for parasite uptake of host cytosolic proteins

10.1101/2021.07.21.453261 ◽

2021 ◽

Author(s):

Yolanda Rivera-Cuevas ◽

Joshua Mayoral ◽

Manlio Di Cristina ◽

Anna-Lisa E. Lawrence ◽

Einar B. Olafsson ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Parasitophorous Vacuole ◽

Dense Granule ◽

Cytosolic Proteins ◽

Endosomal Sorting ◽

Dense Granules ◽

Escrt Machinery ◽

Late Domain ◽

Cytosolic Protein ◽

Hiv Virus

Toxoplasma gondii is a master manipulator capable of effectively siphoning the resources from the host cell for its intracellular subsistence. However, the molecular underpinnings of how the parasite gains resources from its host remain largely unknown. Residing within a non-fusogenic parasitophorous vacuole, the parasite must acquire resources across the limiting membrane of its replicative niche, which is decorated with parasite proteins including those secreted from dense granules. We discovered a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in host cytosolic protein uptake by T. gondii by disrupting host ESCRT function. We identified the transmembrane dense granule protein TgGRA14, which contains motifs homologous to the late domain motifs of HIV-1 Gag, as a candidate for the recruitment of the host ESCRT machinery to the PV membrane. Using an HIV virus-like particle (VLP) release assay, we found that the motif-containing portion of TgGRA14 is sufficient to substitute for HIV Gag late domain to mediate ESCRT-dependent VLP budding. We also show that TgGRA14 is proximal to and interacts with host ESCRT components and other dense granule proteins during infection. Furthermore, analysis of GRA14-deficient parasites revealed a marked reduction in ingestion of a host cytosolic protein compared to WT parasites. Thus, we propose a model in which T. gondii recruits the host ESCRT machinery to the PV where it can interact with TgGRA14 for the internalization of host cytosolic proteins across the PVM. These findings provide new insight into how T. gondii accesses contents of the host cytosol by exploiting a key pathway for vesicular budding and membrane scission.

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Regulated secretion of multi-lamellar vesicles leads to formation of a tubulo-vesicular network in host-cell vacuoles occupied by Toxoplasma gondii

Journal of Cell Science ◽

10.1242/jcs.108.4.1669 ◽

1995 ◽

Vol 108 (4) ◽

pp. 1669-1677 ◽

Cited By ~ 1

Author(s):

L.D. Sibley ◽

I.R. Niesman ◽

S.F. Parmley ◽

M.F. Cesbron-Delauw

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Soluble Protein ◽

Parasitophorous Vacuole ◽

Dense Granule ◽

Host Cell Invasion ◽

Cell Fractionation ◽

Obligate Intracellular ◽

Dense Granules ◽

Storage Granules

Toxoplasma gondii is an obligate intracellular parasite that actively invades virtually all types of nucleated cells, surviving within a specialized vacuole called the parasitophorous vacuole. Shortly after invasion, the parasite modifies this vacuole by secreting a variety of proteins from electron-dense storage granules. Additionally, the parasite forms a network of membranous tubules within the lumen of the vacuole and connecting with the vacuolar membrane. We have used immunolabeling and cell fractionation to examine the secretion of two dense granule proteins, GRA1 and GRA2, which are involved in formation of the intravacuolar network. Following host-cell invasion, GRA1 was secreted into the lumen of the vacuole as a soluble protein that subsequently became peripherally associated with the network. In addition to being secreted as a soluble protein from dense granules, GRA2 was secreted within multi-lamellar vesicles released from a specialized posterior invagination of the parasite. The multi-lamellar vesicles assemble to form the intravacuolar network, which contains an integral membrane form of GRA2. These findings indicate that Toxoplasma has a highly developed regulated exocytosis pathway that modifies the parasitophorous vacuole by secretion of soluble proteins and by a novel process of membrane secretion.

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

Infection and Immunity ◽

10.1128/iai.01667-07 ◽

2008 ◽

Vol 76 (12) ◽

pp. 5853-5861 ◽

Cited By ~ 59

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.

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Toxoplasma gondii dense granule protein 3 (GRA3) is a type I transmembrane protein that possesses a cytoplasmic dilysine (KKXX) endoplasmic reticulum (ER) retrieval motif

Parasitology ◽

10.1017/s0031182005007559 ◽

2005 ◽

Vol 131 (2) ◽

pp. 169-179 ◽

Cited By ~ 22

Author(s):

F. L. HENRIQUEZ ◽

M. B. NICKDEL ◽

R. MCLEOD ◽

R. E. LYONS ◽

K. LYONS ◽

...

Keyword(s):

Molecular Weight ◽

Endoplasmic Reticulum ◽

Toxoplasma Gondii ◽

Transmembrane Domain ◽

Neospora Caninum ◽

Parasitophorous Vacuole ◽

Dense Granule ◽

Type I ◽

Dense Granules ◽

Granule Protein

Studies using antibodies to immunolocalize the Toxoplasma gondii dense granule protein GRA3, have shown that this protein associates strongly with the parasitophorous vacuole membrane (PVM). However, as there was no predicted membrane-spanning domain this highlighted an unanswered paradox. We demonstrate that the previously published sequence for GRA3 is actually an artificial chimera of 2 proteins. One protein, of molecular weight 65 kDa, shares the C-terminus with published GRA3 and possesses no significant sequence similarity with any protein thus far deposited in Genbank. The second, with a predicted molecular weight of 24 kDa shares the N-terminal region, is recognized by the monoclonal antibody 2H11 known to react with the dense granules of T. gondii and is therefore the authentic GRA3. The corrected GRA3 has an N-terminal secretory signal sequence and a transmembrane domain consistent with its insertion into the PVM. Antibodies to recombinant GRA3 recognize a protein of 24 kDa in T. gondii excretory–secretory antigen preparations. The signal peptide is necessary and sufficient to target GFP to the dense granules and parasitophorous vacuole. A homologue was identified in Neospora caninum. Finally, GRA3 possesses a dilysine ‘KKXX’ endoplasmic reticulum (ER) retrieval motif that rationalizes its association with PVM and possibly the host cell ER.

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Intervacuolar Transport and Unique Topology of GRA14, a Novel Dense Granule Protein in Toxoplasma gondii

Infection and Immunity ◽

10.1128/iai.00782-08 ◽

2008 ◽

Vol 76 (11) ◽

pp. 4865-4875 ◽

Cited By ~ 60

Author(s):

Michael E. Rome ◽

Josh R. Beck ◽

Jay M. Turetzky ◽

Paul Webster ◽

Peter J. Bradley

Keyword(s):

Toxoplasma Gondii ◽

Parasitophorous Vacuole ◽

Dense Granule ◽

Obligate Intracellular ◽

Dense Granules ◽

Granule Protein ◽

Host Cytoplasm ◽

C Terminus ◽

Membrane Bound ◽

Host Parasite

ABSTRACT Toxoplasma gondii is an obligate intracellular parasite that resides in the cytoplasm of its host in a unique membrane-bound vacuole known as the parasitophorous vacuole (PV). The membrane surrounding the parasite is remodeled by the dense granules, secretory organelles that release an array of proteins into the vacuole and to the PV membrane (PVM). Only a small portion of the protein constituents of the dense granules have been identified, and little is known regarding their roles in infection or how they are trafficked within the infected host cell. In this report, we identify a novel secreted dense granule protein, GRA14, and show that it is targeted to membranous structures within the vacuole known as the intravacuolar network and to the vacuolar membrane surrounding the parasite. We disrupted GRA14 and exploited the knockout strain to show that GRA14 can be transferred between vacuoles in a coinfection experiment with wild-type parasites. We also show that GRA14 has an unexpected topology in the PVM with its C terminus facing the host cytoplasm and its N terminus facing the vacuolar lumen. These findings have important implications both for the trafficking of GRA proteins to their ultimate destinations and for expectations of functional domains of GRA proteins at the host-parasite interface.

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Transmembrane Insertion of the Toxoplasma gondii GRA5 Protein Occurs after Soluble Secretion into the Host Cell

Molecular Biology of the Cell ◽

10.1091/mbc.10.4.1277 ◽

1999 ◽

Vol 10 (4) ◽

pp. 1277-1287 ◽

Cited By ~ 81

Author(s):

Laurence Lecordier ◽

Corinne Mercier ◽

L. David Sibley ◽

Marie-France Cesbron-Delauw

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Secretory Pathway ◽

Transmembrane Domain ◽

Transmembrane Protein ◽

Parasitophorous Vacuole ◽

Dense Granule ◽

Soluble Form ◽

Host Cell Membrane ◽

Dense Granules

The intracellular parasite Toxoplasma gondii resides within a specialized compartment, the parasitophorous vacuole (PV), that resists fusion with host cell endocytic and lysosomal compartments. The PV is extensively modified by secretion of parasite proteins, including the dense granule protein GRA5 that is specifically targeted to the delimiting membrane of the PV (PVM). We show here that GRA5 is present both in a soluble form and in hydrophobic aggregates. GRA5 is secreted as a soluble form into the PV after which it becomes stably associated with the PVM. Topological studies demonstrated that GRA5 was inserted into the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its C terminus in the vacuole lumen. Deletion of 8 of the 18 hydrophobic amino acids of the single predicted transmembrane domain resulted in the failure of GRA5 to associate with the PVM; yet it remained correctly packaged in the dense granules and was secreted as a soluble protein into the PV. Collectively, these studies demonstrate that the secretory pathway inToxoplasma is unusual in two regards; it allows soluble export of proteins containing typical transmembrane domains and provides a mechanism for their insertion into a host cell membrane after secretion from the parasite.

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Differential targeting of dense granule proteins in the parasitophorous vacuole ofToxoplasma gondii

Parasitology ◽

10.1017/s0031182000059837 ◽

1991 ◽

Vol 103 (3) ◽

pp. 321-329 ◽

Cited By ~ 86

Author(s):

A. Achbarou ◽

O. Mercereau-Puijalon ◽

A. Sadak ◽

B. Fortier ◽

M. A. Leriche ◽

...

Keyword(s):

Molecular Weight ◽

Parasitophorous Vacuole ◽

Dense Granule ◽

Parasitophorous Vacuole Membrane ◽

Vacuole Membrane ◽

Molecular Weights ◽

Dense Granules ◽

Metabolic Labelling ◽

Infected Cells ◽

Granule Proteins

The biosynthesis and fate of 4 different dense granule proteins ofToxoplasma gondiiwere studied with 3 monoclonal antibodies raised against tachyzoites and 1 polyclonal antibody raised against a recombinant protein. These proteins have the following molecular weights: 27 kDa (GRA 1), 28 kDa (GRA 2), 30 kDa (GRA 3) and 40 kDa (GRA 4). All four proteins were found in dense granules by immunoelectron microscopy; inT. gondii-infected cells, they were found in the vacuolar network but, in addition, GRA 3 was also detected on the parasitophorous vacuole membrane. Therefore, dense granule contents undergo differential targeting when exocytosed in the parasitophorous vacuole. Metabolic labelling and immunoprecipitation showed that GRA 2 and GRA 3 were processed from lower molecular weight precursors, and that GRA 2 and GRA 4 incorporated [3H] glucosamine and are thus likely to be glycosylated.

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Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells

mSphere ◽

10.1128/msphere.00858-19 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 9

Author(s):

Alicja M. Cygan ◽

Terence C. Theisen ◽

Alma G. Mendoza ◽

Nicole D. Marino ◽

Michael W. Panas ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Protein Translocation ◽

Affinity Purification ◽

Parasitophorous Vacuole ◽

Dense Granule ◽

Effector Proteins ◽

Host Cells ◽

Content Type ◽

Cyclin E1 ◽

Infected Cells

ABSTRACT Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54), and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16 and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5 processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins. IMPORTANCE Toxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells and provide additional potential drug targets.

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In vivo expression and distribution of dense granule protein 7 (GRA7) in the exoenteric (tachyzoite, bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii

Parasitology ◽

10.1017/s0031182099004692 ◽

1999 ◽

Vol 119 (3) ◽

pp. 259-265 ◽

Cited By ~ 26

Author(s):

D. J. P. FERGUSON ◽

D. JACOBS ◽

E. SAMAN ◽

J-F. DUBREMETZ ◽

S. E. WRIGHT

Keyword(s):

Toxoplasma Gondii ◽

Staining Pattern ◽

Parasitophorous Vacuole ◽

Dense Granule ◽

Parasite Development ◽

Double Labelling ◽

Granule Protein ◽

Specific Variation ◽

Strong Staining

The in vivo expression and distribution of the dense granule protein GRA7 was examined in both the exoenteric (tachyzoite and bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii by immunocytochemistry. There was strong staining of GRA7 in granules within all the infectious stages (tachyzoite, bradyzoite, merozoite and sporozoite). During tachyzoite development, GRA7 was secreted and was associated with the parasitophorous vacuole. In contrast, although there was staining of granules within the bradyzoites of more mature cysts, there appeared to be little staining of the tissue cyst wall or host cell. The apparent stage-specific variation in secretion of GRA7 between tachyzoites and bradyzoites was confirmed by double labelling using stage-specific markers (SAG1 and BAG1). In the enteric forms in the cat gut there was strong labelling of the PV containing early asexual and sexual stages and staining of a few granules in the apical cytoplasm of the merozoite. The positive enteric staining pattern differentiates GRA7 from the other GRA proteins (GRA1–6) which were absent in the merozoites and enteric stages. The staining pattern of GRA7 with strong staining during tachyzoite and enteric development and reduced staining in the tissue cysts is similar to that seen for NTPases. The function of GRA7 is unknown but it is unique among the dense granule proteins in being expressed in all the infectious forms of T. gondii which would point to a basic role in the vacuolar adaptations required for active parasite development.

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