The Toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm (original) (raw)
Related papers
The Journal of Cell Biology, 2001
oxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM-organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH 2 -terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves T like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mitochondria and ER. The 30amino acid (aa) NH 2 -terminal sequence of ROP2hc, when fused to green fluorescent protein (GFP), is sufficient for mitochondrial targeting. Deletion of the 30-aa NH 2 -terminal signal from ROP2hc results in robust localization of the truncated protein to the ER. These results demonstrate a new mechanism for tight association of different membrane-bound organelles within the cell cytoplasm.
Structure and Function of the Parasitophorous Vacuole Membrane Surrounding Toxoplasma gondii
Annals of the New York Academy of Sciences, 1994
Toxoplusma gondii is growing in popularity as a model for studying intracellular parasitism.' The parasite has also gained more attention recently as the most common cause of focal central nervous system infections in patients with the acquired immunodeficiency syndrome,* and it continues to cause more than 3,000 cases per year of congenital birth defects in the United States alone. T. gondii infects nearly all animals and most birds and is one of the most widely distributed of all intracellular parasites. In vitro, tachyzoites of T. gondii can invade and replicate within essentially all nucleated cells, an unusual feat for any intracellular organism. The intracellular tachyzoites reside within a vacuole that is incapable of acidifying or fusing with any membrane-bound organelle within the host cell endocytic system= and as such is effectively hidden from the host, yet the parasite replicates rapidly. The fusion incompetence of the vacuole is established at the time of cell entry and does not depend on secretion by the parasite of a soluble inhibitor of the fusion or acidification events. On the basis of these findings and in conjunction with the known morphology of the newly formed parasitophorous vacuole membrane (PVM), we hypothesized that fusion incompetence results from the absence of protein signals for vesicular fusion events. Nonetheless, the PVM surrounding T. gondii serves a critical additional function which is likely to depend on proteins, by allowing access of necessary nutrients from and exchange of metabolites with the host cell. A second potential function for the PVM, binding of host cell mitochondria and endoplasmic reticulum,7 is also likely to be protein dependent. We believe that proteins that provide or contribute to these additional functions for the PVM are likely to be derived from parasite secretory organelles, but little direct data exist to support this hypothesis.
2001
Toxoplasma gondii replicates within a specialized vacuole surrounded by the parasitophorous vacuole membrane (PVM). The PVM forms intimate interactions with host mitochondria and endoplasmic reticulum (ER) in a process termed PVM–organelle association. In this study we identify a likely mediator of this process, the parasite protein ROP2. ROP2, which is localized to the PVM, is secreted from anterior organelles termed rhoptries during parasite invasion into host cells. The NH2-terminal domain of ROP2 (ROP2hc) within the PVM is exposed to the host cell cytosol, and has characteristics of a mitochondrial targeting signal. In in vitro assays, ROP2hc is partially translocated into the mitochondrial outer membrane and behaves like an integral membrane protein. Although ROP2hc does not translocate across the ER membrane, it does exhibit carbonate-resistant binding to this organelle. In vivo, ROP2hc expressed as a soluble fragment in the cytosol of uninfected cells associates with both mit...
Cell Structure and Function, 1997
Tachyzoites of Toxoplasma gondii multiplies within the parasitophorous vacuole (PV) of the host cell. Simultaneously with parasite division growth of the vacuole takes place. Using immunofluorescence microscopy and antibodies recognizing calreticulin, a nonmuscle functional analogue of calsequestrin, and a 76 kDa glycoprotein localized in the endoplasmic reticulum (ER), we showed the incorporation of ER elements of the host cell into parasitophorous vacuole containing-T. gondii. In addition enzyme cytochemistry showed that glucose-6-phosphatase, an enzyme marker of ER, is also localized within the PV. These observations suggest that growth of T, gondii-containing PV is at least in part due to incorporation of elements of the host cell ER into the vacuole.
Pleiotropic effect due to targeted depletion of secretory rhoptry protein ROP2 in Toxoplasma gondii
Journal of Cell Science, 2003
Long after their discovery, the function and biogenesis of rhoptries remain enigmatic. In Apicomplexan parasites, these organelles discharge and their contents are exocytosed at the time of host cell invasion, and are thus proposed to play an essential role in establishing the parasitophorous vacuole. In Toxoplasma gondii, ROP2 is suspected to serve as the molecular link between host cell mitochondria and parasitophorous vacuole membrane. In this study we addressed the function of ROP2. Targeted depletion of ROP2 using a ribozyme-modified antisense RNA strategy resulted in multiple effects on parasite morphology because of a disruption in the formation of mature rhoptries, and an arrest in cytokinesis. The association of host cell mitochondria with the parasitophorous vacuole membrane was abolished and the ROP2-deficient parasites had a reduced uptake of sterol from the host cell. Furthermore, these parasites invaded human fibroblasts poorly and had markedly attenuated virulence in mice. We conclude that rhoptry discharge, and in particular release of ROP2, are essential for parasite invasion, replication and host cell-parasite interaction.
Structure and Function of the Toxoplasma gondii Vacuole
Annals of the New York Academy of Sciences, 1996
Toxoplasma gondii is an obligate intracellular protozoan that resides within a specialized vacuole inside host cells. The vacuolar space and surrounding parasitophorous vacuole membrane (PVM) are the interface between the parasite and the host cell. Establishment and maintenance of the vacuole and PVM are dependent on temporally regulated secretion from three different secretory organelles, the rhoptries, micronemes, and dense granules. Understanding the process of secretion and the localization and function of secreted parasite proteins provides important insights into the pathogenesis of infections with T. gondii. For this reason, we have been studying the structure and function of the PVM and of secreted parasite proteins in the vacuolar space and membrane. Our initial results showed that the newly formed PVM is fusion incompetent, being incapable of fusing with any vesicular organelle of the host endocytic apparatus.' These experiments extended the pioneering work of Jones and Hirsch2 as well as the later experiments of Sibley et ~l. ,~ showing the absence of fusion of T. gondii vacuoles with lysosomes and the lack of vacuolar acidification. We hypothesized4 that vacuolar fusion incompetence results from the unique entry mechanism of the parasite into cells" and the associated exclusion from the PVM of host cell membrane proteins that could serve as signals for fusion. The PVM, however, is modified during and after invasion by parasite proteins that behave as integral membrane proteins in the PVM and are presumed to play important roles in PVM function. Our focus over the last several years has been the identification and characterization of selected secreted parasite proteins that are associated with the PVM or with the vacuolar space as well as with the basic function that the PVM and vacuolar proteins play in nutrient salvage and protein traffic events.
Identification of three novel Toxoplasma gondii rhoptry proteins
International Journal for Parasitology, 2014
The rhoptries are key secretory organelles from apicomplexan parasites that contain proteins involved in invasion and modulation of the host cell. Some rhoptry proteins are restricted to the posterior bulb (ROPs) and others to the anterior neck (RONs). As many rhoptry proteins have been shown to be key players in Toxoplasma invasion and virulence, it is important to identify, understand and characterize the biological function of the components of the rhoptries. In this report, we identified putative novel rhoptry candidate genes by identifying Toxoplasma genes with similar cyclical expression profiles as known rhoptry protein encoding genes across its cell cycle. Using this approach we identified two new rhoptry bulb (ROP47 and ROP48) and one new rhoptry neck protein (RON12). ROP47 is secreted and traffics to the host cell nucleus, RON12 was not detected at the moving junction during invasion. Deletion of ROP47 or ROP48 in a type II strain did not show major influence in in vitro growth or virulence in mice.
Interrelations between the Parasitophorous Vacuole of Toxoplasma gondii and Host Cell Organelles
Microscopy and Microanalysis, 2005
Toxoplasma gondii, the causative agent of toxoplasmosis, is capable of actively penetrating and multiplying in any nucleated cell of warm-blooded animals. Its survival strategies include escape from fusion of the parasitophorous vacuole with host cell lysosomes and rearrangement of host cell organelles in relation to the parasitophorous vacuole. In this article we report the rearrangement of host cell organelles and elements of the cytoskeleton of LLCMK2 cells, a lineage derived from green monkey kidney epithelial cells, in response to infection by T. gondii tachyzoites. Transmission electron microscopy made on flat embedded monolayers cut horizontally to the apical side of the cells or field emission scanning electron microscopy of monolayers scraped with scotch tape before sputtering showed that association of mitochondria to the vacuole is much less frequent than previously described. On the other hand, all parasitophorous vacuoles were surrounded by elements of the endoplasmic reticulum. These data were complemented by observations by laser scanning microscopy using fluorescent probes from mitochondria and endoplasmic reticulum and reinforced by three-dimensional reconstruction from serial sections observed by transmission electron microscopy and labeling of mitochondria and endoplasmic reticulum by fluorescent probes.
PROTEOMICS, 2006
Four rhoptry proteins (ROP) of Toxoplasma gondii previously identified with mAb have been affinity purified and analyzed by MS; the data obtained allowed the genomic sequences to be assigned to these proteins. As previously suggested for some of them by antibody crossreactivity, these proteins were shown to belong to a family, the prototype of which being ROP2. We describe here the proteins ROP2, 4, 5, and 7. These four proteins correspond to the most abundant products of a gene family that comprises several members which we have identified in genomic and EST libraries. Eight additional sequences were found and we have cloned four of them. All members of the ROP2 family contain a protein-kinase-like domain, but only some of them possess a bona fide kinase catalytic site. Molecular modeling of the kinase domain demonstrates the conservation of residues critical for the stabilization of the protein-kinase fold, especially within a hydrophobic segment described so far as transmembrane and which appears as an helix buried inside the protein. The concomitant synthesis of these ROPs by T. gondii tachyzoites suggests a specific role for each of these proteins, especially in the early interaction with the host cell upon invasion.