Invasion by Toxoplasma gondii Establishes a Moving Junction That Selectively Excludes Host Cell Plasma Membrane Proteins on the Basis of Their Membrane Anchoring (original) (raw)
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Proceedings of the National Academy of Sciences, 1996
Most intracellular pathogens avoid lysing their host cells during invasion by wrapping themselves in a vacuolar membrane. This parasitophorous vacuole membrane (PVM) is often retained, serving as a critical transport interface between the parasite and the host cell cytoplasm. To test whether the PVM formed by the parasite Toxoplasma gondii is derived from host cell membrane or from lipids secreted by the parasite, we used time-resolved capacitance measurements and video microscopy to assay host cell surface area during invasion. We observed no significant change in host cell surface area during PVM formation, demonstrating that the PVM consists primarily of invaginated host cell membrane. Pinching off of the PVM from the host cell membrane occurred after an unexpected delay (34-305 sec) and was seen as a 0.219 ± 0.006 pF drop in capacitance, which corresponds well to the predicted surface area of the entire PVM (30-33 Lim2). The formation and closure ofa fission pore
Molecular Biology of the Cell, 1999
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 in Toxoplasma 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.
Traffic, 2008
Toxoplasma gondii is the formation of a membranebound compartment within which the parasite proliferates. This process relies on a set of secretory organelles that discharge their contents into the host cell upon invasion. Among these organelles, the dense granules are specialized in the export of transmembrane (TM) GRA proteins, which are major components of the mature parasitophorous vacuole (PV) membrane. How eukaryotic pathogens export and sort membrane-bound proteins destined for the host cell is still poorly understood at the mechanistic level. In this study, we show that soluble trafficking of the PV-targeted GRA5 TM protein is parasite specific: when expressed in mammalian cells, GRA5 is targeted to the plasma membrane and behaves as an integral membrane protein with a type I toplogy. We also demonstrate the dual role of the GRA5 N-terminal ectodomain, which is sufficient to prevent membrane integration within the parasite and is essential for both sorting and post-secretory membrane insertion into the vacuolar membrane. These results contrast with the general rule that states that information contained within the cytoplasmic tail and/or the TM domain of integral membrane proteins dictates their cellular localization. They also highlight the diversity of sorting mechanisms that leads to the specialization of secretory processes uniquely adapted to intracellular parasitism.
Experimental Parasitology, 1999
3Ј-3-3Ј-tetramethylindocarbocyanine; DMEM, Dulbecco's modified minimal essential me-Toxoplasma gondii resides in a vacuole that avoids fusion with host cell dium; EM, electron microscopy; Hepes, N-[2-hydroxyethyl] piperaendocytic and exocytic vesicular trafficking pathways. Experimental zine-N Ј-[4-butanesulfonic acid]; HF, human fibroblasts; IF, Parasitology 92, 87-99. Toxoplasma gondii actively penetrates its immunofluorescence; LAMP1, lysosome-associated membrane protein vertebrate host cell to establish a nonfusigenic compartment called the 1; MEM, minimal essential medium; MHC, major histocompatability parasitophorous vacuole (PV) that has previously been characterized complex; C 6-NBD ceramide, N-[6-[(7-nitro-benz-2-oxa-l,3-diazol-4primarily in phagocytic cells. To determine the fate of this unique yl)amino]caproyl] sphingosine; NBD, nitrobenzadiazole; NSF, N-ethyl compartment in nonphagocytic cells, we examined the trafficking of maleamide sensitive factor; PBS, phosphate-buffered saline; PV, parasihost cell proteins and lipids in Toxoplasma-infected fibroblasts using tophorous vacuole; TfR, transferrin receptor; TGN, trans-Golgi netquantitative immunofluorescence and immunoelectron microscopy. work. Toxoplasma-containing vacuoles remained segregated from all levels of the endocytic pathway, as shown by the absence of delivery of transferrin receptors, mannose phosphate receptors, and the lysosomalassociated protein LAMP1 to the vacuole. The PV was also inaccessible to lipids (DiIC 16 , and GM1) that were internalized from the plasma membrane via the endocytic system. In contrast, vacuoles containing
The Journal of cell biology, 1998
All known proteins that accumulate in the vacuolar space surrounding the obligate intracellular protozoan parasite Toxoplasma gondii are derived from parasite dense granules. To determine if constitutive secretory vesicles could also mediate delivery to the vacuolar space, T. gondii was stably transfected with soluble Escherichia coli alkaline phosphatase and E. coli beta-lactamase. Surprisingly, both foreign secretory reporters were delivered quantitatively into parasite dense granules and efficiently secreted into the vacuolar space. Addition of a glycosylphosphatidylinositol membrane anchor rerouted alkaline phosphatase to the parasite surface. Alkaline phosphatase fused to the transmembrane domain and cytoplasmic tail from the endogenous dense granule protein GRA4 localized to dense granules. The protein was secreted into a tuboreticular network in the vacuolar space, in a fashion dependent upon the cytoplasmic tail, but not upon a tyrosine-based motif within the tail. Alkaline ...
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.
Signaling during the invasion of host cells by Toxoplasma gondii
FEMS Microbiology Reviews, 1999
Invasion of host cells is essential for the pathogenicity of Toxoplasma gondii. This review examines the signal transduction pathways that lead to the internalization of T. gondii. We demonstrate that extra-and intracellular Ca 2 mobilization, Ca 2calmodulin complex and phospholipase A 2 activities are required for T. gondii entry. T. gondii also causes the activation of mitogen-activated protein kinase in infected cells and modifies its ionic environment during its intracellular state. Thus, many of the signaling systems found in other eukaryotes are operative in Toxoplasma invasion.
Calcium ionophore-induced egress of Toxoplasma gondii shortly after host cell invasion
Veterinary Parasitology, 2007
Calcium plays crucial roles in important events of Toxoplasma gondii life cycle, including motility, invasion and egress from the host cell. Calcium ionophore has been used to artificially trigger release of the parasites from infected cells. In this report we describe that calcium ionophore A21387 induced T. gondii egress from LLC-MK2 cells at times as early as 2 h after entry. Addition of kinase inhibitors as staurosporine, wortmanine and genistein to the incubation medium significantly reduced ionophore-induced egress. The same occurred when the actin inhibitor cytochalasin D was used. Parasites egressed 2 h post-infection from ionophoretreated cultures were unable of establishing infection in a new cell. S-VHS recording of egressing parasites showed that they assume an hourglass shape as they cross the plasma membrane, similar to the moving junction constriction observed during active invasion, and extrudes the conoid, similarly to what is also observed during invasion. Transmission and high resolution scanning electron microscopy revealed that the egressing tachyzoites are free from host cell derived membranes. These include plasma membrane and parasitophorous vacuole membranes as well as associated endoplasmic reticulum membranes. Taken together, these results indicate that although invasion and egress may share similar signaling pathways, as indicated by the effect of kinase and actin inhibitors, the tachyzoites move freely in the cytosol, a phenomenon very distinctive from invasion and that deserves attention. #
BioMed Research International, 2014
The intracellular parasiteToxoplasma gondiican penetrate any warm-blooded animal cell. Conserved molecular assemblies of host cell plasma membranes should be involved in the parasite-host cell recognition. Lipid rafts are well-conserved membrane microdomains that contain high concentrations of cholesterol, sphingolipids, glycosylphosphatidylinositol, GPI-anchored proteins, and dually acylated proteins such as members of the Src family of tyrosine kinases. Disturbing lipid rafts of mouse peritoneal macrophages and epithelial cells of the lineage LLC-MK2 with methyl-beta cyclodextrin (MβCD) and filipin, which interfere with cholesterol or lidocaine, significantly inhibited internalization ofT. gondiiin both cell types, although adhesion remained unaffected in macrophages and decreased only in LLC-MK2 cells. Scanning and transmission electron microscopy confirmed these observations. Results are discussed in terms of the original role of macrophages as professional phagocytes versus the...