Characterization of a family of rhoptry proteins ofToxoplasma gondii

Sadak, Abderrahim; Taghy, Zoubida; Fortier, B; Dubremetz, Jean‐François

doi:10.1016/0166-6851(88)90075-8

Cited by 166 publications

(117 citation statements)

References 19 publications

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“…A second wave of secretion occurs from apical club-shaped organelles dubbed rhoptries [6] ( Figure 1A). Rhoptry proteins (ROP) were first identified based on monoclonal antibodies generated to cell fractions enriched in this organelle [7]. Rhoptry proteins are trafficked from the ER through the Golgi by a conserved pathway [8] prior to being packaged into the apically located secretory organelles.…”

Section: Rhoptries: An Apical Secretory Organelle Involved In Invasionmentioning

confidence: 99%

Rhoptries: an arsenal of secreted virulence factors

Bradley

Sibley

2007

Current Opinion in Microbiology

177

142

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Apicomplexan parasites use actin-based motility coupled with regulated protein secretion from apical organelles to actively invade host cells. Critical in this process are rhoptries, club-shaped secretory organelles that discharge their contents during parasite invasion into host cells. A proteomic analysis of the rhoptries in Toxoplasma gondii demonstrated that this organelle contains a number of novel rhoptry proteins (ROP) including serine-threonine kinases and protein phosphatases. A subset of ROP proteins have been shown to target to the moving junction, which plays a key role in invasion and parasitophorous vacuole formation. Other ROP proteins have various destinations in the host cell including the host cell nucleus and the parasitophorous vacuole, likely reflecting their distinct targets and roles. Forward genetic analysis recently revealed that secretory ROP kinases dramatically influence host gene expression and are the major parasite virulence factors. Thus, ROP proteins are functionally analogous (although not homologous) to effectors released by type III and IV secretion systems, which are factors that play an important role in bacterial virulence. Deciphering the role of ROP effectors may allow specific disruption of these factors, thus offering new options for preventing disease.

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Section: Rhoptries: An Apical Secretory Organelle Involved In Invasionmentioning

confidence: 99%

Rhoptries: an arsenal of secreted virulence factors

Bradley

Sibley

2007

Current Opinion in Microbiology

177

142

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“…MAbs T4 2F3 and T4 1E5 were isolated during the screening of hybridomas produced against T. gondii as described elsewhere (Couvreur et al, 1988;Sadak et al, 1988). MAb T8 2C10 was obtained independently using similar procedures (Tomavo et al, 1991).…”

Section: Antibodiesmentioning

confidence: 99%

The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite

Lebrun²,

et al. 2000

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SummaryAssay of the adhesion of cultured cells on Toxoplasma gondii tachyzoite protein Western blots identified a major adhesive protein, that migrated at 90 kDa in non-reducing gels. This band comigrated with the previously described microneme protein MIC3. Cellular binding on Western blots was abolished by MIC3-specific monoclonal and polyclonal antibodies. The MIC3 protein affinity purified from tachyzoite lysates bound to the surface of putative host cells. In addition, T. gondii tachyzoites also bound to immobilized MIC3. Immunofluorescence analysis of T. gondii tachyzoite invasion showed that MIC3 was exocytosed and relocalized to the surface of the parasite during invasion. The cDNA encoding MIC3 and the corresponding gene have been cloned, allowing the determination of the complete coding sequence. The MIC3 sequence has been confirmed by affinity purification of the native protein and N-terminal sequencing. The deduced protein sequence contains five partially overlapping EGF-like domains and a chitin binding-like domain, which can be involved in protein±protein or protein± carbohydrate interactions. Taken together, these results suggest that MIC3 is a new microneme adhesin of T. gondii.

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“…Cells were washed in PBS, permeabilized for 5 min in 0.25% Triton X-100 in PBS, and washed three additional times in PBS. Cells were incubated for 1 h with primary antibody diluted in PBS with 3% bovine serum albumin using anti-ROP2/3/4 mAb T3 4A7 (14) or antihemagglutinin (HA) epitope tag monoclonal antibody for overexpressing wild-type Tg1-HA, dominant negative Tg1-HA(D176A) 2 or ROP2⌬-HA mutant parasites (24). Toxopain-1 localization was performed with mAb CP1.7 (2 mg/ml) directly labeled with Alexa 594 per kit instructions (Molecular Probes, Eugene, OR) before adding the second antibody.…”

Section: Methodsmentioning

confidence: 99%

“…How rhoptry proteins directly contribute to the invasion process is still not clear. T. gondii rhoptry proteins are synthesized as prepro proteins that are subject to proteolytic cleavage to remove the presequence and the proregion at the N terminus and then processed to their mature forms (11)(12)(13)(14). Rhoptries and pre-rhoptries are the only acidified organelles identified in the parasite (15), contain a high lipid to protein ratio (16), and scavenge sterols from the host cell (17).…”

mentioning

confidence: 99%

The Cathepsin B of Toxoplasma gondii,Toxopain-1, Is Critical for Parasite Invasion and Rhoptry Protein Processing

Que¹,

Ngô²,

Lawton³

et al. 2002

Journal of Biological Chemistry

100

130

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Cysteine proteinases play a major role in invasion and intracellular survival of a number of pathogenic parasites. We cloned a single copy gene, tgcp1, from Toxoplasma gondii and refolded recombinant enzyme to yield active proteinase. Substrate specificity of the enzyme and homology modeling identified the proteinase as a cathepsin B. Specific cysteine proteinase inhibitors interrupted invasion by tachyzoites. The T. gondii cathepsin B localized to rhoptries, secretory organelles required for parasite invasion into cells. Processing of the pro-rhoptry protein 2 to mature rhoptry proteins was delayed by incubation of extracellular parasites with a cathepsin B inhibitor prior to pulse-chase immunoprecipitation. Delivery of cathepsin B to mature rhoptries was impaired in organisms with disruptions in rhoptry formation by expression of a dominant negative 1-adaptin. Similar disruption of rhoptry formation was observed when infected fibroblasts were treated with a specific inhibitor of cathepsin B, generating small and poorly developed rhoptries. This first evidence for localization of a cysteine proteinase to the unusual rhoptry secretory organelle of an apicomplexan parasite suggests that the rhoptries may be a prototype of a lysosome-related organelle and provides a critical link between cysteine proteinases and parasite invasion for this class of organism.The protozoan, Toxoplasma gondii, is an obligate intracellular parasite that can invade and replicate within any nucleated cell of vertebrate hosts, including humans (1-3). Invasion by T. gondii tachyzoites is mediated by the sequential regulated release of specialized secretory organelles of the parasite including the micronemes, rhoptries, and dense granules (4). In early observations, the penetration of host cells by tachyzoites was enhanced by the addition of partially purified lysosomal enzymes (5). In addition, proteinases have been implicated in host cell invasion in other members of the Apicomplexa such as Plasmodium (6) and Eimeria tenella (7). We now show that a cathepsin B, toxopain-1, is strongly implicated in T. gondii invasion and that infection can be interrupted with specific cathepsin B inhibitors.Another unusual feature of Toxoplasma is the absence of a morphologically identifiable lysosomal system. In higher eukaryotic cells, acidic cathepsins in lysosomes are important in protein processing and breakdown. The mammalian precursor of cathepsin B is targeted to the lysosomal compartment by mannose 6-P for proteolytic activation (8). Thus, the apparent lack of a lysosomal system in Toxoplasma raises a number of questions regarding cellular proteinase functions within the parasite. The rhoptries are club-shaped organelles located at the apical end of the parasites with no known counterpart outside of the phylum Apicomplexa (9). Although nine rhoptry proteins (ROP1-9) 1 have been identified to date (10), a definitive function has only been determined for ROP2, which mediates binding of host mitochondria to the parasitophorous membrane (11). How rho...

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Characterization of a family of rhoptry proteins ofToxoplasma gondii

Cited by 166 publications

References 19 publications

Rhoptries: an arsenal of secreted virulence factors

Rhoptries: an arsenal of secreted virulence factors

The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite

The Cathepsin B of Toxoplasma gondii,Toxopain-1, Is Critical for Parasite Invasion and Rhoptry Protein Processing

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