Functional domains of the Toxoplasma GRA2 protein in the formation of the membranous nanotubular network of the parasitophorous vacuole

Travier, Laetitia; Mondragón, Ricardo; Dubremetz, Jean‐François; Musset, Karine; Mondragón, Margarita; González, Sirenia; Cesbron-Delauw, Marie-France; Mercier, Corinne

doi:10.1016/j.ijpara.2007.10.010

Cited by 50 publications

(66 citation statements)

References 45 publications

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“…1A) in E. coli and isolated the Pop oligomers by gel filtration at pH 5.0, as previously described (26). These samples were subsequently incubated with artificial liposomes prepared with a 2:3 ratio of ␣-phosphatidyl-L-serine and 1,2-dipalmitoyl-snglycero-3-phosphocholine in the presence/absence of agents known to release peripheral membrane proteins (37,38). Experiments were performed with (a) PBS, pH 7.0, as a negative control, (b) 1 M KCl, (c) 6 M urea, and (d) 0.425 mM DDM (which corresponds to 2.5 ϫ critical micelle concentration (CMC)).…”

Section: Resultsmentioning

confidence: 99%

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa

Discola

Förster

Boulay

et al. 2014

Journal of Biological Chemistry

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Background:The type III secretion system allows bacteria to inject effectors directly into the host cytoplasm through a translocation pore. Results: Translocator proteins were characterized in lipid-bound and chaperone-associated forms. Conclusion: Pseudomonas translocators use different regions to recognize their common chaperone and the eukaryotic membrane. Significance: Mapping of key regions in translocators provides a starting point for the potential development of novel antibacterials.

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Section: Resultsmentioning

confidence: 99%

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa

Discola

Förster

Boulay

et al. 2014

Journal of Biological Chemistry

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“…This dual targeting is also seen with the dense granule proteins GRA3 and GRA7, both of which colocalize with GRA14 in the PV (29). Specific signals for targeting to the IVN or PVM have not been identified, but such signals are likely to exist, as some GRA proteins are found exclusively in one compartment or the other (e.g., GRA8 is detected only on the PVM and GRA2 is found only on the IVN) (8,45).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2008

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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.Capable of infecting essentially any warm-blooded vertebrate, Toxoplasma gondii is one of the most successful pathogens on the planet (20, 39). Toxoplasma infects nearly onethird of the human population and causes potentially fatal disease in immunocompromised individuals and congenitally infected neonates (20). This protozoan parasite also causes ocular disease in immunocompetent individuals who are either congenitally or postnatally infected (46). As an obligate intracellular parasite, Toxoplasma enters the host cell into a nonfusogenic vacuole (the parasitophorous vacuole [PV]), in which the parasite replicates in the cytoplasm of its host. The PV membrane (PVM) is porous to small molecules (less than 1,300 Da) but otherwise serves as a boundary between the host and parasite during its intracellular survival (36).Toxoplasma invasion is mediated by a trio of specialized secretory organelles, named the micronemes, rhoptries, and dense granules, which contribute to the parasite's ability to initiate and sustain infection within its host. The first proteins secreted are from the micronemes, which release molecular adhesins that interact with the parasite's actin-myosin motor to provide the driving force for invasion (24). The rhoptries are then released and help to establish the nascent PV and modulate host cell processes (4). Lastly, proteins from the dense granules that are implicated in the remodeling and maintenance of the PV for intracellular survival are secreted (29).The precise role of dense granule proteins (GRAs) in the T. gondii life cycle is still largely unknown. To date, two groups of GRA proteins have been identified. The first group contains proteins that lack homology to organisms other than closely relat...

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“…YFP tagging of this protein showed localization in the parasitophorous vacuole and, in particular, an overlap with the intravacuolar membranous nanotubular network (42), as revealed by colocalization with the dense granule protein GRA2 (24) (Fig. 4A, panel ii).…”

Section: Vol 8 2009 Endogenous Tagging Of T Gondii Genes 533mentioning

confidence: 93%

Tagging of Endogenous Genes in aToxoplasma gondiiStrain Lacking Ku80

2009

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As with other organisms with a completed genome sequence, opportunities for performing large-scale studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a ⌬ku80 strain. Ku80 is involved in DNA strand repair and nonhomologous DNA end joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a dihydrofolate reductasethymidylate synthase selectable marker. The YFP is preceded by a ligation-independent cloning (LIC) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrated that the ⌬ku80 strain is more effective and efficient in integrating the YFP-tagged constructs into the correct locus than wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and showed expression by immunoblotting. Our findings demonstrate that the combination of the ⌬ku80 strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should allow the opportunity for performing larger-scale studies of novel T. gondii genes.Toxoplasma gondii is an obligate intracellular protozoan in the phylum Apicomplexa that has garnered more intense study in recent decades. This is due in part to the genetic tractability and ease of growth of T. gondii and also because knowledge obtained for this parasite is often relevant to its kin, including other pathogens of medical and veterinary importance, such as Plasmodium falciparum, the parasite that causes malaria (11). The genome sequence of Toxoplasma was recently completed (10), opening the door to identifying novel genes involved in a variety of events, such as cell invasion, replication, gliding motility, metabolism, stage conversion, and virulence. With a large number of new genes of interest, it is essential to have tools that enable investigators to perform studies on a larger scale. One bottleneck for the analysis of novel genes is protein localization, since this generally necessitates time-consuming production of antibodies that often require additional affinity purification. Protein localization by ectopic expression of a tagged construct can also be problematic due to overexpression or mistiming of expression from a heterologous promoter. If genes could be tagged directly on the chromosome, this would better mimic natural expression and allow the use of fluorescent protein tags or standardized antibodies to monitor localization.DNA double-strand breaks...

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Functional domains of the Toxoplasma GRA2 protein in the formation of the membranous nanotubular network of the parasitophorous vacuole

Cited by 50 publications

References 45 publications

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa

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

Tagging of Endogenous Genes in aToxoplasma gondiiStrain Lacking Ku80

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