Toxoplasma gondii: Molecular Cloning and Characterization of a Novel 18-kDa Secretory Antigen, TgMIC10

Hoff, Eleanor F; Cook, Susan H.; Sherman, Gale D.; Harper, Jill M.; Ferguson, David J. P.; Dubremetz, Jean‐François; Carruthers, Vern B.

doi:10.1006/expr.2000.4585

Cited by 42 publications

(17 citation statements)

References 37 publications

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“…5A). In extracellular sporozoites, sporoAMA1 localizes to the apical end of the parasite, similar to the pattern seen with MIC10 and similar to what has been observed in tachyzoites [32], [33], [34]. Attempts to stain unpermeabilized sporozoites gave highly variable patterns that were not considered reliable (data not shown).…”

Section: Resultssupporting

confidence: 69%

Toxoplasma gondii Sporozoites Invade Host Cells Using Two Novel Paralogues of RON2 and AMA1

et al. 2013

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Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. The interaction of two well-studied proteins, Apical Membrane Antigen 1 (AMA1) and Rhoptry Neck protein 2 (RON2), has been shown to be critical for invasion by the asexual tachyzoite stage. Recently, two paralogues of these proteins, dubbed sporoAMA1 and sporoRON2 (or RON2L2), respectively, have been identified but not further characterized in proteomic and transcriptomic analyses of Toxoplasma sporozoites. Here, we show that sporoAMA1 and sporoRON2 localize to the apical region of sporozoites and that, in vitro, they interact specifically and exclusively, with no detectable interaction of sporoAMA1 with generic RON2 or sporoRON2 with generic AMA1. Structural studies of the interacting domains of sporoRON2 and sporoAMA1 indicate a novel pairing that is similar in overall form but distinct in detail from the previously described interaction of the generic pairing. Most notably, binding of sporoRON2 domain 3 to domains I/II of sporoAMA1 results in major alterations in the latter protein at the site of binding and allosterically in the membrane-proximal domain III of sporoAMA1 suggesting a possible role in signaling. Lastly, pretreatment of sporozoites with domain 3 of sporoRON2 substantially impedes their invasion into host cells while having no effect on tachyzoites, and vice versa for domain 3 of generic RON2 (which inhibits tachyzoite but not sporozoite invasion). These data indicate that sporozoites and tachyzoites each use a distinct pair of paralogous AMA1 and RON2 proteins for invasion into host cells, possibly due to the very different environment in which they each must function.

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Section: Resultssupporting

confidence: 69%

Toxoplasma gondii Sporozoites Invade Host Cells Using Two Novel Paralogues of RON2 and AMA1

et al. 2013

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“…The presence of an N-terminal signal peptide sequence and the fact that NcMIC1 was sequestered almost exclusively into the Triton X-114 hydrophilic fraction following detergent extraction suggested that, in analogy to other microneme proteins, NcMIC1 was secreted and released by the parasite into the environment as a soluble molecule. In T. gondii, it has been shown that initial contact of tachyzoites to host cells is followed by Ca 2ϩ signaling and that this rise in intracellular Ca 2ϩ is required for microneme secretion and invasion (2,4,9,22,25,36). We found that NcMIC1 is indeed secreted into the medium as a soluble molecule upon incubation of tachyzoites at 37°C (Fig.…”

Section: Discussionmentioning

confidence: 59%

“…Binding of NcMIC3 onto the host cell surface was shown to be mediated through its four consecutive epidermal growth factor-like domains (27). Third, NcMIC10 was identified by Hoff et al (22) according to its sequence similarity to TgMIC10. These two proteins do not possess any adhesive domains, and their putative function is largely unknown.…”

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confidence: 99%

Identification of a Neospora caninum Microneme Protein (NcMIC1) Which Interacts with Sulfated Host Cell Surface Glycosaminoglycans

et al. 2002

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The invasive stages of apicomplexan parasites enter their host cells through mechanisms which are largely conserved throughout the phylum. Host cell invasion is divided into two distinct events, namely, adhesion onto the host cell surface and the actual host cell entry process. The former is mediated largely through microneme proteins which are secreted at the onset of establishing contact with the host cell surface. Many of the microneme proteins identified so far contain adhesive domains. We here present the genomic and corresponding cDNA sequences coding for a 460-amino-acid (aa) microneme protein in Neospora caninum tachyzoites which, due to its homology to MIC1 in Toxoplasma gondii (TgMIC1), was named NcMIC1. The deduced NcMIC1 polypeptide sequence contains an N-terminal signal peptide of 20 aa followed by two tandemly internal repeats of 48 and 44 aa, respectively. Integrated into each repeat is a CXXXCG sequence motif reminiscent of the thrombospondin-related family of adhesive proteins. The positioning of this motif is strictly conserved in TgMIC1 and NcMIC1. The C-terminal part, comprised of 278 aa, was expressed in Escherichia coli, and antibodies affinity purified on recombinant NcMIC1 were used to confirm the localization within the micronemes by immunofluorescence and immunogold transmission electron microscopy of tachyzoites. Immunohistochemistry of mouse brains infected with tissue cysts showed that expression of this protein is reduced in the bradyzoite stage. Upon initiation of secretion by elevating the temperature to 37°C, NcMIC1 is released into the medium supernatant. NcMIC1 binds to trypsinized, rounded Vero cells, as well as to Vero cell monolayers. Removal of glycosaminoglycans from the host cell surface and modulation of host cell surface glycosaminoglycan sulfation significantly reduces the binding of NcMIC1 to the host cell surface. Solid-phase binding assays employing defined glycosaminoglycans confirmed that NcMIC1 binds to sulfated glycosaminoglycans.

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“…Southern blotting. Southern blots were performed essentially as described previously (25), with the following modifications. The MIC5 probe was amplified from a T. gondii cDNA library with primers MIC5.45.f (GCTGGTGTGGGTT CTTGATGTAGT) and MIC5.379.r (CCGGGATGTTCTGCCTGTC).…”

Section: Methodsmentioning

confidence: 99%

Targeted Deletion of MIC5 Enhances Trimming Proteolysis of Toxoplasma Invasion Proteins

et al. 2006

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Limited proteolysis of proteins transiently expressed on the surface of the opportunistic pathogen Toxoplasma gondii accompanies cell invasion and facilitates parasite migration across cell barriers during infection. However, little is known about what factors influence this specialized proteolysis or how these proteolytic events are regulated. Here we show that genetic ablation of the micronemal protein MIC5 enhances the normal proteolytic processing of several micronemal proteins secreted by Toxoplasma tachyzoites. Restoring MIC5 expression by genetic complementation reversed this phenotype, as did treatment with the protease inhibitor ALLN, which was previously shown to block the activity of a hypothetical parasite surface protease called MPP2. We show that, despite its lack of obvious membrane association signals, MIC5 occupies the parasite surface during invasion in the vicinity of the proteins affected by enhanced processing. Proteolysis of other secretory proteins, including GRA1, was also enhanced in MIC5 knockout parasites, indicating that the phenotype is not strictly limited to proteins derived from micronemes. Together, our findings suggest that MIC5 either directly regulates MPP2 activity or it influences MPP2's ability to access substrate cleavage sites on the parasite surface.Members of the phylum Apicomplexa are obligate intracellular parasites that replicate in a variety of cell types. Some, including the human pathogens Plasmodium and Babesia, replicate primarily in the bloodstream, whereas others such as Cryptosporidium, a cause of chronic gastritis among the immune-compromised, and Eimeria, an agricultural parasite, replicate in the intestinal epithelium. Only parasites in the isosporoid coccidian clade, which includes Toxoplasma gondii, the causative agent of toxoplasmosis, replicate in deep tissues (2). Thus, it can be expected that each clade has its own complement of proteins facilitating survival in a specific habitat, along with conserved proteins that play fundamental roles in events common to all apicomplexans.Many invasion studies have been performed in T. gondii, as it is more amenable to in vitro manipulation than other members of the Apicomplexa. Upon contact with a host cell, T. gondii tachyzoites discharge the contents of apically localized microneme (MIC) organelles (13). MIC adhesive proteins contribute to binding host cell receptors, and blocking micronemal secretion dramatically reduces invasion (9). These proteins, which often cluster into multiunit complexes, are transiently deployed to the apical surface during apical attachment and invasion. Transmembrane (TM) MIC proteins, such as MIC2, MIC6, and MIC8, are thought to act as bridging molecules that connect host receptors with the parasite's motility apparatus, the glideosome (37). By translocating MIC-receptor complexes backwards toward the posterior end, the parasite "pulls" itself into the target cell, invaginating the host plasma membrane to form the nascent parasitophorous vacuole (PV). As they treadmill posteriorly,...

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Toxoplasma gondii: Molecular Cloning and Characterization of a Novel 18-kDa Secretory Antigen, TgMIC10

Cited by 42 publications

References 37 publications

Toxoplasma gondii Sporozoites Invade Host Cells Using Two Novel Paralogues of RON2 and AMA1

Toxoplasma gondii Sporozoites Invade Host Cells Using Two Novel Paralogues of RON2 and AMA1

Identification of a Neospora caninum Microneme Protein (NcMIC1) Which Interacts with Sulfated Host Cell Surface Glycosaminoglycans

Targeted Deletion of MIC5 Enhances Trimming Proteolysis of Toxoplasma Invasion Proteins

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