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Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii.

Reiss M, Viebig N, Brecht S, Fourmaux MN, Soete M, Di Cristina M, Dubremetz JF, Soldati D - J. Cell Biol. (2001)

Bottom Line: MIC4 binds directly to MIC1 and behaves as a passive cargo molecule.MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act.We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Biology, University of Heidelberg, Heidelberg D-63120, Germany.

ABSTRACT
The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. Micronemes are apical secretory organelles releasing their contents upon contact with host cells. We have identified a transmembrane micronemal protein MIC6, which functions as an escorter for the accurate targeting of two soluble proteins MIC1 and MIC4 to the micronemes. Disruption of MIC1, MIC4, and MIC6 genes allowed us to precisely dissect their contribution in sorting processes. We have mapped domains on these proteins that determine complex formation and targeting to the organelle. MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. In contrast, MIC1 is linked to a quality control system and is absolutely required for the complex to leave the early compartments of the secretory pathway. MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act. We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.

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MIC1 and MIC4 are mistargeted in mic6ko and accumulate in the dense granules and the parasitophorous vacuole. (A) Mistargeting of MIC1 and MIC4 was analyzed by confocal microscopy of HFF cells infected with mic6ko and compared with RH. In wild-type parasites, T101F7 mAb anti-MIC1 and polyclonal anti-MIC4 showed perfect colocalization of the two proteins and a typical apical micronemes staining. In contrast, MIC4 did not colocalize with the other micronemal protein MIC2 (mAb T34A11) in mic6ko parasites but instead accumulated in dense granules and in the parasitophorous vacuolar space. Under mild fixation conditions, the vacuolar signal of MIC4 is lost, and the remaining MIC4 colocalizes with the dense granule marker GRA3 (mAb T26H11, in red). (B) The vacuolar accumulation of MIC4 in mic6ko was confirmed by immunolocalization of MIC4 in mic6ko. The label is over the dense material found at the posterior end of a tachyzoite fixed early after invasion, typical of dense granules exocytosis. T, tachyzoite; V, parasitophorous vacuole; H, host cell. (C) Schematic representation of the secretory pathway in a tachyzoite. The nucleus (gray), the ER (dark green), the Golgi apparatus (light green), the apicoplast (pink), the dense granules (blue), the rhoptries (yellow), and the micronemes (red). (D) Schematic representation eight parasites arranged in rosette within the parasitophorous vacuole after three cell divisions. Bars: (A) 1 μm; (B) 0.2 μm.
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Figure 3: MIC1 and MIC4 are mistargeted in mic6ko and accumulate in the dense granules and the parasitophorous vacuole. (A) Mistargeting of MIC1 and MIC4 was analyzed by confocal microscopy of HFF cells infected with mic6ko and compared with RH. In wild-type parasites, T101F7 mAb anti-MIC1 and polyclonal anti-MIC4 showed perfect colocalization of the two proteins and a typical apical micronemes staining. In contrast, MIC4 did not colocalize with the other micronemal protein MIC2 (mAb T34A11) in mic6ko parasites but instead accumulated in dense granules and in the parasitophorous vacuolar space. Under mild fixation conditions, the vacuolar signal of MIC4 is lost, and the remaining MIC4 colocalizes with the dense granule marker GRA3 (mAb T26H11, in red). (B) The vacuolar accumulation of MIC4 in mic6ko was confirmed by immunolocalization of MIC4 in mic6ko. The label is over the dense material found at the posterior end of a tachyzoite fixed early after invasion, typical of dense granules exocytosis. T, tachyzoite; V, parasitophorous vacuole; H, host cell. (C) Schematic representation of the secretory pathway in a tachyzoite. The nucleus (gray), the ER (dark green), the Golgi apparatus (light green), the apicoplast (pink), the dense granules (blue), the rhoptries (yellow), and the micronemes (red). (D) Schematic representation eight parasites arranged in rosette within the parasitophorous vacuole after three cell divisions. Bars: (A) 1 μm; (B) 0.2 μm.

Mentions: Recombinant parasites lacking MIC6 (mic6ko) showed normal growth and appeared to bind and invade HFF cells like the parent strain in the cell culture conditions tested so far. Upon analysis of other micronemal proteins by IFA, we observed that two soluble proteins MIC1 and MIC4 failed to accumulate in the micronemes in absence of MIC6. Instead, as shown in Fig. 3 A (and see Fig. 5 A), both MIC1 and MIC4 are rerouted into the default secretory pathway, which in T. gondii traffics through the dense granules and ends in the parasitophorous vacuole (Karsten et al. 1998). Mild fixation protocols were used to visualize the presence of MIC4 and its colocalization with GRA3 in the dense granules (Bermudes et al. 1994). In contrast, the transmembrane protein MIC2 and two other micronemal proteins exhibiting no putative transmembrane domain, MIC3 (Garcia-Réguet et al. 2001), or MIC5 (Brydges et al. 2000) were faithfully targeted to the micronemes in mic6ko (data not shown). Analysis of the mic6ko by transmission electron microscopy confirmed the unprecedented localization of the micronemal proteins MIC4 (Fig. 3 B) and MIC1 (not shown) in the parasitophorous vacuolar space. The distinct compartments of the secretory pathway in T. gondii are schematically drawn in Fig. 3 C. After few divisions by endodyogeny, the parasites are organized in rosette within the parasitophorous vacuole (Fig. 3 D).


Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii.

Reiss M, Viebig N, Brecht S, Fourmaux MN, Soete M, Di Cristina M, Dubremetz JF, Soldati D - J. Cell Biol. (2001)

MIC1 and MIC4 are mistargeted in mic6ko and accumulate in the dense granules and the parasitophorous vacuole. (A) Mistargeting of MIC1 and MIC4 was analyzed by confocal microscopy of HFF cells infected with mic6ko and compared with RH. In wild-type parasites, T101F7 mAb anti-MIC1 and polyclonal anti-MIC4 showed perfect colocalization of the two proteins and a typical apical micronemes staining. In contrast, MIC4 did not colocalize with the other micronemal protein MIC2 (mAb T34A11) in mic6ko parasites but instead accumulated in dense granules and in the parasitophorous vacuolar space. Under mild fixation conditions, the vacuolar signal of MIC4 is lost, and the remaining MIC4 colocalizes with the dense granule marker GRA3 (mAb T26H11, in red). (B) The vacuolar accumulation of MIC4 in mic6ko was confirmed by immunolocalization of MIC4 in mic6ko. The label is over the dense material found at the posterior end of a tachyzoite fixed early after invasion, typical of dense granules exocytosis. T, tachyzoite; V, parasitophorous vacuole; H, host cell. (C) Schematic representation of the secretory pathway in a tachyzoite. The nucleus (gray), the ER (dark green), the Golgi apparatus (light green), the apicoplast (pink), the dense granules (blue), the rhoptries (yellow), and the micronemes (red). (D) Schematic representation eight parasites arranged in rosette within the parasitophorous vacuole after three cell divisions. Bars: (A) 1 μm; (B) 0.2 μm.
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Figure 3: MIC1 and MIC4 are mistargeted in mic6ko and accumulate in the dense granules and the parasitophorous vacuole. (A) Mistargeting of MIC1 and MIC4 was analyzed by confocal microscopy of HFF cells infected with mic6ko and compared with RH. In wild-type parasites, T101F7 mAb anti-MIC1 and polyclonal anti-MIC4 showed perfect colocalization of the two proteins and a typical apical micronemes staining. In contrast, MIC4 did not colocalize with the other micronemal protein MIC2 (mAb T34A11) in mic6ko parasites but instead accumulated in dense granules and in the parasitophorous vacuolar space. Under mild fixation conditions, the vacuolar signal of MIC4 is lost, and the remaining MIC4 colocalizes with the dense granule marker GRA3 (mAb T26H11, in red). (B) The vacuolar accumulation of MIC4 in mic6ko was confirmed by immunolocalization of MIC4 in mic6ko. The label is over the dense material found at the posterior end of a tachyzoite fixed early after invasion, typical of dense granules exocytosis. T, tachyzoite; V, parasitophorous vacuole; H, host cell. (C) Schematic representation of the secretory pathway in a tachyzoite. The nucleus (gray), the ER (dark green), the Golgi apparatus (light green), the apicoplast (pink), the dense granules (blue), the rhoptries (yellow), and the micronemes (red). (D) Schematic representation eight parasites arranged in rosette within the parasitophorous vacuole after three cell divisions. Bars: (A) 1 μm; (B) 0.2 μm.
Mentions: Recombinant parasites lacking MIC6 (mic6ko) showed normal growth and appeared to bind and invade HFF cells like the parent strain in the cell culture conditions tested so far. Upon analysis of other micronemal proteins by IFA, we observed that two soluble proteins MIC1 and MIC4 failed to accumulate in the micronemes in absence of MIC6. Instead, as shown in Fig. 3 A (and see Fig. 5 A), both MIC1 and MIC4 are rerouted into the default secretory pathway, which in T. gondii traffics through the dense granules and ends in the parasitophorous vacuole (Karsten et al. 1998). Mild fixation protocols were used to visualize the presence of MIC4 and its colocalization with GRA3 in the dense granules (Bermudes et al. 1994). In contrast, the transmembrane protein MIC2 and two other micronemal proteins exhibiting no putative transmembrane domain, MIC3 (Garcia-Réguet et al. 2001), or MIC5 (Brydges et al. 2000) were faithfully targeted to the micronemes in mic6ko (data not shown). Analysis of the mic6ko by transmission electron microscopy confirmed the unprecedented localization of the micronemal proteins MIC4 (Fig. 3 B) and MIC1 (not shown) in the parasitophorous vacuolar space. The distinct compartments of the secretory pathway in T. gondii are schematically drawn in Fig. 3 C. After few divisions by endodyogeny, the parasites are organized in rosette within the parasitophorous vacuole (Fig. 3 D).

Bottom Line: MIC4 binds directly to MIC1 and behaves as a passive cargo molecule.MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act.We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.

View Article: PubMed Central - PubMed

Affiliation: Center for Molecular Biology, University of Heidelberg, Heidelberg D-63120, Germany.

ABSTRACT
The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. Micronemes are apical secretory organelles releasing their contents upon contact with host cells. We have identified a transmembrane micronemal protein MIC6, which functions as an escorter for the accurate targeting of two soluble proteins MIC1 and MIC4 to the micronemes. Disruption of MIC1, MIC4, and MIC6 genes allowed us to precisely dissect their contribution in sorting processes. We have mapped domains on these proteins that determine complex formation and targeting to the organelle. MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. In contrast, MIC1 is linked to a quality control system and is absolutely required for the complex to leave the early compartments of the secretory pathway. MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act. We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.

Show MeSH
Related in: MedlinePlus