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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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Disruption of MIC1, MIC4, and MIC6 genes in T. gondii tachyzoites by double homologous recombination. Western blot analysis of an equal loading of whole cell lysates corresponding to 5 × 106 tachyzoites from RH, mic1ko, mic4ko, and mic6ko. In A–C, membranes were probed with rabbit antibodies anti-MIC4, anti-MIC6, and mAb anti-MIC-1, respectively.
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Figure 2: Disruption of MIC1, MIC4, and MIC6 genes in T. gondii tachyzoites by double homologous recombination. Western blot analysis of an equal loading of whole cell lysates corresponding to 5 × 106 tachyzoites from RH, mic1ko, mic4ko, and mic6ko. In A–C, membranes were probed with rabbit antibodies anti-MIC4, anti-MIC6, and mAb anti-MIC-1, respectively.

Mentions: To investigate the function of the micronemal proteins in T. gondii, we have generated mutant parasite cell lines in which MIC1, MIC4, or MIC6 genes have been disrupted by gene replacement. The vectors used to generate these knockouts contained ≥1,500 bp of homologous sequences corresponding to the 5′ and 3′ flanking sequences of MICs genes. The selectable marker gene cassette used in those experiments was HXGPRT controlled by DHFRTS flanking sequences (Donald and Roos 1998). Parasite clones resistant to mycophenolic acid were examined for the absence of MIC1, MIC4, or MIC6, respectively, by IFA. 10–20% of the resistant clones appeared to have recombined homologously in the case of MIC4 and MIC6. One clone of mic1ko, mic4ko, and mic6ko were kept for further analysis. The absence of MIC1, MIC4, and MIC6 proteins in mutant cell lines was confirmed by Western blot analysis. The rabbit serum anti-MIC4 and the antisera raised against the CD of MIC6 recognized two bands corresponding to processed forms of MIC4 and MIC6, respectively. In the knockout strains, both signals disappeared, confirming that the two forms are indeed processed forms of the same gene products (Fig. 2). Southern blot analysis established that disruption of these three micronemal genes occurred by double homologous recombination events (data not shown).


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)

Disruption of MIC1, MIC4, and MIC6 genes in T. gondii tachyzoites by double homologous recombination. Western blot analysis of an equal loading of whole cell lysates corresponding to 5 × 106 tachyzoites from RH, mic1ko, mic4ko, and mic6ko. In A–C, membranes were probed with rabbit antibodies anti-MIC4, anti-MIC6, and mAb anti-MIC-1, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196004&req=5

Figure 2: Disruption of MIC1, MIC4, and MIC6 genes in T. gondii tachyzoites by double homologous recombination. Western blot analysis of an equal loading of whole cell lysates corresponding to 5 × 106 tachyzoites from RH, mic1ko, mic4ko, and mic6ko. In A–C, membranes were probed with rabbit antibodies anti-MIC4, anti-MIC6, and mAb anti-MIC-1, respectively.
Mentions: To investigate the function of the micronemal proteins in T. gondii, we have generated mutant parasite cell lines in which MIC1, MIC4, or MIC6 genes have been disrupted by gene replacement. The vectors used to generate these knockouts contained ≥1,500 bp of homologous sequences corresponding to the 5′ and 3′ flanking sequences of MICs genes. The selectable marker gene cassette used in those experiments was HXGPRT controlled by DHFRTS flanking sequences (Donald and Roos 1998). Parasite clones resistant to mycophenolic acid were examined for the absence of MIC1, MIC4, or MIC6, respectively, by IFA. 10–20% of the resistant clones appeared to have recombined homologously in the case of MIC4 and MIC6. One clone of mic1ko, mic4ko, and mic6ko were kept for further analysis. The absence of MIC1, MIC4, and MIC6 proteins in mutant cell lines was confirmed by Western blot analysis. The rabbit serum anti-MIC4 and the antisera raised against the CD of MIC6 recognized two bands corresponding to processed forms of MIC4 and MIC6, respectively. In the knockout strains, both signals disappeared, confirming that the two forms are indeed processed forms of the same gene products (Fig. 2). Southern blot analysis established that disruption of these three micronemal genes occurred by double homologous recombination events (data not shown).

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