Targeting of soluble proteins to the rhoptries and micronemes in Toxoplasma gondii.

Rhoptry and microneme organelles of the protozoan parasite Toxoplasma gondii are closely associated with host cell adhesion/invasion and establishment of the intracellular parasitophorous vacuole. In order to study the targeting of proteins to these specialized secretory organelles, we have engineered green fluorescent protein (GFP) fusions to the rhoptry protein ROP1 and the microneme protein MIC3. Both chimeras are correctly targeted to the appropriate organelles, permitting deletion analysis to map protein subdomains critical for targeting. The propeptide and a central 146 amino acid region of ROP1 are sufficient to target GFP to the rhoptries. More extensive deletions result in a loss of rhoptry targeting; the GFP reporter is diverted into the parasitophorous vacuole via dense granules. Certain MIC3 deletion mutants were also secreted into the parasitophorous vacuole via dense granules, supporting the view that this route constitutes the default pathway in T. gondii, and that specific signals are required for sorting to rhoptries and micronemes. Deletions within the cysteine-rich central region of MIC3 cause this protein to be arrested at various locations within the secretory pathway, presumably due to improper folding. Although correctly targeted to the appropriate organelles in living parasites, ROP1-GFP and MIC3-GFP fusion proteins were not secreted during invasion. GFP fusion proteins were readily secreted from dense granules, however, suggesting that protein secretion from rhoptries and micronemes might involve more than a simple release of organellar contents.

[Identification and molecular characterization of a Toxoplasma gondii microneme]. / Mise en évidence et caractérisation moléculaire d'une adhésine (TgMIC3) des micronèmes de Toxoplasma gondii.

Protozoan of the phylum Apicomplexa are of high medical and veterinary importance, causing diseases such as malaria, toxoplasmosis and cryptosporidiosis. Invasive stages of apicomplexans possess organelles named micronemes, which are involved in the invasion process. We have recently characterized a protein in micronemes of Toxoplasma gondii, TgMIC3, which possess adhesive properties to host cell surface. Immunofluorescence analysis of T. gondii tachyzoite invasion showed that TgMIC3 is exocytosed and re-localised on the surface of the parasite during invasion. By being able to bind both the putative host cells and the parasites, TgMIC3 could be involved in invasion by acting as a bridge between the parasite and the host cell. Gene sequence analysis of TgMIC3 has revealed 5 partially overlapping EGF-like domains and a lectin binding-like domain, which can be involved in protein-protein or protein-carbohydrate interactions respectively. TgMIC3 is a homodimer synthetized with a N-terminal propeptide that is cleaved during trafficking to the organelle, presumably in the trans-Golgi network. The processing involves a serine protease and is required for correct binding function of TgMIC3. The exact role of this propeptide remains unexplained. It may be involved in the targetting of the protein to the micronemes by masking the region involved in interaction with membranes to avoid binding of the protein in the trafficking pathway.

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.

Assay 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.

Apical organelles and host-cell invasion by Apicomplexa.

Host-cell invasion by apicomplexan parasites involves the successive exocytosis of three different secretory organelles; namely micronemes, rhoptries and dense granules. The findings of recent studies have extended the structural homologies of each set of organelles between most members of the phylum and suggest shared functions of each set. Micronemes are apparently used for host-cell recognition, binding, and possibly motility; rhoptries for parasitophorous vacuole formation; and dense granules for remodeling the vacuole into a metabolically active compartment. In addition, gene cloning and sequencing have demonstrated conserved domains, which are likely to serve similar functions in the invasion process. This is especially true for microneme proteins containing thrombospondin-like domains, which are likely to be involved in binding to sulphated glycoconjugates. One such protein was recently shown to be required for the motility of Plasmodium sporozoites. These molecules have been shown to be shed on the parasite and/or cell surfaces during the invasion process in Plasmodium, Toxoplasma and Eimeria. For rhoptries and dense granules, the association between exocytosed proteins and the parasitophorous vacuole membrane had been analyzed extensively in Toxoplasma, as these proteins are likely to play a crucial role in metabolic interactions between the parasites and their host cells. The development of parasite transformation by gene transfection has provided powerful tools to analyze the fate and function(s) of the corresponding proteins.