SummaryAssay 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.

Apicomplexan parasites invade cells by a unique mechanism involving discharge of secretory vesicles called micronemes. Microneme proteins (MICs) include transmembrane and soluble proteins expressing different adhesive domains. Although the transmembrane protein TRAP and its homologues are thought to bridge cell surface receptors and the parasite submembranous motor, little is known about the function of other MICs. We have addressed the role of MIC1 and MIC3, two soluble adhesins of Toxoplasma gondii, in invasion and virulence. Single deletion of the MIC1 gene decreased invasion in fibroblasts, whereas MIC3 deletion had no effect either alone or in the mic1KO context. Individual disruption of MIC1 or MIC3 genes slightly reduced virulence in the mouse, whereas doubly depleted parasites were severely impaired in virulence and conferred protection against subsequent challenge. Single substitution of two critical amino acids in the chitin binding–like (CBL) domain of MIC3 abolished MIC3 binding to cells and generated the attenuated virulence phenotype. Our findings identify the CBL domain of MIC3 as a key player in toxoplasmosis and reveal the synergistic role of MICs in virulence, supporting the idea that parasites have evolved multiple ligand–receptor interactions to ensure invasion of different cells types during the course of infection.

It was previously demonstrated that immunizing mice with spleen dendritic cells (DCs) that had been pulsed ex vivo with Toxoplasma gondii antigens triggers a systemic Th1-biased specific immune response and induces protection against infection. T. gondii can cause severe sequelae in the fetuses of mothers who acquire the infection during pregnancy, as well as life-threatening neuropathy in immunocompromised patients, in particular those with AIDS. Here, we investigate the efficacy of a novel cell-free vaccine composed of DC exosomes, which are secreted antigen-presenting vesicles that express functional major histocompatibility complex class I and II and T-cell-costimulatory molecules. They have already been shown to induce potent antitumor immune responses. We investigated the potential of DC2.4 cell line-derived exosomes to induce protective immunity against toxoplasmosis. Our data show that most adoptively transferred T. gondii-pulsed DC-derived exosomes were transferred to the spleen, elicited a strong systemic Th1-modulated Toxoplasma-specific immune response in vivo, and conferred good protection against infection. These findings support the possibility that DC-derived exosomes can be used for T. gondii immunoprophylaxis and for immunoprophylaxis against many other pathogens.

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Toxoplasma gondii is an obligate intracellular parasite that infects a wide variety of nucleated cells in its numerous intermediate hosts including man. The oral route is the natural portal of entry of T. gondii. Ingested organisms are released from cysts or oocysts within the gastrointestinal tract and initially invade the intestinal epithelium. We show that T. gondii invades and proliferates in cultured primary rat enterocytes, obtained with an original procedure. Activation of the enterocytes with rat recombinant interferon-gamma (IFN-gamma) inhibits T. gondii replication, the inhibition being dose dependent. Neither nitrogen and oxygen derivatives nor tryptophan starvation appear to be involved in the inhibition of parasite replication by IFN-gamma. Experiments using Fe2+ salt, carrier and chelator indicate that intracellular T. gondii replication is iron dependent, suggesting that IFN-gamma-treated enterocytes inhibit T. gondii replication by limiting the availability of intracellular iron to the parasite. Our data show that enterocytes probably play a major role on mucosal surfaces as a first line of defence against this coccidia, and possibly other pathogens, through an immune mechanism. The results suggest that limiting the availability of iron could represent a broad antimicrobial mechanism through which the activated enterocytes exert control over intracellular pathogens.

Attachment and invasion of host cells by apicomplexan parasites involve the exocytosis of the micronemal proteins (MICs). Most MICs are adhesins, which show homology with adhesive domains from higher eukaryote proteins and undergo proteolytic processing of unknown biological signi®cance during their transport to micronemes. In Toxoplasma gondii, the micronemal homodimeric protein MIC3 is a potent adhesin that displays features shared by most Apicomplexa MICs. We have developed an original MIC3-binding assay by transfection of mammalian cells with complete or truncated MIC3 gene sequences and demonstrated that the receptor binding site of MIC3 is located in the N-terminal chitin-binding-like domain, which remains poorly accessible until the adjacent pro-peptide has been cleaved, and that binding requires dimerization. We have localized the dimerization domain in the C-terminal end of the protein and shown that it is able to convert MIC8, a monomeric micronemal protein sharing the MIC3 lectin-like domain, into a dimer able to interact with host cell receptors. These ®ndings shed new light on molecular mechanisms that control functional maturation of MICs.

Oral infection with Toxoplasma gondii leads to transient systemic hyporesponsiveness. In this report, we characterized the presence in the lungs of GR1 ؉ CD11b ؉ myeloid cells that have potent nitric oxide-dependent immunoregulatory properties. We also demonstrated the interleukin 2-reversible anergy of both pulmonary CD8؉ and CD4 ؉ activated T lymphocytes with infection.

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. The micronemal protein MIC3, which is a potent adhesin of T. gondii, could be a significant candidate vaccine against toxoplasmosis. In this study, all CBA/J mice intramuscularly vaccinated with a plasmid encoding the immature form of the MIC3 protein (pMIC3i) produced specific anti-MIC3 immunoglobulin G (IgG) antibodies, and their sera displayed high antibody titers. This response was increased by the coadministration of a plasmid encoding the granulocyte-macrophage colony-stimulating factor (pGM-CSF). Similarly, a specific and significant cellular immune response was obtained in mice immunized with pMIC3i, and this response was markedly enhanced by pGM-CSF coadministration. The cellular immune response was associated with the production of gamma interferon IFN-␥ and interleukin-2 (IL-2), indicating that this was a Th1-type response. This was confirmed by the production of large amounts of IgG2a. Mice immunized with pMIC3i displayed significant protection against an oral challenge with T. gondii 76K cysts, exhibiting fewer brain cysts than did the control mice. Coadministration of pGM-CSF enhanced this protection. In conclusion, this study describes the design of a potent DNA vaccine encoding the novel T. gondii target antigen, MIC3 protein, that elicits a strong specific immune response as well as providing effective protection against T. gondii infection. In the attempt to achieve complete protection against toxoplasmosis, MIC3 is a good candidate vaccine which could be combined with other relevant and previously described candidates, such as SAG1 and GRA4.