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.

Toxoplasma gondii is a worldwide protozoan parasite which causes severe disease in congenitally infected children and in immunocompromised patients. Besides the well-defined cytoplasmic and membrane antigens of tachyzoites, we felt that excreted/secreted antigens could play a major role in the immune response. We first report the development of a well-controlled procedure for obtaining tachyzoite excreted/secreted antigens (E/SA) in cell-free incubation media. The E/SA immunogenic in human, rat and mouse toxoplasmosis were then characterized. The major E/SA recognized by human sera from the chronic phase of toxoplasmosis had molecular weights of 108, 97, 86, 69, 60, 57, 42, 39, 28.5, 27 and 26 kD. When injected into +/+ Fischer rats, E/SA elicited high antibody titres. In addition, passive transfer of these sera to highly susceptible nu/nu littermates induced a significant degree of protection towards the virulent RH strain of T. gondii. This work, which demonstrates the key role played by E/SA in the protective immune response, suggests that these antigens should be of value both for diagnostic purposes and for the development of new strategies for immunization against toxoplasmosis.