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.

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.

The protozoan parasite Toxoplasma gondii is equipped with a sophisticated secretory apparatus, including three distinct exocytic organelles, named micronemes, rhoptries, and dense granules. We have dissected the requirements for targeting the microneme protein MIC3, a key component of T. gondii infection. We have shown that MIC3 is processed in a post-Golgi compartment and that the MIC3 propeptide and epidermal growth factor (EGF) modules contain microneme-targeting information. The minimal requirement for microneme delivery is defined by the propeptide plus any one of the three EGF domains. We have demonstrated that the cleavage of the propeptide, the dimerization of MIC3, and the chitin binding-like sequence, which are crucial for host cell binding and virulence, are dispensable for proper targeting. Finally, we have shown that part of MIC3 is withheld in the secretory pathway in a cell cycle-dependent manner.The apicomplexa are a group of mostly obligate intracellular parasites that are responsible for diseases such as toxoplasmosis, malaria, neosporosis, coccidiosis, and cryptosporidiosis. Host cell invasion is a prerequisite for the establishment and maintenance of infection for these parasites, and although the range of host cell specificity can vary greatly between different apicomplexan species, the machinery they use to invade their host(s) is strikingly conserved. Toxoplasma gondii, the parasite responsible for congenital infections in the developing fetus and for severe neurological complications in immunocompromised individuals, is relatively easy to manipulate genetically, invades virtually all nucleated cells, and, thus, is a good model for studying motility and invasion. Host cell invasion is accompanied by the secretion of parasite proteins from two distinct secretory organelles that are associated with attachment/motility (microneme proteins [MICs]) (23, 26), parasitophorous vacuole biogenesis (rhoptry proteins) (17), and moving junction formation (cooperative role of MICs and rhoptry neck proteins) (2, 27). Exocytosis from dense granules, a third type of specialized secretory granules, occurs during and following the invasion of the host cell and is believed to take part in the maturation of the parasitophorous vacuole, although most of the GRA proteins have not yet been attributed a function (30).The endoplasmic reticulum (ER) is the site of synthesis and the processing of proteins destined for secretion; thus, almost all secretory proteins contain a signal peptide. Most of protein sorting takes place either in the trans-Golgi network (TGN) (or its equivalent) or in immature secretory vesicles beyond the TGN. In T. gondii, soluble foreign reporter proteins, lacking targeting information, are delivered to the parasitophorous vacuole via the dense granules, which are the default route for secreted proteins (22). Similarly to other eukaryotic sorting mechanisms, a system of tyrosine-dependent signals exists in T. gondii that allows for the specific targeting of transmembrane proteins to the rhopt...