The obligate intracellular parasite Toxoplasma gondii, a member of the phylum Apicomplexa that includes Plasmodium spp., is one of the most widespread parasites and the causative agent of toxoplasmosis. Micronemal proteins (MICs) are released onto the parasite surface just before invasion of host cells and play important roles in host cell recognition, attachment and penetration. Here, we report the atomic structure for a key MIC, TgMIC1, and reveal a novel cell-binding motif called the microneme adhesive repeat (MAR). Using glycoarray analyses, we identified a novel interaction with sialylated oligosaccharides that resolves several prevailing misconceptions concerning TgMIC1. Structural studies of various complexes between TgMIC1 and sialylated oligosaccharides provide high-resolution insights into the recognition of sialylated oligosaccharides by a parasite surface protein.We observe that MAR domains exist in tandem repeats, which provide a highly specialized structure for glycan discrimination. Our work uncovers new features of parasite-receptor interactions at the early stages of host cell invasion, which will assist the design of new therapeutic strategies.

Numerous intracellular pathogens exploit cell surface glycoconjugates for host cell recognition and entry. Unlike bacteria and viruses, Toxoplasma gondii and other parasites of the phylum Apicomplexa actively invade host cells, and this process critically depends on adhesins (microneme proteins) released onto the parasite surface from intracellular organelles called micronemes (MIC). The microneme adhesive repeat (MAR) domain of T. gondii MIC1 (TgMIC1) recognizes sialic acid (Sia), a key determinant on the host cell surface for invasion by this pathogen. By complementation and invasion assays, we demonstrate that TgMIC1 is one important player in Sia-dependent invasion and that another novel Sia-binding lectin, designated TgMIC13, is also involved. Using BLAST searches, we identify a family of MAR-containing proteins in enteroparasitic coccidians, a subclass of apicomplexans, including T. gondii, suggesting that all these parasites exploit sialylated glycoconjugates on host cells as determinants for enteric invasion. Furthermore, this protein family might provide a basis for the broad host cell range observed for coccidians that form tissue cysts during chronic infection. Carbohydrate microarray analyses, corroborated by structural considerations, show that TgMIC13, TgMIC1, and its homologue Neospora caninum MIC1 (NcMIC1) share a preference for ␣2-3-over ␣2-6-linked sialyl-N-acetyllactosamine sequences. However, the three lectins also display differences in binding preferences. Intense binding of TgMIC13 to ␣2-9-linked disialyl sequence reported on embryonal cells and relatively strong binding to 4-O-acetylated-Sia found on gut epithelium and binding of NcMIC1 to 6sulfo-sialyl Lewis x might have implications for tissue tropism. Sialic acids (Sias)6 occur abundantly in glycoproteins and glycolipids on the cell surface and are exploited by many viruses and bacteria for attachment and host cell entry. Recognition of carbohydrates and in particular sialylated glycoconjugates is important also for host cell invasion by the Apicomplexa (1-4), a phylum that includes several thousand species of obligate intracellular parasites, among them the Plasmodium spp. causing malaria. Enteroparasitic coccidians are a subclass of Apicomplexa comprising Eimeria spp. responsible for coccidiosis in poultry, Neospora spp. causing neosporosis in cattle, and Toxoplasma, the causative agent of toxoplasmosis in warmblooded animals and humans.The host range and cell type targeted by these parasites vary widely across the phylum. Whereas Plasmodium falciparum merozoites exclusively invade erythrocytes of humans and great apes (5), Toxoplasma gondii tachyzoites (the form of the parasite associated with acute infection) invade an extremely broad range of cell types in humans and virtually all warmblooded animals, enabling rapid establishment of infection in the host and dissemination into deep tissues (6). Information is emerging on the involvement of carbohydrate-protein interactions in this broad host cell recognition (1).Many intracellular...

Immediately prior to invasion Toxoplasma gondii tachyzoites release a large number of micronemal proteins (TgMICs) that participate in host cell attachment and penetration. The TgMIC4-MIC1-MIC6 complex was the first to be identified in T. gondii and has been recently shown to be critical in invasion. This study establishes that the N-terminal throm-bospondin type I repeat-like domains (TSR1-like) from TgMIC1 function as an independent adhesin as well as promoting association with TgMIC4. Using the newly solved three-dimensional structure of the C-terminal domain of TgMIC1 we have identified a novel Galectin-like fold that does not possess carbohydrate binding properties and redefines the architecture of TgMIC1. Instead, the TgMIC1 Galectin-like domain interacts and stabilizes TgMIC6, which provides the basis for a highly specific quality control mechanism for successful exit from the early secretory compartments and for subsequent trafficking of the complex to the micronemes.Toxoplasma gondii is a protozoan parasite of the phylum Apicomplexa, which infects virtually all warm-blooded animals and invades almost any cell type. Host cell invasion by this obligate intracellular parasite is an active process initiated by the formation of a tight association/junction with the host cell plasma membrane and leading to the creation of a parasitophorous vacuole. Contact with the host cell results in an increase in parasite intracellular calcium ions, which trigger apical organelles called micronemes to discharge their contents (1). Several micronemal proteins act as ligands for host cell receptors (2), while TgMIC2 and other transmembrane proteins establish a connection with the parasite actinomyosin system via their cytoplasmic tail (3), thus providing the motive force for penetration (4). It is becoming increasingly apparent that many microneme proteins are found in stable adhesive complexes, which are formed in the endoplasmic reticulum, and normally comprise an escorter protein, which is responsible for correct micronemal targeting, and one or more soluble effector proteins. The first such complex to be discovered in T. gondii was TgMIC4-MIC1-MIC6, in which TgMIC6 fulfils the role of the escorter protein, whereas TgMIC1 and TgMIC4 function as adhesins (5). Although TgMIC4-MIC1-MIC6 and the recently identified micronemal complex, TgMIC3-MIC8 (5, 6), are individually dispensable, the generation of double knock-outs for TgMIC1 and TgMIC3 renders the parasites avirulent in vivo, demonstrating functional synergy between these complexes (7). Deletion of the mic1 gene in T. gondii also confirmed the specific and critical role played by TgMIC1 in host cell attachment and invasion in vitro.Micronemal proteins have a modular structure with common themes in domain organization, for example many possess thrombospondin type-1 repeat domains (TSR1), 4 apple (or PAN) domains, and epidermal growth factor-like (EGF) domains (8). A schematic representation of the organization within the TgMIC4-MIC1-MIC6 complex is depicted in Fig. 1. Tg...

The intracellular protozoan Toxoplasma gondii is among the most widespread parasites. The broad host cell range of the parasite can be explained by carbohydrate microarray screening analyses that have demonstrated the ability of the T. gondii adhesive protein, TgMIC1, to bind to a wide spectrum of sialyl oligosaccharide ligands. Here, we investigate by further microarray analyses in a dose-response format the differential binding of TgMIC1 to 2-3-and 2-6-linked sialyl carbohydrates. Interestingly, two novel synthetic fluorinated analogs of 3 0 SiaLacNAc 1-4 and 3 0 SiaLacNAc 1-3 were identified as highly potent ligands. To understand the structural basis of the carbohydrate binding specificity of TgMIC1, we have determined the crystal structures of TgMIC1 micronemal adhesive repeat (MAR)-region (TgMIC1-MARR) in complex with five sialyl-Nacetyllactosamine analogs. These crystal structures have revealed a specific, water-mediated hydrogen bond network that accounts for the preferential binding of TgMIC1-MARR to arrayed 2-3-linked sialyl oligosaccharides and the high potency of the fluorinated analogs. Furthermore, we provide strong evidence for the first observation of a CAFÁÁÁHAO hydrogen bond within a lectin-carbohydrate complex. Finally, detailed comparison with other oligosaccharide-protein complexes in the Protein Data Bank (PDB) reveals a new family of sialic-acid binding sites from lectins in parasites, bacteria, and viruses.