Members of a Novel Protein Family Containing Microneme Adhesive Repeat Domains Act as Sialic Acid-binding Lectins during Host Cell Invasion by Apicomplexan Parasites

Abstract

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...

Sialic acids: Key determinants for invasion by the Apicomplexa

“…NcMIC1 was originally characterised as a sulphated glycosaminoglycan (GAG) binding protein (Keller et al, 2004). However carbohydrate microarray analyses demonstrated specific binding for NcMIC1 to sialyl-oligosaccharides with the special feature of a strong binding to two sulphated sialyl Le xrelated probes (Friedrich et al, 2010). Neospora caninum also possesses a homologue of TgMIC13 and hence it corroborates the idea that these proteins fulfil conserved functions, possibly in the capacity of these two cyst-forming parasites to invade a broad range of host cells.…”

“…No carbohydrate-binding activity or cell-adhesive properties were detected for TgMCP3 and TgMCP4, hence their functions remain a mystery (Friedrich et al, 2010). Epitope-tagging of TgMCP3 and TgMCP4 suggest that these proteins are stored in the dense granules and accumulate in the PV.…”

“…Database mining of the apicomplexan genomes revealed the existence of a family of MAR domain-containing proteins that are restricted to coccidians (Friedrich et al, 2010). Interestingly, the apple domain is specific to coccidians as well (Chen et al, 2008) although a divergent fold belonging to the plasminogen, apple, nematode (PAN) superfamily is present in PfAMA1 and this domain is conserved in AMA-1 homologues from all apicomplexans (Chesne-Seck et al, 2005).…”

“…Analyses of data from carbohydrate microarrays revealed that gangliosides GD1a and GT1b are bound by both TgMIC1 and TgMIC13 (Blumenschein et al, 2007;Friedrich et al, 2010). In order to determine whether gangliosides are able to compete with the authentic host cell receptor(s) for binding of TgMIC1 and TgMIC13, cell-binding assays were performed using gangliosides GD1a and GT1b incorporated into liposomes as competitors.…”

Diversity of extracellular proteins during the transition from the ‘proto-apicomplexan’ alveolates to the apicomplexan obligate parasites

“…Examples of carbohydrate-binding receptors include recognition of erythrocyte surface sialic acid by the Plasmodium merozoite protein EBA-175 during invasion (reviewed in Gaur and Chitnis, 2011); recognition of sialic acid via an unrelated saccharide-binding module, as well as a gal-lectin domain, within MIC1 participating in host cell invasion by Toxoplasma (TGME49_291890 in Fig. 2A; Friedrich et al 2010); and recognition of host carbohydrates mediated by an N-terminal domain within the microneme-secreted proteins MIC3 and MIC8 (CBL domain shown in TGME49_286740, Fig. 2A) during host cell invasion by Toxoplasma tachyzoites (Céréde et al 2005).…”