Proteomics and Glycomics Analyses of N-Glycosylated Structures Involved in Toxoplasma gondii-Host Cell Interactions

Abstract

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man5–8(GlcNAc)2) and paucimannosidic (Man3–4(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii. The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man5–8(GlcNAc)2) and paucimannosidic (Man3–4(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii. Apicomplexans are protozoan parasites defined by the presence of a complex of specialized organelles at their apical end (1Kim K. Weiss L.M. Toxoplasma gondii: the model apicomplexan.Int. J. Parasitol. 2004; 34: 423-432Crossref PubMed Scopus (203) Google Scholar). The phylum Apicomplexa contains many important pathogens of humans and animals, including the causative agents of malaria (Plasmodium spp.) and chicken coccidiosis (Eimeria spp.) as well as some of the opportunistic infections associated with AIDS patients such as Cryptosporidium parvum and Toxoplasma gondii. T. gondii is uniquely adapted to infect a wide range of hosts, including virtually all warm blooded animals and up to 50% of the world's human population. The primary transmission route in humans is via ingestion of undercooked contaminated meat, particularly lamb, and contact with feces from infected domestic cats. The parasite causes a serious opportunistic disease (toxoplasmosis) in congenitally infected infants that can lead to severe syndromes including congenital malformations such as and in in to T. gondii is a opportunistic in with AIDS and in patients Google in the of Toxoplasma J. Parasitol. PubMed Scopus Google Scholar). the parasite a in in mammalian hosts, T. gondii is in the and the the is in the host by the which can a range of cell other protozoan pathogens that the host cell T. gondii host is of for and to In to that in the and other the of infected The of and is the of in AIDS patients J. J. in patients with the of the J. 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