Date Published: May 2, 2013
Publisher: Public Library of Science
Author(s): Carolina E. Caffaro, Anita A. Koshy, Li Liu, Gusti M. Zeiner, Carlos B. Hirschberg, John C. Boothroyd, Boris Striepen.
Toxoplasma gondii is an intracellular parasite that transitions from acute infection to a chronic infective state in its intermediate host via encystation, which enables the parasite to evade immune detection and clearance. It is widely accepted that the tissue cyst perimeter is highly and specifically decorated with glycan modifications; however, the role of these modifications in the establishment and persistence of chronic infection has not been investigated. Here we identify and biochemically and biologically characterize a Toxoplasma nucleotide-sugar transporter (TgNST1) that is required for cyst wall glycosylation. Toxoplasma strains deleted for the TgNST1 gene (Δnst1) form cyst-like structures in vitro but no longer interact with lectins, suggesting that Δnst1 strains are deficient in the transport and use of sugars for the biosynthesis of cyst-wall structures. In vivo infection experiments demonstrate that the lack of TgNST1 activity does not detectably impact the acute (tachyzoite) stages of an infection or tropism of the parasite for the brain but that Δnst1 parasites are severely defective in persistence during the chronic stages of the infection. These results demonstrate for the first time the critical role of parasite glycoconjugates in the persistence of Toxoplasma tissue cysts.
The Apicomplexa are a phylum of protozoan organisms, many of which are pathogens with human and veterinary relevance. The Apicomplexan parasites share the remarkable ability to invade and propagate within host cells. Among this group, Toxoplasma gondii is an exemplar of the ability to undergo efficient transmission and persist within immunocompetent hosts without causing significant disease . Furthermore, while the complex life cycle of these parasites includes sexual and asexual stages, Toxoplasma has the unusual capacity to circumvent the sexual stage and clonally disseminate through intermediate hosts . Key to this clonal proliferation is Toxoplasma’s ability to interconvert between a rapidly proliferating tachyzoite and a slow-dividing form, the bradyzoite, which encysts within host cells. The bradyzoite form is critical for the establishment of a chronic infection, persistence within the host and transmission to other hosts via ingestion of raw meat or other tissue from an infected animal. Under circumstances that cause immunosuppression, Toxoplasma bradyzoites that differentiate back to tachyzoites can reinitiate extensive rounds of proliferation causing significant disease , .
Here we have identified and characterized a Toxoplasma nucleotide-sugar transporter, TgNST1, that is required for the synthesis of cyst wall glycoconjugates and whose deletion results in lower cyst numbers during chronic infection. Our results in vitro show that Δnst1 parasites are not defective in the formation of otherwise normal cyst walls, as we demonstrated that the glycosylated cyst wall protein GRA2 and the cyst wall proteins GRA9 and BPK1 are efficiently secreted and incorporated into the cyst matrix surrounding such parasites. This suggests that the glycan moieties of Toxoplasma tissue cysts (protein or otherwise) play a particular role in parasite persistence and the maintenance of a chronic infection. Our results do not address the identities of the key glycan moieties responsible for this phenotype, a complete inventory of which has not been determined for bradyzoites or the cyst wall within which they reside. We cannot exclude, therefore, the possibility that NST1 is involved in modifications involving other glycan residues not tested here. Based on glycoconjugates identified in tachyzoites, however, there are four types of molecules that could be involved: glycosylphosphatidylinositols (GPIs), N-glycans, O-glycans and glycosphingolipids. Toxoplasma GPIs consists of a conserved core phosphatidylinositol-lipid structure linked to a glucosamine and three mannose residues. In addition, Toxoplasma GPIs can be modified by a glucose-α1-4-N-Acetylglucosamine side chain attached to the first mannose. Although we did not specifically examine GPI biosynthesis in the Δnst1 parasites, we saw no differences in the surface staining with antibodies to abundant GPI-anchored proteins like SRS9 (Figures 4 and 5), arguing against a role for TgNST1 in GPI biosynthesis. Such is also suggested from the fact the GPIs are important in the pathogenesis of the acute stage of an infection with Toxoplasma,  and we saw no such phenotype with the Δnst1 parasites. While studies of the topology of GPI biosynthesis in Toxoplasma indicate that most of the GPI intermediates, including those with a side chain modification, localize to the cytosolic phase of the ER, these results don’t completely rule out their lumenal synthesis and thus the requirement of a nucleotide-sugar transporter . A thorough analysis of GPI structures in Δnst1 parasites compared to wild type will be needed to definitively determine if NST1 has a role in GPI biosynthesis.