UC Merced

Toxoplasma gondii is an intracellular protozoan parasite that infects nearly 1 in 3 individuals in the world. In immunocompromised individuals T. gondii infection can disseminate and cause serious disease and even death. Manifestations of the disease caused by T. gondii, toxoplasmosis, can include ocular lesions, vision loss, miscarriage, hydrocephaly, mental retardation, and even death. T. gondii is closely related to the malaria-causing parasite, Plasmodium sp., and both of which are without effective protective vaccines. For the development of an effective treatment or vaccine against parasitic infections, more about the battle between host and pathogen must be understood. What allows for some mouse strains to survive infections, while others succumb? What antigens on T. gondii elicit the strongest antibody response? What receptors and pathways must be triggered to provide protection?

The glycosylphosphatidylinositol (GPI)-anchor is a highly conserved glycolipid that anchors proteins to the external membrane of T. gondii and is also found on all protozoan pathogens. Most of the major surface antigens (SAGs) of T. gondii are GPI-anchored proteins, and express GPI without attached proteins on their surface called GIPLs. The GPI is known to be recognized by innate pattern recognition receptors Toll-like Receptors (TLR) -2 and -4 and is robustly targeted by IgM antibodies after infection. While the glycan portion of the GPI consists of a conserved set of 3 mannose sugars, a GlcNAc, and an inositol, species differ in additional sugars or sidechains added to the mannose backbone. The functional significance of GPI sidechains has remained largely unknown because only a few GPI sidechain modifying enzymes have been identified. Since the GPI is such a potent antigen and in high abundance, we sought out to address the role of the GPI sidechain in parasite pathogenesis.

We have successfully identified and knocked out both of the enzymes responsible for GPI sidechain modification in T. gondii. Named here PIGJ and PIGE, the former adds the first GalNAc sugar to the core mannose backbone of the GPI, while the latter adds a terminal Glc to the GalNAc as confirmed by mass spectrometry. Parasites lacking the entire GPI sidechain exhibit increased virulence during primary and secondary infections, demonstrating the GPI sidechain as an important pathogenesis factor. To elucidate the mechanism for increased virulence we characterized innate immune responses, parasite burden in various organs, early cytokine responses, antibody recognition, antigen expression, parasite growth and invasion of PIGJ mutant parasites that lack a GPI sidechain. Our findings reveal intact cytokine responses, antibody recognition of GPI-anchored SAGs, and complement binding to PIGJ mutants. In contrast, the scavenger receptor CD36 shows enhanced binding to parasites that lack GPI sidechains, potentially explaining the tropism for macrophages observed early in infection. Galecitn-3, which binds to GIPL of T. gondii, exhibits enhanced binding to PIGJ mutants and infection of galectin-3 deficient mice results in their protection from lethal outcomes, suggesting the enhanced virulence of sidechain deficient parasites is galectin-3 dependent. Loss of the sidechain has no bearing on parasite numbers early in the infection, suggest a breakdown of tolerance is occurring, the exact mechanism for which is still unclear. In contrast, we see increased tissue cysts in the brains of PIGJ mutant infected mice, indicating some advantage over the wildtype is involved. Finally, vaccination of TLR2 or TLR4 deficient mice are susceptible to challenge with virulent T. gondii strains. Collectively, our data point to an important role for the GPI and its sidechain in host-parasite interactions, and that the presence of the sidechain prevents T. gondii pathogenesis promoting survival of its host.