Abstract
Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of any warm-blooded animal. In a previous screen to identify virulence determinants, disruption of gene TgME49_305140 generated a T. gondii mutant that could not establish a chronic infection in mice. The protein product of TgME49_305140, here named TgPL3, is a 277 kDa protein with a patatin-like phospholipase (PLP) domain and a microtubule binding domain. Antibodies generated against TgPL3 show that it is localized to the apical cap. Using a rapid selection FACS-based CRISPR/Cas-9 method, a TgPL3 deletion strain (ΔTgPL3) was generated. ΔTgPL3 parasites have defects in host cell invasion, which may be caused by reduced rhoptry secretion. We generated complementation clones with either wild type TgPL3 or an active site mutation in the PLP domain by converting the catalytic serine to an alanine, ΔTgPL3::TgPL3S1409A (S1409A). Complementation of ΔTgPL3 with wild type TgPL3 restored all phenotypes, while S1409A did not, suggesting that phospholipase activity is necessary for these phenotypes. ΔTgPL3 and S1409A parasites are also virtually avirulent in vivo but induce a robust antibody response. Vaccination with ΔTgPL3 and S1409A parasites protected mice against subsequent challenge with a lethal dose of Type I T. gondii parasites, making ΔTgPL3 a compelling vaccine candidate. These results demonstrate that TgPL3 has a role in rhoptry secretion, host cell invasion and survival of T. gondii during acute mouse infection.
Highlights
Toxoplasma gondii is an obligate intracellular parasite that can infect all warm-blooded animals and any nucleated cell within the host
Sequence and structural alignment of the 277kDa TgPL3 protein revealed a PLP domain predicted to have phospholipase A2 (PLA2) activity and a MIP-T3 microtubule-binding domain (MtBD) surrounded by polyserine stretches (Fig 1A), which may act as flexible linkers allowing the binding domain to be accessible to its target [15]
These results suggest that ΔTgPL3 and S1409A mutant parasites were capable of low levels of replication in mice at or near the site of injection, but that they were unable to disseminate beyond the peritoneal cavity
Summary
Toxoplasma gondii is an obligate intracellular parasite that can infect all warm-blooded animals and any nucleated cell within the host. T. gondii infects an estimated 30–50% of humans [1], which are dead-end intermediate hosts. The asexual cycle of T. gondii can occur in any warm-blooded animal and has two developmental stages: a rapidly replicating form called the tachyzoite and a slow growing stage called the bradyzoite. T. gondii is acquired orally either by ingestion of oocyst-contaminated vegetables or other foods, or by eating bradyzoite cyst-harboring meat products. Upon infection of an intermediate host, cyst and oocyst stages differentiate into the tachyzoite form and disseminate throughout the host before transitioning to the bradyzoite form. Bradyzoite-containing cysts reside in the brain and muscle tissue, and represent the long-term chronic phase of infection. No medications are available that can clear the chronic stage of T. gondii
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