AToxoplasma gondiipatatin-like phospholipase contributes to host cell invasion

Author summary Toxoplasma gondiiis a eukaryotic parasite commonly found in warm-blooded animals worldwide. Successful replication ofT.gondiiwithin the host relies on its sophisticated cell invasion mechanism. Previous studies found that disruption of theT.gondiigene TgME49_305140 generated a mutant that could not establish a chronic infection in mice. The protein product of this gene, named TgPL3, is large with both patatin-like phospholipase and microtubule binding domains. Here we show that TgPL3 is localized to the apical end ofT.gondii, which is used for invasion. A mutantT.gondiistrain with a deletion of TgPL3 (Delta TgPL3) was generated. Delta TgPL3 parasites are defective for host cell invasion and do not cause disease in mice, even at high doses. Complementation of the TgPL3 gene back into Delta TgPL3 parasites repairs the invasion and mice infection defects. Moreover, complementation of Delta TgPL3 with a point mutation in the active site of the phospholipase domain (S1409A) did not rescue the invasion and mouse infection defects, suggesting the PLP domain is responsible for these phenotypes. Vaccination of mice with Delta TgPL3 and S1409A parasites protected them against lethal challenge withT.gondii, highlighting the potential of Delta TgPL3 as a vaccine strain. Toxoplasma gondiiis 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 aT.gondiimutant 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 (Delta TgPL3) was generated. Delta TgPL3 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, Delta TgPL3::TgPL3(S1409A)(S1409A). Complementation of Delta TgPL3 with wild type TgPL3 restored all phenotypes, while S1409A did not, suggesting that phospholipase activity is necessary for these phenotypes. Delta TgPL3 and S1409A parasites are also virtually avirulentin vivobut induce a robust antibody response. Vaccination with Delta TgPL3 and S1409A parasites protected mice against subsequent challenge with a lethal dose of Type IT.gondiiparasites, making Delta TgPL3 a compelling vaccine candidate. These results demonstrate that TgPL3 has a role in rhoptry secretion, host cell invasion and survival ofT.gondiiduring acute mouse infection.