A Toxoplasma gondii patatin-like phospholipase contributes to host cell invasion

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::TgPL3 S1409A (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.

Past and present giant viruses diversity explored through permafrost metagenomics

“…6). Its role in viral infection is still unclear but such proteins participate to cell invasion in parasitic bacteria and eukaryotes (40,41). Also, according to our data, the A32-like packaging ATPase is no longer a universal Nucleocytoviricota marker gene, as it is not only lacking from the reference Pithoviridae genomes but also absent from all clades ranging from Pitho-orpheo_div8 to Pithoviridae (Fig.…”

Unraveling the Elusive Rhoptry Exocytic Mechanism of Apicomplexa

“…The past 3 years have seen spectacular progress in our understanding of rhoptry secretion in the apicomplexan model Toxoplasma and, to some extent, in P. falciparum, at both molecular and structural levels [45,68,[72][73][74]77]. These recent studies strongly suggest that Toxoplasma rhoptries do not directly contact the PPM but instead do so via an AV tightly connected to a rosette of eight IMPs embedded in the PPM.…”

“…Another protein recently associated with rhoptry secretion in Toxoplasma is TgPL3, a patatin-like phospholipase [77]. TgPL3 contains a phospholipase A2 (PLA2) domain whose activity is essential for rhoptry secretion, and also a microtubule-binding domain that might determine its localization at the apical cap of the parasite, just beneath the conoid, which becomes even more pronounced in extracellular parasites.…”

Investigation of Antiparasitic Activity of Two Marine Natural Products, Estradiol Benzoate, and Octyl Gallate, on Toxoplasma gondii In Vitro

“…The invasion assay was performed as described previously ( Wilson et al, 2020 ) with minor modifications. RH-2F parasites were inoculated in HFFs and pretreated with 5 μM of each compound for 8 h. Then the freshly released tachyzoites (5 × 10 6 ) were added to a new 24-well plate of HFFs with coverslips and each compound.…”

“…The egress assay was performed as described previously ( Wilson et al, 2020 ) with minor modifications. Monolayers of HFF in 24-well plates were infected with 1 × 10 4 tachyzoites followed by incubation at 37°C and 5% CO 2 in a medium containing 5 μM of each compound for 24 h. Prior to egress assays, infected wells were washed three times with prewarmed PBS.…”