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Abstract Programmed-cell death is an antimicrobial defense mechanism that promotes clearance of intracellular pathogens. Toxoplasma counteracts host immune defenses by secreting effector proteins into host cells; however, how the parasite evades lytic cell death and the effectors involved remain poorly characterized. We identified ROP55, a rhoptry protein that promotes parasite survival by preventing lytic cell death in absence of IFN-γ stimulation. RNA-Seq analysis revealed that ROP55 acts as a repressor of host pro-inflammatory responses. In THP-1 monocytes ΔROP55 infection increased NF-κB p65 nuclear translocation, IL-1β production, and GSDMD cleavage compared to wild type or complemented parasites. ΔROP55 infection also induced RIPK3-dependent necroptosis in human and mouse primary macrophages. Moreover, ΔROP55 parasites were significantly impaired in virulence in female mice and prevented NF-κB activation and parasite clearance in mBMDM. These findings place ROP55 as a major virulence factor, dampening lytic cell death and enabling Toxoplasma to evade clearance from infected cells.

ABSTRACT Toxoplasma gondii is a single-celled parasitic eukaryote that evolved to successfully propagate in any nucleated cell. As with any other eukaryote, its life cycle is regulated by signaling pathways controlled by kinases and phosphatases. T. gondii encodes an atypical bacterial-like phosphatase absent from mammalian genomes, named Shelph, after its first identification in the psychrophilic bacterium Schewanella sp. Here, we demonstrate that Toxoplasma Shelph is an active phosphatase localized in the parasite endoplasmic reticulum. The phenotyping of a shelph knockout (KO) line showed a minor impairment in invasion on human fibroblasts, while the other steps of the parasite lytic cycle were not affected. In contrast with Plasmodium ortholog Shelph1, this invasion deficiency was not correlated with any default in the biogenesis of secretory organelles. However, Shelph-KO parasites displayed a much-pronounced defect in virulence in vivo. These phenotypes could be rescued by genetic complementation, thus supporting an important function for Shelph in the context of a natural infection. IMPORTANCE Toxoplasma gondii belongs to the Apicomplexa phylum, which comprises more than 5,000 species, among which is Plasmodium falciparum, the notorious agent of human malaria. Intriguingly, the Apicomplexa genomes encode at least one phosphatase closely related to the bacterial Schewanella phosphatase, or Shelph. To better understand the importance of these atypical bacterial enzymes in eukaryotic parasites, we undertook the functional characterization of T. gondii Shelph. Our results uncovered its subcellular localization and its enzymatic activity, revealed its subtle involvement during the tachyzoite invasion step of the lytic cycle, and more importantly, highlighted a critical requirement of this phosphatase for parasite propagation in mice. Overall, this study revealed an unexpected role for T. gondii Shelph in the maintenance of parasite virulence in vivo.