Highlights d Toxoplasma gondii has a widespread impact on rodent behavior d Infection-induced loss of fear is not specific to feline predators d Behavioral alterations directly correlate with cyst burden and neuroinflammation d Cysts are predominantly localized in cortical areas of the brain

The obligate intracellular parasite Toxoplasma gondii possesses a repertoire of 11 myosins. Three class XIV motors participate in motility, invasion and egress, whereas the class XXII myosin F is implicated in organelle positioning and inheritance of the apicoplast. Here we provide evidence that TgUNC acts as a chaperone dedicated to the folding, assembly and function of all Toxoplasma myosins. The conditional ablation of TgUNC recapitulates the phenome of the known myosins and uncovers two functions in parasite basal complex constriction and synchronized division within the parasitophorous vacuole. We identify myosin J and centrin 2 as essential for the constriction. We demonstrate the existence of an intravacuolar cell–cell communication ensuring synchronized division, a process dependent on myosin I. This connectivity contributes to the delayed death phenotype resulting from loss of the apicoplast. Cell–cell communication is lost in activated macrophages and during bradyzoite differentiation resulting in asynchronized, slow division in the cysts.

Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite’s ability to modulate host signalling pathways and immune responses.

Micronemes and rhoptries are specialized secretory organelles that deploy their contents at the apical tip of apicomplexan parasites in a regulated manner. The secretory proteins participate in motility, invasion, and egress and are subjected to proteolytic maturation prior to organellar storage and discharge. Here we establish that Toxoplasma gondii aspartyl protease 3 (ASP3) resides in the endosomal-like compartment and is crucially associated to rhoptry discharge during invasion and to host cell plasma membrane lysis during egress. A comparison of the N-terminome, by terminal amine isotopic labelling of substrates between wild type and ASP3 depleted parasites identified microneme and rhoptry proteins as repertoire of ASP3 substrates. The role of ASP3 as a maturase for previously described and newly identified secretory proteins is confirmed in vivo and in vitro. An antimalarial compound based on a hydroxyethylamine scaffold interrupts the lytic cycle of T. gondii at submicromolar concentration by targeting ASP3.

Introductory paragraph The protozoan parasite Toxoplasma gondii has co-evolved with its homeothermic hosts, human included, strategies that drive its quasi asymptomatic persistence in hosts, hence optimizing the chance of transmission to new hosts. Persistence which starts with a small subset of parasites that escape from the host immune killing and colonize the so-called immune privileged tissues where they differentiate into a low replicating stage, is driven by the IL-12 and IFN-γ axis. The recent characterization of a Toxoplasma effector family delivered into the host cell where it rewires the host cell gene expression has allowed identifying regulators of the IL-12-IFN-γ axis including repressors. We now report on the dense granule-resident effector, called TEEGR (Toxoplasma E2F4-associated EZH2-inducing Gene Regulator) that counteracts the NF-κB signaling pathway. Once exported in the host cell TEEGR ends up in the nucleus where it not only complexes with E2F3 and E2F4 host transcription factors to induce gene expression but also promotes shaping of a nonpermissive chromatin through its capacity to switch on EZH2. Remarkably, EZH2 fosters the epigenetic silencing of a subset of NF-κB-regulated cytokines thereby strongly contributing to the host immune equilibrium that influencing the host immune response and in mice promotes parasite persistence.