Toxoplasma gondii HOOK-FTS-HIP Complex is Critical for Secretory Organelle Discharge during Motility, Invasion, and Egress.

Members of the Apicomplexa phylum possess specialized secretory organelles that discharge, apically and in a timely regulated manner, key factors implicated in parasite motility, host cell invasion, egress and subversion of host cellular functions. The mechanisms regulating trafficking and apical docking of these secretory organelles are only partially elucidated. Here, we characterized two conserved endosomal trafficking regulators known to promote vesicle transport and/or fusion, HOOK and Fused Toes (FTS), in the context of organelle discharge in Toxoplasma gondii. TgHOOK and TgFTS form a complex with a coccidian-specific partner, named HOOK interacting partner (HIP). TgHOOK displays an apically enriched vesicular pattern and concentrates at the parasite apical tip where it colocalizes with TgFTS and TgHIP. Functional investigations revealed that TgHOOK is dispensable but fitness conferring. The protein regulates the apical positioning and secretion of micronemes and contributes to egress, motility, host cell attachment, and invasion. Conditional depletion of TgFTS or TgHIP impacted on the same processes but led to more severe phenotypes. This study provides evidence of endosomal trafficking regulators involved in the apical exocytosis of micronemes and possibly as a consequence or directly on the discharge of the rhoptries. IMPORTANCE Toxoplasma gondii affects between 30 and 80% of the human population, poses a life-threatening risk to immunocompromised individuals, and is a cause of abortion and birth defects following congenital transmission. T. gondii belongs to the phylum of Apicomplexa characterized by a set of unique apical secretory organelles called the micronemes and rhoptries. Upon host cell recognition, this obligatory intracellular parasite secretes specific effectors contained in micronemes and rhoptries to promote parasite invasion of host cells and subsequent persistence. Here, we identified novel T. gondii endosomal trafficking regulators and demonstrated that they regulate microneme organelle apical positioning and exocytosis, thereby strongly contributing to host cell invasion and parasite virulence.

The UPR sensor IRE1α promotes dendritic cell responses to control Toxoplasma gondii infection.

The unfolded protein response (UPR) has emerged as a central regulator of immune cell responses in several pathologic contexts including infections. However, how intracellular residing pathogens modulate the UPR in dendritic cells (DCs) and thereby affect T cell-mediated immunity remains uncharacterized. Here, we demonstrate that infection of DCs with Toxoplasma gondii (T. gondii) triggers a unique UPR signature hallmarked by the MyD88-dependent activation of the IRE1α pathway and the inhibition of the ATF6 pathway. Induction of XBP1s controls pro-inflammatory cytokine secretion in infected DCs, while IRE1α promotes MHCI antigen presentation of secreted parasite antigens. In mice, infection leads to a specific activation of the IRE1α pathway, which is restricted to the cDC1 subset. Mice deficient for IRE1α and XBP1 in DCs display a severe susceptibility to T. gondii and succumb during the acute phase of the infection. This early mortality is correlated with increased parasite burden and a defect in splenic T-cell responses. Thus, we identify the IRE1α/XBP1s branch of the UPR as a key regulator of host defense upon T. gondii infection.

Rab11A regulates dense granule transport and secretion during Toxoplasma gondii invasion of host cells and parasite replication.

Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of dense granules and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile and invading parasites suggesting that apically polarized Rab11A-dependent delivery of cargo regulates early secretory events during parasite entry into host cells.