Treatment of mice with S4B6 IL‑2 complex prevents lethal toxoplasmosis via IL‑12‑ and IL‑18‑dependent interferon‑gamma production by non‑CD4 immune cells

Toxoplasmic encephalitis is an AIDS-defining condition. The decline of IFN-γ-producing CD4⁺ T cells in AIDS is a major contributing factor in reactivation of quiescent Toxoplasma gondii to an actively replicating stage of infection. Hence, it is important to characterize CD4-independent mechanisms that constrain acute T. gondii infection. We investigated the in vivo regulation of IFN-γ production by CD8⁺ T cells, DN T cells and NK cells in response to acute T. gondii infection. Our data show that processing of IFN-γ by these non-CD4 cells is dependent on both IL-12 and IL-18 and the secretion of bioactive IL-18 in response to T. gondii requires the sensing of viable parasites by multiple redundant inflammasome sensors in multiple hematopoietic cell types. Importantly, our results show that expansion of CD8+ T cells, DN T cells and NK cell by S4B6 IL-2 complex pre-treatment increases survival rates of mice infected with T. gondii and this is dependent on IL-12, IL-18 and IFN-γ. Increased survival is accompanied by reduced pathology but is independent of expansion of TReg cells or parasite burden. This provides evidence for a protective role of IL2C-mediated expansion of non-CD4 cells and may represent a promising lead to adjunct therapy for acute toxoplasmosis

From virulence to persistence: role of an aspartyl protease maturing secretory proteins in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that infects all warm-blooded animals and chronically persists in one-third of world's human population. Two invasive life stages of this apicomplexan parasite are found in intermediate hosts: a fast replicating tachyzoite, responsible for acute infection that is rapidly controlled by the immune system and a cyst-forming, slow growing bradyzoite that persists during the entire life span of the host. Both stages presumably export effector proteins to hijack host cellular functions, enabling the acquisition of nutrients and evasion of the host cellular and immune defenses. My project was directed toward the functional dissection of the aspartic protease 5 (TgASP5) contribution to the cleavage and export of effector proteins. An in depth analysis of the knock-out of TgASP5 revealed its essential role for the export of dense granules proteins (GRAs) and for the cleavage of those that harbor a TEXEL (Toxoplasma EXport ELement) motif

Insights into the molecular basis of host behaviour manipulation by <i>Toxoplasma gondii</i> infection

Typically illustrating the ‘manipulation hypothesis', Toxoplasma gondii is widely known to trigger sustainable behavioural changes during chronic infection of intermediate hosts to enhance transmission to its feline definitive hosts, ensuring survival and dissemination. During the chronic stage of infection in rodents, a variety of neurological dysfunctions have been unravelled and correlated with the loss of cat fear, among other phenotypic impacts. However, the underlying neurological alteration(s) driving these behavioural modifications is only partially understood, which makes it difficult to draw more than a correlation between T. gondii infection and changes in brain homeostasis. Moreover, it is barely known which among the brain regions governing fear and stress responses are preferentially affected during T. gondii infection. Studies aiming at an in-depth dissection of underlying molecular mechanisms occurring at the host and parasite levels will be discussed in this review. Addressing this reminiscent topic in the light of recent technical progress and new discoveries regarding fear response, olfaction and neuromodulator mechanisms could contribute to a better understanding of this complex host–parasite interaction

Toxoplasma gondii TFP1 is an essential transporter family protein critical for microneme maturation and exocytosis

Invasion and egress are two key steps in the lytic cycle of Apicomplexa that are governed by the sequential discharge of proteins from two apical secretory organelles called micronemes and rhoptries. In Toxoplasma gondii, the biogenesis of these specialized organelles depends on the post Golgi trafficking machinery, forming an endosomal-like compartment (ELC) resembling endomembrane systems found in eukaryotes. In this study, we have characterized four phylogenetically related Transporter Facilitator Proteins (TFPs) conserved among the apicomplexans. TFP1 localises to the micronemes and ELC, TFP2 and TFP3 to the rhoptries and TFP4 to the Golgi. TFP1 crucially contributes to parasite fitness and survival while the other members of this family are dispensable. Conditional depletion of TFP1 impairs microneme biogenesis and leads to a complete block in exocytosis, which hampers gliding motility, attachment, invasion and egress. Morphological investigations revealed that TFP1 participates in the condensation of the microneme content, suggesting the transport of a relevant molecule for maintaining the intraluminal microenvironment necessary for organelle maturation and exocytosis. In absence of TFP2, rhoptries adopt a considerable elongated shape, but the abundance, processing or secretion of the rhoptry content are not affected. These findings establish the relevance of TFPs in organelle maturation of T. gondii. This article is protected by copyright. All rights reserved

Myosin-dependent cell-cell communication controls synchronicity of division in acute and chronic stages of Toxoplasma gondii

AbstractThe 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.</jats:p

Structural basis of phosphatidic acid sensing by APH in apicomplexan parasites

Plasmodium falciparum and Toxoplasma gondii are obligate intracellular parasites that belong to the phylum of Apicomplexa and cause major human diseases. Their access to an intracellular lifestyle is reliant on the coordinated release of proteins from the specialized apical organelles called micronemes and rhoptries. A specific phosphatidic acid effector, the acylated pleckstrin homology domain-containing protein (APH) plays a central role in microneme exocytosis and thus is essential for motility, cell entry, and egress. TgAPH is acylated on the surface of the micronemes and recruited to phosphatidic acid (PA)-enriched membranes. Here, we dissect the atomic details of APH PA-sensing hub and its functional interaction with phospholipid membranes. We unravel the key determinant of PA recognition for the first time and show that APH inserts into and clusters multiple phosphate head-groups at the bilayer binding surface

A druggable secretory protein maturase of Toxoplasma essential for invasion and egress

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

Toxoplasma gondii GRA60 is an effector protein that modulates host cell autonomous immunity and contributes to virulence

Toxoplasma gondii infects virtually any nucleated cell and resides inside a non‐phagocytic vacuole surrounded by a parasitophorous vacuolar membrane (PVM). Pivotal to the restriction of T. gondii dissemination upon infection in murine cells is the recruitment of Immunity Regulated GTPases (IRGs) and Guanylate Binding Proteins (GBPs) to the PVM that leads to pathogen elimination. The virulent T. gondii type I RH strain secretes a handful of effectors including the dense granule protein GRA7, the serine‐threonine kinases ROP17 and ROP18, and a pseudo‐kinase ROP5, that synergistically inhibit the recruitment of IRGs to the PVM. Here, we characterize GRA60, a novel dense granule effector which localizes to the vacuolar space and PVM and contributes to virulence of RH in mice suggesting a contribution to the subversion of host cell defense mechanisms. Members of the host cell IRG defense system Irgb10 and Irga6 are recruited to the PVM of RH parasites lacking GRA60 as observed previously for the avirulent RHΔrop5 mutant, with RH preventing such recruitment. Deletion of GRA60 in RHΔrop5 leads to a recruitment of IRGs comparable to the single knockouts. GRA60 therefore represents a novel parasite effector conferring resistance to IRGs in type I parasites, and is found to associate with ROP18, a member of the virulence complex