Toxoplasma gondii possesses a highly polarized secretory system, which efficiently assembles de novo micronemes and rhoptries during parasite replication. These apical secretory organelles release their contents into host cells promoting parasite invasion and survival. Using a CreLox-based inducible knock-out strategy and the ddFKBP over-expression system, we unraveled novel functions of the clathrin adaptor complex TgAP1. First, our data indicate that AP1 in T. gondii likely functions as a conserved heterotetrameric complex composed of the four subunits γ, β, μ1, σ1 and interacts with known regulators of clathrin-mediated vesicular budding such as the unique ENTH-domain containing protein, which we named Epsin-like protein (TgEpsL). Disruption of the μ1 subunit resulted in the mis-sorting of microneme proteins at the level of the Trans-Golgi-Network (TGN). Furthermore, we demonstrated that TgAP1 regulates rhoptry biogenesis by activating rhoptry protein exit from the TGN, but also participates in the post-Golgi maturation process of preROP compartments into apically anchored club-shaped mature organelles. For this latter activity, our data indicate a specific functional relationship between TgAP1 and the Rab5A-positive endosome-like compartment. In addition, we unraveled an original role for TgAP1 in the regulation of parasite division. APμ1-depleted parasites undergo normal daughter cell budding and basal complex assembly but fail to segregate at the end of cytokinesis.

SummaryUnattached kinetochores generate an anaphase inhibitor, through the spindle assembly checkpoint (SAC), that allows cells more time to establish proper kinetochore-microtubule (K-MT) linkages and thus avoid aneuploidy. Mad1 is the receptor for Mad2 at kinetochores, where it catalyzes the formation of Mad2-Cdc20 complexes, an essential part of the anaphase inhibitor, but whether it has any other mitotic function is unknown. We have generated a mad1-null mutation in Drosophila. This mutant is SAC defective and Mad2 is no longer localized to either nuclear envelope or kinetochores, but it displays normal basal mitotic timing. Unlike mad2 mutants, which have relatively normal mitoses, mad1 anaphases show high frequencies of lagging chromatids, at least some of which are caused by persistent merotelic linkages. A transgene expressing GFP-Mad1 rescues both the SAC and the anaphase defects. In an attempt to separate the SAC function from the mitotic function, we made a mad1 transgene with a mutated Mad2-binding domain. Surprisingly, this transgene failed to complement the anaphase phenotype. Thus, Mad1 has activity promoting proper K-MT attachments in addition to its checkpoint function. This activity does not require the presence of Mad2, but it does depend in some unknown way on key residues in the Mad2-binding domain of Mad1.

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Mycobacterium tuberculosis (Mtb), the pathogen causing human tuberculosis, has evolved multiple strategies to successfully prevent clearance by immune cells and to establish dissemination and long-term survival in the host. The modulation of host immunity to maximize pathogen elimination while minimizing inflammation-mediated tissue damage may provide another tool to fight drug-resistant Mtb strains.Metabolic reprogramming of immune cell populations can dramatically influence the outcome of immune responses and modulate antimicrobial properties of infected host cells, nicely demonstrating that metabolites are tightly linked to immune cell effector functions. One important endogenous metabolite of the Krebs cycle is itaconate, which has potent bactericidal activity by inhibiting isocitrate lyase and the glyoxylate shunt within prokaryotes including mycobacteria. Recent findings show that itaconate and the catalytic enzyme responsible for its generation in mammalian cells, i.e. IRG1 (immune-responsive gene 1), also modify inflammatory signaling of infected cells enhancing host defense pathways.Here, we demonstrate that IRG1 is recruited to Mtb-containing phagosomes and that it influences the host response controlling Mtb infection. While IRG1 deficiency does not affect uptake of Mtb by macrophages and dendritic cells (DCs) in vitro, it increases the intracellular replication of Mtb. Concomitantly, in comparison to wild type cells, IRG1-deficient macrophages and DCs have increased levels of lipid droplets, a correlate of inflammation. These intracellular organelles store triacylglycerol and phospholipids that are hijacked by Mtb as reservoir of host nutrients. Exposure of IRG1-deficient mice to M. bovis BCG via the intranasal route induced neither lethality nor severe lung immunopathology, while IRG1-deficient mice were highly susceptible to Mtb infection resulting in animal death three weeks post-infection linked to exacerbated inflammation and high mycobacterial burden. The lungs of infected IRG1-deficient mice displayed large areas of necrotizing granulomatous inflammation and neutrophil infiltration, accompanied by reduced levels of B and T lymphocytes and increased levels of alveolar and interstitial macrophage populations, compared to their wild type counterparts. Therefore, our findings demonstrate that IRG1 is a major player in controlling the acute phase of Mtb infection with a specific effect on pathogenic mycobacteria.

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.

Biallelic gene defects in MFSD8 are not only a cause of the late-infantile form of neuronal ceroid lipofuscinosis, but also of rare isolated retinal degeneration. We report clinical and genetic data of seven patients compound heterozygous or homozygous for variants in MFSD8, issued from a French cohort with inherited retinal degeneration, and two additional patients retrieved from a Swiss cohort. Next-generation sequencing of large panels combined with whole-genome sequencing allowed for the identification of twelve variants from which seven were novel. Among them were one deep intronic variant c.998+1669A>G, one large deletion encompassing exon 9 and 10, and a silent change c.750A>G. Transcript analysis performed on patients’ lymphoblastoid cell lines revealed the creation of a donor splice site by c.998+1669A>G, resulting in a 140 bp pseudoexon insertion in intron 10. Variant c.750A>G produced exon 8 skipping. In silico and in cellulo studies of these variants allowed us to assign the pathogenic effect, and showed that the combination of at least one severe variant with a moderate one leads to isolated retinal dystrophy, whereas the combination in trans of two severe variants is responsible for early onset severe retinal dystrophy in the context of late-infantile neuronal ceroid lipofuscinosis.

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