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Gamma-aminobutyric acid (GABA) serves diverse biological functions in prokaryotes and eukaryotes, including neurotransmission in vertebrates. Yet, the role of GABA in the immune system has remained elusive. Here, a comprehensive characterization of human and murine myeloid mononuclear phagocytes revealed the presence of a conserved and tightly regulated GABAergic machinery with expression of GABA metabolic enzymes and transporters, GABA-A receptors and regulators, and voltage-dependent calcium channels. Infection challenge with the common coccidian parasites Toxoplasma gondii and Neospora caninum activated GABAergic signaling in phagocytes. Using gene silencing and pharmacological modulators in vitro and in vivo in mice, we identify the functional determinants of GABAergic signaling in parasitized phagocytes and demonstrate a link to calcium responses and migratory activation. The findings reveal a regulatory role for a GABAergic signaling machinery in the host-pathogen interplay between phagocytes and invasive coccidian parasites. The co-option of GABA underlies colonization of the host by a Trojan horse mechanism.

Toxoplasma gondii ist ein obligater intrazellulärer Parasit, der bei Menschen und Nutztieren Toxoplasmose verursacht. Die Phospholipid-Biosynthese ist entscheidend für das erfolgreiche intrazelluläre Überleben und die Replikation des Parasiten, da sie eine wichtige Rolle bei der Biogenese von Membranorganellen, der Signal- transduktion und anderen zellulären Prozessen spielt. Hier untersuchten wir die physiologische Bedeutung von zwei Synthesewegen, die für PtdEtn und PtdIns verantwortlich sind. Wir zeigen das Vorhandensein einer neuartigen PtdIns-Synthase (PIS) in T. gondii, die als TgPIS bezeichnet wird und ein funktionelles Enzym mit einem katalytisch wichtigen CDP-Alkohol- Phosphotransferase-Motiv codiert, das sich ausschließlich im Golgi-Apparat befindet. Der Parasit importiert Myoinosit aus dem Milieu und verwendet es zusammen mit de novo synthetisiertem CDP- Diacylglycerin, um PtdIns zu produzieren. Eine durch Auxin induzierbare bedingte Unterdrückung von TgPIS schaltet den Lysezyklus des Parasiten aufgrund von Defekten in der Replikation, Motilität und Austritt in Säugetierzellen aus. Das Lipidom-Profiling der PIS-Mutante zeigt eine selektive Reduktion bestimmter PtdIns- und PtdThr-Spezies, wohingegen ausgewählte PtdGro-, PtdSer- und BMP-Spezies erhöht sind, was auf eine enge Interregulation und Homöostase von anionischen Phospholipiden zur Aufrechterhaltung der Membranintegrität hindeutet. Zusätzlich identifizierten wir eine Ethanolamin-Cytidyltransferase (TgECT), das geschwindigkeitsbestimmende Enzym des Kennedy-Signalwegs, das im Cytosol lokalisiert ist. Das Enzym ist eindeutig für den Lysezyklus essentiell, da seine genetische Ablation nicht durchführbar ist und der durch Auxin meditierte bedingte Abbau des Proteins das Parasitenwachstum in Plaqueassays stark beeinträchtigt. Die Lipidomanalyse der Mutante identifizierte eine wichtige Rolle bei der Biogenese ausgewählter Arten von PtdEtn, PtdSer und PtdThr. Darüber hinaus haben wir fest, Toxoplasma gondii is an obligate intracellular parasite that causes Toxoplasmosis in human and livestock. Phospholipid biosynthesis is crucial for the successful intracellular survival and replication of the parasites, as the phospholipids have important roles in the biogenesis of membrane organelles, signal transduction and other cellular processes. Here, we dissected the physiological importance of two pathways accounting for the synthesis of PtdEtn and PtdIns. We demonstrated the presence of a novel PtdIns synthase (PIS) in T.gondii termed TgPIS, expressing a functional enzyme with a catalytically vital CDP-alcohol phosphotransferase motif, which resides exclusively in the Golgi body. The parasite imports myo-inositol from milieu, and co-utilizes de novo-synthesized CDP-diacylglycerol to produce PtdIns. An auxin-inducible conditional repression of TgPIS abrogated the lytic cycle of the parasite in mammalian cells due to defects in the replication, motility and egress. Lipidomic profiling of the PIS mutant demonstrated selective reduction of certain PtdIns and PtdThr species, whereas selected PtdGro, PtdSer and BMP species were increased, which suggested a tight inter-regulation and homeostasis of anionic phospholipids to maintain the membrane integrity. In addition, we identified an ethanolamine cytidyltransferase (TgECT), the rate-limiting enzyme of Kennedy pathway, which is localized in the cytosol. The enzyme is clearly essential for the lytic cycle as its genetic ablation was not feasible, and auxin-meditated conditional knockdown severely impaired the parasite growth in plaque assays. Similarly, lipidomic analysis of the mutant identified an important role in the biogenesis of selected species of PtdEtn, PtdSer and PtdThr. Moreover, we discovered that TgECT is required for the generation of ethanolamine-phosphory ceramide (EPC), a rare sphingolipid present only a limited number of organisms.

Toxoplasma gondii ist ein obligater intrazellulärer Parasit, der bei Menschen und Nutztieren Toxoplasmose verursacht. Die Phospholipid-Biosynthese ist entscheidend für das erfolgreiche intrazelluläre Überleben und die Replikation des Parasiten, da sie eine wichtige Rolle bei der Biogenese von Membranorganellen, der Signal- transduktion und anderen zellulären Prozessen spielt. Hier untersuchten wir die physiologische Bedeutung von zwei Synthesewegen, die für PtdEtn und PtdIns verantwortlich sind. Wir zeigen das Vorhandensein einer neuartigen PtdIns-Synthase (PIS) in T. gondii, die als TgPIS bezeichnet wird und ein funktionelles Enzym mit einem katalytisch wichtigen CDP-Alkohol- Phosphotransferase-Motiv codiert, das sich ausschließlich im Golgi-Apparat befindet. Der Parasit importiert Myoinosit aus dem Milieu und verwendet es zusammen mit de novo synthetisiertem CDP- Diacylglycerin, um PtdIns zu produzieren. Eine durch Auxin induzierbare bedingte Unterdrückung von TgPIS schaltet den Lysezyklus des Parasiten aufgrund von Defekten in der Replikation, Motilität und Austritt in Säugetierzellen aus. Das Lipidom-Profiling der PIS-Mutante zeigt eine selektive Reduktion bestimmter PtdIns- und PtdThr-Spezies, wohingegen ausgewählte PtdGro-, PtdSer- und BMP-Spezies erhöht sind, was auf eine enge Interregulation und Homöostase von anionischen Phospholipiden zur Aufrechterhaltung der Membranintegrität hindeutet. Zusätzlich identifizierten wir eine Ethanolamin-Cytidyltransferase (TgECT), das geschwindigkeitsbestimmende Enzym des Kennedy-Signalwegs, das im Cytosol lokalisiert ist. Das Enzym ist eindeutig für den Lysezyklus essentiell, da seine genetische Ablation nicht durchführbar ist und der durch Auxin meditierte bedingte Abbau des Proteins das Parasitenwachstum in Plaqueassays stark beeinträchtigt. Die Lipidomanalyse der Mutante identifizierte eine wichtige Rolle bei der Biogenese ausgewählter Arten von PtdEtn, PtdSer und PtdThr. Darüber hinaus haben wir fest, Toxoplasma gondii is an obligate intracellular parasite that causes Toxoplasmosis in human and livestock. Phospholipid biosynthesis is crucial for the successful intracellular survival and replication of the parasites, as the phospholipids have important roles in the biogenesis of membrane organelles, signal transduction and other cellular processes. Here, we dissected the physiological importance of two pathways accounting for the synthesis of PtdEtn and PtdIns. We demonstrated the presence of a novel PtdIns synthase (PIS) in T.gondii termed TgPIS, expressing a functional enzyme with a catalytically vital CDP-alcohol phosphotransferase motif, which resides exclusively in the Golgi body. The parasite imports myo-inositol from milieu, and co-utilizes de novo-synthesized CDP-diacylglycerol to produce PtdIns. An auxin-inducible conditional repression of TgPIS abrogated the lytic cycle of the parasite in mammalian cells due to defects in the replication, motility and egress. Lipidomic profiling of the PIS mutant demonstrated selective reduction of certain PtdIns and PtdThr species, whereas selected PtdGro, PtdSer and BMP species were increased, which suggested a tight inter-regulation and homeostasis of anionic phospholipids to maintain the membrane integrity. In addition, we identified an ethanolamine cytidyltransferase (TgECT), the rate-limiting enzyme of Kennedy pathway, which is localized in the cytosol. The enzyme is clearly essential for the lytic cycle as its genetic ablation was not feasible, and auxin-meditated conditional knockdown severely impaired the parasite growth in plaque assays. Similarly, lipidomic analysis of the mutant identified an important role in the biogenesis of selected species of PtdEtn, PtdSer and PtdThr. Moreover, we discovered that TgECT is required for the generation of ethanolamine-phosphory ceramide (EPC), a rare sphingolipid present only a limited number of organisms.

Neosporosis caused by the apicomplexan parasite Neospora caninum is an economically important disease that induces abortion in dairy and beef cattle. There are no vaccines or drugs available on the market for control or treatment of the disease in bovines. The peroxide artemisinin and its derivatives used clinically for treatment of malaria are active against N. caninum and other apicomplexan parasites. We have now evaluated the activities of the readily accessible and chemically robust 11-azaartemisinin 5 and selected N-sulfonyl derivatives prepared as described in the accompanying paper against N. caninum tachyzoites grown in infected human foreskin fibroblasts. Azaartemisinin elicited an IC50 value of 150 nm, and the 2′,5′-dichloro-3′-thienylsulfonyl-11-azaartemisinin 17 was found to be the most active, with an IC50 value of 40 nm. Comparison with normal human fetal lung fibroblasts HFLF WI-38 revealed relatively benign cytotoxicity. The compounds were also screened in vitro against TK-10 (renal), UACC-62 (melanoma) and MCF-7 (breast) cancer cell lines; overall, in line with activities against HFLF cells, most compounds in the series were found to be inactive

Neosporosis caused by the apicomplexan parasite Neospora caninum is an economically important disease that induces abortion in dairy and beef cattle. There are no vaccines or drugs available on the market for control or treatment of the disease in bovines. The peroxide artemisinin and its derivatives used clinically for treatment of malaria are active against N. caninum and other apicomplexan parasites. We have now evaluated the activities of the readily accessible and chemically robust 11-azaartemisinin 5 and selected N-sulfonyl derivatives prepared as described in the accompanying paper against N. caninum tachyzoites grown in infected human foreskin fibroblasts. Azaartemisinin elicited an IC50 value of 150 nm, and the 2′,5′-dichloro-3′-thienylsulfonyl-11-azaartemisinin 17 was found to be the most active, with an IC50 value of 40 nm. Comparison with normal human fetal lung fibroblasts HFLF WI-38 revealed relatively benign cytotoxicity. The compounds were also screened in vitro against TK-10 (renal), UACC-62 (melanoma) and MCF-7 (breast) cancer cell lines; overall, in line with activities against HFLF cells, most compounds in the series were found to be inactive

Prokaryotic histone-like proteins (Hlps) are abundant proteins found in bacterial and plastid nucleoids. Hlps are also found in the eukaryotic dinoflagellates and the apicomplexans, two major lineages of the Alveolata. It may be expected that Hlps of both groups were derived from the same ancestral Alveolates. However, our phylogenetic analyses suggest different origins for the dinoflagellate and the apicomplexan Hlps. The apicomplexan Hlps are affiliated with the cyanobacteria and probably originated from Hlps of the plastid genome. The dinoflagellate Hlps and the proteobacterial long Hlps form a clade that branch off from the node with the proteobacterial short Hlps.

Prokaryotic histone-like proteins (Hlps) are abundant proteins found in bacterial and plastid nucleoids. Hlps are also found in the eukaryotic dinoflagellates and the apicomplexans, two major lineages of the Alveolata. It may be expected that Hlps of both groups were derived from the same ancestral Alveolates. However, our phylogenetic analyses suggest different origins for the dinoflagellate and the apicomplexan Hlps. The apicomplexan Hlps are affiliated with the cyanobacteria and probably originated from Hlps of the plastid genome. The dinoflagellate Hlps and the proteobacterial long Hlps form a clade that branch off from the node with the proteobacterial short Hlps.

Toxoplasma gondii and Cryptosporidium spp. can cause devastating pathological effects in humans and livestock, and in particular to young or immunocompromised individuals. The current treatment plans for these enteric parasites are limited due to long drug courses, severe side effects, or simply a lack of efficacy. The study of the early interactions between the parasites and the site of infection in the small intestinal epithelium has been thwarted by the lack of accessible, physiologically relevant, and species-specific models. Increasingly, 3D stem cell-derived enteroid models are being refined and developed into sophisticated models of infectious disease. In this review we shall illustrate the use of enteroids to spearhead research into enteric parasitic infections, bridging the gap between cell line cultures and in vivo experiments.

Toxoplasma gondii and Cryptosporidium spp. can cause devastating pathological effects in humans and livestock, and in particular to young or immunocompromised individuals. The current treatment plans for these enteric parasites are limited due to long drug courses, severe side effects, or simply a lack of efficacy. The study of the early interactions between the parasites and the site of infection in the small intestinal epithelium has been thwarted by the lack of accessible, physiologically relevant, and species-specific models. Increasingly, 3D stem cell-derived enteroid models are being refined and developed into sophisticated models of infectious disease. In this review we shall illustrate the use of enteroids to spearhead research into enteric parasitic infections, bridging the gap between cell line cultures and in vivo experiments.