A highly conserved Toxo1 haplotype directs resistance to toxoplasmosis and its associated caspase-1 dependent killing of parasite and host macrophage.

Natural immunity or resistance to pathogens most often relies on the genetic make-up of the host. In a LEW rat model of refractoriness to toxoplasmosis, we previously identified on chromosome 10 the Toxo1 locus that directs toxoplasmosis outcome and controls parasite spreading by a macrophage-dependent mechanism. Now, we narrowed down Toxo1 to a 891 kb interval containing 29 genes syntenic to human 17p13 region. Strikingly, Toxo1 is included in a haplotype block strictly conserved among all refractory rat strains. The sequencing of Toxo1 in nine rat strains (5 refractory and 4 susceptible) revealed resistant-restricted conserved polymorphisms displaying a distribution gradient that peaks at the bottom border of Toxo1, and highlighting the NOD-like receptor, Nlrp1a, as a major candidate. The Nlrp1 inflammasome is known to trigger, upon pathogen intracellular sensing, pyroptosis programmed-cell death involving caspase-1 activation and cleavage of IL-1ß. Functional studies demonstrated that the Toxo1-dependent refractoriness in vivo correlated with both the ability of macrophages to restrict T. gondii growth and a T. gondii-induced death of intracellular parasites and its host macrophages. The parasite-induced cell death of infected macrophages bearing the LEW-Toxo1 alleles was found to exhibit pyroptosis-like features with ROS production, the activation of caspase-1 and IL1-ß secretion. The pharmacological inactivation of caspase-1 using YVAD and Z-VAD inhibitors prevented the death of both intravacuolar parasites and host non-permissive macrophages but failed to restore parasite proliferation. These findings demonstrated that the Toxo1-dependent response of rat macrophages to T. gondii infection may trigger two pathways leading to the control of parasite proliferation and the death of parasites and host macrophages. The NOD-like receptor NLRP1a/Caspase-1 pathway is the best candidate to mediate the parasite-induced cell death. These data represent new insights towards the identification of a major pathway of innate resistance to toxoplasmosis and the prediction of individual resistance.

ALOX12 in human toxoplasmosis.

ALOX12 is a gene encoding arachidonate 12-lipoxygenase (12-LOX), a member of a nonheme lipoxygenase family of dioxygenases. ALOX12 catalyzes the addition of oxygen to arachidonic acid, producing 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which can be reduced to the eicosanoid 12-HETE (12-hydroxyeicosatetraenoic acid). 12-HETE acts in diverse cellular processes, including catecholamine synthesis, vasoconstriction, neuronal function, and inflammation. Consistent with effects on these fundamental mechanisms, allelic variants of ALOX12 are associated with diseases including schizophrenia, atherosclerosis, and cancers, but the mechanisms have not been defined. Toxoplasma gondii is an apicomplexan parasite that causes morbidity and mortality and stimulates an innate and adaptive immune inflammatory reaction. Recently, it has been shown that a gene region known as Toxo1 is critical for susceptibility or resistance to T. gondii infection in rats. An orthologous gene region with ALOX12 centromeric is also present in humans. Here we report that the human ALOX12 gene has susceptibility alleles for human congenital toxoplasmosis (rs6502997 [P, <0.000309], rs312462 [P, <0.028499], rs6502998 [P, <0.029794], and rs434473 [P, <0.038516]). A human monocytic cell line was genetically engineered using lentivirus RNA interference to knock down ALOX12. In ALOX12 knockdown cells, ALOX12 RNA expression decreased and levels of the ALOX12 substrate, arachidonic acid, increased. ALOX12 knockdown attenuated the progression of T. gondii infection and resulted in greater parasite burdens but decreased consequent late cell death of the human monocytic cell line. These findings suggest that ALOX12 influences host responses to T. gondii infection in human cells. ALOX12 has been shown in other studies to be important in numerous diseases. Here we demonstrate the critical role ALOX12 plays in T. gondii infection in humans.

Inhibition of p-aminobenzoate and folate syntheses in plants and apicomplexan parasites by natural product rubreserine.

Glutamine amidotransferase/aminodeoxychorismate synthase (GAT-ADCS) is a bifunctional enzyme involved in the synthesis of p-aminobenzoate, a central component part of folate cofactors. GAT-ADCS is found in eukaryotic organisms autonomous for folate biosynthesis, such as plants or parasites of the phylum Apicomplexa. Based on an automated screening to search for new inhibitors of folate biosynthesis, we found that rubreserine was able to inhibit the glutamine amidotransferase activity of the plant GAT-ADCS with an apparent IC(50) of about 8 µM. The growth rates of Arabidopsis thaliana, Toxoplasma gondii, and Plasmodium falciparum were inhibited by rubreserine with respective IC(50) values of 65, 20, and 1 µM. The correlation between folate biosynthesis and growth inhibition was studied with Arabidopsis and Toxoplasma. In both organisms, the folate content was decreased by 40-50% in the presence of rubreserine. In both organisms, the addition of p-aminobenzoate or 5-formyltetrahydrofolate in the external medium restored the growth for inhibitor concentrations up to the IC(50) value, indicating that, within this range of concentrations, rubreserine was specific for folate biosynthesis. Rubreserine appeared to be more efficient than sulfonamides, antifolate drugs known to inhibit the invasion and proliferation of T. gondii in human fibroblasts. Altogether, these results validate the use of the bifunctional GAT-ADCS as an efficient drug target in eukaryotic cells and indicate that the chemical structure of rubreserine presents interesting anti-parasitic (toxoplasmosis, malaria) potential.

NALP1 influences susceptibility to human congenital toxoplasmosis, proinflammatory cytokine response, and fate of Toxoplasma gondii-infected monocytic cells.

NALP1 is a member of the NOD-like receptor (NLR) family of proteins that form inflammasomes. Upon cellular infection or stress, inflammasomes are activated, triggering maturation of proinflammatory cytokines and downstream cellular signaling mediated through the MyD88 adaptor. Toxoplasma gondii is an obligate intracellular parasite that stimulates production of high levels of proinflammatory cytokines that are important in innate immunity. In this study, susceptibility alleles for human congenital toxoplasmosis were identified in the NALP1 gene. To investigate the role of the NALP1 inflammasome during infection with T. gondii, we genetically engineered a human monocytic cell line for NALP1 gene knockdown by RNA interference. NALP1 silencing attenuated progression of T. gondii infection, with accelerated host cell death and eventual cell disintegration. In line with this observation, upregulation of the proinflammatory cytokines interleukin-1ß (IL-1ß), IL-18, and IL-12 upon T. gondii infection was not observed in monocytic cells with NALP1 knockdown. These findings suggest that the NALP1 inflammasome is critical for mediating innate immune responses to T. gondii infection and pathogenesis. Although there have been recent advances in understanding the potent activity of inflammasomes in directing innate immune responses to disease, this is the first report, to our knowledge, on the crucial role of the NALP1 inflammasome in the pathogenesis of T. gondii infections in humans.

Toxoplasma gondii acyl-lipid metabolism: de novo synthesis from apicoplast-generated fatty acids versus scavenging of host cell precursors.

Toxoplasma gondii is an obligate intracellular parasite that contains a relic plastid, called the apicoplast, deriving from a secondary endosymbiosis with an ancestral alga. Metabolic labelling experiments using [14C]acetate led to a substantial production of numerous glycero- and sphingo-lipid classes in extracellular tachyzoites. Syntheses of all these lipids were affected by the herbicide haloxyfop, demonstrating that their de novo syntheses necessarily required a functional apicoplast fatty acid synthase II. The complex metabolic profiles obtained and a census of glycerolipid metabolism gene candidates indicate that synthesis is probably scattered in the apicoplast membranes [possibly for PA (phosphatidic acid), DGDG (digalactosyldiacylglycerol) and PG (phosphatidylglycerol)], the endoplasmic reticulum (for major phospholipid classes and ceramides) and mitochondria (for PA, PG and cardiolipid). Based on a bioinformatic analysis, it is proposed that apicoplast produced acyl-ACP (where ACP is acyl-carrier protein) is transferred to glycerol-3-phosphate for apicoplast glycerolipid synthesis. Acyl-ACP is also probably transported outside the apicoplast stroma and irreversibly converted into acyl-CoA. In the endoplasmic reticulum, acyl-CoA may not be transferred to a three-carbon backbone by an enzyme similar to the cytosolic plant glycerol-3-phosphate acyltransferase, but rather by a dual glycerol-3-phosphate/dihydroxyacetone-3-phosphate acyltransferase like in animal and yeast cells. We further showed that intracellular parasites could also synthesize most of their lipids from scavenged host cell precursors. The observed appearance of glycerolipids specific to either the de novo pathway in extracellular parasites (unknown glycerolipid 1 and the plant like DGDG), or the intracellular stages (unknown glycerolipid 8), may explain the necessary coexistence of both de novo parasitic acyl-lipid synthesis and recycling of host cell compounds.

The rat Toxo1 locus directs toxoplasmosis outcome and controls parasite proliferation and spreading by macrophage-dependent mechanisms.

Toxoplasmosis is a healthcare problem in pregnant women and immunocompromised patients. Like humans, rats usually develop a subclinical chronic infection. LEW rats exhibit total resistance to Toxoplasma gondii infection, which is expressed in a dominant mode. A genome-wide search carried out in a cohort of F(2) progeny of susceptible BN and resistant LEW rats led to identify on chromosome 10 a major locus of control, which we called Toxo1. Using reciprocal BN and LEW lines congenic for chromosome 10 genomic regions from the other strain, Toxo1 was found to govern the issue of T. gondii infection whatever the remaining genome. Analyzes of rats characterized by genomic recombination within Toxo1, reduced the interval down to a 1.7-cM region syntenic to human 17p13. In vitro studies showed that the Toxo1-mediated refractoriness to T. gondii infection is associated with the ability of the macrophage to impede the proliferation of the parasite within the parasitophorous vacuole. In contrast, proliferation was observed in fibroblasts whatever the genomic origin of Toxo1. Furthermore, ex vivo studies indicate that macrophage controls parasitic infection spreading by a Toxo1-mediated mechanism. This forward genetics approach should ultimately unravel a major pathway of innate resistance to toxoplasmosis and possibly to other apicomplexan parasitic diseases.

The Arabidopsis nuclear DAL gene encodes a chloroplast protein which is required for the maturation of the plastid ribosomal RNAs and is essential for chloroplast differentiation.

Altered pigmentation is an easily scored and sensitive monitor of plastid function. We analyzed in detail a yellow colored transposon-tagged mutant (dal1-2) that is allelic to the dal mutant previously identified (Babiychuk et al., 1997). Mesophyll cells of mutant plants possess abnormal nucleoids and more but smaller plastids than wild type cells. Plastid development in dal1-2 is not altered in the dark but is arrested at the early steps of thylakoid assembly. The amino acid sequence of the protein deduced from our cDNA clone is 21 amino acids longer than the previously published DAL sequence (Babiychuk et al., 1997) and allowed us to show that DAL codes for a chloroplast protein. The dal1-2 mutation has a global negative effect on plastid RNA accumulation and on expression of nuclear encoded photosynthetic genes. We show that the plastid RNA polymerases, the nuclear-encoded NEP and the plastid-encoded PEP, are functional in the mutant. Precursor 16S and 23S rRNA species specifically accumulate at a high level in the mutant but the 5'-end and the long 3'-end trailer are not modified. We suggest that the dal mutation is involved in plastid rRNA processing and consequently in translation and early chloroplast differentiation.