Bastien Touquet

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-1β. 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-β 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.

Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-γ–mediated host defenses

Gay et al. identify a Toxoplasma gondii secreted effector that recruits NuRD transcriptional repressor and blocks IFN-γ–stimulated STAT1-dependent gene expression, thus dampening host responses to infection.

Cryptosporidium and Toxoplasma Parasites are Inhibited by a Benzoxaborole Targeting Leucyl-tRNA Synthetase.

ABSTRACT The apicomplexan parasites Cryptosporidium and Toxoplasma are serious threats to human health. Cryptosporidiosis is a severe diarrheal disease in malnourished children and immunocompromised individuals, with the only FDA-approved drug treatment currently being nitazoxanide. The existing therapies for toxoplasmosis, an important pathology in immunocompromised individuals and pregnant women, also have serious limitations. With the aim of developing alternative therapeutic options to address these health problems, we tested a number of benzoxaboroles, boron-containing compounds shown to be active against various infectious agents, for inhibition of the growth of Cryptosporidium parasites in mammalian cells. A 3-aminomethyl benzoxaborole, AN6426, with activity in the micromolar range and with activity comparable to that of nitazoxanide, was identified and further characterized using biophysical measurements of affinity and crystal structures of complexes with the editing domain of Cryptosporidium leucyl-tRNA synthetase (LeuRS). The same compound was shown to be active against Toxoplasma parasites, with the activity being enhanced in the presence of norvaline, an amino acid that can be mischarged by LeuRS. Our observations are consistent with AN6426 inhibiting protein synthesis in both Cryptosporidium and Toxoplasma by forming a covalent adduct with tRNALeu in the LeuRS editing active site and suggest that further exploitation of the benzoxaborole scaffold is a valid strategy to develop novel, much needed antiparasitic agents.

Development of High-Throughput Methods to Quantify Cysts of Toxoplasma gondii

Toxplasma is a protozoan parasite, which forms persistent cysts in tissues of chronically infected animals and humans. Cysts can reactivate leading to severe pathologies. They also contribute to the transmission of Toxoplasma infection in humans by ingestion of undercooked meat. Classically, the quantification of cyst burden in tissues uses microscopy methods, which are laborious and time consuming. Here, we have developed automated protocols to quantify cysts, based on flow cytometry or high‐throughput microscopy. Brains of rodents infected with cysts of Prugniaud strain were incubated with the FITC‐Dolichos biflorus lectin and analyzed by flow cytometry and high‐throughput epifluorescence microscopy. The comparison of cyst counts by manual epifluorescence microscopy to flow cytometry or to high‐throughput epifluorescence microscopy revealed a good correlation (r = 0.934, r = 0.993, P < 0.001 respectively). High‐throughput epifluorescence microscopy was found to be more specific and sensitive than flow cytometry and easier to use for large series of samples. This reliable and easy protocol allow the specific detection of Toxoplasma cysts in brain, even at low concentrations; it could be a new way to detect them in water and in contaminate food.

Phenotypes Associated with Knockouts of Eight Dense Granule Gene Loci (GRA2-9) in Virulent Toxoplasma gondii

Toxoplasma gondii actively invades host cells and establishes a parasitophorous vacuole (PV) that accumulates many proteins secreted by the dense granules (GRA proteins). To date, at least 23 GRA proteins have been reported, though the function(s) of most of these proteins still remains unknown. We targeted gene knockouts at ten GRA gene loci (GRA1-10) to investigate the cellular roles and essentiality of these classical GRA proteins during acute infection in the virulent type I RH strain. While eight of these genes (GRA2-9) were successfully knocked out, targeted knockouts at the GRA1 and GRA10 loci were not obtained, suggesting these GRA proteins may be essential. As expected, the Δgra2 and Δgra6 knockouts failed to form an intravacuolar network (IVN). Surprisingly, Δgra7 exhibited hyper-formation of the IVN in both normal and lipid-free growth conditions. No morphological alterations were identified in parasite or PV structures in the Δgra3, Δgra4, Δgra5, Δgra8, or Δgra9 knockouts. With the exception of the Δgra3 and Δgra8 knockouts, all of the GRA knockouts exhibited defects in their infection rate in vitro. While the single GRA knockouts did not exhibit reduced replication rates in vitro, replication rate defects were observed in three double GRA knockout strains (Δgra4Δgra6, Δgra3Δgra5 and Δgra3Δgra7). However, the virulence of single or double GRA knockout strains in CD1 mice was not affected. Collectively, our results suggest that while the eight individual GRA proteins investigated in this study (GRA2-9) are not essential, several GRA proteins may provide redundant and potentially important functions during acute infection.

Investigation of a new 1,3-diarylpropenone as a potential antimitotic agent targeting bladder carcinoma

1-(2,4-dimethoxyphenyl)-3-(1-methylindolyl) propenone, namely IPP51, was identified by screening a library of 3-indolyl-1-phenylpropenones. IPP51 was investigated for its ability to inhibit proliferation and/or to induce apoptosis of human bladder cancer cell lines and to assess its potential use in bladder carcinoma treatment. After treating the cells with IPP51 for 24 h, the title compound induced a predominant and reversible G2+M accumulation at the prometaphase stage of mitosis. However, when used for a longer period, it leads to cell apoptosis. These results suggest that the compound has potential anticancer activities, which could be useful in bladder cancer treatment.

High-content imaging assay to evaluate Toxoplasma gondii infection and proliferation: A multiparametric assay to screen new compounds

Toxoplasma gondii is an intracellular protozoan parasite widely distributed in animals and humans. Infection of host cells and parasite proliferation are essential steps in Toxoplasma pathology. The objective of this study was to develop and validate a novel automatic High Content Imaging (HCI) assay to study T. gondii infection and proliferation. We tested various fluorescent markers and strategies of image analysis to obtain an automated method providing results comparable to those from gold standard infection and proliferation assays. No significant difference was observed between the results obtained from the HCI assay and the standard assays (manual fluorescence microscopy and incorporation of [3H]-uracil). We developed here a robust and time-saving assay. This automated technology was then used to screen a library of compounds belonging to four classes of either natural compounds or synthetic derivatives. Inhibition of parasite proliferation and host cell toxicity were measured in the same assay and led to the identification of one hit, a thiosemicarbazone that allows important inhibition of Toxoplasma proliferation while being relatively safe for the host cells.

Specific Targeting of Plant and Apicomplexa Parasite Tubulin through Differential Screening Using In Silico and Assay-Based Approaches

Dinitroanilines are chemical compounds with high selectivity for plant cell α-tubulin in which they promote microtubule depolymerization. They target α-tubulin regions that have diverged over evolution and show no effect on non-photosynthetic eukaryotes. Hence, they have been used as herbicides over decades. Interestingly, dinitroanilines proved active on microtubules of eukaryotes deriving from photosynthetic ancestors such as Toxoplasma gondii and Plasmodium falciparum, which are responsible for toxoplasmosis and malaria, respectively. By combining differential in silico screening of virtual chemical libraries on Arabidopsis thaliana and mammal tubulin structural models together with cell-based screening of chemical libraries, we have identified dinitroaniline related and non-related compounds. They inhibit plant, but not mammalian tubulin assembly in vitro, and accordingly arrest A. thaliana development. In addition, these compounds exhibit a moderate cytotoxic activity towards T. gondii and P. falciparum. These results highlight the potential of novel herbicidal scaffolds in the design of urgently needed anti-parasitic drugs.