In vitro production of cat-restricted Toxoplasma pre-sexual stages.

Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described1,2. Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. 1), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.

In vitro production of cat-restricted Toxoplasma pre-sexual stages by epigenetic reprogramming.

Sexual reproduction of Toxoplasma gondii , which is restricted to the small intestine of felids, is sparsely documented, due to ethical concerns surrounding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described 1 . In this study, we found that transcription factors AP2XII-1 and AP2XI-2, expressed in tachyzoite stage that causes acute toxoplasmosis, can silence genes necessary for merozoites, a developmental stage critical for sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a drastic change in the transcriptional program, promoting a complete transition from tachyzoites to merozoites. Pre-gametes produced in vitro under these conditions are characterized by specific protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit the epigenitors MORC and HDAC3 1 , which in turn restrict the accessibility of chromatin to the transcriptional machinery. Thus, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. This effective in vitro culture of merozoites paves the way to explore Toxoplasma sexual reproduction without the need to infect kittens and has potential for the development of therapeutics to block parasite transmission.

Altiratinib blocks Toxoplasma gondii and Plasmodium falciparum development by selectively targeting a spliceosome kinase.

The Apicomplexa comprise a large phylum of single-celled, obligate intracellular protozoa that include Toxoplasma gondii, Plasmodium, and Cryptosporidium spp., which infect humans and animals and cause severe parasitic diseases. Available therapeutics against these diseases are limited by suboptimal efficacy and frequent side effects, as well as the emergence and spread of resistance. We use a drug repurposing strategy and identify altiratinib, a compound originally developed to treat glioblastoma, as a promising drug candidate with broad spectrum activity against apicomplexans. Altiratinib is parasiticidal and blocks the development of intracellular zoites in the nanomolar range and with a high selectivity index when used against T. gondii. We have identified TgPRP4K of T. gondii as the primary target of altiratinib using genetic target deconvolution, which highlighted key residues within the kinase catalytic site that conferred drug resistance when mutated. We have further elucidated the molecular basis of the inhibitory mechanism and species selectivity of altiratinib for TgPRP4K and for its Plasmodium falciparum counterpart, PfCLK3. Our data identified structural features critical for binding of the other PfCLK3 inhibitor, TCMDC-135051. Consistent with the splicing control activity of this kinase family, we have shown that altiratinib can cause global disruption of splicing, primarily through intron retention in both T. gondii and P. falciparum. Thus, our data establish parasitic PRP4K/CLK3 as a potential pan-apicomplexan target whose repertoire of inhibitors can be expanded by the addition of altiratinib.

The BCC7 Protein Contributes to the Toxoplasma Basal Pole by Interfacing between the MyoC Motor and the IMC Membrane Network.

T. gondii is a eukaryotic parasite that has evolved a stage called tachyzoite which multiplies in host cells by producing two daughter cells internally. These nascent tachyzoites bud off their mother and repeat the division process until the expanding progenies escape to settle and multiply in other host cells. Over these intra- and extra-cellular phases, the tachyzoite maintains an essential apicobasal polarity that emerges through a unique bidirectional budding process of the elongating cells. This process requires the assembly of several molecular complexes that, at the nascent pole, encompass structural and myosin motor elements. To characterize a recently identified basal pole marker named BCC7 with respect to the posterior myosin J and myosin C motors, we used conventional biochemistry as well as advanced proteomic and in silico analysis in conjunction with live and super resolution microscopy of transgenic fluorescent tachyzoites. We document that BCC7 forms a ribbed ring below which myosin C motor entities distribute regularly. In addition, we identified-among 13 BCC7 putative partners-two novel and five known members of the inner membrane complex (IMC) family which ends at the apical side of the ring. Therefore, BCC7 could assist the stabilization of the IMC plaques and contribute to the parasite biomechanical properties.

A brain cyst load-associated antigen is a Toxoplasma gondii biomarker for serodetection of persistent parasites and chronic infection.

BACKGROUND: Biomarker discovery remains a major challenge for predictive medicine, in particular, in the context of chronic diseases. This is true for the widespread protozoan Toxoplasma gondii which establishes long-lasting parasitism in metazoans, humans included. This microbe successively unfolds distinct genetic programs that direct the transition from high to low replicative potential inside host cells. As a slow-replicating cell, the T. gondii bradyzoite developmental stage persists enclosed in a cyst compartment within tissues including the nervous system, being held by a sustained immune equilibrium which accounts for the prolonged clinically silent phase of parasitism. Serological surveys indicate that nearly one third of the human population has been exposed to T. gondii and possibly host bradyzoites. Because any disruption of the immune balance drives the reverse transition from bradyzoite to fast replicating tachyzoite and uncontrolled growth of the latter, these people are at risk for life-threatening disease. While serological tests for discriminating recent from past infection are available, there is yet no immunogenic biomarker used in the serological test to allow ascertaining the presence of persistent bradyzoites. RESULTS: Capitalizing on genetically engineered parasites induced to produce mature bradyzoites in vitro, we have identified the BCLA/MAG2 protein being restricted to the bradyzoite and the cyst envelope. Using laboratory mice as relevant T. gondii host models, we demonstrated that BCLA/MAG2 drives the generation of antibodies that recognize bradyzoite and the enveloping cyst structure. We have designed an ELISA assay based on a bacterially produced BCLA recombinant polypeptide, which was validated using a large collection of sera from mice of different genetic backgrounds and infected with bcla+ or bcla-null cystogenic and non-cystogenic T. gondii strains. To refine the design of the ELISA assay, we applied high-resolution BCLA epitope mapping and identified a specific combination of peptides and accordingly set up a selective and sensitive ELISA assay which allowed the detection of anti-BCLA/MAG2 antibodies in the sera of human patients with various forms of toxoplasmosis. CONCLUSIONS: We brought proof of principle that anti-BCLA/MAG2 antibodies serve as specific and sensitive serological markers in the perspective of a combinatorial strategy for detection of persistent T. gondii parasitism.

A MORC-driven transcriptional switch controls Toxoplasma developmental trajectories and sexual commitment.

Toxoplasma gondii has a complex life cycle that is typified by asexual development that takes place in vertebrates, and sexual reproduction, which occurs exclusively in felids and is therefore less studied. The developmental transitions rely on changes in the patterns of gene expression, and recent studies have assigned roles for chromatin shapers, including histone modifications, in establishing specific epigenetic programs for each given stage. Here, we identified the T. gondii microrchidia (MORC) protein as an upstream transcriptional repressor of sexual commitment. MORC, in a complex with Apetala 2 (AP2) transcription factors, was shown to recruit the histone deacetylase HDAC3, thereby impeding the accessibility of chromatin at the genes that are exclusively expressed during sexual stages. We found that MORC-depleted cells underwent marked transcriptional changes, resulting in the expression of a specific repertoire of genes, and revealing a shift from asexual proliferation to sexual differentiation. MORC acts as a master regulator that directs the hierarchical expression of secondary AP2 transcription factors, and these transcription factors potentially contribute to the unidirectionality of the life cycle. Thus, MORC plays a cardinal role in the T. gondii life cycle, and its conditional depletion offers a method to study the sexual development of the parasite in vitro, and is proposed as an alternative to the requirement of T. gondii infections in cats.

The Toxoplasma effector TEEGR promotes parasite persistence by modulating NF-κB signalling via EZH2.

The protozoan parasite Toxoplasma gondii has co-evolved with its homeothermic hosts (humans included) strategies that drive its quasi-asymptomatic persistence in hosts, hence optimizing the chance of transmission to new hosts. Persistence, which starts with a small subset of parasites that escape host immune killing and colonize the so-called immune privileged tissues where they differentiate into a low replicating stage, is driven by the interleukin 12 (IL-12)-interferon-γ (IFN-γ) axis. Recent characterization of a family of Toxoplasma effectors that are delivered into the host cell, in which they rewire the host cell gene expression, has allowed the identification of regulators of the IL-12-IFN-γ axis, including repressors. We now report on the dense granule-resident effector, called TEEGR (Toxoplasma E2F4-associated EZH2-inducing gene regulator) that counteracts the nuclear factor-κB (NF-κB) signalling pathway. Once exported into the host cell, TEEGR ends up in the nucleus where it not only complexes with the E2F3 and E2F4 host transcription factors to induce gene expression, but also promotes shaping of a non-permissive chromatin through its capacity to switch on EZH2. Remarkably, EZH2 fosters the epigenetic silencing of a subset of NF-κB-regulated cytokines, thereby strongly contributing to the host immune equilibrium that influences the host immune response and promotes parasite persistence in mice.

Modifications at K31 on the lateral surface of histone H4 contribute to genome structure and expression in apicomplexan parasites.

An unusual genome architecture characterizes the two related human parasitic pathogens Plasmodium falciparum and Toxoplasma gondii. A major fraction of the bulk parasite genome is packaged as transcriptionally permissive euchromatin with few loci embedded in silenced heterochromatin. Primary chromatin shapers include histone modifications at the nucleosome lateral surface close to the DNA but their mode of action remains unclear. We now identify versatile modifications at Lys31 within the globular domain of histone H4 that crucially determine genome organization and expression in Apicomplexa parasites. H4K31 acetylation at the promoter correlates with, and perhaps directly regulates, gene expression in both parasites. By contrast, monomethylated H4K31 is enriched in the core body of T. gondii active genes but inversely correlates with transcription, whereas it is unexpectedly enriched at transcriptionally inactive pericentromeric heterochromatin in P. falciparum, a region devoid of the characteristic H3K9me3 histone mark and its downstream effector HP1.

AMA1-deficient Toxoplasma gondii parasites transiently colonize mice and trigger an innate immune response that leads to long-lasting protective immunity.

The apical membrane antigen 1 (AMA1) protein was believed to be essential for the perpetuation of two Apicomplexa parasite genera, Plasmodium and Toxoplasma, until we genetically engineered viable parasites lacking AMA1. The reduction in invasiveness of the Toxoplasma gondii RH-AMA1 knockout (RH-AMA1(KO)) tachyzoite population, in vitro, raised key questions about the outcome associated with these tachyzoites once inoculated in the peritoneal cavity of mice. In this study, we used AMNIS technology to simultaneously quantify and image the parasitic process driven by AMA1(KO) tachyzoites. We report their ability to colonize and multiply in mesothelial cells and in both resident and recruited leukocytes. While the RH-AMA1(KO) population amplification is rapidly lethal in immunocompromised mice, it is controlled in immunocompetent hosts, where immune cells in combination sense parasites and secrete proinflammatory cytokines. This innate response further leads to a long-lasting status immunoprotective against a secondary challenge by high inocula of the homologous type I or a distinct type II T. gondii genotypes. While AMA1 is definitively not an essential protein for tachyzoite entry and multiplication in host cells, it clearly assists the expansion of parasite population in vivo.

miR-146a and miR-155 delineate a MicroRNA fingerprint associated with Toxoplasma persistence in the host brain.

microRNAs were recently found to be regulators of the host response to infection by apicomplexan parasites. In this study, we identified two immunomodulatory microRNAs, miR-146a and miR-155, that were coinduced in the brains of mice challenged with Toxoplasma in a strain-specific manner. These microRNAs define a characteristic fingerprint for infection by type II strains, which are the most prevalent cause of human toxoplasmosis in Europe and North America. Using forward genetics, we showed that strain-specific differences in miR-146a modulation were in part mediated by the rhoptry kinase, ROP16. Remarkably, we found that miR-146a deficiency led to better control of parasite burden in the gut and most likely of early parasite dissemination in the brain tissue, resulting in the long-term survival of mice.