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.

Targeting prolyl-tRNA synthetase via a series of ATP-mimetics to accelerate drug discovery against toxoplasmosis.

The prolyl-tRNA synthetase (PRS) is a validated drug target for febrifugine and its synthetic analog halofuginone (HFG) against multiple apicomplexan parasites including Plasmodium falciparum and Toxoplasma gondii. Here, a novel ATP-mimetic centered on 1-(pyridin-4-yl) pyrrolidin-2-one (PPL) scaffold has been validated to bind to Toxoplasma gondii PRS and kill toxoplasma parasites. PPL series exhibited potent inhibition at the cellular (T. gondii parasites) and enzymatic (TgPRS) levels compared to the human counterparts. Cell-based chemical mutagenesis was employed to determine the mechanism of action via a forward genetic screen. Tg-resistant parasites were analyzed with wild-type strain by RNA-seq to identify mutations in the coding sequence conferring drug resistance by computational analysis of variants. DNA sequencing established two mutations, T477A and T592S, proximal to terminals of the PPL scaffold and not directly in the ATP, tRNA, or L-pro sites, as supported by the structural data from high-resolution crystal structures of drug-bound enzyme complexes. These data provide an avenue for structure-based activity enhancement of this chemical series as anti-infectives.

Double drugging of prolyl-tRNA synthetase provides a new paradigm for anti-infective drug development.

Toxoplasmosis is caused by Toxoplasma gondii and in immunocompromised patients it may lead to seizures, encephalitis or death. The conserved enzyme prolyl-tRNA synthetase (PRS) is a validated druggable target in Toxoplasma gondii but the traditional 'single target-single drug' approach has its caveats. Here, we describe two potent inhibitors namely halofuginone (HFG) and a novel ATP mimetic (L95) that bind to Toxoplasma gondii PRS simultaneously at different neighbouring sites to cover all three of the enzyme substrate subsites. HFG and L95 act as one triple-site inhibitor in tandem and form an unusual ternary complex wherein HFG occupies the 3'-end of tRNA and the L-proline (L-pro) binding sites while L95 occupies the ATP pocket. These inhibitors exhibit nanomolar IC50 and EC50 values independently, and when given together reveal an additive mode of action in parasite inhibition assays. This work validates a novel approach and lays a structural framework for further drug development based on simultaneous targeting of multiple pockets to inhibit druggable proteins.

Anti-adaptors provide multiple modes for regulation of the RssB adaptor protein.

RpoS, an RNA polymerase σ factor, controls the response of Escherichia coli and related bacteria to multiple stress responses. During nonstress conditions, RpoS is rapidly degraded by ClpXP, mediated by the adaptor protein RssB, a member of the response regulator family. In response to stress, RpoS degradation ceases. Small anti-adaptor proteins--IraP, IraM, and IraD, each made under a different stress condition--block RpoS degradation. RssB mutants resistant to either IraP or IraM were isolated and analyzed in vivo and in vitro. Each of the anti-adaptors is unique in its interaction with RssB and sensitivity to RssB mutants. One class of mutants defined an RssB N-terminal region close to the phosphorylation site and critical for interaction with IraP but unnecessary for IraM and IraD function. A second class, in the RssB C-terminal PP2C-like domain, led to activation of RssB function. These mutants allowed the response regulator to act in the absence of phosphorylation but did not abolish interaction with anti-adaptors. This class of mutants is broadly resistant to the anti-adaptors and bears similarity to constitutively activated mutants found in a very different PP2C protein. The mutants provide insight into how the anti-adaptors perturb RssB response regulator function and activation.

The aspartyl protease TgASP5 mediates the export of the Toxoplasma GRA16 and GRA24 effectors into host cells.

Toxoplasma gondii and Plasmodium species are obligatory intracellular parasites that export proteins into the infected cells in order to interfere with host-signalling pathways, acquire nutrients or evade host defense mechanisms. With regard to export mechanism, a wealth of information in Plasmodium spp. is available, while the mechanisms operating in T. gondii remain uncertain. The recent discovery of exported proteins in T. gondii, mainly represented by dense granule resident proteins, might explain this discrepancy and offers a unique opportunity to study the export mechanism in T. gondii. Here, we report that GRA16 export is mediated by two protein elements present in its N-terminal region. Because the first element contains a putative Plasmodium export element linear motif (RRLAE), we hypothesized that GRA16 export depended on a maturation process involving protein cleavage. Using both N- and C-terminal epitope tags, we provide evidence for protein proteolysis occurring in the N-terminus of GRA16. We show that TgASP5, the T. gondii homolog of Plasmodium plasmepsin V, is essential for GRA16 export and is directly responsible for its maturation in a Plasmodium export element-dependent manner. Interestingly, TgASP5 is also involved in GRA24 export, although the GRA24 maturation mechanism is TgASP5-independent. Our data reveal different modus operandi for protein export, in which TgASP5 should play multiple functions.

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

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 tRNA(Leu) 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.

Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression.

Toxoplasma gondii is the most widespread apicomplexan parasite and occupies a large spectrum of niches by infecting virtually any warm-blooded animals. As an obligate intracellular parasite, Toxoplasma has evolved a repertoire of strategies to fine-tune the cellular environment in an optimal way to promote growth and persistence in host tissues hence increasing the chance to be transmitted to new hosts. Short and long-term intracellular survival is associated with Toxoplasma ability to both evade the host deleterious immune defences and to stimulate a beneficial immune balance by governing host cell gene expression. It is only recently that parasite proteins responsible for driving these transcriptional changes have been identified. While proteins contained in the apical secretory Rhoptry organelle have already been identified as bona fide secreted effectors that divert host signalling pathways, recent findings revealed that dense granule proteins should be added to the growing list of effectors as they reach the host cell cytoplasm and nucleus and target various host cell pathways in the course of cell infection. Herein, we emphasize on a novel subfamily of dense granule residentproteins, exemplified with the GRA16 and GRA24 members we recently discovered as both are exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genome expression.

Flexible synthesis and evaluation of diverse anti-Apicomplexa cyclic peptides.

A modular approach to synthesize anti-Apicomplexa parasite inhibitors was developed that takes advantage of a pluripotent cyclic tetrapeptide scaffold capable of adjusting appendage and skeletal diversities in only a few steps (one to three steps). The diversification processes make use of selective radical coupling reactions and involve a new example of a reductive carbon-nitrogen cleavage reaction with SmI2. The resulting bioactive cyclic peptides have revealed new insights into structural factors that govern selectivity between Apicomplexa parasites such as Toxoplasma and Plasmodium and human cells.

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.

A DNA damage response in Escherichia coli involving the alternative sigma factor, RpoS.

We isolated an Escherichia coli mutant in the iraD gene, sensitive to various forms of DNA damage. Our data are consistent with the function of IraD to promote accumulation of the alternative transcription sigma factor, RpoS, by binding to the adaptor RssB protein that targets RpoS for degradation. Our results demonstrate the physiological importance of this mode of regulation for DNA damage tolerance. Although RpoS is best known for its regulation of genes induced in stationary phase, our work underscores the importance of the RpoS regulon in a DNA damage response in actively growing cells. We show that iraD transcription is induced by DNA damage by a mechanism independent of the SOS response. The IraD and SOS regulatory pathways appear to act synergistically to ensure survival of cells faced with oxidative or DNA damaging stress during cellular growth.

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 Toxoplasma effector GRA28 promotes parasite dissemination by inducing dendritic cell-like migratory properties in infected macrophages.

Upon pathogen detection, macrophages normally stay sessile in tissues while dendritic cells (DCs) migrate to secondary lymphoid tissues. The obligate intracellular protozoan Toxoplasma gondii exploits the trafficking of mononuclear phagocytes for dissemination via unclear mechanisms. We report that, upon T. gondii infection, macrophages initiate the expression of transcription factors normally attributed to DCs, upregulate CCR7 expression with a chemotactic response, and perform systemic migration when adoptively transferred into mice. We show that parasite effector GRA28, released by the MYR1 secretory pathway, cooperates with host chromatin remodelers in the host cell nucleus to drive the chemotactic migration of parasitized macrophages. During in vivo challenge studies, bone marrow-derived macrophages infected with wild-type T. gondii outcompeted those challenged with MYR1- or GRA28-deficient strains in migrating and reaching secondary organs. This work reveals how an intracellular parasite hijacks chemotaxis in phagocytes and highlights a remarkable migratory plasticity in differentiated cells of the mononuclear phagocyte system.

Chromatin modifications: implications in the regulation of gene expression in Toxoplasma gondii.

The apicomplexan Toxoplasma gondii completes its life cycle by successive processes of parasite differentiation that rely on a tight control of gene expression to ensure appropriate protein profiles on time. During the last 5 years, several groups have pioneered this field of investigation, suggesting that epigenetics could play an important role in the control of parasite gene expression. Histone modifications serve as an effective way to regulate gene transcription but they do not operate alone; rather, they act in concert with other putative epigenetic information carriers (histone variants, small RNAs) and DNA sequence-specific transcription factors to modulate the higher-order structure of the chromatin fibre and govern the on-time recruitment of the transcriptional machinery to specific genes. Regarding the 'histone code' hypothesis, the parasite is endowed with a rich repertoire of histone-modifying enzymes catalysing site-selective modifications, which are subsequently interpreted by effector proteins that recognize specific covalent marks. Still, several peculiarities seem unique to T. gondii. This review is a synthesis of the current knowledge of how epigenetics contribute to the control of gene expression in T. gondii and, likely, other Apicomplexa.

A plant-like mechanism coupling m6A reading to polyadenylation safeguards transcriptome integrity and developmental gene partitioning in Toxoplasma.

Correct 3'end processing of mRNAs is one of the regulatory cornerstones of gene expression. In a parasite that must adapt to the regulatory requirements of its multi-host life style, there is a need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly arranged stage-specific genes. In this study, we report our findings in T. gondii of an m6A-dependent 3'end polyadenylation serving as a transcriptional barrier at these loci. We identify the core polyadenylation complex within T. gondii and establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of the T. gondii CPSF4-YTH in complex with an m6A-modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition is associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant model Arabidopsis thaliana. Nanopore direct RNA sequencing shows the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in both T. gondii and A. thaliana. Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in both T. gondii and A. thaliana, as a guardian against aberrant transcriptional read-throughs.

ATAD2 controls chromatin-bound HIRA turnover.

Taking advantage of the evolutionary conserved nature of ATAD2, we report here a series of parallel functional studies in human, mouse, and Schizosaccharomyces pombe to investigate ATAD2's conserved functions. In S. pombe, the deletion of ATAD2 ortholog, abo1, leads to a dramatic decrease in cell growth, with the appearance of suppressor clones recovering normal growth. The identification of the corresponding suppressor mutations revealed a strong genetic interaction between Abo1 and the histone chaperone HIRA. In human cancer cell lines and in mouse embryonic stem cells, we observed that the KO of ATAD2 leads to an accumulation of HIRA. A ChIP-seq mapping of nucleosome-bound HIRA and FACT in Atad2 KO mouse ES cells demonstrated that both chaperones are trapped on nucleosomes at the transcription start sites of active genes, resulting in the abnormal presence of a chaperone-bound nucleosome on the TSS-associated nucleosome-free regions. Overall, these data highlight an important layer of regulation of chromatin dynamics ensuring the turnover of histone-bound chaperones.

Activity of the histone deacetylase inhibitor FR235222 on Toxoplasma gondii: inhibition of stage conversion of the parasite cyst form and study of new derivative compounds.

Bradyzoite-to-tachyzoite conversion plays a role in the pathogenesis of recrudescence of ocular toxoplasmosis and disease in immunocompromised persons. The currently available medicines are ineffective on cysts and fail to prevent reactivation of latent toxoplasmosis. A previous study showed that the histone deacetylase inhibitor FR235222 has a dramatic effect on tachyzoite growth and induces tachyzoite-to-bradyzoite conversion in vitro. The present study shows that FR235222 can target in vitro-converted cysts and bradyzoites. Moreover, the compound is active on ex vivo T. gondii cysts. Free bradyzoites isolated after lysis of the cell wall did not proliferate in vitro when the cyst was treated with FR235222. The results imply that this compound is able to cross the T. gondii cystic cell wall. Fluorescent labeling shows that the compound impairs the capacity of the bradyzoites to convert without damaging the cyst wall integrity. In vivo inoculation of formerly treated cysts fails to infect mice when these cysts were treated with FR235222. We used our structural knowledge of FR235222 and its target, T. gondii HDAC3, to synthesize new FR235222 derivative compounds. We identified two new molecules that are highly active against tachyzoites. They harbor a better selectivity index that is more suitable for a future in vivo approach. These results identify FR235222 and its derivatives as new lead compounds in the range of therapeutics available for acute and chronic toxoplasmosis.

Target Identification of an Antimalarial Oxaborole Identifies AN13762 as an Alternative Chemotype for Targeting CPSF3 in Apicomplexan Parasites.

Boron-containing compounds represent a promising class of molecules with proven efficacy against a wide range of pathogens, including apicomplexan parasites. Following lead optimization, the benzoxaborole AN13762 was identified as a preclinical candidate against the human malaria parasite, yet the molecular target remained uncertain. Here, we uncovered the parasiticidal mechanisms of AN13762, by combining forward genetics with transcriptome sequencing and computational mutation discovery and using Toxoplasma gondii as a relevant model for Apicomplexa. AN13762 was shown to target TgCPSF3, the catalytic subunit of the pre-mRNA cleavage and polyadenylation complex, as the anti-pan-apicomplexan benzoxaborole compound, AN3661. However, unique mutations within the TgCPSF3 catalytic site conferring resistance to AN13762 do not confer cross-protection against AN3661, suggesting a divergent resistance mechanism. Finally, in agreement with the high sequence conservation of CPSF3 between Toxoplasma and Cryptosporidium, AN13762 shows oral efficacy in cryptosporidiosis mouse model, a disease for which new drug development is of high priority.

Coupling Polar Adhesion with Traction, Spring, and Torque Forces Allows High-Speed Helical Migration of the Protozoan Parasite Toxoplasma.

Among the eukaryotic cells that navigate through fully developed metazoan tissues, protozoans from the Apicomplexa phylum have evolved motile developmental stages that move much faster than the fastest crawling cells owing to a peculiar substrate-dependent type of motility, known as gliding. Best-studied models are the Plasmodium sporozoite and the Toxoplasma tachyzoite polarized cells for which motility is vital to achieve their developmental programs in the metazoan hosts. The gliding machinery is shared between the two parasites and is largely characterized. Localized beneath the cell surface, it includes actin filaments, unconventional myosin motors housed within a multimember glideosome unit, and apically secreted transmembrane adhesins. In contrast, less is known about the force mechanisms powering cell movement. Pioneered biophysical studies on the sporozoite and phenotypic analysis of tachyzoite actin-related mutants have added complexity to the general view that force production for parasite forward movement directly results from the myosin-driven rearward motion of the actin-coupled adhesion sites. Here, we have interrogated how forces and substrate adhesion-de-adhesion cycles operate and coordinate to allow the typical left-handed helical gliding mode of the tachyzoite. By combining quantitative traction force and reflection interference microscopy with micropatterning and expansion microscopy, we unveil at the millisecond and nanometer scales the integration of a critical apical anchoring adhesion with specific traction and spring-like forces. We propose that the acto-myoA motor directs the traction force which allows transient energy storage by the microtubule cytoskeleton and therefore sets the thrust force required for T. gondii tachyzoite vital helical gliding capacity.

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.

Multiple pathways for regulation of sigmaS (RpoS) stability in Escherichia coli via the action of multiple anti-adaptors.

SigmaS, the stationary phase sigma factor of Escherichia coli and Salmonella, is regulated at multiple levels. The sigmaS protein is unstable during exponential growth and is stabilized during stationary phase and after various stress treatments. Degradation requires both the ClpXP protease and the adaptor RssB. The small antiadaptor protein IraP is made in response to phosphate starvation and interacts with RssB, causing sigmaS stabilization under this stress condition. IraP is essential for sigmaS stabilization in some but not all starvation conditions, suggesting the existence of other anti-adaptor proteins. We report here the identification of new regulators of sigmaS stability, important under other stress conditions. IraM (inhibitor of RssB activity during Magnesium starvation) and IraD (inhibitor of RssB activity after DNA damage) inhibit sigmaS proteolysis both in vivo and in vitro. Our results reveal that multiple anti-adaptor proteins allow the regulation of sigmaS stability through the regulation of RssB activity under a variety of stress conditions.