IL-1β is produced by myeloid cells and acts as a critical mediator of host defense during infection and injury. We found that the intracellular protozoan parasite Toxoplasma gondii induced an early IL-1β response (within 4 h) in primary human peripheral blood monocytes isolated from healthy donors. This process involved upregulation of IL-1β, IL-1RN (IL-1R antagonist), and NLRP3 transcripts, de novo protein synthesis, and the release of pro- and mature IL-1β from infected primary monocytes. The released pro-IL-1β was cleavable to mature bioactive IL-1β in the extracellular space by the protease caspase-1. Treatment of primary monocytes with the NLRP3 inhibitor MCC950 or with extracellular potassium significantly reduced IL-1β cleavage and release in response to T. gondii infection, without affecting the release of TNF-α, and indicated a role for the inflammasome sensor NLRP3 and for potassium efflux in T. gondii-induced IL-1β production. Interestingly, T. gondii infection did not induce an IL-1β response in primary human macrophages derived from the same blood donors as the monocytes. Consistent with this finding, NLRP3 was downregulated during the differentiation of monocytes to macrophages and was not induced in macrophages during T. gondii infection. To our knowledge, these findings are the first to identify NLRP3 as an inflammasome sensor for T. gondii in primary human peripheral blood cells and to define an upstream regulator of its activation through the release of intracellular potassium.
One third of humans are infected lifelong with the brain-dwelling, protozoan parasite, Toxoplasma gondii. Approximately fifteen million of these have congenital toxoplasmosis. Although neurobehavioral disease is associated with seropositivity, causality is unproven. To better understand what this parasite does to human brains, we performed a comprehensive systems analysis of the infected brain: We identified susceptibility genes for congenital toxoplasmosis in our cohort of infected humans and found these genes are expressed in human brain. Transcriptomic and quantitative proteomic analyses of infected human, primary, neuronal stem and monocytic cells revealed effects on neurodevelopment and plasticity in neural, immune, and endocrine networks. These findings were supported by identification of protein and miRNA biomarkers in sera of ill children reflecting brain damage and T. gondii infection. These data were deconvoluted using three systems biology approaches: "Orbital-deconvolution" elucidated upstream, regulatory pathways interconnecting human susceptibility genes, biomarkers, proteomes, and transcriptomes. "Cluster-deconvolution" revealed visual protein-protein interaction clusters involved in processes affecting brain functions and circuitry, including lipid metabolism, leukocyte migration and olfaction. Finally, "disease-deconvolution" identified associations between the parasite-brain interactions and epilepsy, movement disorders, Alzheimer's disease, and cancer. This "reconstruction-deconvolution" logic provides templates of progenitor cells' potentiating effects, and components affecting human brain parasitism and diseases.
Micronemes and rhoptries are specialized secretory organelles that deploy their contents at the apical tip of apicomplexan parasites in a regulated manner. The secretory proteins participate in motility, invasion, and egress and are subjected to proteolytic maturation prior to organellar storage and discharge. Here we establish that Toxoplasma gondii aspartyl protease 3 (ASP3) resides in the endosomal-like compartment and is crucially associated to rhoptry discharge during invasion and to host cell plasma membrane lysis during egress. A comparison of the N-terminome, by terminal amine isotopic labelling of substrates between wild type and ASP3 depleted parasites identified microneme and rhoptry proteins as repertoire of ASP3 substrates. The role of ASP3 as a maturase for previously described and newly identified secretory proteins is confirmed in vivo and in vitro. An antimalarial compound based on a hydroxyethylamine scaffold interrupts the lytic cycle of T. gondii at submicromolar concentration by targeting ASP3.
Apicomplexa; Toxoplasma gondii; aspartyl protease; infectious disease; invasion and egress; microbiology; micronemes and rhoptries; peptidomimetic inhibitor
Toxoplasmosis represents one of the most common zoonosis worldwide. Its agent, Toxoplasma gondii, causes a severe innate pro-inflammatory response. The indigenous intestinal microbiota promotes host animal homoeostasis and may protect the host against pathogens. Germ-free (GF) animals provide an important tool for the study of interactions between host and microbiota. In this study, we assessed the role of indigenous microorganisms in disease development utilizing a murine toxoplasmosis model, which includes conventional (CV) and GF NIH Swiss mice. CV and GF mice orally inoculated with T. gondii had similar survival curves. However, disease developed differently in the two animal groups. In CV mice, intestinal permeability increased and levels of intestinal pro-inflammatory cytokines were altered. In GF animals, there were discrete epithelial degenerative changes and mucosal oedema, but the liver and lungs displayed significant lesions. We conclude that, despite similar survival curves, CV animals succumb to an exaggerated inflammatory response, whereas GF mice fail to produce an adequate systemic response.
IL-17A is known to be involved in the host defense against pathogens and pathogenesis of autoimmune diseases. Previously, we showed that excessive IFN-γ plays an important role in the pathogenesis of lethal effect of Toxoplasma gondii (T. gondii) by inducing anaphylactic responses. In this report, we examine the effects of an IL-17A deficiency on murine host defense against oral T. gondiiinfection. IL-17A-deficient C57BL/6 (B6) mice exhibited higher mortality than wild type (WT) mice to T. gondii at the acute phase of infection. CD4+ T cells in mesenteric lymph nodes (mLNs) and ileum of T. gondii-infected IL-17A-deficient mice produced higher levels of IFN-γ than did those in WT mice. In addition, T. gondii HSP70 (T.g.HSP70) expression was also significantly increased in the ileum, mLNs, liver and spleen of infected IL-17A-deficient mice as compared with WT mice. These elevated expressions of T.g.HSP70 and IFN-γ in infected IL-17A-deficient mice were presumably linked to the IL-17A defect since they decreased to WT levels after treatment with recombinant IL-17A. Furthermore, IL-17A-deficient mice were highly susceptible to anaphylactic effect of T.g.HSP70, and acute phase survival of IL-17A-deficient mice was improved by the treatment with anti-T.g.HSP70 monoclonal antibody. These results suggest that IL-17A plays an important role in host survival against T. gondii infection by protecting host from anaphylactic reaction via downregulating T.g.HSP70 and IFN-γ production.
Apicomplexan parasites such as Babesia, Theileria, Eimeria, Cryptosporidium and Toxoplasma greatly impact animal health globally, and improved, cost-effective measures to control them are urgently required. These parasites have complex multi-stage life cycles including obligate intracellular stages. Major gaps in our understanding of the biology of these relatively poorly characterized parasites and the diseases they cause severely limit options for designing novel control methods. Here we review potentially important shared aspects of the biology of these parasites, such as cell invasion, host cell modification, and asexual and sexual reproduction, and explore the potential of the application of relatively well-established or newly emerging genetic manipulation methods (GMMs), such as classical transfection or gene editing, respectively, for closing important gaps in our knowledge of the function of specific genes and proteins, and the biology of these parasites. In addition, GMMs impact the development of novel methods of control of the diseases caused by these economically important parasites. Transient and stable transfection methods, in conjunction with whole and deep genome sequencing, were initially instrumental in improving our understanding of the molecular biology of apicomplexan parasites and paved the way for the application of the more recently developed gene editing methods. The increasingly efficient and more recently developed gene editing methods, in particular those based on the CRISPR/Cas9 system and previous conceptually similar techniques, are already contributing to additional gene function discovery using reverse genetics and related approaches. However, gene editing methods are only possible due to the increasing availability of in vitro culture, transfection, and genome sequencing and analysis techniques. We envisage that rapid progress in the development of novel gene editing techniques applied to apicomplexan parasites of veterinary interest will ultimately lead to the development of novel and more efficient methods for disease control.
Toxoplasma gondii is a widespread zoonotic parasite that is relevant for veterinary and public health. The domestic cat, the definitive host species with the largest worldwide population, has become evolutionarily and epidemiologically the most important host of T. gondii. The outcome of T. gondii infection is influenced by congenital and acquired host characteristics. We detected differences in T. gondii seroprevalence by cat breed in our previous studies. The aims of this study were to estimate T. gondii seroprevalence in selected domestic cat breeds, and to evaluate whether being of a certain breed is associated with T. gondii seropositivity, when the age and lifestyle of the cat are taken into account. The studied breeds were the Birman, British Shorthair, Burmese, Korat, Norwegian Forest Cat, Ocicat, Persian, and Siamese. Plasma samples were analyzed for the presence of immunoglobulin G antibodies against T. gondii with a commercial direct agglutination test at dilution 1:40. The samples were accompanied by owner-completed questionnaires that provided background data on the cats. Overall, 41.12% of the 1121 cats tested seropositive, and the seroprevalence increased with age. The Burmese had the lowest seroprevalence (18.82%) and the Persian had the highest (60.00%). According to the final multivariable logistic regression model, the odds to test seropositive were four to seven times higher in Birmans, Ocicats, Norwegian Forest Cats, and Persians when compared with the Burmese, while older age and receiving raw meat were also risk factors for T. gondii seropositivity. This study showed that T. gondii seroprevalence varies by cat breed and identified being of certain breeds, older age, and receiving raw meat as risk factors for seropositivity.
Perturbations of the physiological status of the endoplasmic reticulum (ER) trigger a specific response known as the ER stress response or unfolded protein response (UPR). In mammalian cells, the UPR is mediated by three ER transmembrane proteins (IRE1, PERK and ATF6) which activate three signaling cascades to restore ER homeostasis. In recent years, a cross-talk between UPR, inflammatory and microbial sensing pathways has been elucidated. Pathogen infection can lead to UPR activation; moreover, several pathogens subvert the UPR to promote their survival and replication. While the UPR in viral and bacterial infection has been characterized, little is known about the role of UPR in intracellular parasite infection. Here, we review recent findings on UPR induction/modulation by intracellular parasites in host cells.
Cryptosporidium; ER stress; Leishmania; Plasmodium; Toxoplasma; immunity; protozoan parasites; unfolded protein response
Monensin (Mon) is an anticoccidial polyether ionophore widely used to control coccidiosis. The extensive use of polyether ionophores on poultry farms resulted in widespread resistance, but the underlying resistance mechanisms are unknown in detail. For analysing the mode of action by which resistance against polyether ionophores is obtained, we induced in vitro Mon resistance in Toxoplasma gondii-RH strain (MonR-RH) and compared it with the sensitive parental strain (Sen-RH). The proteome assessment of MonR-RH and Sen-RH strains was obtained after isotopic labelling using stable isotope labelling by amino acid in cell culture. Relative proteomic quantification between resistant and sensitive strains was performed using liquid chromatography-mass spectrometry/mass spectrometry. Overall, 1024 proteins were quantified and 52 proteins of them were regulated. The bioinformatic analysis revealed regulation of cytoskeletal and transmembrane proteins being involved in transport mechanisms, metal ion-binding and invasion. During invasion, actin and microneme protein 8 (MIC8) are seem to be important for conoid extrusion and forming moving junction with host cells, respectively. Actin was significantly upregulated, while MIC8 was downregulated, which indicate an invasion reduction in the resistant strain. Resistance against Mon is not a simple process but it involves reduced invasion and egress activity of T. gondii tachyzoites while intracellular replication is enhanced.
Toxoplasma gondii ; SILAC; monensin; resistance mechanism; stable isotope labelling by amino acid in cell culture
Protozoan parasites have developed elaborate motility systems that facilitate infection and dissemination. For example, amoebae use actin-rich membrane extensions called pseudopodia, whereas Kinetoplastida are propelled by microtubule-containing flagella. By contrast, the motile and invasive stages of the Apicomplexa - a phylum that contains the important human pathogens Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis) - have a unique machinery called the glideosome, which is composed of an actomyosin system that underlies the plasma membrane. The glideosome promotes substrate-dependent gliding motility, which powers migration across biological barriers, as well as active host cell entry and egress from infected cells. In this Review, we discuss the discovery of the principles that govern gliding motility, the characterization of the molecular machinery involved, and its impact on parasite invasion and egress from infected cells.
Developing anti-parasitic lead compounds that act on key vulnerabilities are necessary for new anti-infectives. Malaria, leishmaniasis, toxoplasmosis, cryptosporidiosis and coccidiosis together kill >500,000 humans annually. Their causative parasites Plasmodium, Leishmania, Toxoplasma, Cryptosporidium and Eimeria display high conservation in many housekeeping genes, suggesting that these parasites can be attacked by targeting invariant essential proteins. Here, we describe selective and potent inhibition of prolyl-tRNA synthetases (PRSs) from the above parasites using a series of quinazolinone-scaffold compounds. Our PRS-drug co-crystal structures reveal remarkable active site plasticity that accommodates diversely substituted compounds, an enzymatic feature that can be leveraged for refining drug-like properties of quinazolinones on a per parasite basis. A compound we termed In-5 exhibited a unique double conformation, enhanced drug-like properties, and cleared malaria in mice. It thus represents a new lead for optimization. Collectively, our data offer insights into the structure-guided optimization of quinazolinone-based compounds for drug development against multiple human eukaryotic pathogens.
Environmental and genetic perturbations of endoplasmic reticulum (ER) function can lead to the accumulation of unfolded proteins. In these conditions, eukaryotic cells can activate a complex signaling network called the unfolded protein response (UPR) to reduce ER stress and restore cellular homeostasis. Autophagy, a degradation and recycling process, is part of this response. The parasitic protist Toxoplasma gondii is known to be able to activate the UPR upon ER stress, and we now show that this pathway leads to autophagy activation, supporting the idea of a regulated function for canonical autophagy as part of an integrated stress response in the parasites.
Latent infection with Toxoplasma gondii has repeatedly been shown to be associated with behavioral changes that are commonly attributed to a presumed increase in dopaminergic signaling. Yet, virtually nothing is known about its effects on dopamine-driven reward processing. We therefore assessed behavior and event-related potentials in individuals with vs. without latent toxoplasmosis performing a rewarded control task. The data show that otherwise healthy young adults with latent toxoplasmosis show a greatly diminished response to monetary rewards as compared to their non-infected counterparts. While this selective effect eliminated a toxoplasmosis-induced speed advantage previously observed for non-rewarded behavior, Toxo-positive subjects could still be demonstrated to be superior to Toxo-negative subjects with respect to response accuracy. Event-related potential (ERP) and source localization analyses revealed that this advantage during rewarded behavior was based on increased allocation of processing resources reflected by larger visual late positive component (LPC) amplitudes and associated activity changes in the right temporo-parietal junction (BA40) and left auditory cortex (BA41). Taken together, individuals with latent toxoplasmosis show superior behavioral performance in challenging cognitive control situations but may at the same time have a reduced sensitivity towards motivational effects of rewards, which might be explained by the presumed increase in dopamine.
In immunocompromised hosts, latent infection with Toxoplasma gondii can reactivate from tissue cysts, leading to encephalitis. A characteristic of T. gondii bradyzoites in tissue cysts is the presence of amylopectin granules. The regulatory mechanisms and role of amylopectin accumulation in this organism are not fully understood. The T. gondii genome encodes a putative glycogen phosphorylase (TgGP), and mutants were constructed to manipulate the activity of TgGP and to evaluate the function of TgGP in amylopectin storage. Both a stop codon mutant (Pru/TgGPS25stop [expressing a Ser-to-stop codon change at position 25 in TgGP]) and a phosphorylation null mutant (Pru/TgGPS25A [expressing a Ser-to-Ala change at position 25 in TgGp]) mutated at Ser25 displayed amylopectin accumulation, while the phosphorylation-mimetic mutant (Pru/TgGPS25E [expressing a Ser-to-Glu change at position 25 in TgGp]) had minimal amylopectin accumulation under both tachyzoite and bradyzoite growth conditions. The expression of active TgGPS25S or TgGPS25E restored amylopectin catabolism in Pru/TgGPS25A To understand the relation between GP and calcium-dependent protein kinase 2 (CDPK2), which was recently reported to regulate amylopectin consumption, we knocked out CDPK2 in these mutants. PruΔcdpk2/TgGPS25E had minimal amylopectin accumulation, whereas the Δcdpk2 phenotype in the other GP mutants and parental lines displayed amylopectin accumulation. Both the inactive S25A and hyperactive S25E mutant produced brain cysts in infected mice, but the numbers of cysts produced were significantly less than the number produced by the S25S wild-type GP parasite. Complementation that restored amylopectin regulation restored brain cyst production to the control levels seen in infected mice. These data suggest that T. gondii requires tight regulation of amylopectin expression for efficient production of cysts and persistent infections and that GP phosphorylation is a regulatory mechanism involved in amylopectin storage and utilization.
IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that causes disease in immune-suppressed individuals, as well as a fetopathy in pregnant women who acquire infection for the first time during pregnancy. This parasite can differentiate between tachyzoites (seen in acute infection) and bradyzoites (seen in latent infection), and this differentiation is associated with disease relapse. A characteristic of bradyzoites is that they contain cytoplasmic amylopectin granules. The regulatory mechanisms and the roles of amylopectin granules during latent infection remain to be elucidated. We have identified a role of T. gondii glycogen phosphorylase (TgGP) in the regulation of starch digestion and a role of posttranslational modification of TgGP, i.e., phosphorylation of Ser25, in the regulation of amylopectin digestion. By manipulating TgGP activity in the parasite with genome editing, we found that the digestion and storage of amylopectin due to TgGP activity are both important for latency in the brain.
RNA contains over 100 modified nucleotides that are created post-transcriptionally, among which pseudouridine (Ψ) is one of the most abundant. Although it was one of the first modifications discovered, the biological role of this modification is still not fully understood. Recently, we reported that a pseudouridine synthase (TgPUS1) is necessary for differentiation of the single-celled eukaryotic parasite Toxoplasma gondii from active to chronic infection. To better understand the biological role of pseudouridylation we report here gel-based and deep-sequencing methods to identify TgPUS1-dependent Ψs in Toxoplasma RNA, and the use of TgPUS1 mutants to examine the effect of this modification on mRNAs. In addition to identifying conserved sites of pseudouridylation in Toxoplasma rRNA, tRNA, and snRNA, we also report extensive pseudouridylation of Toxoplasma mRNAs, with the Ψs being relatively depleted in the 3'-UTR but enriched at position 1 of codons. We show that many of the Ψs in tRNA and mRNA are dependent on the action of TgPUS1 and that TgPUS1-dependent mRNA Ψs are enriched in developmentally regulated transcripts. RNA-Seq data obtained from wild-type and TgPUS1-mutant parasites shows that genes containing a TgPUS1-dependent Ψ are relatively more abundant in mutant parasites while pulse/chase labeling of RNA with 4-thiouracil shows that mRNAs containing TgPUS1-dependent Ψ have a modest but statistically significant increase in half-life in the mutant parasites. These data are some of the first evidence suggesting that mRNA Ψs play an important biological role.
Published by Cold Spring Harbor Laboratory Press for the RNA Society.