Translational Control in the Latency of Apicomplexan Parasites

Trends Parasitol. 2017 Sep 20. pii: S1471-4922(17)30211-8. doi: 10.1016/j.pt.2017.08.006. [Epub ahead of print]

Holmes MJ¹, Augusto LDS¹, Zhang M², Wek RC³, Sullivan WJ Jr⁴.

Apicomplexan parasites Toxoplasma gondii and Plasmodium spp. use latent stages to persist in the host, facilitate transmission, and thwart treatment of infected patients. Therefore, it is important to understand the processes driving parasite differentiation to and from quiescent stages. Here, we discuss how a family of protein kinases that phosphorylate the eukaryotic initiation factor-2 (eIF2) function in translational control and drive differentiation. This translational control culminates in reprogramming of the transcriptome to facilitate parasite transition towards latency. We also discuss how eIF2 phosphorylation contributes to the maintenance of latency and provides a crucial role in the timing of reactivation of latent parasites towards proliferative stages.

Copyright © 2017 Elsevier Ltd. All rights reserved.

KEYWORDS:

Plasmodium; Toxoplasma; eIF2; latency; translational control

PMID:

28942109

DOI:

10.1016/j.pt.2017.08.006

Apicomplexan actin polymerization depends on nucleation

Sci Rep. 2017 Sep 22;7(1):12137. doi: 10.1038/s41598-017-11330-w.

Kumpula EP¹, Pires I¹, Lasiwa D¹, Piirainen H¹, Bergmann U¹, Vahokoski J^1,2, Kursula I^3,4.

Filamentous actin is critical for apicomplexan motility and host cell invasion. Yet, parasite actin filaments are short and unstable. Their kinetic characterization has been hampered by the lack of robust quantitative methods. Using a modified labeling method, we carried out thorough biochemical characterization of malaria parasite actin. In contrast to the isodesmic polymerization mechanism suggested for Toxoplasma gondii actin, Plasmodium falciparum actin I polymerizes via the classical nucleation-elongation pathway, with kinetics similar to canonical actins. A high fragmentation rate, governed by weak lateral contacts within the filament, is likely the main reason for the short filament length. At steady state, Plasmodium actin is present in equal amounts of short filaments and dimers, with a small proportion of monomers, representing the apparent critical concentration of ~0.1 µM. The dimers polymerize but do not serve as nuclei. Our work enhances understanding of actin evolution and the mechanistic details of parasite motility, serving as a basis for exploring parasite actin and actin nucleators as drug targets against malaria and other apicomplexan parasitic diseases.

PMID:

28939886

DOI:

10.1038/s41598-017-11330-w

Inhibition of calcium dependent protein kinase 1 (CDPK1) by pyrazolopyrimidine analogs decreases establishment and reoccurrence of central nervous system disease by Toxoplasma gondii

J Med Chem. 2017 Sep 21. doi: 10.1021/acs.jmedchem.7b01192. [Epub ahead of print]

Rutaganira FU, Barks J, Dhason MS, Wang Q, Lopez MS, Long S, Radke JB, Jones NG, Maddirala AR, Janetka JW, El Bakkouri M, Hui R, Shokat KM, Sibley LD.

Abstract

Calcium dependent protein kinase 1 (CDPK1) is an essential enzyme in the opportunistic pathogen Toxoplasma gondii. CDPK1 controls multiple processes that are essential to the intracellular replicative cycle of T. gondii including secretion of adhesins, motility, invasion, and egress. Remarkably, CDPK1 contains a small glycine gatekeeper residue in the ATP binding pocket making it sensitive to ATP-competitive inhibitors with bulky substituents that complement this expanded binding pocket. Here we explored structure-activity relationships of a series of pyrazolopyrimidine inhibitors of CDPK1 with the goals of increasing selectivity over host enzymes, improving anti-parasite potency, and improving metabolic stability. The resulting lead compound 24 exhibits excellent enzyme inhibition and selectivity for CDPK1 and potently inhibited parasite growth in vitro. Compound 24 was also effective at treating acute toxoplasmosis in the mouse, reducing dissemination to the central nervous system, decreasing reactivation of chronic infection in severely immunocompromised mice. These findings provide proof of concept for the development of small molecule inhibitors of CDPK1 for treatment of CNS toxoplasmosis.

PMID:

28933846

DOI:

10.1021/acs.jmedchem.7b01192

Characterization of a cytoplasmic glucosyltransferase that extends the core trisaccharide of the Toxoplasma Skp1 E3 ubiquitin ligase subunit

J Biol Chem. 2017 Sep 19. pii: jbc.M117.809301. doi: 10.1074/jbc.M117.809301. [Epub ahead of print]

Rahman K¹, Mandalasi M¹, Zhao P¹, Sheikh MO¹, Taujale R¹, Kim HW¹, van der Wel H¹, Matta K², Kannan N¹, Glushka JN¹, Wells L¹, West CM³.

Skp1 is a subunit of the SCF (Skp1/Cullin-1/F-box protein) class of E3 ubiquitin ligases that are important for eukaryotic protein degradation. Unlike its animal counterparts, Skp1 from Toxoplasma gondii is hydroxylated by an O2-dependent prolyl-4-hydroxylase (PhyA), and the resulting hydroxyproline can subsequently be modified by a five-sugar chain. A similar modification is found in the social amoeba Dictyostelium, where it regulates SCF assembly and O2-dependent development. Homologous glycosyltransferases assemble a similar core trisaccharide in both organisms, and a bifunctional α-galactosyltransferase from CAZy family GT77 mediates addition of the final two sugars in Dictyostelium, generating Galα1,3Galα1,3Fucα1,2Galβ1,3GlcNAcα1-. Here, we found that Toxoplasma utilizes a cytoplasmic glycosyltransferase from an ancient clade of CAZy family GT32 to catalyze transfer of the fourth sugar. Catalytically active Glt1 was required for addition of the terminal disaccharide in cells, and cytosolic extracts catalyzed transfer of [3H]glucose from UDP-[3H]glucose to the trisaccharide form of Skp1 in a glt1-dependent fashion. Recombinant Glt1 catalyzed the same reaction, confirming that it directly mediates Skp1 glucosylation, and NMR demonstrated formation of a Glcα1,3Fuc linkage. Recombinant Glt1 strongly preferred the full core trisaccharide attached to Skp1, and labeled only Skp1 in glt1Δ extracts, suggesting specificity for Skp1. glt1-knockout parasites exhibited a growth defect not rescued by catalytically inactive Glt1, indicating that the glycan acts in concert with the first enzyme in the pathway, PhyA, in cells. A genomic bioinformatics survey suggested that Glt1 belongs to the ancestral Skp1 glycosylation pathway in protists and evolved separately from related Golgi-resident GT32 glycosyltransferases.

Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

KEYWORDS:

E3 ubiquitin ligase; Toxoplasma gondii; carbohydrate structure; cytoplasmic glycosylation; evolution; glycobiology; glycosyltransferase; mass spectrometry (MS)

PMID:

28928220

DOI:

10.1074/jbc.M117.809301

NLRP3 and Potassium Efflux Drive Rapid IL-1β Release from Primary Human Monocytes during Toxoplasma gondii Infection

J Immunol. 2017 Sep 13. pii: ji1700245. doi: 10.4049/jimmunol.1700245. [Epub ahead of print]

Gov L¹, Schneider CA¹, Lima TS¹, Pandori W¹, Lodoen MB².

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.

Copyright © 2017 by The American Association of Immunologists, Inc.

PMID:

28904126

DOI:

10.4049/jimmunol.1700245

Toxoplasma Modulates Signature Pathways of Human Epilepsy, Neurodegeneration & Cancer

Sci Rep. 2017 Sep 13;7(1):11496. doi: 10.1038/s41598-017-10675-6.

Ngô HM^1,2,3, Zhou Y¹, Lorenzi H⁴, Wang K⁵, Kim TK⁵, Zhou Y⁵, Bissati KE¹, Mui E¹, Fraczek L¹, Rajagopala SV⁴, Roberts CW⁶, Henriquez FL^1,7, Montpetit A⁸, Blackwell JM^9,10, Jamieson SE¹⁰, Wheeler K¹, Begeman IJ¹, Naranjo-Galvis C¹, Alliey-Rodriguez N¹, Davis RG¹¹, Soroceanu L¹², Cobbs C¹², Steindler DA¹³, Boyer K¹⁴, Noble AG², Swisher CN², Heydemann PT¹⁴, Rabiah P¹⁵, Withers S¹, Soteropoulos P¹⁶, Hood L⁵, McLeod R¹⁷.

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.

PMID:

28904337

DOI:

10.1038/s41598-017-10675-6

A druggable secretory protein maturase of Toxoplasma essential for invasion and egress

Elife. 2017 Sep 12;6. pii: e27480. doi: 10.7554/eLife.27480.

Dogga SK1, Mukherjee B1, Jacot D1, Kockmann T2, Molino L1, Hammoudi PM1, Hartkoorn RC1,3, Hehl AB4, Soldati-Favre D1.

Author information

Abstract

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.

KEYWORDS: 

Apicomplexa; Toxoplasma gondii; aspartyl protease; infectious disease; invasion and egress; microbiology; micronemes and rhoptries; peptidomimetic inhibitor

PMID:

 

28898199

 

DOI:

 

10.7554/eLife.27480

Influence of indigenous microbiota on experimental toxoplasmosis in conventional and germ-free mice

Int J Exp Pathol. 2017 Sep 11. doi: 10.1111/iep.12236. [Epub ahead of print]

Nascimento BB1, Cartelle CT1, Noviello ML1, Pinheiro BV2, de Almeida Vitor RW2, Souza DDG3, de Vasconcelos Generoso S4, Cardoso VN4, Martins FDS3, Nicoli JR3, Arantes RME1.

Author information

Abstract

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.

© 2017 The Authors. International Journal of Experimental Pathology © 2017 International Journal of Experimental Pathology.

KEYWORDS: 

Toxoplasma gondii ; germ-free mice; gut inflammation; microbiota; toxoplasmosis

PMID:

 

28895246

 

DOI:

 

10.1111/iep.12236

IL17A-deficient mice are highly susceptible to Toxoplasma gondii infection due to excessively induced T. gondii HSP70 and IFN-γ production

Infect Immun. 2017 Sep 11. pii: IAI.00399-17. doi: 10.1128/IAI.00399-17. [Epub ahead of print]

Moroda M1, Takamoto M2, Iwakura Y3, Nakayama J2, Aosai F4,2.

Author information

Abstract

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.

Copyright © 2017 American Society for Microbiology.

PMID:

 

28893913

 

DOI:

 

10.1128/IAI.00399-17

Advances in the application of genetic manipulation methods to Apicomplexan parasites

Int J Parasitol. 2017 Sep 8. pii: S0020-7519(17)30246-1. doi: 10.1016/j.ijpara.2017.08.002. [Epub ahead of print]

Suarez CE1, Bishop RP2, Alzan HF3, Poole WA4, Cooke BM5.

Author information

Abstract

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.

Published by Elsevier Ltd.

KEYWORDS: 

Apicomplexan; CRISPR/Cas9; Gene editing; Genetic manipulation; Transfection

PMID:

 

28893636

 

DOI:

 

10.1016/j.ijpara.2017.08.002

Toxoplasma gondii seroprevalence varies by cat breed

PLoS One. 2017 Sep 8;12(9):e0184659. doi: 10.1371/journal.pone.0184659. eCollection 2017.

Must K1, Hytönen MK2,3,4, Orro T1, Lohi H2,3,4, Jokelainen P1,4,5.

Author information

Abstract

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.

PMID:

 

28886182

 

DOI:

 

10.1371/journal.pone.0184659

Endoplasmic reticulum stress and unfolded protein response in infection by intracellular parasites

Future Sci OA. 2017 May 12;3(3):FSO198. doi: 10.4155/fsoa-2017-0020. eCollection 2017 Aug.

Galluzzi L1, Diotallevi A1, Magnani M1.

Author information

Abstract

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.

KEYWORDS: 

Cryptosporidium; ER stress; Leishmania; Plasmodium; Toxoplasma; immunity; protozoan parasites; unfolded protein response

PMID:

 

28883998

 

PMCID:

 

PMC5583660

 

DOI:

 

10.4155/fsoa-2017-0020

Resistance towards monensin is proposed to be acquired in a Toxoplasma gondii model by reduced invasion and egress activities, in addition to increased intracellular replication

Parasitology. 2017 Sep 5:1-13. doi: 10.1017/S0031182017001512. [Epub ahead of print]

Thabet A¹, Schmidt J², Baumann S², Honscha W³, VON Bergen M², Daugschies A¹, Bangoura B¹.

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.

KEYWORDS:

Toxoplasma gondii ; SILAC; monensin; resistance mechanism; stable isotope labelling by amino acid in cell culture

PMID:

28870270

DOI:

10.1017/S0031182017001512