Antimicrob Agents Chemother. 2014 Feb 24. [Epub ahead of print]

Identification of Cryptosporidium parvum active chemical series by repurposing the Open Access Malaria Box

Bessoff K1, Spangenberg T, Foderaro J, Jumani RS, Ward GE, Huston CD.

Author information

Abstract

The apicomplexan parasites Cryptosporidium parvum and C. hominis are major etiologic agents of human cryptosporidiosis. Infection is typically self-limited in immunocompetent adults, but can cause chronic, fulminant diarrhea in immunocompromised patients, and malnutrition and stunting in children. Nitazoxanide, the current standard-of-care for cryptosporidiosis, is only partially efficacious for children and is no more effective than placebo in AIDS patients. Unfortunately, financial obstacles to drug discovery for diseases that disproportionately affect low-income countries and technical limitations associated with studies of Cryptosporidium biology both impede development of better drugs for cryptosporidiosis. Using a cell-based high-throughput screen, we queried the Medicines for Malaria Venture's (MMV) "Open Access Malaria Box" for activity against C. parvum. We identified 3 novel chemical series derived from the quinolin-8-ol, allopurinol-based, and 2,4-diamino-quinazoline chemical scaffolds that exhibited sub-micromolar potency against C. parvum. Potency was conserved in a sub-set of compounds from each scaffold with variable physicochemical properties, and two of the scaffolds identified exhibit more rapid inhibition of C. parvum growth than nitazoxanide, making them excellent candidates for further development. The 2,4-diamino-quinazoline and allopurinol-based compounds were also potent growth inhibitors of the related apicomplexan parasite Toxoplasma gondii, and a good correlation was observed in the relative activities of compounds in the allopurinol-based series against T. gondii and C. parvum. Taken together, these data illustrate the utility of the Open Access Malaria Box as a source of both potential leads for drug development and chemical probes to elucidate basic biological processes in C. parvum and other apicomplexan parasites.

PMID:

 

24566188

 

[PubMed - as supplied by publisher]

Parasitology. 2014 Feb 21:1-10. [Epub ahead of print]

Toxoplasma gondii Hsp90: potential roles in essential cellular processes of the parasite

Angel SO1, Figueras MJ1, Alomar ML1, Echeverria PC2, Deng B3.

Author information

Abstract

SUMMARY Hsp90 is a widely distributed and highly conserved molecular chaperone that is ubiquitously expressed throughout nature, being one of the most abundant proteins within non-stressed cells. This chaperone is up-regulated following stressful events and has been involved in many cellular processes. In Toxoplasma gondii, Hsp90 could be linked with many essential processes of the parasite such as host cell invasion, replication and tachyzoite-bradyzoite interconversion. A Protein-Protein Interaction (PPI) network approach of TgHsp90 has allowed inferring how these processes may be altered. In addition, data mining of T. gondii phosphoproteome and acetylome has allowed the generation of the phosphorylation and acetylation map of TgHsp90. This review focuses on the potential roles of TgHsp90 in parasite biology and the analysis of experimental data in comparison with its counterparts in yeast and humans.

PMID:

 

24560345

 

[PubMed - as supplied by publisher]

Role of Mouse and Human Autophagy Proteins in IFN-γ-Induced Cell-Autonomous Responses against Toxoplasma

J Immunol. 2014 Feb 21. [Epub ahead of print]

Role of Mouse and Human Autophagy Proteins in IFN-γ-Induced Cell-Autonomous Responses against Toxoplasma gondii

Ohshima J1, Lee Y, Sasai M, Saitoh T, Su Ma J, Kamiyama N, Matsuura Y, Pann-Ghill S, Hayashi M, Ebisu S, Takeda K, Akira S,Yamamoto M.

Author information

Abstract

IFN-γ mediates cellular innate immunity against an intracellular parasite, Toxoplasma gondii, by inducing immunity-related GTPases such as p47 IFN-γ-regulated GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), which also participate in antibacterial responses via autophagy. An essential autophagy protein, Atg5, was previously shown to play a critical role in anti-T. gondii cell-autonomous immunity. However, the involvement of other autophagy proteins remains unknown. In this study, we show that essential autophagy proteins differentially participate in anti-T. gondii cellular immunity by recruiting IFN-γ-inducible GTPases. IFN-γ-induced suppression of T. gondii proliferation and recruitment of an IRG Irgb6 and GBPs are profoundly impaired in Atg7- or Atg16L1-deficient cells. In contrast, cells lacking other essential autophagy proteins, Atg9a and Atg14, are capable of mediating the anti-T. gondii response and recruiting Irgb6 and GBPs to the parasites. Although IFN-γ also stimulates anti-T. gondii cellular immunity in humans, whether this response requires GBPs and human autophagy proteins remains to be seen. To analyze the role of human ATG16L1 and GBPs in IFN-γ-mediated anti-T. gondii responses, human cells lacking ATG16L1 or GBPs were generated by the Cas9/CRISPR genome-editing technique. Although both ATG16L1 and GBPs are dispensable for IFN-γ-induced inhibition of T. gondii proliferation in the human cells, human ATG16L1 is also required for the recruitment of GBPs. Taken together, human ATG16L1 and mouse autophagy components Atg7 and Atg16L1, but not Atg9a and Atg14, participate in the IFN-γ-induced recruitment of the immunity-related GTPases to the intracellular pathogen.

PMID:

 

24563254

 

[PubMed - as supplied by publisher]

Antimicrob Agents Chemother. 2014 Feb 18. [Epub ahead of print]

Expression of the essential kinase PfCDPK1 from Plasmodium falciparum in Toxoplasma gondii facilitates the discovery of novel antimalarial drugs

Gaji RY¹, Checkley L, Reese M, Ferdig MT, Arrizabalaga G.

Author information

• ¹Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN, USA.

Abstract

We have previously shown that genetic disruption of Toxoplasma gondii calcium dependent protein kinase 3 (TgCDPK3) affects calcium ionophore induced egress. We examined whether PfCDPK1, the closest homolog of TgCDPK3 in the malaria parasite Plasmodium falciparum, could complement a TgCDPK3 mutant strain. PfCDPK1 is essential and plays critical roles in merozoite development, motility and secretion. We show that expression of PfCDPK1 in the TgCDPK3 mutant strain rescues the egress defect. This phenotypic complementation requires PfCDPK1's localization to the plasma membrane and its kinase activity. Interestingly, Toxoplasma expressing PfCDPK1 become more sensitive to egress inhibition by purfalcamine, a potent inhibitor of PfCDPK1 with low activity against TgCDPK3. Based on this result, we tested eight small molecules previously determined to inhibit kinase activity of recombinant PfCDPK1, for their ability to inhibit ionophore induced Egress in the PfCDPK1-expressing strain. While two of these chemicals did not inhibit egress, we find that six drugs affected this process selectively in PfCDPK1 expressing Toxoplasma. Using mutant versions of PfCDPK1 and TgCDPK3, we show that the selectivity of Dasatinib and PLX-4720 is regulated by the gatekeeper residue in the ATP binding site. Importantly, we have confirmed that the three most potent inhibitors of egress in the PfCDPK1-expressing strain effectively kill P. falciparum. Thus, we have established and validated a recombinant strain of Toxoplasma that can be used as a surrogate for the discovery and analysis of PfCDPK1 specific inhibitors that can be developed as antimalarials.

PMID:

24550330

[PubMed - as supplied by publisher]

Proc Natl Acad Sci U S A. 2014 Feb 18. [Epub ahead of print]

Toxoplasma aldolase is required for metabolism but dispensable for host-cell invasion

Shen B¹, Sibley LD.

Author information

• ¹Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.

Abstract

Gliding motility and host-cell invasion by apicomplexan parasites depend on cell-surface adhesins that are translocated via an actin-myosin motor beneath the membrane. The current model posits that fructose-1,6-bisphosphate aldolase (ALD) provides a critical link between the cytoplasmic tails of transmembrane adhesins and the actin-myosin motor. Here we tested this model using the Toxoplasma gondii apical membrane protein 1 (TgAMA1), which binds to aldolase in vitro. TgAMA1 cytoplasmic tail mutations that disrupt ALD binding in vitro showed no correlation with host-cell invasion, indicating this interaction is not essential. Furthermore, ALD-depleted parasites were impaired when grown in glucose, yet they showed normal gliding and invasion in glucose-free medium. Depletion of ALD in the presence of glucose led to accumulation of fructose-1,6-bisphosphate, which has been associated with toxicity in other systems. Finally, TgALD knockout parasites and an ALD mutant that specifically disrupts adhesin binding in vitro also supported normal invasion when cultured in glucose-free medium. Taken together, these results suggest that ALD is primarily important for energy metabolism rather than interacting with microneme adhesins, challenging the current model for apicomplexan motility and invasion.

KEYWORDS:

glycolysis, micronemal adhesin, motor complex

PMID:

24550496

[PubMed - as supplied by publisher]

PLoS Negl Trop Dis. 2014 Feb 13;8(2):e2567. doi: 10.1371/journal.pntd.0002567. eCollection 2014.

Impairment of T cell function in parasitic infections

Rodrigues V¹, Cordeiro-da-Silva A², Laforge M³, Ouaissi A⁴, Akharid K⁵, Silvestre R⁴, Estaquier J⁶.

Abstract

In mammals subverted as hosts by protozoan parasites, the latter and/or the agonists they release are detected and processed by sensors displayed by many distinct immune cell lineages, in a tissue(s)-dependent context. Focusing on the T lymphocyte lineage, we review our present understanding on its transient or durable functional impairment over the course of the developmental program of the intracellular parasites Leishmania spp., Plasmodium spp., Toxoplasma gondii, and Trypanosoma cruzi in their mammalian hosts. Strategies employed by protozoa to down-regulate T lymphocyte function may act at the initial moment of naïve T cell priming, rendering T cells anergic or unresponsive throughout infection, or later, exhausting T cells due to antigen persistence. Furthermore, by exploiting host feedback mechanisms aimed at maintaining immune homeostasis, parasites can enhance T cell apoptosis. We will discuss how infections with prominent intracellular protozoan parasites lead to a general down-regulation of T cell function through T cell anergy and exhaustion, accompanied by apoptosis, and ultimately allowing pathogen persistence.

PMID:

24551250

[PubMed - in process]

Discovery of compounds blocking the proliferation of Toxoplasma gondii and Plasmodium falciparum

Antimicrob Agents Chemother. 2014 Feb 18. [Epub ahead of print]

Discovery of compounds blocking the proliferation of Toxoplasma gondii and Plasmodium falciparum in a chemical space based on piperidinyl-benzimidazolidinone analogs

Saïdani N¹, Botté CY, Deligny M, Bonneau AL, Reader J, Lasselin R, Merer G, Niepceron A, Brossier F, Cintrat JC, Rousseau B, Birkholtz LM, Cesbron-Delauw MF, Dubremetz JF, Mercier C, Vial H, Lopez R, Maréchal E.

Author information

• ¹Commissariat à l'Energie Atomique, Centre National de la Recherche Scientifique, Université Joseph Fourier Grenoble I, Institut National de la Recherche Agronomique, Unité Mixte de Recherche 5168; Institut de Recherches en Technologies et Sciences pour le Vivant, Grenoble, France.

Abstract

A piperidinyl-benzimidazolidinone scaffold has been found in the structure of different inhibitors of membrane glycerolipid metabolism, acting on enzymes manipulating diacylglycerol or phosphatidic acid. Screening a focus library of piperidinyl-benzimidazolidinone analogs might therefore identify compounds acting against infectious parasites. We first evaluated the in vitro effects of (S)-2-(dibenzylamino)-3-phenylpropyl 4-(1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)piperidine-1-carboxylate (compound 1) on Toxoplasma gondii and Plasmodium falciparum. In T. gondii, motility and apical complex integrity appeared unaffected, whereas cell division was inhibited at concentrations of compound 1 in the micromolar range. In P. falciparum, proliferation of erythrocytic stages was inhibited, without any delayed death phenotype. We then explored a library of 250 analogs in two steps. We selected 114 compounds with an IC50 cutoff of 2 μM on at least one species and determined in vitro selectivity indexes (SI), based on toxicity against K-562 human cells. We identified compounds with high gains in IC50 (in the 100 nM range) and SI (up to 1,000-2,000). Isobole analyses of two of the most active compounds against P. falciparum indicated that their interaction with artemisin was additive. We propose structure activity relationship (SAR) models, which will be useful for the design of probes to identify the compounds' target(s), and optimizations for mono- or combined-therapeutic strategies.

PMID:

24550329

[PubMed - as supplied by publisher]

MBio. 2014 Feb 18;5(1). pii: e01117-13. doi: 10.1128/mBio.01117-13.

Dual Role for Inflammasome Sensors NLRP1 and NLRP3 in Murine Resistance to Toxoplasma gondii

Gorfu G, Cirelli KM, Melo MB, Mayer-Barber K, Crown D, Koller BH, Masters S, Sher A, Leppla SH, Moayeri M, Saeij JP, Grigg ME.

Abstract

ABSTRACT Induction of immunity that limits Toxoplasma gondii infection in mice is critically dependent on the activation of the innate immune response. In this study, we investigated the role of cytoplasmic nucleotide-binding domain and leucine-rich repeat containing a pyrin domain (NLRP) inflammasome sensors during acute toxoplasmosis in mice. We show that in vitro Toxoplasma infection of murine bone marrow-derived macrophages activates the NLRP3 inflammasome, resulting in the rapid production and cleavage of interleukin-1β (IL-1β), with no measurable cleavage of IL-18 and no pyroptosis. Paradoxically, Toxoplasma-infected mice produced large quantities of IL-18 but had no measurable IL-1β in their serum. Infection of mice deficient in NLRP3, caspase-1/11, IL-1R, or the inflammasome adaptor protein ASC led to decreased levels of circulating IL-18, increased parasite replication, and death. Interestingly, mice deficient in NLRP1 also displayed increased parasite loads and acute mortality. Using mice deficient in IL-18 and IL-18R, we show that this cytokine plays an important role in limiting parasite replication to promote murine survival. Our findings reveal T. gondii as a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis. IMPORTANCE Inflammasomes are multiprotein complexes that are a major component of the innate immune system. They contain "sensor" proteins that are responsible for detecting various microbial and environmental danger signals and function by activating caspase-1, an enzyme that mediates cleavage and release of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Toxoplasma gondii is a highly successful protozoan parasite capable of infecting a wide range of host species that have variable levels of resistance. We report here that T. gondii is a novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host resistance to toxoplasmosis. Using mice deficient in IL-18 and IL-18R, we show that the IL-18 cytokine plays a pivotal role by limiting parasite replication to promote murine survival.

PMID:

24549849

[PubMed - in process]

Cell Host Microbe. 2014 Feb 12;15(2):132-138. doi: 10.1016/j.chom.2014.01.004.

Innate Resistance against Toxoplasma gondii: An Evolutionary Tale of Mice, Cats, and Men

Gazzinelli RT1, Mendonça-Neto R2, Lilue J3, Howard J4, Sher A5.

Author information

Abstract

Recent studies have revealed remarkable species specificity of the Toll-like receptors (TLRs) TLR11 and TLR12 and the immunity-related GTPase (IRG) proteins that are essential elements for detection and immune control of Toxoplasma gondii in mice, but not in humans. The biological and evolutionary implications of these findings for the T. gondii host-pathogen relationship and for human disease are discussed.

Copyright © 2014 Elsevier Inc. All rights reserved.

PMID:

 

24528860

 

[PubMed - as supplied by publisher]

Eukaryot Cell. 2014 Feb 14. [Epub ahead of print]

Infection by Toxoplasma gondii specifically induces host c-Myc and the genes this pivotal transcription factor regulates

Franco M, Shastri AJ, Boothroyd JC.

Author information

Abstract

Toxoplasma gondii infection has previously been described to cause dramatic changes in the host transcriptome by manipulating key regulators including STATs, NF-κB, and microRNAs. Here, we report that Toxoplasma tachyzoites also mediate rapid and sustained induction of another pivotal regulator of host cell transcription, c-Myc. This induction is seen in cells infected with all three canonical Types of Toxoplasma but not the closely related Apicomplexan parasite, Neospora caninum. Coinfection of cells with both Toxoplasma and Neospora still results in an increase in host c-Myc, showing that c-Myc is actively upregulated by Toxoplasma infection (rather than repressed by Neospora). We further demonstrate that this upregulation may be mediated through JNK kinase and is unlikely to be a non-specific host response as heat-killed Toxoplasma parasites do not induce this increase and neither do nonviable parasites inside the host cell. Finally, we show that the induced c-Myc is active and that transcripts dependent on its function are upregulated, as predicted. Hence, c-Myc represents an additional way in which Toxoplasma tachyzoites have evolved to specifically alter host cell functions during intracellular growth.

PMID:

 

24532536

 

[PubMed - as supplied by publisher]

Differentially expressed proteins in sulfadiazine resistant and sensitive strains of Toxoplasma gondii

Int J Parasitol Drugs Drug Resist. 2013 Jan 5;3:35-44. doi: 10.1016/j.ijpddr.2012.12.002. eCollection 2013.

Identification of differentially expressed proteins in sulfadiazine resistant and sensitive strains of Toxoplasma gondii using difference-gel electrophoresis (DIGE)

Doliwa C1, Xia D2, Escotte-Binet S3, Newsham EL2, Sanya J S2, Aubert D3, Randle N2, Wastling JM2, Villena I3.

Author information

Abstract

Treatment options for toxoplasmosis in humans are generally limited to the use of sulfonamide and/or pyrimethamine-based compounds. However, there is increasing evidence for clinical therapy failures in patients suggesting the existence of drug resistance in these classes of drug. In vitro resistance to sulfadiazine has been detected in three strains of Toxoplasma gondii isolated from clinical cases. In order to begin to understand the mechanisms of resistance, we undertook a difference-gel electrophoresis (DIGE) approach combined with mass spectrometry to identify proteins that are differentially expressed in sulfadiazine-resistance strains of the parasite. Naturally resistant strains TgA 103001 (Type I), TgH 32006 (Type II) and TgH 32045 (Type II variant) were compared to sensitive strains RH (Type I) and ME-49 (Type II) using DIGE and the modulated proteins analyzed using LC-MS/MS. In total, 68 differentially expressed protein spots were analyzed by mass spectrometer and 31 unique proteins, including four hypothetical proteins, were identified. Among the differentially expressed proteins, 44% were over-expressed in resistant strains and 56% were over-expressed in sensitive strains. The virulence-associated rhoptry protein, ROP2A, was found in greater abundance in both naturally resistant Type II strains TgH 32006 and TgH 32045 compared to the sensitive strain ME-49. Enolase 2 and IMC1 were found to be in greater abundance in sensitive strains RH and ME-49, and MIC2 was found to be more abundant in the sensitive strain ME-49. Proteins regulation of ROP2, MIC2, ENO2, IMC1 and GRA7 were confirmed by Western blot analysis. In addition, gene expression patterns of ROP2, MIC2, ENO2 and IMC1 were analyzed with qRT-PCR. This study provides the first proteomics insights into sulfadiazine resistance in T. gondii resistant strains isolated from clinical cases.

KEYWORDS:

DIGE, Drug resistance, EF1-α, elongation factor 1 alpha, ENO2, enolase 2, G3PDH, glyceraldehyde-3-phosphate dehydrogenase, GRA2, dense granule protein 2, GRA7, dense granule protein 7, Hsp70, heat shock protein 70, Hsp90, heat shock protein 90, MIC1, microneme protein 1, MIC2, microneme protein 2, PP2C, protein phosphatase 2C, ROP2, rhoptry protein 2, ROP9, rhoptry protein 9, Sulfadiazine, TgCDPK1, Toxoplasma gondii calcium-dependent protein kinase 1, Toxoplasma gondii, eIF-5A, translation initiation factor 5A, small Hsp20, small heat shock protein 20

PMID:

 

24533291

 

[PubMed]

Int J Parasitol Drugs Drug Resist. 2013 May 18;3:93-101. doi: 10.1016/j.ijpddr.2013.04.001. eCollection 2013.

Identification of mutations in TgMAPK1 of Toxoplasma gondii conferring resistance to 1NM-PP1

Sugi T1, Kobayashi K2, Takemae H1, Gong H1, Ishiwa A1, Murakoshi F1, Recuenco FC1, Iwanaga T1, Horimoto T1, Akashi H1, Kato K3.

Author information

Abstract

Toxoplasma gondii is an important food and waterborne pathogen that causes severe disease in immunocompromised patients. Bumped kinase inhibitors (BKIs) have an antiparasitic effect on T. gondii tachyzoite growth by targeting T. gondii calmodulin-domain protein kinase 1 (TgCDPK1). To identify mutations that confer resistance to BKIs, chemical mutagenesis was performed, followed by selection in media containing either 250 or 1000 nM 1NM-PP1. Whole-genome sequence analysis of resistant clones revealed single nucleotide mutations in T. gondii mitogen-activated protein kinase 1 (TgMAPK1) at amino acids 162 (L162Q) and 171 (I171N). Plasmid constructs having the TgMAPK1 L162Q mutant sequence successfully replaced native TgMAPK1 genome locus in the presence of 1000 nM 1NM-PP1. The inhibitory effect of 1NM-PP1 on cell division observed in the parent clone was decreased in 1NM-PP1-resistant clones; however, effects on parasite invasion and calcium-induced egress were similar in both parent and resistant clones. A plasmid construct expressing the full length TgMAPK1 splicing isoform with L162Q mutation successfully complemented TgMAPK1 function in the pressure of 250 nM 1NM-PP1 in plaque assay. 1NM-PP1-resistant clones showed resistance to other BKIs (3MB-PP1 and 3BrB-PP1) with different levels. Here we identify TgMAPK1 as a novel target for 1NM-PP1 activity. This inhibitory effect is mediated through inhibition of tachyzoite cell division, and can be overcome through mutations at multiple residues in TgMAPK1.

KEYWORDS:

1NM-PP1, Bumped kinase inhibitor, Drug resistance, TgMAPK1, Toxoplasma gondii

PMID:

 

24533298

 

[PubMed]

Dis Model Mech. 2014 Feb 13. [Epub ahead of print]

Chronic murine toxoplasmosis is defined by subtle changes in the neuronal connectivity

Parlog A, Harsan LA, Zagrebelsky M, Weller M, von Elverfeldt D, Mawrin C, Korte M, Dunay IR.

Author information

Abstract

AbstractRecent studies correlate chronic Toxoplasma gondii (T. gondii) infection with behavioral changes in rodents, besides the seropositivity in humans is reported to be associated with behavioral and neuropsychiatric diseases. In this study we investigated if the described behavioral changes in a murine model of chronic toxoplasmosis are associated with changes in synaptic plasticity and brain neuronal circuitry. In mice chronically infected with T. gondii, Magnetic Resonance Imaging (MRI) data analysis displayed the presence of heterogeneous lesions scattered throughout all brain areas. However, a higher density of lesions could be observed within specific regions such as the somatosensory cortex (SSC). Further histopathological examination of these brain areas indicated the presence of activated resident glia and recruited immune cells accompanied by limited alterations of neuronal viability. In vivo Diffusion Tensor MRI analysis of neuronal fiber density within the infected regions revealed connectivity abnormalities in the SSC. Altered fiber density was confirmed by morphological analysis of individual, pyramidal and granule neurons showing a reduction in dendritic arbor and spine density within the SSC, as well as in the hippocampus. Evaluation of synapse efficacy revealed diminished levels of two key synaptic proteins, PSD95 and Synaptophysin, within the same brain areas indicating deficits in functionality of the synaptic neurotransmission in infected mice. Our results demonstrate that persistent T. gondii infection in a murine model results in synaptic deficits within brain structures leading to disturbances in the morphology of noninfected neurons and modified brain connectivity, suggesting a potential explanation for the behavioral and neuropsychiatric alterations.

PMID:

 

24524910

 

[PubMed - as supplied by publisher]

PLoS One. 2014 Feb 5;9(2):e88398. doi: 10.1371/journal.pone.0088398. eCollection 2014.

Differential Induction of TLR3-Dependent Innate Immune Signaling by Closely Related Parasite Species

Beiting DP1, Peixoto L1, Akopyants NS2, Beverley SM2, Wherry EJ3, Christian DA4, Hunter CA4, Brodsky IE4, Roos DS1.

Author information

Abstract

The closely related protozoan parasites Toxoplasma gondii and Neospora caninum display similar life cycles, subcellular ultrastructure, invasion mechanisms, metabolic pathways, and genome organization, but differ in their host range and disease pathogenesis. Type II (γ) interferon has long been known to be the major mediator of innate and adaptive immunity to Toxoplasma infection, but genome-wide expression profiling of infected host cells indicates that Neospora is a potent activator of the type I (α/β) interferon pathways typically associated with antiviral responses. Infection of macrophages from mice with targeted deletions in various innate sensing genes demonstrates that host responses to Neospora are dependent on the toll-like receptor Tlr3 and the adapter protein Trif. Consistent with this observation, RNA from Neospora elicits TLR3-dependent type I interferon responses when targeted to the host endo-lysosomal system. Although live Toxoplasma fail to induce type I interferon, heat-killed parasites do trigger this response, albeit much weaker than Neospora, and co-infection studies reveal that T. gondii actively suppresses the production of type I interferon. These findings reveal that eukaryotic pathogens can be potent inducers of type I interferon and that related parasite species interact with this pathway in distinct ways.

PMID:

 

24505488

 

[PubMed - in process]

MBio. 2014 Feb 4;5(1). pii: e01003-13. doi: 10.1128/mBio.01003-13.

Differential Locus Expansion Distinguishes Toxoplasmatinae Species and Closely Related Strains of Toxoplasma gondii

Adomako-Ankomah Y, Wier GM, Borges AL, Wand HE, Boyle JP.

Author information

Abstract

ABSTRACT Toxoplasma gondii is a human obligate intracellular parasite that has infected over 20% of the world population and has a vast intermediate host range compared to those of its nearest relatives Neospora caninum and Hammondia hammondi. While these 3 species have highly syntenic genomes (80 to 99%), in this study we examined and compared species-specific structural variations, specifically at loci that have undergone local (i.e., tandem) duplication and expansion. To do so, we used genomic sequence coverage analysis to identify and curate T. gondii and N. caninum loci that have undergone duplication and expansion (expanded loci [ELs]). The 53 T. gondii ELs are significantly enriched for genes with predicted signal sequences and single-exon genes and genes that are developmentally regulated at the transcriptional level. We validated 24 T. gondii ELs using comparative genomic hybridization; these data suggested significant copy number variation at these loci. High-molecular-weight Southern blotting for 3 T. gondii ELs revealed that copy number varies across T. gondii lineages and also between members of the same clonal lineage. Using similar methods, we identified 64 N. caninum ELs which were significantly enriched genes belonging to the SAG-related surface (SRS) antigen family. Moreover, there is significantly less overlap (30%) between the expanded gene sets in T. gondii and N. caninum than would be predicted by overall genomic synteny (81%). Consistent with this finding, only 59% of queried T. gondii ELs are similarly duplicated/expanded in H. hammondi despite over 99% genomic synteny between these species. IMPORTANCE Gene duplication, expansion, and diversification are a basis for phenotypic differences both within and between species. This study represents the first characterization of both the extent and degree of overlap in gene duplication and locus expansion across multiple apicomplexan parasite species. The most important finding of this study is that the locus duplications/expansions are quantitatively and qualitatively distinct, despite the high degree of genetic relatedness between the species. Given that these differential expansions are prominent species-specific genetic differences, they may also contribute to some of the more striking phenotypic differences between these species. More broadly, this work is important in providing further support for the idea that postspeciation selection events may have a dramatic impact on locus structure and copy number that overshadows selection on single-copy genes.

PMID:

 

24496792

 

[PubMed - in process] 

PLoS One. 2014 Jan 28;9(1):e85386. doi: 10.1371/journal.pone.0085386. eCollection 2014.

Virulent and Avirulent Strains of Toxoplasma gondii Which Differ in Their Glycosylphosphatidylinositol Content Induce Similar Biological Functions in Macrophages

Niehus S1, Smith TK2, Azzouz N3, Campos MA4, Dubremetz JF5, Gazzinelli RT6, Schwarz RT7, Debierre-Grockiego F8.

Author information

Abstract

Glycosylphosphatidylinositols (GPIs) from several protozoan parasites are thought to elicit a detrimental stimulation of the host innate immune system aside their main function to anchor surface proteins. Here we analyzed the GPI biosynthesis of an avirulent Toxoplasma gondii type 2 strain (PTG) by metabolic radioactive labeling. We determined the biological function of individual GPI species in the PTG strain in comparison with previously characterized GPI-anchors of a virulent strain (RH). The GPI intermediates of both strains were structurally similar, however the abundance of two of six GPI intermediates was significantly reduced in the PTG strain. The side-by-side comparison of GPI-anchor content revealed that the PTG strain had only ∼34% of the protein-free GPIs as well as ∼70% of the GPI-anchored proteins with significantly lower rates of protein N-glycosylation compared to the RH strain. All mature GPIs from both strains induced comparable secretion levels of TNF-α and IL-12p40, and initiated TLR4/MyD88-dependent NF-κBp65 activation in macrophages. Taken together, these results demonstrate that PTG and RH strains differ in their GPI biosynthesis and possess significantly different GPI-anchor content, while individual GPI species of both strains induce similar biological functions in macrophages.

PMID:

 

24489660

 

[PubMed - in process] 

PLoS One. 2014 Jan 29;9(1):e85763. doi: 10.1371/journal.pone.0085763. eCollection 2014.

Disruption of TgPHIL1 Alters Specific Parameters of Toxoplasma gondii Motility Measured in a Quantitative, Three-Dimensional Live Motility Assay

Leung JM1, Rould MA2, Konradt C3, Hunter CA3, Ward GE4.

Author information

Abstract

T. gondii uses substrate-dependent gliding motility to invade cells of its hosts, egress from these cells at the end of its lytic cycle and disseminate through the host organism during infection. The ability of the parasite to move is therefore critical for its virulence. T. gondii engages in three distinct types of gliding motility on coated two-dimensional surfaces: twirling, circular gliding and helical gliding. We show here that motility in a three-dimensional Matrigel-based environment is strikingly different, in that all parasites move in irregular corkscrew-like trajectories. Methods developed for quantitative analysis of motility parameters along the smoothed trajectories demonstrate a complex but periodic pattern of motility with mean and maximum velocities of 0.58±0.07 µm/s and 2.01±0.17 µm/s, respectively. To test how a change in the parasite's crescent shape might affect trajectory parameters, we compared the motility of Δphil1 parasites, which are shorter and wider than wild type, to the corresponding parental and complemented lines. Although comparable percentages of parasites were moving for all three lines, the Δphil1 mutant exhibited significantly decreased trajectory lengths and mean and maximum velocities compared to the parental parasite line. These effects were either partially or fully restored upon complementation of the Δphil1 mutant. These results show that alterations in morphology may have a significant impact on T. gondii motility in an extracellular matrix-like environment, provide a possible explanation for the decreased fitness of Δphil1 parasites in vivo, and demonstrate the utility of the quantitative three-dimensional assay for studying parasite motility.

PMID:

 

24489670

 

[PubMed - in process] 

Free PMC Article