Saturday, March 29, 2014

The inner membrane complex sub-compartment proteins critical for replication of the apicomplexan parasite Toxoplasma gondii adopt a Pleckstrin homology fold

 2014 Mar 27. [Epub ahead of print]

The inner membrane complex sub-compartment proteins critical for replication of the apicomplexan parasite Toxoplasma gondii adopt a Pleckstrin homology fold

Abstract

Toxoplasma gondii, an apicomplexan parasite prevalent in developed nations, infects up to one third of the human population. The success of this parasite depends on several unique structures including an Inner Membrane Complex (IMC) that lines the interior of the plasma membrane and contains proteins important for gliding motility and replication. Of these proteins, the IMC Sub-compartment Proteins (ISPs) have recently been shown to play a role in asexual T. gondii daughter cell formation, yet the mechanism is unknown. Complicating mechanistic characterization of the ISPs is a lack of sequence identity with proteins of known structure or function. In support of elucidating the function of ISPs, we first determined the crystal structures of representative members TgISP1 and TgISP3 to a resolution of 2.10Å and 2.32Å, respectively. Structural analysis revealed that both ISPs adopt a Pleckstrin homology (PH) fold often associated with phospholipid binding or protein-protein interactions. Substitution of basic for hydrophobic residues in the region that overlays with phospholipid binding in related PH domains, however, suggests that ISPs do not retain phospholipid binding activity. Consistent with this observation, biochemical assays revealed no phospholipid binding activity. Interestingly, mapping of conserved surface residues combined with crystal packing analysis indicates that TgISPs have functionally repurposed the phospholipid-binding site likely to coordinate protein partners. Recruitment of larger protein complexes may also be aided through avidity-enhanced interactions resulting from multimerization of the ISPs. Overall, we propose a model where TgISPs recruit protein partners to the IMC to ensure correct progression of daughter cell formation.

KEYWORDS:

Apicomplexa, Cell division, Inner Membrane Complex, Parasite, Pleckstrin Homology Fold, Protein-protein interactions, Structural biology, Toxoplasma gondii, X-ray crystallography, endodyogeny
PMID:
 
24675080
 
[PubMed - as supplied by publisher]

Thursday, March 27, 2014

Proteomic characterization of the subpellicular cytoskeleton of Toxoplasma gondii tachyzoites

2014 Mar 21. pii: S1874-3919(14)00130-4. doi: 10.1016/j.jprot.2014.03.008. [Epub ahead of print]

Proteomic characterization of the subpellicular cytoskeleton of Toxoplasma gondii tachyzoites

 
Toxoplasma, the causative agent of toxoplasmosis in animals and humans, has a subpellicular cytoskeleton that is involved in motility, cell shape and invasion. Knowledge of components of the cytoskeleton is necessary to understand the invasion mechanisms as well as for the identification of possible therapeutic targets. To date, most cytoskeletal components of Toxoplasma remain unidentified due mainly to the lack of reproducible methods for their isolation. Based on the successful isolation of the cytoskeleton, it was possible to report by the first time, the proteomic characterization of the subpellicular cytoskeleton of Toxoplasma formed by 95 cytoskeletal proteins through proteomic analysis by tandem mass spectrometry of one dimension SDS PAGE. By bioinformatic analysis of the data, proteins were classified as: Eighteen conventional cytoskeletal proteins; 10 inner membrane complex proteins, included 7 with alveolin repeats; 5 new proteins with alveolin like repeats; 37 proteins associated with other organelles and 25 novel proteins of unknown function. One of the alveolin like protein not previously described in Toxoplasma named as TgArticulin was partially characterized with a specific monoclonal antibody. Presence of TgArticulin was exclusively associated to the cytoskeleton fraction with a cortical distribution. Functions for the several molecules identified are proposed. This article is part of a Special Issue entitled: Proteomics, mass spectrometry and peptidomics, Cancun 2013. Industrial significance This manuscript describes, for the first time, the proteome of the subpellicular cytoskeleton of Toxoplasma gondii. The importance of this study is related to the role of the cytoskeleton in the highly invasive capability of a parasite that causes abortion, blindness, and death by encephalitis in immunocompromised patients. Proteomic characterization of the cytoskeleton of T. gondii tachyzoites was possible by the development of a successful procedure for the isolation of the subpellicular cytoskeleton. Knowledge of the composition of the cytoskeleton of Toxoplasma is fundamental for the understanding of the motility and host cell invasion mechanisms, and for the future design and development of toxoplasmicidal drugs with effects against specific components of the cytoskeleton of this parasite that are absent in mammal host cells.
Copyright © 2014 Elsevier B.V. All rights reserved.

KEYWORDS:

Toxoplasma, articulin, proteome, subpellicular cytoskeleton, tandem mass spectrometry
PMID:
24662527
[PubMed - as supplied by publisher]

Immune-profiling of pregnant Toxoplasma-infected US and Colombia patients reveals surprising impacts of infection on peripheral blood cytokines

2014 Mar 23. [Epub ahead of print]

Immune-profiling of pregnant Toxoplasma-infected US and Colombia patients reveals surprising impacts of infection on peripheral blood cytokines

Abstract

In North America (NA) and Europe, the majority of toxoplasmosis cases are benign and generally asymptomatic, while in South America (SA) toxoplasmosis is associated with much more severe symptoms in adults and congenitally infected children. The reasons for these differences remain unknown and, currently, there is little information from patients in either region on how the immune system responds to infection with Toxoplasma gondii. Here, we report the relative abundance of 51 serum cytokines from acute and chronic toxoplasmosis cohorts of pregnant women from the United States (US), where approximately one-half of clinical isolates are Type II, and Colombia, where clinical isolates are generally "atypical" or Type I-like strains. Surprisingly, the results showed notably lower levels of 23 cytokines in acutely infected patients from the US, relative to uninfected US controls. In acutely infected Colombian patients, however, only 8 cytokine levels differed detectably with four being lower and four higher relative to uninfected controls. Strikingly, there were also differences in the cytokine profiles of the chronically infected patients relative to uninfected controls in the US cohort. Hence, Toxoplasma appears to specifically impact levels of circulating cytokines and our results may partly explain region-specific differences in the clinical spectrum of toxoplasmosis.
PMID:
24664173
[PubMed - as supplied by publisher]

Overproduction of Toxoplasma gondii cyclophilin-18 regulates host cell migration and enhances parasite dissemination in a CCR5-independent manner

2014 Mar 25;14(1):76. [Epub ahead of print]

Overproduction of Toxoplasma gondii cyclophilin-18 regulates host cell migration and enhances parasite dissemination in a CCR5-independent manner

Abstract

BACKGROUND:

Toxoplasma gondii hijacks host cells to allow it to disseminate throughout a host animal; however, the migratory machinery involved in this process has not been well characterized. We examined the functional role of T. gondii cyclophilin 18 (TgCyp18) in host cell recruitment using recombinant parasites transfected with TgCyp18.

RESULTS:

High levels of TgCyp18 enhanced IL-12 production in cysteine-cysteine chemokine receptor 5 (CCR5) knockout mice (CCR5-/-) that had been infected peritoneally with T. gondii. Recruitment of CD11b+ cells to the infection site was enhanced in a CCR5-independent manner. T. gondii spread to several organs, particularly the liver, in a TgCyp18-dependent and CCR5-independent manner. Additionally, CCL5 levels were upregulated in macrophages treated with recombinant protein TgCyp18 and in the peritoneal fluids of the infected CCR5-/- mice. Furthermore, the chemokines involved in macrophage migration, CCL2 and CXCL10, were upregulated in the livers of CCR5-/- mice infected with recombinant parasites that had been transfected with TgCyp18.

CONCLUSION:

TgCyp18 may play a crucial role in macrophage migration, and in assisting with transport of T. gondii via CCR5-independent mechanisms. TgCyp18 may also play a role with CCL5 in the migration of macrophages to the site of infection, and with CCL2 and CXCL10 in the transport of T. gondii-infected cells to the liver.
PMID:
24661782
[PubMed - as supplied by publisher]

Wednesday, March 26, 2014

Ed Yong: Suicidal wasps, zombie 'roaches and other parasite tales

MicroRNA-132 dysregulation in Toxoplasma gondii infection has implications for dopamine signaling pathway

2014 Mar 18. pii: S0306-4522(14)00221-8. doi: 10.1016/j.neuroscience.2014.03.015. [Epub ahead of print]

MicroRNA-132 dysregulation in Toxoplasma gondii infection has implications for dopamine signaling pathway

 
Congenital toxoplasmosis and toxoplasmic encephalitis can be associated with severe neuropsychiatric symptoms. However, which host cell processes are regulated and how Toxoplasma gondii affects these changes remain unclear. MicroRNAs (miRNAs) are small noncoding RNA sequences critical to neurodevelopment and adult neuronal processes by coordinating the activity of multiple genes within biological networks. We examined the expression of over 1000 miRNAs in human neuroepithelioma cells in response to infection with Toxoplasma. MiR-132, a cyclic AMP-responsive element binding (CREB)-regulated miRNA, was the only miRNA that was substantially upregulated by all three prototype Toxoplasma strains. The increased expression of miR-132 was also documented in mice following infection with Toxoplasma. To identify cellular pathways regulated by miR-132, we performed target prediction followed by pathway enrichment analysis in the transcriptome of Toxoplasma-infected mice. This led us to identify 20 genes and dopamine receptor signaling was their strongest associated pathway. We then examined myriad aspects of the dopamine pathway in the striatum of Toxoplasma infected mice 5 days after infection. Here we report decreased expression of D1-like dopamine receptors (DRD1, DRD5), metabolizing enzyme (MAOA) and intracellular proteins associated with the transduction of dopamine-mediated signaling (DARPP-32 phosphorylation at Thr34 and Ser97). Increased concentrations of dopamine and its metabolites, serotonin and 5-hydroxyindoleacetic acid were documented by HPLC analysis; however, the metabolism of dopamine was decreased and serotonin metabolism was unchanged. Our data show that miR-132 is upregulated following infection with Toxoplasma and is associated with changes in dopamine receptor signaling. Our findings provide a possible mechanism for how the parasite contributes to the neuropathology of infection.
Copyright © 2014. Published by Elsevier Ltd.

KEYWORDS:

Alteration in expression, Dopamine receptor pathway, Mouse striatum, Toxoplasma gondii, miR-132
PMID:
24657774
[PubMed - as supplied by publisher]

The effect of artemether on psychotic symptoms and cognitive impairment in first-episode, antipsychotic drug-naive persons with schizophrenia seropositive to Toxoplasma

2014 Feb 28. pii: S0022-3956(14)00056-9. doi: 10.1016/j.jpsychires.2014.02.016. [Epub ahead of print]

The effect of artemether on psychotic symptoms and cognitive impairment in first-episode, antipsychotic drug-naive persons with schizophrenia seropositive to Toxoplasma gondii

 

Abstract

The objective was to evaluate the efficacy and safety of add-on artemether in first-episode, untreated people with schizophrenia, who were Toxoplasma gondii seropositive, and explore the change in T. gondii antibodies during treatment. In this eight-week, double-blind, randomized, placebo-controlled trial, 100 T. gondii seropositive participants with schizophrenia were randomized to either the artemether or placebo group. Participants in the artemether group received 80 mg artemether once per day during the second week (days 8-14) and the fourth week (days 22-28). Participants in the placebo group received identical looking placebo capsules. Psychopathology, adverse side effects and cognitive function were measured using standardized instruments. The group × time interaction effects for the scores of the Positive and Negative Syndrome Scale (PANSS) subscales and performances on all cognitive components were not significant, only the main effect of group was significant. Compared to the placebo group, artemether group participants showed significantly greater reduction in the PANSS negative symptom scale (F(1,46) = 4.7, p = 0.03) and the Clinical Global Impressions Scale (F(1,96) = 6.2, p = 0.01) scores, but there were no significant differences in the PANSS positive symptom and general psychopathology scales (p > 0.05). There were also no significant differences between the two groups in performance on any of the Brief Assessment of Cognition in Schizophrenia (BACS) cognitive domains. The artemether-risperidone combination is safe and well tolerated, but artemether as an adjunct to risperidone does not appear to alleviate cognitive deficits of schizophrenia. Trial Registration Chinese Clinical Trial Register (ChiCTR) TRC-13003145.
Copyright © 2014 Elsevier Ltd. All rights reserved.

KEYWORDS:

Artemether, Cognition, Psychopathology, Schizophrenia, Toxoplasma gondii
PMID:
24656425
[PubMed - as supplied by publisher]

Saturday, March 22, 2014

RON5 Is Critical for Organization and Function of the Toxoplasma Moving Junction Complex

 2014 Mar 20;10(3):e1004025. doi: 10.1371/journal.ppat.1004025. eCollection 2014.

RON5 Is Critical for Organization and Function of the Toxoplasma Moving Junction Complex

Abstract

Apicomplexans facilitate host cell invasion through formation of a tight-junction interface between parasite and host plasma membranes called the moving junction (MJ). A complex of the rhoptry neck proteins RONs 2/4/5/8 localize to the MJ during invasion where they are believed to provide a stable anchoring point for host penetration. During the initiation of invasion, the preformed MJ RON complex is injected into the host cell where RON2 spans the host plasma membrane while RONs 4/5/8 localize to its cytosolic face. While much attention has been directed toward an AMA1-RON2 interaction supposed to occur outside the cell, little is known about the functions of the MJ RONs positioned inside the host cell. Here we provide a detailed analysis of RON5 to resolve outstanding questions about MJ complex organization, assembly and function during invasion. Using a conditional knockdown approach, we show loss of RON5 results in complete degradation of RON2 and mistargeting of RON4 within the parasite secretory pathway, demonstrating that RON5 plays a key role in organization of the MJ RON complex. While RON8 is unaffected by knockdown of RON5, these parasites are unable to invade new host cells, providing the first genetic demonstration that RON5 plays a critical role in host cell penetration. Although invasion is not required for injection of rhoptry effectors into the host cytosol, parasites lacking RON5 also fail to form evacuoles suggesting an intact MJ complex is a prerequisite for secretion of rhoptry bulb contents. Additionally, while the MJ has been suggested to function in egress, disruption of the MJ complex by RON5 depletion does not impact this process. Finally, functional complementation of our conditional RON5 mutant reveals that while proteolytic separation of RON5 N- and C-terminal fragments is dispensable, a portion of the C-terminal domain is critical for RON2 stability and function in invasion.
PMID:
 
24651769
 
[PubMed - in process]

Toxoplasma gondii virulence factor ROP18 inhibits the host NF-kappaB pathway by promoting p65 degradation

 2014 Mar 19. [Epub ahead of print]

Toxoplasma gondii virulence factor ROP18 inhibits the host NF-kappaB pathway by promoting p65 degradation

Abstract

The obligate intracellular parasite Toxoplasma gondii secretes effector molecules into the host cell to modulate host immunity. Previous studies have shown that T. gondii could interfere with host NF-κB signaling to promote their survival, but the effectors of type I strains remains unclear. The polymorphic rhoptry protein ROP18 is a key serine/threonine kinase that phosphorylates host proteins to modulate acute virulence. Our data demonstrated that the N-terminal portion of ROP18 is associated with the dimerization domain of p65. ROP18 phosphorylates p65 at Ser468 and targets this protein to the ubiquitin-dependent degradation pathway. The kinase activity of ROP18 is required for p65 degradation and suppresses NF-κB activation. Consistently, compared with wild-type ROP18 strain, ROP18 kinase-deficient type I parasites displayed a severe inability to inhibit NF-κB, culminating in the enhanced production of IL-6, IL-12 and TNF-α in infected macrophages. In addition, studies have shown that transgenic parasites carrying kinase-deficient ROP18 induce M1-biased activation. These results demonstrate for the first time that the virulence factor ROP18 in T. gondii type I strains is responsible for inhibiting the host NF-κB pathway and for suppressing proinflammatory cytokine expression, thus providing a survival advantage to the infectious agent.

KEYWORDS:

Cell biology, NF-kB transcription factor, NF-kappa B (NF-KB), Parasite, Parasitology, Pathogenesis, Signal transduction
PMID:
 
24648522
 
[PubMed - as supplied by publisher]

Wednesday, March 19, 2014

Structures of the Toxoplasma gliding motility adhesin

2014 Mar 17. [Epub ahead of print]

Structures of the Toxoplasma gliding motility adhesin

 

Abstract

Micronemal protein 2 (MIC2) is the key adhesin that supports gliding motility and host cell invasion by Toxoplasma gondii. With a von Willebrand factor A (VWA) domain and six thrombospondin repeat domains (TSR1-6) in its ectodomain, MIC2 connects to the parasite actomyosin system through its cytoplasmic tail. MIC2-associated protein (M2AP) binds noncovalently to the MIC2 ectodomain. MIC2 and M2AP are stored in micronemes as proforms. We find that the MIC2-M2AP ectodomain complex is a highly elongated 1:1 monomer with M2AP bound to the TSR6 domain. Crystal structures of N-terminal fragments containing the VWA and TSR1 domains for proMIC2 and MIC2 reveal a closed conformation of the VWA domain and how it associates with the TSR1 domain. A long, proline-rich, disulfide-bonded pigtail loop in TSR1 overlaps the VWA domain. Mannose α-C-linked to Trp-276 in TSR1 has an unusual 1C4 chair conformation. The MIC2 VWA domain includes a mobile α5-helix and a 22-residue disordered region containing two disulfide bonds in place of an α6-helix. A hydrophobic residue in the prodomain binds to a pocket adjacent to the α7-helix that pistons in opening of the VWA domain to a putative high-affinity state.
PMID:
24639528
[PubMed - as supplied by publisher]

Toxoplasma Acto-MyoA Motor Complex Is Important but Not Essential for Gliding Motility and Host Cell Invasion

2014 Mar 14;9(3):e91819. doi: 10.1371/journal.pone.0091819. eCollection 2014.

The Toxoplasma Acto-MyoA Motor Complex Is Important but Not Essential for Gliding Motility and Host Cell Invasion

 

Abstract

Apicomplexan parasites are thought to actively invade the host cell by gliding motility. This movement is powered by the parasite's own actomyosin system, and depends on the regulated polymerisation and depolymerisation of actin to generate the force for gliding and host cell penetration. Recent studies demonstrated that Toxoplasma gondii can invade the host cell in the absence of several core components of the invasion machinery, such as the motor protein myosin A (MyoA), the microneme proteins MIC2 and AMA1 and actin, indicating the presence of alternative invasion mechanisms. Here the roles of MyoA, MLC1, GAP45 and Act1, core components of the gliding machinery, are re-dissected in detail. Although important roles of these components for gliding motility and host cell invasion are verified, mutant parasites remain invasive and do not show a block of gliding motility, suggesting that other mechanisms must be in place to enable the parasite to move and invade the host cell. A novel, hypothetical model for parasite gliding motility and invasion is presented based on osmotic forces generated in the cytosol of the parasite that are converted into motility.
PMID:
24632839
[PubMed - in process]

Identification and genetic characterization of a new Brazilian genotype of Toxoplasma gondii from sheep intended for human consumption

2014 Mar 11. pii: S1383-5769(14)00032-4. doi: 10.1016/j.parint.2014.03.001. [Epub ahead of print]

Identification and genetic characterization of a new Brazilian genotype of Toxoplasma gondii from sheep intended for human consumption

 

Abstract

Recent studies have demonstrated that strains of Toxoplasma gondii in Brazil are frequently different from those detected in other countries, thus making accurate phylogenetic analysis difficult. The aim of this study was to genetically characterize T. gondii samples from sheep raised in southern Bahia and intended for human consumption, by means of PCR-RFLP and sequencing techniques. Experimental samples were obtained from 200 sheep brains purchased at butcher's shops in Itabuna, Bahia, Brazil. In total, three samples (#54, #124 and #127) were Toxoplasma gondii-positive. Application of multilocus PCR-RFLP using ten molecular markers (SAG1, SAG2, SAG3, BTUB, c22-8, PK1, GRA6, L358, c-29-2 and Apico) revealed a single genotype common to all samples of this study, which differed from any other published T. gondii genotypes. An atypical allele was detected in the L358 genetic marker; this has not previously been shown in any other South American T. gondii isolates. Phylogenetic analysis on the sequences from multilocus PCR sequencing revealed that these three samples were classified into the same lineage. Extensive indel regions were detected in the Apico genetic marker. Together, our findings revealed a new Brazilian T. gondii genotype. Further research should be conducted to enrich the database of Brazilian T. gondii genotypes from different regions. This will make it possible to understand the phylogenetic relationship between isolates.
Copyright © 2014. Published by Elsevier Ireland Ltd.

KEYWORDS:

Genetic diversity, PCR-RFLP, genotype, population structure, sequencing, toxoplasmosis
PMID:
24631791
[PubMed - as supplied by publisher]

Monday, March 17, 2014

Inflammasome Sensor NLRP1 Controls Rat Macrophage Susceptibility to Toxoplasma gondii

2014 Mar 13;10(3):e1003927. doi: 10.1371/journal.ppat.1003927. eCollection 2014.

Inflammasome Sensor NLRP1 Controls Rat Macrophage Susceptibility to Toxoplasma gondii

 

Abstract

Toxoplasma gondii is an intracellular parasite that infects a wide range of warm-blooded species. Rats vary in their susceptibility to this parasite. The Toxo1 locus conferring Toxoplasma resistance in rats was previously mapped to a region of chromosome 10 containing Nlrp1. This gene encodes an inflammasome sensor controlling macrophage sensitivity to anthrax lethal toxin (LT) induced rapid cell death (pyroptosis). We show here that rat strain differences in Toxoplasma infected macrophage sensitivity to pyroptosis, IL-1β/IL-18 processing, and inhibition of parasite proliferation are perfectly correlated with NLRP1 sequence, while inversely correlated with sensitivity to anthrax LT-induced cell death. Using recombinant inbred rats, SNP analyses and whole transcriptome gene expression studies, we narrowed the candidate genes for control of Toxoplasma-mediated rat macrophage pyroptosis to four genes, one of which was Nlrp1. Knockdown of Nlrp1 in pyroptosis-sensitive macrophages resulted in higher parasite replication and protection from cell death. Reciprocally, overexpression of the NLRP1 variant from Toxoplasma-sensitive macrophages in pyroptosis-resistant cells led to sensitization of these resistant macrophages. Our findings reveal Toxoplasma as a novel activator of the NLRP1 inflammasome in rat macrophages.
PMID:
24626226
[PubMed - in process]

Single nucleotide polymorphisms in the interferon gamma gene are associated with distinct types of retinochoroidal scar lesions presumably caused by Toxoplasma

2014 Feb 1;109(1):99-107. doi: 10.1590/0074-0276140539.

Single nucleotide polymorphisms in the interferon gamma gene are associated with distinct types of retinochoroidal scar lesions presumably caused by Toxoplasma gondii infection

Abstract

The association of single nucleotide polymorphisms (SNPs) in the interferon (IFN)-γ gene ( IFNG ) with different types of retinal scar lesions presumably caused by toxoplasmosis were investigated in a cross-sectional population-based genetic study. Ten SNPs were investigated and after Bonferroni correction, only the associations between SNPs rs2069718 and rs3181035 with retinal/retinochoroidal scar lesions type A (most severe scar lesions) and C (least severe scar lesions), respectively, remained significant. The associations of two different IFNG SNPs with two different types of retinal lesions attributable to toxoplasmosis support the hypothesis that different inflammatory mechanisms underlie the development of these lesions. The in vitro analysis of IFN-γ secretion by peripheral blood mononuclear cells stimulated with Toxoplasma gondii antigens was also investigated. The association between SNP rs2069718 and type A scar lesions revealed that differential IFN-γ levels are correlated with distinct genotypes. However, no correlation was observed with IFN-γ secretion levels and the SNP rs3181035 , which was significantly associated with type C scar lesions. Our findings strongly suggest that immunogenetic studies of individuals with congenital or postnatally acquired infection are needed to better understand the role of IFN-γ and its polymorphisms in the pathogenesis of ocular toxoplasmosis.
PMID:
24626309
[PubMed - in process]

Friday, March 14, 2014

The inner membrane complex through development of Toxoplasma gondii and Plasmodium

2014 Feb 26. doi: 10.1111/cmi.12285. [Epub ahead of print]

The inner membrane complex through development of Toxoplasma gondii and Plasmodium

Abstract

Plasmodium spp and Toxoplasma gondii are important human and veterinary pathogens. These parasites possess an unusual double membrane structure located directly below the plasma membrane named the inner membrane complex (IMC). First identified in early electron micrograph studies, huge advances in genetic manipulation of the Apicomplexa have allowed the visualisation of a dynamic, highly structured cellular compartment with important roles in maintaining the structure and motility of these parasites. This review summarises recent advances in the field and highlights the changes the IMC undergoes during the complex life cycles of the Apicomplexa.
This article is protected by copyright. All rights reserved.
PMID:
24612102
[PubMed - as supplied by publisher]

Toxoplasma gondii induce apoptosis of neural stem cells via endoplasmic reticulum stress pathway

2014 Mar 10:1-8. [Epub ahead of print]

Toxoplasma gondii induce apoptosis of neural stem cells via endoplasmic reticulum stress pathway

 

Abstract

SUMMARY Toxoplasma gondii is a major cause of congenital brain disease; however, the underlying mechanism of neuropathogenesis in brain toxoplasmosis remains elusive. To explore the role of T. gondii in the development of neural stem cells (NSCs), NSCs were isolated from GD14 embryos of ICR mice and were co-cultured with tachyzoites of T. gondii RH strain. We found that apoptosis levels of the NSCs co-cultured with 1×106 RH tachyzoites for 24 and 48 h significantly increased in a dose-dependent manner, as compared with the control. Western blotting analysis displayed that the protein level of C/EBP homologous protein (CHOP) was up-regulated, and caspase-12 and c-Jun N-terminal kinase (JNK) were activated in the NSCs co-cultured with the parasites. Pretreatment with endoplasmic reticulum stress (ERS) inhibitor (TUDCA) and caspase-12 inhibitor (Z-ATAD-FMK) inhibited the expression or activation of the key molecules involved in the ERS-mediated apoptotic pathway, and subsequently decreased the apoptosis levels of the NSCs induced by the T. gondii. The findings here highlight that T. gondii induced apoptosis of the NSCs through the ERS signal pathway via activation of CHOP, caspase-12 and JNK, which may constitute a potential molecular mechanism responsible for the cognitive disturbance in neurological disorders of T. gondii.
PMID:
24612639
[PubMed - as supplied by publisher]

Sunday, March 09, 2014

Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites

 2013 Nov 11;4(1):1-13. eCollection 2014.

Aminoacyl-tRNA synthetases as drug targets in eukaryotic parasites

Abstract

Aminoacyl-tRNA synthetases are central enzymes in protein translation, providing the charged tRNAs needed for appropriate construction of peptide chains. These enzymes have long been pursued as drug targets in bacteria and fungi, but the past decade has seen considerable research on aminoacyl-tRNA synthetases in eukaryotic parasites. Existing inhibitors of bacterial tRNA synthetases have been adapted for parasite use, novel inhibitors have been developed against parasite enzymes, and tRNA synthetases have been identified as the targets for compounds in use or development as antiparasitic drugs. Crystal structures have now been solved for many parasite tRNA synthetases, and opportunities for selective inhibition are becoming apparent. For different biological reasons, tRNA synthetases appear to be promising drug targets against parasites as diverse as Plasmodium (causative agent of malaria), Brugia (causative agent of lymphatic filariasis), and Trypanosoma (causative agents of Chagas disease and human African trypanosomiasis). Here we review recent developments in drug discovery and target characterisation for parasite aminoacyl-tRNA synthetases.

KEYWORDS:

Aminoacyl-tRNA synthetase, Drug target, Parasite, Protein translation
PMID:
 
24596663
 
[PubMed - as supplied by publisher]

Tuesday, March 04, 2014

miR-146a and miR-155 Delineate a microRNA Fingerprint Associated with Toxoplasma Persistence in the Host Brain

2014 Feb 25. pii: S2211-1247(14)00081-3. doi: 10.1016/j.celrep.2014.02.002. [Epub ahead of print]

miR-146a and miR-155 Delineate a microRNA Fingerprint Associated with Toxoplasma Persistence in the Host Brain

 
microRNAs were recently found to be regulators of the host response to infection by apicomplexan parasites. In this study, we identified two immunomodulatory microRNAs, miR-146a and miR-155, that were coinduced in the brains of mice challenged with Toxoplasma in a strain-specific manner. These microRNAs define a characteristic fingerprint for infection by type II strains, which are the most prevalent cause of human toxoplasmosis in Europe and North America. Using forward genetics, we showed that strain-specific differences in miR-146a modulation were in part mediated by the rhoptry kinase, ROP16. Remarkably, we found that miR-146a deficiency led to better control of parasite burden in the gut and most likely of early parasite dissemination in the brain tissue, resulting in the long-term survival of mice.
Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
PMID:
24582962
[PubMed - as supplied by publisher]

Toxoplasma gondii Inhibits Apoptosis via A Novel STAT3-miR-17~92-Bim Pathway in Macrophages

2014 Feb 27. pii: S0898-6568(14)00073-4. doi: 10.1016/j.cellsig.2014.02.013. [Epub ahead of print]

Toxoplasma gondii Inhibits Apoptosis via A Novel STAT3-miR-17~92-Bim Pathway in Macrophages


In order to accomplish their life cycles, intracellular pathogens, including the apicomplexan Toxoplasma gondii, subvert the innate apoptotic response of infected host cells. However, the precise mechanisms of parasite interference with the apoptotic pathway remain unclear. MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level. Using T. gondii strain TgCtwh3, which was isolated from felids and possesses the predominant genotype China 1 (ToxoDB#9) in China, we analyzed the miRNA expression profile of human macrophages challenged with TgCtwh3. The results showed that miR-17~92 miRNAs expression was significantly increased and Bim was decreased in TgCtwh3-infected cells. Database analysis of miR-17~92 miRNAs revealed the potential binding sites in the 3'UTR of Bim, one of the crucial effectors of pro-apoptosis. Furthermore, we demonstrated that promoter of the miR-17~92 gene cluster which encodes miRNAs was transactivated through the promoter binding of the STAT3 following TgCtwh3 infection. Taken together, we describe a novel STAT3-miR-17~92-Bim pathway, thus providing a mechanistic explanation for inhibition of apoptosis of host cells following Toxoplasma infection.
Copyright © 2014. Published by Elsevier Inc.

KEYWORDS:

Bim, STAT3, apoptosis, macrophage, microRNA
PMID:
24583285
[PubMed - as supplied by publisher]

INHIBITION AND STRUCTURE OF TOXOPLASMA GONDII PURINE NUCLEOSIDE PHOSPHORYLASE

2014 Feb 28. [Epub ahead of print]

INHIBITION AND STRUCTURE OF TOXOPLASMA GONDII PURINE NUCLEOSIDE PHOSPHORYLASE

Abstract

The intracellular pathogen Toxoplasma gondii is a purine auxotroph that relies on purine salvage for proliferation. We have optimized T. gondii purine nucleoside phosphorylase (TgPNP) stability and crystallized TgPNP with phosphate and Immucillin-H, a transition-state analogue that has high affinity for the enzyme. Immucillin-H bound to TgPNP with a dissociation constant of 370 pM, the highest affinity of eleven immucillins selected to probe the catalytic site. The specificity for transition-state analogues indicated an early dissociative transition state for TgPNP. Compared with PfPNP, large substituents surrounding the 5' -hydroxyl group of inhibitors demonstrate reduced capacity for TgPNP inhibition. Catalytic discrimination against large 5' groups is consistent with the inability of TgPNP to catalyze the phosphorylysis of 5' -methylthioinosine to hypoxanthine. In contrast to mammalian PNP, the 2' -hydroxyl group is crucial for inhibitor binding in the catalytic site of TgPNP. This first crystal structure of TgPNP describes the basis for discrimination against 5' -methylthioinosine and similarly 5' -hydroxy-substituted immucillins; structural differences reflect the unique adaptations of purine salvage pathways of Apicomplexa.
PMID:
24585883
[PubMed - as supplied by publisher]

Overlapping Toxoplasma gondii Genotypes Circulating in Domestic Animals and Humans in Southeastern Brazil

2014 Feb 27;9(2):e90237. doi: 10.1371/journal.pone.0090237. eCollection 2014.

Overlapping Toxoplasma gondii Genotypes Circulating in Domestic Animals and Humans in Southeastern Brazil

Abstract

Although several Toxoplasma gondii genotyping studies have been performed in Brazil, studies of isolates from animals in the state of Minas Gerais are rare. The objective of this study was to conduct a genotypic characterization of T. gondii isolates obtained from dogs, free-range chickens, and humans in Minas Gerais and to verify whether the T. gondii genotypes circulating in domestic animals correspond to the genotypes detected in humans. Genetic variability was assessed by restricted fragment length polymorphism at 11 loci (SAG1, 5'+3'SAG2, SAG2 alt, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Twelve different genotypes were identified among the 24 isolates studied, including 8 previously identified genotypes and 4 new genotypes. The genetic relationship of the 24 T. gondii isolates, together with the genotypes previously described from 24 human newborns with congenital toxoplasmosis, revealed a high degree of similarity among the genotypes circulating in humans and animals in Minas Gerais. The most common genotypes among these species were BrII, BrIII, ToxoDB #108, and ToxoDB #206. Restricted fragment length polymorphism at the CS3 locus of these 48 isolates showed that the majority of isolates presented alleles I (50%) or II (27%). Isolates harboring allele III at the CS3 locus presented low virulence for mice, whereas those harboring alleles I or II presented higher virulence. These results confirm the utility of marker CS3 for predicting the virulence of Brazilian isolates of T. gondii in mice. No association was found between the allele type and clinical manifestations of human congenital toxoplasmosis. This is the first report of T. gondii genotyping that verifies the overlapping genotypes of T. gondii from humans and animals in the same geographic region of Brazil. Our results suggest that there is a common source of infection to the species studied, most likely oocysts contaminating the environment.
PMID:
24587295
[PubMed - in process]

Assembly of the Novel Five-Component Apicomplexan Multi-Aminoacyl-tRNA Synthetase Complex Is Driven by the Hybrid Scaffold Protein Tg-p43

2014 Feb 20;9(2):e89487. doi: 10.1371/journal.pone.0089487. eCollection 2014.

Assembly of the Novel Five-Component Apicomplexan Multi-Aminoacyl-tRNA Synthetase Complex Is Driven by the Hybrid Scaffold Protein Tg-p43

 
In Toxoplasma gondii, as in other eukaryotes, a subset of the amino-acyl-tRNA synthetases are arranged into an abundant cytoplasmic multi-aminoacyl-tRNA synthetase (MARS) complex. Through a series of genetic pull-down assays, we have identified the enzymes of this complex as: methionyl-, glutaminyl-, glutamyl-, and tyrosyl-tRNA synthetases, and we show that the N-terminal GST-like domain of a partially disordered hybrid scaffold protein, Tg-p43, is sufficient for assembly of the intact complex. Our gel filtration studies revealed significant heterogeneity in the size and composition of isolated MARS complexes. By targeting the tyrosyl-tRNA synthetases subunit, which was found exclusively in the complete 1 MDa complex, we were able to directly visualize MARS particles in the electron microscope. Image analyses of the negative stain data revealed the observed heterogeneity and instability of these complexes to be driven by the intrinsic flexibility of the domain arrangements within the MARS complex. These studies provide unique insights into the assembly of these ubiquitous but poorly understood eukaryotic complexes.
PMID:
24586818
[PubMed - in process]