Thursday, February 28, 2013

Biochemical and immunological characterisation of Toxoplasma gondii macrophage migration inhibitory factor

J Biol Chem. 2013 Feb 26. [Epub ahead of print]

Biochemical and immunological characterisation of Toxoplasma gondii macrophage migration inhibitory factor

Sommerville C, Richardson JM, Williams RA, Mottram JC, Roberts CW, Alexander J, Henriquez FL.

University of Strathclyde, United Kingdom

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory molecule in mammals, which unusually, for a cytokine exhibits tautomerase and oxidoreductase enzymatic activities. Homologues of this well conserved protein are found within diverse phyla including a number of parasitic organisms. Herein, we produced recombinant histidine-tagged Toxoplasma gondii MIF (TgMIF), a 12kDa protein that lacks oxidoreductase activity, but exhibits tautomerase activity with a specific activity of 19.3μmol/min/mg that cannot be inhibited by the human MIF inhibitor ISO-1. The crystal structure of the TgMIF homotrimer has been determined to 1.82 Å and, although it has close structural homology with mammalian MIFs, it has critical differences in the tautomerase active site that account for the different inhibitor sensitivity. We also demonstrate that TgMIF can elicit IL-8 production from human peripheral blood mononuclear cells whilst also activating ERK-MAPK pathways in murine bone marrow derived macrophages. TgMIF may therefore play an immunomodulatory role during T. gondii infection in mammals.

PMID: 23443656 [PubMed - as supplied by publisher]

Tuesday, February 26, 2013

Discovery of a splicing regulator required for cell cycle progression

PLoS Genet. 2013 Feb;9(2):e1003305. doi: 10.1371/journal.pgen.1003305. Epub 2013 Feb 21.

Discovery of a splicing regulator required for cell cycle progression

Suvorova ES, Croken M, Kratzer S, Ting LM, de Felipe MC, Balu B, Markillie ML, Weiss LM, Kim K, White MW.

Departments of Molecular Medicine and Global Health and the Florida Center for Drug Discovery and Innovation, University of South Florida, Tampa, Florida, United States of America.

In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

PMID: 23437009 [PubMed - in process]

How and why Toxoplasma makes us crazy

Trends Parasitol. 2013 Feb 19. pii: S1471-4922(13)00020-2. doi: 10.1016/ [Epub ahead of print]

How and why Toxoplasma makes us crazy

Flegr J

Faculty of Science, Charles University in Prague, Prague 128 44, Czech Republic. Electronic address:

For a long time, a latent toxoplasmosis, the lifelong presence of dormant stages of Toxoplasma in various tissues, including the brain, was considered harmless for immunocompetent persons. Within the past 10 years, however, many independent studies have shown that this parasitic disease, with a worldwide prevalence of about 30%, could be indirectly responsible for hundreds of thousands of deaths due to its effects on the rate of traffic and workplace accidents, and also suicides. Moreover, latent toxoplasmosis is probably one of the most important risk factors for schizophrenia. At least some of these effects, possibly mediated by increased dopamine and decreased tryptophan, are products of manipulation activity by Toxoplasma aiming to increase the probability of transmission from intermediate to definitive host through predation.

PMID: 23433494 [PubMed - as supplied by publisher]

Toxoplasma gondii Migration within and Infection of Human Retina

PLoS One. 2013;8(2):e54358. doi: 10.1371/journal.pone.0054358. Epub 2013 Feb 21.

Toxoplasma gondii Migration within and Infection of Human Retina

Furtado JM, Ashander LM, Mohs K, Chipps TJ, Appukuttan B, Smith JR.

Casey Eye Institute, Oregon Health & Science University, Portland, Oregon, United States of America

Toxoplasmic retinochoroiditis is a common blinding retinal infection caused by the parasite, Toxoplasma gondii. Basic processes relating to establishment of infection in the human eye by T. gondii tachyzoites have not been investigated. To evaluate the ability of tachyzoites to navigate the human retina, we developed an ex vivo assay, in which a suspension containing 1.5×10(7) parasites replaced vitreous in a posterior eyecup. After 8 hours, the retina was formalin-fixed and paraffin-embedded, and sections were immunostained to identify tachyzoites. To determine the preference of tachyzoites for human retinal neuronal versus glial populations, we infected dissociated retinal cultures, subsequently characterized by neuron-specific enolase or glial fibrillary acidic protein expression, and retinal cell lines, with YFP-expressing tachyzoites. In migration assays, retinas contained 110-250 live tachyzoites; 64.5-95.2% (mean  = 79.6%) were localized to the nerve fiber layer, but some were detected in the outer retina. Epifluorescence imaging of dissociated retinal cultures 24 hours after infection indicated preferential infection of glia. This observation was confirmed in growth assays, with significantly higher (p≤0.005) numbers of tachyzoites measured in glial verus neuronal cell lines. Our translational studies indicate that, after entering retina, tachyzoites may navigate multiple tissue layers. Tachyzoites preferentially infect glial cells, which exist throughout the retina. These properties may contribute to the success of T. gondii as a human pathogen.

PMID: 23437042 [PubMed - in process]

Monday, February 25, 2013

Association between Intracellular Infectious Agents and Schizophrenia

Clin Psychopharmacol Neurosci. 2012 Aug;10(2):117-23. doi: 10.9758/cpn.2012.10.2.117. Epub 2012 Aug 31.

Association between Intracellular Infectious Agents and Schizophrenia

Park MH, Kwon YJ, Jeong HY, Lee HY, Hwangbo Y, Yoon HJ, Shim SH.

Department of Psychiatry, Soon Chun Hyang University Cheonan Hospital, Soon Chun Hyang University College of Medicine, Cheonan, Korea.

A number of studies have reported association between Toxoplasma gondii (T. gondii) and Chlamydia infection and the risk of schizophrenia. The aim of the present study was to compare the prevalence of T. gondii and Chlamydia infection between the schizophrenia and normal control subjects and to compare the clinical features between seropositive and seronegative schizophrenia patients.
The rate of serum reactivity to T. gondii, Chlamydia trachomatis (C. trachomatis), Chlamydia pneumonia in 96 schizophrenia and 50 control subjects was investigated using enzyme-linked immunosorbent assay and indirect fluorescent antibody technique. The clinical symptoms of the schizophrenia patients were scored with Positive and Negative Syndrome Scale and a comparative analysis was carried out.
A significant positive association between immunoglobulin G (IgG) antibodies to T. gondii and C. trachomatis in schizophrenia was found, and the odds ratio of schizophrenia associated with IgG antibody was found to be 3.22 and 2.86, respectively. The Toxoplasma-seropositive schizophrenia patient had higher score on the negative subscale N1 and N7 and general psychopathology subscale G13, while C. trachomatis-seropositive schizophrenia patient had higher score on the general psychopathology subscale G10.
The results from the present study suggest significant association between T. gondii, C. trachomatis infection and schizophrenia. In future, further studies are needed to elucidate the correlation between the two types of infection and schizophrenia.

PMID: 23430959 [PubMed - in process]

Expression, characterization and inhibition of Toxoplasma gondii 1-deoxy-d-xylulose-5-phosphate reductoisomerase

Bioorg Med Chem Lett. 2013 Jan 30. pii: S0960-894X(13)00130-3. doi: 10.1016/j.bmcl.2013.01.097. [Epub ahead of print]

Expression, characterization and inhibition of Toxoplasma gondii 1-deoxy-d-xylulose-5-phosphate reductoisomerase

Cai G, Deng L, Xue J, Moreno SN, Striepen B, Song Y.

Department of Pharmacology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030, US

The apicomplexan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, is an important human pathogen. 1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) in the non-mevalonate isoprene biosynthesis pathway is essential to the organism and therefore a target for developing anti-toxoplasmosis drugs. In order to find potent inhibitors, we expressed and purified recombinant T. gondii DXR (TgDXR). Biochemical properties of this enzyme were characterized and an enzyme activity/inhibition assay was developed. A collection of 11 compounds with a broad structural diversity were tested against TgDXR and several potent inhibitors were identified with K(i) values as low as 48nM. Analysis of the results as well as those of Escherichia coli and Plasmodium falciparum DXR enzymes revealed a different structure-activity relationship profile for the inhibition of TgDXR.

PMID: 23428849 [PubMed - as supplied by publisher]

Tuesday, February 19, 2013

IL-10 Reduces Levels of Apoptosis in Toxoplasma gondii-Infected Trophoblasts

PLoS One. 2013;8(2):e56455. Epub 2013 Feb 13.

IL-10 Reduces Levels of Apoptosis in Toxoplasma gondii-Infected Trophoblasts

Zhao M, Zhang R, Xu X, Liu Y, Zhang H, Zhai X, Hu X.

Department of Radiology, Yantai Affiliated Hospital of Binzhou Medical University, Yantai, People's Republic of China.


To analyze the effects of IL-10 on the HLA-G expression and the apoptosis of trophoblasts infected with Toxoplasma gondii.
T. gondii-infected or uninfected human trophoblasts and immortalized human placental BeWo cells were cultured with or without human IL-10. Uninfected and infected cells without IL-10 cells served as controls. HLA-G expression was measured by real-time PCR and flow cytometry, respectively. Cells apoptosis were analyzed by flow cytometry. Apoptosis associated moleculars were measured by real-time PCR and Western bolt.
HLA-G expression was increased in the infected trophoblasts and BeWo cells compared to uninfected cells. Treatment of infected cells with IL-10 decreased HLA-G expression compared to infected cells while no change in treatment of uninfected cells compared with uninfected cells. Levels of apoptosis and apoptosis associated caspase-3 and caspase-8 decreased and c-FLIP levels increased in treated infected cells with IL-10 compared to infected cells and no difference in IL-10 treated uninfected cells compared to uninfected cells.
IL-10 regulates HLA-G expression in T. gondii-infected trophoblasts. IL-10 treatment of infected trophoblasts reduced levels of apoptosis. This may contribute to the improvement in pregnancy outcomes when women infected with T. gondii treated with IL-10.

PMID: 23418570 [PubMed - as supplied by publisher]

Adaptive host manipulation by Toxoplasma gondii: fact or fiction?

Trends Parasitol. 2013 Feb 14. pii: S1471-4922(13)00017-2. doi: 10.1016/ [Epub ahead of print]

Adaptive host manipulation by Toxoplasma gondii: fact or fiction?

Worth AR, Lymbery AJ, Thompson RC.

School of Veterinary and Biomedical Science, Murdoch University, South Street, Murdoch 6150, Australia. Electronic address:

It is widely accepted that behavioural changes induced by Toxoplasma gondii are an adaptation of the parasite to enhance transmission to its cat definitive host. In our opinion, this explanation requires a rethink. We argue that the experimental evidence that observed behavioural changes will enhance transmission to cats is not convincing. We also argue that cats and sexual reproduction may not be essential for transmission and maintenance of this parasite. Thus, the selection pressure to infect a cat may not be sufficiently strong for the evolution of adaptive host manipulation to have occurred in order to enhance predation by cats.

 PMID: 23415732 [PubMed - as supplied by publisher]

Friday, February 15, 2013

Protein intrinsic disorder in the acetylome of intracellular and extracellular Toxoplasma gondii

Mol Biosyst. 2013 Feb 12. [Epub ahead of print]

Protein intrinsic disorder in the acetylome of intracellular and extracellular Toxoplasma gondii

Xue B, Jeffers V, Sullivan WJ, Uversky VN.

Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA.

Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa, which includes a number of species of medical and veterinary importance. Inhibitors of lysine deacetylases (KDACs) exhibit potent antiparasitic activity, suggesting that interference with lysine acetylation pathways holds promise for future drug targeting. Using high resolution LC-MS/MS to identify parasite peptides enriched by immunopurification with acetyl-lysine antibody, we recently produced an acetylome of the proliferative intracellular stage of Toxoplasma. In this study, we used similar approaches to greatly expand the Toxoplasma acetylome by identifying acetylated proteins in non-replicating extracellular tachyzoites. The functional breakdown of acetylated proteins in extracellular parasites is similar to intracellular parasites, with an enrichment of proteins involved in metabolism, translation, and chromatin biology. Altogether, we have now detected over 700 acetylation sites on a wide variety of parasite proteins of diverse function in multiple subcellular compartments. We found 96 proteins uniquely acetylated in intracellular parasites, 216 uniquely acetylated in extracellular parasites, and 177 proteins acetylated in both states. Our findings suggest that dramatic changes occur at the proteomic level as tachyzoites transition from the intracellular to the extracellular environment, similar to reports documenting significant changes in gene expression during this transition. The expanded dataset also allowed a thorough analysis of the degree of protein intrinsic disorder surrounding lysine residues targeted for this post-translational modification. These analyses indicate that acetylated lysines in proteins from extracellular and intracellular tachyzoites are largely located within similar local environments, and that lysine acetylation preferentially occurs in intrinsically disordered or flexible regions.

PMID: 23403842 [PubMed - as supplied by publisher]

A Toxoplasma Palmitoyl Acyl Transferase and the Palmitoylated Armadillo Repeat Protein TgARO Govern Apical Rhoptry Tethering

PLoS Pathog. 2013 Feb;9(2):e1003162. doi: 10.1371/journal.ppat.1003162. Epub 2013 Feb 7.

A Toxoplasma Palmitoyl Acyl Transferase and the Palmitoylated Armadillo Repeat Protein TgARO Govern Apical Rhoptry Tethering and Reveal a Critical Role for the Rhoptries in Host Cell Invasion but Not Egress
Beck JR, Fung C, Straub KW, Coppens I, Vashisht AA, Wohlschlegel JA, Bradley PJ.

Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, United States of America.

Apicomplexans are obligate intracellular parasites that actively penetrate their host cells to create an intracellular niche for replication. Commitment to invasion is thought to be mediated by the rhoptries, specialized apical secretory organelles that inject a protein complex into the host cell to form a tight-junction for parasite entry. Little is known about the molecular factors that govern rhoptry biogenesis, their subcellular organization at the apical end of the parasite and subsequent release of this organelle during invasion. We have identified a Toxoplasma palmitoyl acyltransferase, TgDHHC7, which localizes to the rhoptries. Strikingly, conditional knockdown of TgDHHC7 results in dispersed rhoptries that fail to organize at the apical end of the parasite and are instead scattered throughout the cell. While the morphology and content of these rhoptries appears normal, failure to tether at the apex results in a complete block in host cell invasion. In contrast, attachment and egress are unaffected in the knockdown, demonstrating that the rhoptries are not required for these processes. We show that rhoptry targeting of TgDHHC7 requires a short, highly conserved C-terminal region while a large, divergent N-terminal domain is dispensable for both targeting and function. Additionally, a point mutant lacking a key residue predicted to be critical for enzyme activity fails to rescue apical rhoptry tethering, strongly suggesting that tethering of the organelle is dependent upon TgDHHC7 palmitoylation activity. We tie the importance of this activity to the palmitoylated Armadillo Repeats-Only (TgARO) rhoptry protein by showing that conditional knockdown of TgARO recapitulates the dispersed rhoptry phenotype of TgDHHC7 knockdown. The unexpected finding that apicomplexans have exploited protein palmitoylation for apical organelle tethering yields new insight into the biogenesis and function of rhoptries and may provide new avenues for therapeutic intervention against Toxoplasma and related apicomplexan parasites.

PMID: 23408890 [PubMed - in process]

Monday, February 11, 2013

Possible role of Toxoplasma gondii in brain cancer through modulation of host microRNAs

Infect Agent Cancer. 2013 Feb 8;8(1):8. [Epub ahead of print]

Possible role of Toxoplasma gondii in brain cancer through modulation of host microRNAs

Thirugnanam S, Rout N, Gnanasekar M.

BACKGROUND: The obligate intracellular protozoan parasite Toxoplasma gondii infects humans and other warm-blooded animals and establishes a chronic infection in the central nervous system after invasion. Studies showing a positive correlation between anti-Toxoplasma antibodies and incidences of brain cancer have led to the notion that Toxoplasma infections increase the risk of brain cancer. However, molecular events involved in Toxoplasma induced brain cancers are not well understood.
Toxoplasma gains the control of host cell functions including proliferation and apoptosis by channelizing parasite proteins into the cell cytoplasm and some of the proteins are targeted to the host nucleus. Recent studies have shown that Toxoplasma is capable of manipulating host micro RNAs (miRNAs) which play a central role in post-transcriptional regulation of gene expression. Therefore, we hypothesize that Toxoplasma promotes brain carcinogenesis by altering the host miRNAome using parasitic proteins and/or miRNAs.
The miRNA expression profiles of brain cancer specimens obtained from patients that are infected with Toxoplasma could be analyzed and compared with that of normal tissues as well as brain cancer tissues from Toxoplasma uninfected individuals to identify dysregulated miRNAs in Toxoplasma-driven brain cancer cells. Identified miRNAs will be further confirmed by studying cancer related miRNA profiles of the different types of brain cells before and after Toxoplasma infection using cell lines and experimental animals.Expected outcome: The miRNAs specifically associated with brain cancers that are caused by Toxoplasma infection will be identified.
Toxoplasma infection may promote initiation and progression of cancer by modifying the miRNAome in brain cells. If this hypothesis is true, the outcome of this research would lead to the development of novel biomarkers and therapeutic tools against Toxoplasma driven brain cancers.

 PMID: 23391314 [PubMed - as supplied by publisher]

Toxoplasma gondii Infection Regulates the Balance of Activating and Inhibitory Receptors on Decidual Natural Killer Cells

PLoS One. 2013;8(2):e55432. doi: 10.1371/journal.pone.0055432. Epub 2013 Feb 5.

Toxoplasma gondii Infection Regulates the Balance of Activating and Inhibitory Receptors on Decidual Natural Killer Cells

Xu X, Zhao M, Liu X, Jiang Y, Zhang H, Zhai X, Zhang L, Hu X.

Department of Immunology, Binzhou Medical University, Shandong, People's Republic of China.

Inhibitory receptors and activating receptor expressed on decidual natural killer (dNK) cells are generally believed to be important in abnormal pregnancy outcomes and induced adverse pregnancy. However, if Toxoplasma gondii (T. gondii) infection induced abnormal pregnancy was related to dNK cells changes is not clear. In this study, we used human dNK cells co-cultured with human extravillous cytotrophoblast (EVT) cells following YFP-Toxoplasma gondii (YFP-T. gondii) infection in vitro and established animal pregnant infection model. Levels of inhibitory receptors KIR2DL4 and ILT-2, their ligand HLA-G, and activating receptor NKG2D in human decidua, and NKG2A and its ligand Qa-1 and NKG2D in mice uterine were analyzed by real-time PCR and flow cytometry with levels of NKG2D significantly higher than those of KIR2DL4 and ILT-2 in vitro and in invo. The level of NKG2D was positively correlated with cytotoxic activity of dNK cells in vitro. Numbers of abnormal pregnancies were significantly greater in the infected group than in the control group. This result demonstrated that the increased NKG2D expression and imbalance between inhibitory receptors of dNK cells and HLA-G may contribute to abnormal pregnancy outcomes observed upon maternal infection with T. gondii.

PMID: 23393578 [PubMed - in process]

Friday, February 08, 2013

Sexual transmission of Toxoplasma gondii in sheep

Vet Parasitol. 2013 Jan 9. pii: S0304-4017(13)00008-3. doi: 10.1016/j.vetpar.2012.12.056. [Epub ahead of print]

Sexual transmission of Toxoplasma gondii in sheep

Lopes WD, Rodriguez JD, Souza FA, Dos Santos TR, Dos Santos RS, Rosanese WM, Lopes WR, Sakamoto CA, da Costa AJ.

Faculdade de Ciências Agrárias e Veterinárias, UNESP/CPPAR, Via de acesso prof. Paulo Donatto Castellani, s/n CEP: 14884-900, Jaboticabal, São Paulo, Brazil.

Male sheep of reproductive age were distributed into three groups: GI, a sheep inoculated (oral) with 2.0×10(5) oocysts of the P strain of Toxoplasma gondii; GII, a sheep infected (subcutaneous) with 1.0×10(6) tachyzoites of the RH strain of T. gondii; and GIII, a sheep kept as a control (not infected). After the inoculation of the males, 12 breeding ewes, which were not pregnant and which were serologically negative for reproductive diseases (particularly toxoplasmosis), were distributed into three groups, synchronized, and subsequently exposed to natural mating with previously inoculated males. The distribution was as follows: five ewes that underwent natural mating with the GI male, five ewes that were exposed to natural mating with the GII male, and two ewes that were mated with the non-infected male (control). Serum samples of all the ewes were collected on days -30, -14, -7, -1, and 0 (days before natural mating) and on days 1, 3, 5, 7, 11, 14, and weekly until birth; the presence of serum antibodies against T. gondii was assessed by IFAT. Using a bioassay and PCR, T. gondii was isolated from the semen of the infected reproducing sheep before mating. Following natural mating, 5 of the 12 females displayed antibodies specific for T. gondii; of these animals, two of the ewes underwent natural mating with the male inoculated with oocysts (GI) and three with the male infected with tachyzoites (GII). One of the females that displayed antibodies specific to this coccidian and that underwent natural mating with the GII sheep had a macerated fetus on the 70th day following coverage. Using a bioassay after the birth, it was possible to isolate T. gondii from samples of the "pool" of tissues from the five females that seroconverted after natural mating and from their respective lambs. Using PCR, the DNA of T. gondii was isolated from the "pool" of tissues from one and two females exposed to natural mating with the reproductive males infected with the oocysts and tachyzoites, respectively. Using this technique, it was also possible to diagnose the presence of the parasite in the "pool" of tissues from the lambs of one female that underwent natural mating with the male sheep infected with oocysts. These results demonstrated the sexual transmission of T. gondii in the sheep species with consequent vertical transmission to their lambs.

 PMID: 23384578 [PubMed - as supplied by publisher]

Wednesday, February 06, 2013

Inhibitors of eIF2α dephosphorylation slow replication and stabilize latency in Toxoplasma gondii

Antimicrob Agents Chemother. 2013 Feb 4. [Epub ahead of print]

Inhibitors of eIF2α dephosphorylation slow replication and stabilize latency in Toxoplasma gondii

Konrad C, Queener SF, Wek RC, Sullivan WJ Jr.

Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Toxoplasma gondii is an obligate intracellular parasite that permanently infects warm-blooded vertebrates through its ability to convert into a latent tissue cyst form. The latent form (bradyzoite) can reinitiate a life-threatening acute infection if host immunity wanes, most commonly in AIDS or organ transplant patients. We have previously shown that bradyzoite development is accompanied by phosphorylation of the parasite eukaryotic initiation factor-2 alpha subunit (eIF2α), which dampens global protein synthesis and reprograms gene expression. In this study, we analyzed the activities of two specific inhibitors of eIF2α dephosphorylation, salubrinal (SAL) and guanabenz (GA). We establish that these drugs are able to inhibit the dephosphorylation of Toxoplasma eIF2α. Our results show that SAL and GA reduce tachyzoite replication in vitro and in vivo. Furthermore, both drugs induce bradyzoite formation and inhibit the reactivation of latent bradyzoites in vitro. To address whether the antiparasitic activities of SAL and GA involve host eIF2α phosphorylation, we infected mutant MEF cells incapable of phosphorylating eIF2α, which had no impact on the efficacy of SAL and GA against Toxoplasma infection. Our findings suggest that SAL and GA may serve as potential new drugs for the treatment of acute and chronic toxoplasmosis.

PMID: 23380722 [PubMed - as supplied by publisher]

Prevalence of active convulsive epilepsy in sub-Saharan Africa and associated risk factors: cross-sectional and case-control studies

Lancet Neurol. 2013 Jan 30. pii: S1474-4422(13)70003-6. doi: 10.1016/S1474-4422(13)70003-6. [Epub ahead of print]

Prevalence of active convulsive epilepsy in sub-Saharan Africa and associated risk factors: cross-sectional and case-control studies

Ngugi AK, Bottomley C, Kleinschmidt I, Wagner RG, Kakooza-Mwesige A, Ae-Ngibise K, Owusu-Agyei S, Masanja H, Kamuyu G, Odhiambo R, Chengo E, Sander JW, Newton CR; for the SEEDS group.

Studies of Epidemiology of Epilepsy in Demographic Surveillance Systems, International Network for the Demographic Evaluation of Populations and Their Health (INDEPTH), Accra, Ghana; Kenya Medical Research Institute/Wellcome Trust Research Programme, Centre for Geographic Medicine Research-Coast, Kilifi, Kenya; Department of Infectious Disease Epidemiology, London School of Hygiene and Tropical Medicine, London, UK. Electronic address:

The prevalence of epilepsy in sub-Saharan Africa seems to be higher than in other parts of the world, but estimates vary substantially for unknown reasons. We assessed the prevalence and risk factors of active convulsive epilepsy across five centres in this region.
We did large population-based cross-sectional and case-control studies in five Health and Demographic Surveillance System centres: Kilifi, Kenya (Dec 3, 2007-July 31, 2008); Agincourt, South Africa (Aug 4, 2008-Feb 27, 2009); Iganga-Mayuge, Uganda (Feb 2, 2009-Oct 30, 2009); Ifakara, Tanzania (May 4, 2009-Dec 31, 2009); and Kintampo, Ghana (Aug 2, 2010-April 29, 2011). We used a three-stage screening process to identify people with active convulsive epilepsy. Prevalence was estimated as the ratio of confirmed cases to the population screened and was adjusted for sensitivity and attrition between stages. For each case, an age-matched control individual was randomly selected from the relevant centre's census database. Fieldworkers masked to the status of the person they were interviewing administered questionnaires to individuals with active convulsive epilepsy and control individuals to assess sociodemographic variables and historical risk factors (perinatal events, head injuries, and diet). Blood samples were taken from a randomly selected subgroup of 300 participants with epilepsy and 300 control individuals from each centre and were screened for antibodies to Toxocara canis, Toxoplasma gondii, Onchocerca volvulus, Plasmodium falciparum, Taenia solium, and HIV. We estimated odds ratios (ORs) with logistic regression, adjusted for age, sex, education, employment, and marital status.
The prevalence of active convulsive epilepsy varies in sub-Saharan Africa and that the variation is probably a result of differences in risk factors. Programmes to control parasitic diseases and interventions to improve antenatal and perinatal care could substantially reduce the prevalence of epilepsy in this region.

PMID: 23375964 [PubMed - as supplied by publisher]

Characterization of a second sterol-esterifying enzyme in Toxoplasma highlights the importance of cholesterol storage pathways for the parasite

Mol Microbiol. 2013 Feb 3. doi: 10.1111/mmi.12142. [Epub ahead of print]

Characterization of a second sterol-esterifying enzyme in Toxoplasma highlights the importance of cholesterol storage pathways for the parasite

Lige B, Sampels V, Coppens I.

Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, 21205, USA.

Lipid bodies are eukaryotic structures for temporary storage of neutral lipids such as acylglycerols and steryl esters. Fatty acyl-CoA and cholesterol are two substrates for cholesteryl ester (CE) synthesis via the ACAT reaction. The intracellular parasite Toxoplasma gondii is incapable of sterol synthesis and unremittingly scavenges cholesterol from mammalian host cells. We previously demonstrated that the parasite expresses a cholesteryl ester-synthesizing enzyme, TgACAT1. In this article, we identified and characterized a second ACAT-like enzyme, TgACAT2, which shares 56% identity with TgACAT1. Both enzymes are endoplasmic reticulum-associated and contribute to CE formation for storage in lipid bodies. While TgACAT1 preferentially utilizes palmitoyl-CoA, TgACAT2 has broader fatty acid specificity and produces more CE. Genetic ablation of each individual ACAT results in parasite growth impairment whereas dual ablation of ACAT1 and ACAT2 is not tolerated by Toxoplasma. ΔACAT1 and ΔACAT2 parasites have reduced CE levels, fewer lipid bodies, and accumulate free cholesterol, which causes injurious membrane effects. Mutant parasites are particularly vulnerable to ACAT inhibitors. This study underlines the important physiological role of ACAT enzymes to store cholesterol in a sterol-auxotrophic organism such as Toxoplasma, and furthermore opens up possibilities of exploiting TgACAT as targets for the development of antitoxoplasmosis drugs.

 PMID: 23374239 [PubMed - as supplied by publisher]

MyD88 is crucial for the development of a protective CNS immune response to Toxoplasma gondii infection

J Neuroinflammation. 2013 Feb 1;10(1):19. [Epub ahead of print]

MyD88 is crucial for the development of a protective CNS immune response to Toxoplasma gondii infection

Torres M, Guiton R, Lacroix-Lamandé S, Ryffel B, Leman S, Dimier-Poisson I.

BACKGROUND: Toxoplasmosis is one of the most common parasitic infections in humans. It can establish chronic infection and is characterized by the formation of tissue cysts in the brain. The cysts remain largely quiescent for the life of the host, but can reactivate and cause life-threatening toxoplasmic encephalitis in immunocompromised patients, such as those with AIDS, neoplastic diseases and organ transplants. Toll-like receptor (TLR) adaptor MyD88 activation is required for the innate sensing of Toxoplasma gondii. Mice deficient in MyD88 have defective IL-12 and Th1 effector responses, and are highly susceptible to the acute phase of T. gondii infection. However, the role of this signaling pathway during cerebral infection is poorly understood and requires examination.
MyD88-deficient mice and control mice were orally infected with T. gondii cysts. Cellular and parasite infiltration in the peripheral organs and in the brain were determined by histology and immunohistochemistry. Cytokine levels were determined by ELISA and chemokine mRNA levels were quantified by real-time PCR (qPCR).
Thirteen days after infection, a higher parasite burden was observed but there was no histological change in the liver, heart, lungs and small intestine of MyD88-/- and MyD88+/+ mice. However, MyD88-/- mice compared to MyD88+/+ mice were highly susceptible to cerebral infection, displayed high parasite migration to the brain, severe neuropathological signs of encephalitis and succumbed within 2 weeks of oral infection. Susceptibility was primarily associated with lower expression of Th1 cytokines, especially IL-12, IFN-gamma and TNF-alpha, significant decrease in the expression of CCL3, CCL5, CCL7 and CCL19 chemokines, marked defect of CD8+ T cells, and infiltration of CD11b+ and F4/80+ cells in the brain.
MyD88 is essential for the protection of mice during the cerebral installation of T. gondii infection. These results establish a role for MyD88 in T cell-mediated control of T. gondii in the central nervous system (CNS).

 PMID: 23374751 [PubMed - as supplied by publisher]

Friday, February 01, 2013

Cover story: Non-canonical maturation of two papain-family proteases in Toxoplasma gondii

J Biol Chem. 2012 Dec 18. [Epub ahead of print]

Non-canonical maturation of two papain-family proteases in Toxoplasma gondii

Dou Z, Coppens I, Carruthers VB

University of Michigan, United States

Proteases regulate key events during infection by the pervasive intracellular parasite Toxoplasma gondii. Understanding how parasite proteases mature from an inactive zymogen to an active enzyme is expected to inform new strategies for blocking their actions. Herein, we show that T. gondii cathepsin B protease (TgCPB) does not undergo self-maturation, but instead requires the expression of a second papain-family cathepsin protease, TgCPL. Using recombinant enzymes we also show that TgCPL is capable of partially maturing TgCPB in vitro. Consistent with this interrelationship, antibodies with validated specificity detected TgCPB in the lysosome-like vacuolar compartment along with TgCPL. Our findings also establish that TgCPB does not localize to the rhoptries as previously reported. Accordingly, rhoptry morphology and rhoptry protein maturation are normal in TgCPB knockout parasites. Finally, we show that while maturation of TgCPL is independent of TgCPB, it may involve an additional protease(s) in conjunction with self-maturation.

PMID: 23250753 [PubMed - as supplied by publisher]