Friday, January 27, 2012

Impaired Chromatin Remodelling at STAT1-Regulated Promoters Leads to Global Unresponsiveness of Toxoplasma gondii-Infected Macrophages to IFN-γ

PLoS Pathog. 2012 Jan;8(1):e1002483. Epub 2012 Jan 19.

Impaired Chromatin Remodelling at STAT1-Regulated Promoters Leads to Global Unresponsiveness of Toxoplasma gondii-Infected Macrophages to IFN-γ.

Lang C, Hildebrandt A, Brand F, Opitz L, Dihazi H, Lüder CG.

Institute for Medical Microbiology, University Medical Center, Georg-August-University, Göttingen, Germany.

Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites' replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors.

PMID: 22275866 [PubMed - in process]

Proteomic analysis of fractionated toxoplasma oocysts reveals clues to their environmental resistance

PLoS One. 2012;7(1):e29955. Epub 2012 Jan 18.

Proteomic analysis of fractionated toxoplasma oocysts reveals clues to their environmental resistance.

Fritz HM, Bowyer PW, Bogyo M, Conrad PA, Boothroyd JC.

Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California Davis, Davis, California, United States of America.

Toxoplasma gondii is an obligate intracellular parasite that is unique in its ability to infect a broad range of birds and mammals, including humans, leading to an extremely high worldwide prevalence and distribution. This work focuses on the environmentally resistant oocyst, which is the product of sexual replication in felids and an important source of human infection. Due to the difficulty in producing and working with oocysts, relatively little is known about how this stage is able to resist extreme environmental stresses and how they initiate a new infection, once ingested. To fill this gap, the proteome of the wall and sporocyst/sporozoite fractions of mature, sporulated oocysts were characterized using one-dimensional gel electrophoresis followed by LC-MS/MS on trypsin-digested peptides. A combined total of 1021 non-redundant T. gondii proteins were identified in the sporocyst/sporozoite fraction and 226 were identified in the oocyst wall fraction. Significantly, 172 of the identified proteins have not previously been identified in Toxoplasma proteomic studies. Among these are several of interest for their likely role in conferring environmental resistance including a family of small, tyrosine-rich proteins present in the oocyst wall fractions and late embryogenesis abundant domain-containing (LEA) proteins in the cytosolic fractions. The latter are known from other systems to be key to enabling survival against desiccation.

PMID: 22279555 [PubMed - in process]

A Novel PAN/Apple Domain-Containing Protein from Toxoplasma gondii: Characterization and Receptor Identification

PLoS One. 2012;7(1):e30169. Epub 2012 Jan 19.

A Novel PAN/Apple Domain-Containing Protein from Toxoplasma gondii: Characterization and Receptor Identification.

Gong H, Kobayashi K, Sugi T, Takemae H, Kurokawa H, Horimoto T, Akashi H, Kato K.

Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan.

Toxoplasma gondii is an intracellular parasite that invades nucleated cells, causing toxoplasmosis in humans and animals worldwide. The extremely wide range of hosts susceptible to T. gondii is thought to be the result of interactions between T. gondii ligands and receptors on its target cells. In this study, a host cell-binding protein from T. gondii was characterized, and one of its receptors was identified. P104 (GenBank Access. No. CAJ20677) is 991 amino acids in length, containing a putative 26 amino acid signal peptide and 10 PAN/apple domains, and shows low homology to other identified PAN/apple domain-containing molecules. A 104-kDa host cell-binding protein was detected in the T. gondii lysate. Immunofluorescence assays detected P104 at the apical end of extracellular T. gondii. An Fc-fusion protein of the P104 N-terminus, which contains two PAN/apple domains, showed strong affinity for the mammalian and insect cells evaluated. This binding was not related to protein-protein or protein-lipid interactions, but to a protein-glycosaminoglycan (GAG) interaction. Chondroitin sulfate (CS), a kind of GAG, was shown to be involved in adhesion of the Fc-P104 N-terminus fusion protein to host cells. These results suggest that P104, expressed at the apical end of the extracellular parasite, may function as a ligand in the attachment of T. gondii to CS or other receptors on the host cell, facilitating invasion by the parasite.

PMID: 22276154 [PubMed - in process]

Wednesday, January 25, 2012

High tissue burden of Toxoplasma gondii is the hallmark of acute virulence in mice

Vet Parasitol. 2012 Jan 8. [Epub ahead of print]

High tissue burden of Toxoplasma gondii is the hallmark of acute virulence in mice.

Hill RD, Su C.

Department of Microbiology, University of Tennessee, Knoxville, TN 37996, United States.

Toxoplasma gondii virulence is commonly determined by mortality rate of infected mice. Limited data showed that virulent T. gondii strains had increased parasite growth in mice compared to that of less virulent strains. To determine if this is a common phenomenon for a variety of strains and to develop an alternative assay to test acute virulence in mice, we measured parasite burdens in experimentally infected outbred CD-1 mice for 19 T. gondii isolates, in which the virulence phenotypes had previously been determined by mortality assay. Our results showed that parasite concentrations in spleen tissues were two orders of magnitude higher in the virulent than the intermediately and non-virulent isolates at day 7 post infection. In competition assays, mice inoculated with mixed tachyzoites of virulent and intermediately virulent strains or virulent and non-virulent strains showed that the former always reached a higher concentration at day 7 post infection. In mixed infection of intermediate and non-virulent strains, both strains were detectable in mice at day 7 post infection. In conclusion, our data showed that the virulence of T. gondii can be predicted by parasite load in the spleen tissue of infected mice at 7 days post infection, providing an alternative method to determine virulence of Toxoplasma.

Copyright © 2012 Elsevier B.V. All rights reserved.

PMID: 22270034 [PubMed - as supplied by publisher]

Significant reduction of Toxoplasma gondii brain cysts after treatment with spiramycin coadministered with metronidazole in a mouse model

Antimicrob Agents Chemother. 2012 Jan 23. [Epub ahead of print]

Significant reduction of Toxoplasma gondii brain cysts after treatment with spiramycin coadministered with metronidazole in a mouse model of chronic toxoplasmosis.

Chew WK, Segarra I, Ambu S, Mak JW.

Dep. of Human Biology, School of Medicine, International Medical University; No 126 Jalan 19/155B, Bukit Jalil, 57000 Kuala Lumpur; Malaysia.

Toxoplasma gondii is a parasite that generates latent cysts in brain which reactivation may lead to fatal toxoplasmic encephalitis for which treatment remains unsuccessful. We assessed spiramycin pharmacokinetics coadministered with metronidazole, the eradication of brain cysts and the in vitro reactivation. Male Balb/c mice were fed 1000 tachyzoites orally to develop chronic toxoplasmosis. Four weeks later infected mice underwent different treatments: infected untreated group (n=9) which received vehicle, spiramycin only group (n=9), 400 mg/kg daily for 7 days, metronidazole only group (n=9) 500 mg/kg daily for 7 days, and combination group (n=9) which received spiramycin (400 mg/kg) and metronidazole (500 mg/kg) daily for 7 days. Uninfected control group (n=10) was administered vehicle only. After treatment, the brain cysts were counted, brain homogenate cultured in confluent Vero cells and cysts and tachyzoites counted after 1 week. Separately, pharmacokinetic profiles (plasma and brain) were assessed after a single dose of spiramycin (400 mg/kg) or metronidazole (500 mg/kg) or both. Metronidazole increased 67% brain spiramycin AUC(0-∞) without affecting its plasma disposition. Metronidazole plasma and brain AUC(0-∞) were reduced 9% and 62% respectively after spiramycin coadministration. Enhanced spiramycin brain exposure after coadministration reduced 15-fold brain cysts (79±23 combination treatment versus 1198±153 untreated control group, p<0.05), and 10-fold versus spiramycin only group (768±125). Metronidazole alone showed no effect (1028±149). Tachyzoites were absent in brain. Spiramycin reduced in vitro reactivation. Metronidazole increased spiramycin brain penetration causing a significant reduction of T. gondii brain cysts with potential clinical translatability for chronic toxoplasmosis treatment.

PMID: 22271863 [PubMed - as supplied by publisher]

Friday, January 20, 2012

Virulence without catalysis: how can a pseudokinase affect host cell signaling?

Trends Parasitol. 2012 Jan 16. [Epub ahead of print]

Virulence without catalysis: how can a pseudokinase affect host cell signaling?

Reese ML, Boyle JP.

Stanford University, Department of Microbiology and Immunology, 299 Campus Drive, Stanford, CA 94305-5124, USA.

A hallmark of the pathogenic lifestyle is the secretion of enzymes and other effectors that dysregulate host signaling. Intriguingly, the most potent virulence locus identified in the intracellular parasite Toxoplasma gondii encodes a family of related catalytically inactive protein kinases, or pseudokinases. Toxoplasma has in its kinome among the highest percentage of pseudokinases among all sequenced organisms, and the majority of these appear to be secreted into the host cell. We posit that the pseudokinase fold represents a particularly well-suited domain for functional diversification, discuss the relevance of gene expansion at these loci, and outline potential mechanisms by which a pseudokinase might affect host signaling.

Copyright © 2012. Published by Elsevier Ltd.

PMID: 22257555 [PubMed - as supplied by publisher]

Wednesday, January 18, 2012

A DOC2 protein identified by mutational profiling is essential for apicomplexan parasite exocytosis

Science. 2012 Jan 13;335(6065):218-21.

A DOC2 protein identified by mutational profiling is essential for apicomplexan parasite exocytosis.

Farrell A, Thirugnanam S, Lorestani A, Dvorin JD, Eidell KP, Ferguson DJ, Anderson-White BR, Duraisingh MT, Marth GT, Gubbels MJ.

Department of Biology, Boston College, Chestnut Hill, MA 02467, USA.

Exocytosis is essential to the lytic cycle of apicomplexan parasites and required for the pathogenesis of toxoplasmosis and malaria. DOC2 proteins recruit the membrane fusion machinery required for exocytosis in a Ca(2+)-dependent fashion. Here, the phenotype of a Toxoplasma gondii conditional mutant impaired in host cell invasion and egress was pinpointed to a defect in secretion of the micronemes, an apicomplexan-specific organelle that contains adhesion proteins. Whole-genome sequencing identified the etiological point mutation in TgDOC2.1. A conditional allele of the orthologous gene engineered into Plasmodium falciparum was also defective in microneme secretion. However, the major effect was on invasion, suggesting that microneme secretion is dispensable for Plasmodium egress.

PMID: 22246776 [PubMed - in process]

Toxoplasma gondii: Protective immunity against toxoplasmosis with recombinant actin depolymerizing factor protein in BALB/c mice

Exp Parasitol. 2012 Jan 10. [Epub ahead of print]

Toxoplasma gondii: Protective immunity against toxoplasmosis with recombinant actin depolymerizing factor protein in BALB/c mice.

Huang X, Li J, Zhang G, Gong P, Yang J, Zhang X.

College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi'an Road, Changchun 130062, China.

Toxoplasmosis is one of the most world-wide spread zoonosis representing a very serious clinical and veterinary problem. There is still need for vaccines for toxoplasmosis. In the present study, we evaluated the protective efficacy of a recombinant actin depolymerizing factor (ADF) subunit vaccine against Toxoplasma gondii infection in BALB/c mice. The recombinant T. gondii ADF protein (rADF) was expressed in Escherichia coli and used as antigens for BALB/c mice immunization. The results indicated that specific antibody and the increased percentage of CD4(+) T lymphocyte were found in vaccinated BALB/c mice with rADF, when compared with adjuvant or PBS groups. After challenged with T. gondii (RH strain) tachyzoites, the survival time of the mice in rADF group was longer than those in the control group. The numbers of brain cysts of the mice in rADF group reduced significantly when compared with those in control groups (P<0.05), and the rate of reduction could reach to around 30%. These results suggest that rADF can generate protective immunity against T. gondii infection in BALB/c mice.

Copyright © 2012. Published by Elsevier Inc.

PMID: 22248986 [PubMed - as supplied by publisher]

Saturday, January 14, 2012

Sex-specific changes in gene expression and behavior induced by chronic Toxoplasma infection in mice

Neuroscience. 2012 Jan 3. [Epub ahead of print]

Sex-specific changes in gene expression and behavior induced by chronic Toxoplasma infection in mice.

Xiao J, Kannan G, Jones-Brando L, Brannock C, Krasnova IN, Cadet JL, Pletnikov M, Yolken RH.

Stanley Division of Developmental Neurovirology, Department of Pediatrics, Johns Hopkins School of Medicine, Baltimore, MD 21287, USA.

There is growing evidence that Toxoplasma gondii modifies the behavior of its intermediate hosts. We investigated the molecular basis of these infection-induced behavioral changes, followed by five related behavioral tests to assess the extent of biological relevance. Gene expression signatures were generated in the frontal cortex of male and female mice during the latent stage of infection. We found marked sex-dependent expression differences in mice. In female mice, Toxoplasma infection altered the expression of genes involved in the development of the forebrain, neurogenesis, and sensory and motor coordination (i.e. downregulation of fatty acid-binding protein 7 and eyes absent homolog 1, upregulation of semaphorin 7A). In male mice, infection led mainly to modulation of genes associated with olfactory function (i.e. downregulation of a number of olfactory receptors and dopamine receptor D4, upregulation of slit homolog 1). Although infection appears to affect the olfactory function in male mice, it is the female but not male mice that exhibited attraction to cat odor. In contrast, infected male mice showed a deficit in social transmission of food preference. In contrast to males, infected females displayed locomotor hyperactivity in open field. General olfaction and sensorimotor gating were normal in both male and female infection. Our results indicate that the sex of the host plays a major role in determining variable brain and behavior changes following Toxoplasma infection. These observations are consistent with heterogeneity of neuropsychiatric outcomes of the infection in humans.

Copyright © 2012. Published by Elsevier Ltd.

PMID: 22240252 [PubMed - as supplied by publisher]

Monday, January 09, 2012

Behavioral changes in mice caused by Toxoplasma gondii invasion of brain

Parasitol Res. 2012 Jan 6. [Epub ahead of print]

Behavioral changes in mice caused by Toxoplasma gondii invasion of brain.

Gatkowska J, Wieczorek M, Dziadek B, Dzitko K, Dlugonska H.

Department of Immunoparasitology, University of Lodz, Banacha 12/16, 90-237, Lodz, Poland,

Toxoplasma gondii, a protozoan parasite, is capable of infecting a broad range of intermediate warm-blooded hosts including humans. The parasite undergoes sexual reproduction resulting in genetic variability only in the intestine of the definitive host (a member of the cat family). The parasite seems to be capable of altering the natural behavior of the host to favor its transmission in the environment. The aim of this study was to evaluate the number of parasite cysts formed in the hippocampus and amygdala of experimentally infected mice as these regions are involved in defense behaviors control and emotion processing, and to assess the influence of the infection on mice behavior. The obtained results revealed the presence of parasite cysts both in the hippocampus and the amygdala of infected mice; however, no clear region-dependent distribution was observed. Furthermore, infected mice showed significantly diminished exploratory activity described by climbing and rearing, smaller preference for the central, more exposed part of the OF arena and engaged in less grooming behavior compared to uninfected controls.

PMID: 22223035 [PubMed - as supplied by publisher]

Thursday, January 05, 2012

A Single Chromosome Unexpectedly Links Highly Divergent Isolates of Toxoplasma gondii

MBio. 2012 Jan 3;3(1). pii: e00284-11. doi: 10.1128/mBio.00284-11. Print 2012.

A Single Chromosome Unexpectedly Links Highly Divergent Isolates of Toxoplasma gondii.

Walzer KA, Boyle JP.

Department of Biological Sciences, Kenneth P. Dietrich School of Arts and Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

ABSTRACT Toxoplasma gondii is an obligate intracellular parasite that can cause disease in all warm-blooded animals studied to date, including humans. Over a billion people have been infected with this parasite worldwide. In Europe and North America, Toxoplasma has a clonal population structure, where only three lineages are highly dominant (strain types I, II, and III). Khan et al. [mBio 2(6): e00228-11, 2011] have carried out phylogenetic analyses on a large number of diverse strains from outside of these lineages and found evidence for a significant split between the clonal North American/European lineages and those in South America. In contrast to most of the genome, nearly all North American/European strains sampled, and the majority of South American strains sampled, harbored at least portions of a monomorphic chromosome Ia (Ia*). In contrast to previous models, these data suggest that the monomorphic haplotype originated in South America and migrated to the North. These authors propose that South American haplotype 12 was a precursor to modern-day type II, while South American haplotypes 6 and 9 crossed with haplotype 12 to give rise to the type I and III lineages, respectively. However, the findings reported by Khan et al. complicate the origin of chromosome Ia, since there are members of haplotypes 9 and 12 with nearly complete versions of Ia* and members of haplotypes 6 and 12 with over 50% of Ia*. This unexpected finding raises exciting new questions about how an entire common chromosome can be found within strains that are highly divergent at most other genomic loci.

PMID: 22215568 [PubMed - in process]

1-(Fluoroalkylidene)-1,1-bisphosphonic acids are potent and selective inhibitors of Toxoplasma farnesyl pyrophosphate synthase

Org Biomol Chem. 2012 Jan 3. [Epub ahead of print]

1-(Fluoroalkylidene)-1,1-bisphosphonic acids are potent and selective inhibitors of the enzymatic activity of Toxoplasma gondii farnesyl pyrophosphate synthase.

Szajnman SH, Rosso VS, Malayil L, Smith A, Moreno SN, Docampo R, Rodriguez JB.

Departamento de Química Orgánica and UMYMFOR (CONICET-FCEyN), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, C1428EHA, Buenos Aires, Argentina.

α-Fluorinated-1,1-bisphosphonic acids derived from fatty acids were designed, synthesized and biologically evaluated against Trypanosoma cruzi, the etiologic agent of Chagas disease, and against Toxoplasma gondii, the agent responsible for toxoplasmosis, and also towards the target parasitic enzymes farnesyl pyrophosphate synthase of T. cruzi (TcFPPS) and T. gondii (TgFPPS). Interestingly, 1-fluorononylidene-1,1-bisphosphonic acid (compound 43) proved to be an extremely potent inhibitor of the enzymatic activity of TgFPPS at the low nanomolar range, exhibiting an IC(50) of 30 nM. This compound was two-fold more potent than risedronate (IC(50) = 74 nM) that was taken as a positive control. This enzymatic activity was associated with a strong cell growth inhibition against tachyzoites of T. gondii, with an IC(50) value of 2.7 μM.

PMID: 22215028 [PubMed - as supplied by publisher]

Foxp3(+) Regulatory T cells, Immune Stimulation and Host Defense against Infection

Immunology. 2011 Dec 29. doi: 10.1111/j.1365-2567.2011.03551.x. [Epub ahead of print]

Foxp3(+) Regulatory T cells, Immune Stimulation and Host Defense against Infection.

Rowe JH, Ertelt JM, Way SS.

Departments of Pediatrics and Microbiology University of Minnesota School of Medicine Center for Infectious Disease and Microbiology Translational Research.

The immune system is intricately regulated allowing potent effectors to expand and become rapidly mobilized after infection, while simultaneously silencing potentially detrimental responses that averts immune-mediated damage to host tissues. This relies in large part on the delicate interplay between immune suppressive regulatory CD4(+) T cells (Tregs) and immune effectors that without active suppression by Tregs cause systemic and organ-specific autoimmunity. Although these beneficial roles have been classically described to be counter-balanced by impaired host defense against infection, newfound protective roles for Tregs against specific viral pathogens (e.g. Herpes simplex-2, Lymphocytic choriomeningitis, West Nile virus) have been uncovered using transgenic mice that allow in vivo Treg-ablation based on Foxp3-expression. In turn, Foxp3(+) Tregs also provide protection against some parasitic (Plasmodium sp., Toxoplasma gondii) and fungal (Candida albicans) pathogens. By contrast for bacterial and mycobacterial infections (e.g. Listeria monocytogenes, Salmonella enterica, Mycobacterium tuberculosis), experimental manipulation of Foxp3(+) cells continue to indicate detrimental roles for Tregs in host defense. This discordance is likely related to functional plasticity in Treg suppression that shifts discordantly following infection with different types of pathogens. Furthermore, the efficiency whereby Tregs silence immune activation coupled with the plasticity in Foxp3(+) cell activity suggest overriding Treg-mediated suppression represents a prerequisite "signal zero" that together with other stimulation signals (T cell receptor [signal 1], co-stimulation [signal 2], inflammatory cytokines [signal 3]) are essential for T cell activation in vivo. Herein, the importance of Foxp3(+) Tregs in host defense against infection, and the significance of infection-induced shifts Treg suppression are summarized.

Journal compilation © 2011 Blackwell Publishing Ltd.

PMID: 22211994 [PubMed - as supplied by publisher]

Autophagy is a cell death mechanism in Toxoplasma gondii

Cell Microbiol. 2011 Dec 30. doi: 10.1111/j.1462-5822.2011.01745.x. [Epub ahead of print]

Autophagy is a cell death mechanism in Toxoplasma gondii.

Ghosh D, Walton JL, Roepe PD, Sinai AP.


Department of Microbiology, Immunology and Molecular Genetics; University of Kentucky College of Medicine, Lexington KY 40536, USA. Departments of Chemistry, Biochemistry and Cellular and Molecular Biology, Georgetown University, Washington DC. 20057, USA.

Nutrient sensing and the capacity to respond to starvation is tightly regulated as a means of cell survival. Among the features of the starvation response are induction of both translational repression and autophagy. Despite the fact that intracellular parasite like Toxoplasma gondii within a host cell predicted to be nutrient rich, they encode genes involved both in translational repression and autophagy. We therefore examined the consequence of starvation, a classic trigger of autophagy, on intracellular parasites. As expected, starvation results in the activation of the translational repression system as evidenced by elevation of phosphorylated TgIF2α (TgIF2α-P). Surprisingly, we also observe a rapid and selective fragmentation of the single parasite mitochondrion that leads irreversibly to parasite death. This profound effect was dependent primarily on the limitation of amino acids and involved signaling by the parasite TOR homolog. Notably, the effective blockade of mitochondrial fragmentation by the autophagy inhibitor 3-methyl adenine (3-MA) suggests an autophagic mechanism. In the absence of a documented apoptotic cascade in T. gondii, the data suggest that autophagy is the primary mechanism of programmed cell death in T. gondii and potentially other related parasites.

© 2011 Blackwell Publishing Ltd.

PMID: 22212386 [PubMed - as supplied by publisher]

Wednesday, January 04, 2012

Molecular characterization of Toxoplasma gondii formin 3, an actin nucleator dispensable for tachyzoite growth and motility

Eukaryot Cell. 2011 Dec 30. [Epub ahead of print]

Molecular characterization of Toxoplasma gondii formin 3, an actin nucleator dispensable for tachyzoite growth and motility.

Daher W, Klages N, Carlier MF, Soldati-Favre D.

Department of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland.

Toxoplasma gondii belongs to the phylum of Apicomplexa, a group of obligate intracellular parasites that relies on gliding motility to enter host cells. Drugs interfering with the actin cytoskeleton block parasite motility, host cell invasion and egress from infected cells. Myosin A, profilin, formin 1, formin 2 and actin depolymerizing factor have all been implicated in parasite motility, yet little is known regarding the importance of actin polymerization and other myosins for the remaining steps of the parasite lytic cycle. Here we establish that T. gondii formin 3 (TgFRM3), a newly described Formin Homology 2 domain (FH2) containing protein, binds to Toxoplasma actin and nucleates rabbit actin assembly in vitro. TgFRM3 expressed as a transgene exhibits a patchy localization at several distinct structures within the parasite. Disruption of the TgFRM3 gene by double homologous recombination in ku80-ko strain reveals no vital function for tachyzoite propagation in vitro, which is consistent with its weak level of expression in this life stage. Conditional stabilization of truncated forms of TgFRM3 suggests that different regions of the molecule contribute to distinct localizations. Moreover, expression of TgFRM3 lacking the C-terminal domain severely impacts on parasite growth and replication. This work provides a first insight into how this specialized formin, restricted to the group of Coccidia, completes its actin nucleating activity.

PMID: 22210829 [PubMed - as supplied by publisher]

Toxoplasma gondii Sis1-like J-domain protein is a cytosolic chaperone associated to HSP90/HSP70 complex

Int J Biol Macromol. 2011 Dec 23. [Epub ahead of print]

Toxoplasma gondii Sis1-like J-domain protein is a cytosolic chaperone associated to HSP90/HSP70 complex.

Figueras MJ, Martin OA, Echeverria PC, de Miguel N, Naguleswaran A, Sullivan WJ Jr, Corvi MM, Angel SO.

Laboratorio de Parasitología Molecular, IIB-INTECH, CONICET-UNSAM, Av. Intendente Marino Km. 8.2, C.C 164 (B7130IIWA), Chascomús, Prov. Buenos Aires, Argentina.

Toxoplasma gondii is an obligate intracellular protozoan parasite in which 36 predicted Hsp40 family members were identified by searching the T. gondii genome. The predicted protein sequence from the gene ID TGME49_065310 showed an amino acid sequence and domain structure similar to Saccharomyces cerevisiae Sis1. TgSis1 did not show differences in its expression profile during alkaline stress by microarray analysis. Furthermore, TgSis1 showed to be a cytosolic Hsp40 which co-immunoprecipitated with T. gondii Hsp70 and Hsp90. Structural modeling of the TgSis1 peptide binding fragment revealed structural and electrostatic properties different from the experimental model of human Sis1-like protein (Hdj1). Based on these differences; we propose that TgSis1 may be a potentially attractive drug target for developing a novel anti-T. gondii therapy.

Copyright © 2011. Published by Elsevier B.V.

PMID: 22209934 [PubMed - as supplied by publisher]

Toxoplasma gondii infection causes morphological changes in caecal myenteric neurons

Exp Parasitol. 2011 Dec 22. [Epub ahead of print]

Toxoplasma gondii infection causes morphological changes in caecal myenteric neurons.

Zaniolo LM, da Silva AV, Sant'ana DD, Araújo EJ.

Mestrado em Ciência Animal, Universidade Paranaense (UNIPAR), Umuarama, Paraná, Brazil.

The aim of this study was to evaluate the effects of chronic infection of Toxoplasma gondii (with genotype I and genotype III strains) on the population density and morphometry of caecum myenteric neurons in rats. Fifteen, 60-day-old, male Wistar rats (Rattus norvegicus) were used. The animals were assigned into three groups: Control Group (CG), Experimental Group 1 (EG1) and Experimental Group 2 (EG2). EG1 animals received 10(5) tachyzoites of the genotype I (BTU IV) T. gondii strain orally, and the EG2 animals received 10(5) tachyzoites of the genotype III (BTU II) strain orally. Thirty days after inoculation, caecal whole-mount preparations were stained by Giemsa technique. The caecal preparations were then analysed by assessing the population density and morphometry of myenteric neurons in specific regions of the caecum: mesenteric apical (MA), antimesenteric apical (AA), antimesenteric basal (AB) and next to caecal ampulla (NA). Myenteric neurons from the AA region were more clustered in EG1 animals (P<0.05). The EG1 animals presented a 16.8% reduction in the area of the nucleus, whereas the EG2 animals showed 18.4% increase (P<0.05). There was a more marked reduction in the cytoplasm of the animals in EG1 (↓23.2%) compared to EG2 (↓6.2%). There was 35.8% neuronal atrophy in the AB region and 16.8% in the region NA of the EG1 animals (P<0.05). In conclusion, different strains of T. gondii cause morphometric changes in caecal myenteric neurons of rats. Only the genotype I strain was able to cause neuronal density changes.

Copyright © 2011. Published by Elsevier Inc.

PMID: 22210156 [PubMed - as supplied by publisher]

Roles of Apicomplexan protein kinases at each life cycle stage

Parasitol Int. 2011 Dec 24. [Epub ahead of print]

Roles of Apicomplexan protein kinases at each life cycle stage.

Kato K, Sugi T, Iwanaga T.

Inhibitors of cellular protein kinases have been reported to inhibit the development of Apicomplexan parasites, suggesting that the functions of protozoan protein kinases are critical for their life cycle. However, the specific roles of these protein kinases cannot be determined using only these inhibitors without molecular analysis, including gene disruption. In this report, we describe the functions of Apicomplexan protein kinases in each parasite life stage and the potential of pre-existing protein kinase inhibitors as Apicomplexan drugs against, mainly, Plasmodium and Toxoplasma.

Copyright © 2011. Published by Elsevier Ireland Ltd.

PMID: 22209882 [PubMed - as supplied by publisher]