Regions of intrinsic disorder help identify a novel nuclear localization signal in Toxoplasma gondii histone acetyltransferase TgGCN5-B

Mol Biochem Parasitol. 2010 Nov 2. [Epub ahead of print]

Regions of intrinsic disorder help identify a novel nuclear localization signal in Toxoplasma gondii histone acetyltransferase TgGCN5-B

Dixon SE, Bhatti MM, Uversky VN, Dunker AK, Sullivan WJ Jr.

Department of Pharmacology and Toxicology.

Abstract
We have previously shown that protozoan parasites, such as Toxoplasma gondii, contain a high prevalence of intrinsically disordered regions in their predicted proteins. Here, we determine that both TgGCN5-family histone acetyltransferases (HATs) contain unusually high levels of intrinsic disorder. A previously identified basic-rich nuclear localization signal (NLS) in the N-terminus of TgGCN5-A is located within such a region of predicted disorder, but this NLS is not conserved in TgGCN5-B. We therefore analyzed the intrinsically disordered regions of TgGCN5-B for basic-rich sequences that could be indicative of a functional NLS, and this led to the identification of a novel NLS for TgGCN5-B, RPAENKKRGR. The functionality of the GCN5-B NLS was validated experimentally and has predictive value. These studies demonstrate that basic-rich sequences within regions predicted to be intrinsically disordered constitute criteria for a candidate NLS.

Copyright © 2010. Published by Elsevier B.V.
PMID: 21055425 [PubMed - as supplied by publisher]

Full-parasites: database of full-length cDNAs of apicomplexa parasites, 2010 update

Nucleic Acids Res. 2010 Nov 4. [Epub ahead of print]

Full-parasites: database of full-length cDNAs of apicomplexa parasites, 2010 update

Tuda J, Mongan AE, Tolba ME, Imada M, Yamagishi J, Xuan X, Wakaguri H, Sugano S, Sugimoto C, Suzuki Y.

Faculty of Medicine, Sam Ratulangi University, Kampus Unsrat, Bahu Manado, 95115, Indonesia, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan, Department of Parasitology, Assiut University, Assiut, 71515, Egypt, Department of Tropical Medicine and Parasitology School of Medicine, Keio University, 35 Shinanomachi Sinjuku, Tokyo 160-8582, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho west 2-13, Obihiro, Hokkaido 080-8555 and Research Center for Zoonosis Control, Hokkaido University, North 20, West 10 Kita-ku, Sapporo 001-0020, Japan.

Abstract
Full-Parasites (http://fullmal.hgc.jp/) is a transcriptome database of apicomplexa parasites, which include Plasmodium and Toxoplasma species. The latest version of Full-Parasites contains a total of 105 786 EST sequences from 12 parasites, of which 5925 full-length cDNAs have been completely sequenced. Full-Parasites also contain more than 30 million transcription start sites (TSS) for Plasmodium falciparum (Pf) and Toxoplasma gondii (Tg), which were identified using our novel oligo-capping-based protocol. Various types of cDNA data resources were interconnected with our original database functionalities. Specifically, in this update, we have included two unique RNA-Seq data sets consisting of 730 million mapped RNA-Seq tags. One is a dataset of 16 time-lapse experiments of cultured bradyzoite differentiation for Tg. The other dataset includes 31 clinical samples of Pf. Parasite RNA was extracted together with host human RNA, and the extracted mixed RNA was used for RNA sequencing, with the expectation that gene expression information from the host and parasite would be simultaneously represented. By providing the largest unique full-length cDNA and dynamic transcriptome data, Full-Parasites is useful for understanding host-parasite interactions and will help to eventually elucidate how monophyletic organisms have evolved to become parasites by adopting complex life cycles.

PMID: 21051343 [PubMed - as supplied by publisher]

The immunity-related GTPases in mammals: a fast-evolving cell-autonomous resistance system against intracellular pathogens

Mamm Genome. 2010 Oct 30. [Epub ahead of print]

The immunity-related GTPases in mammals: a fast-evolving cell-autonomous resistance system against intracellular pathogens

Hunn JP, Feng CG, Sher A, Howard JC.

Institute for Genetics, University of Cologne, Zuelpicher Str. 47a, 50674, Cologne, Germany.

Abstract
The immunity-related GTPases (IRGs) belong to the family of large, interferon-inducible GTPases and constitute a cell-autonomous resistance system essential for the control of vacuolar pathogens like Toxoplasma gondii in mice. Recent results demonstrated that numerous IRG members accumulate collaboratively at the parasitophorous vacuole of invading T. gondii leading to the destruction of the vacuole and the parasite and subsequent necrotic host cell death. Complex regulatory interactions between different IRG proteins are necessary for these processes. Disturbance of this finely balanced system, e.g., by single genetic deficiency for the important negative regulator Irgm1 or the autophagic regulator Atg5, leads to spontaneous activation of the effector IRG proteins when induced by IFNγ. This activation has cytotoxic consequences resulting in a severe lymphopenia, macrophage defects, and failure of the adaptive immune system in Irgm1-deficient mice. However, alternative functions in phagosome maturation and induction of autophagy have been proposed for Irgm1. The IRG system has been studied primarily in mice, but IRG genes are present throughout the mammalian lineage. Interestingly, the number, type, and diversity of genes present differ greatly even between closely related species, probably reflecting intimate host-pathogen coevolution driven by an armed race between the IRG resistance proteins and pathogen virulence factors. IRG proteins are targets for polymorphic T. gondii virulence factors, and genetic variation in the IRG system between different mouse strains correlates with resistance and susceptibility to virulent T. gondii strains.

PMID: 21052678 [PubMed - as supplied by publisher]

Dysregulation of the inflammatory response to the parasite, Toxoplasma gondii, in P2X(7) receptor-deficient mice

Int J Parasitol. 2010 Oct 30. [Epub ahead of print]

Dysregulation of the inflammatory response to the parasite, Toxoplasma gondii, in P2X(7) receptor-deficient mice

Miller CM, Zakrzewski AM, Ikin RJ, Boulter NR, Katrib M, Lees MP, Fuller SJ, Wiley JS, Smith NC.

Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Broadway, NSW, 2007, Australia.

Abstract
The P2X(7) receptor (P2X(7)R) is a two transmembrane receptor that is highly expressed on the surface of immune cells. Loss of function polymorphisms in this receptor have been linked to increased susceptibility to intracellular pathogens. P2X(7)R gene knockout (P2X(7)R(-/-); on a C57Bl/6J background), C57Bl/6J and BALB/c mice were infected with the avirulent ME49 strain of the intracellular parasite, Toxoplasma gondii, and susceptibility determined by monitoring weight loss. P2X(7)R(-/-) mice lost significantly more weight than C57Bl/6J mice from day 8 p.i.. C57Bl/6J, in turn, lost significantly more weight than BALB/c mice. Thus, by day 10 p.i., P2X(7)R(-/-) mice had lost 5.7 ± 0.7% of their weight versus 2.4 ± 0.6% for C57Bl/6J mice, whereas BALB/c mice had gained 1.9 ± 0.5%; by day 12 p.i., P2X(7)R(-/-) mice had lost 15.1 ± 0.6%, C57Bl/6J had lost 10.1 ± 0.8% and BALB/c had lost 4.8±0.8% of their weight. Neither parasite burden nor liver pathology was greater in the P2X(7)R(-/-) mice than in C57Bl/6J mice but BALB/c mice had significantly smaller numbers of parasites and less pathology in their livers than these strains. Absence of the P2X(7) receptor did not affect IFN-γ, IL-12, IL-1β, monocyte chemoattractant protein-1 (MCP-1) or TNF production. However, both P2X(7)R(-/-) and C57Bl/6J mice produced more IL-1 β and TNF than BALB/c mice. There was one important point of differentiation between the P2X(7)R(-/-) and C57Bl/6J mice, namely the significantly enhanced and prolonged production of nitric oxide, accompanied by delayed production of IL-10 in the P2X(7)R-deficient mice.

Copyright © 2010. Published by Elsevier Ltd.
PMID: 21044631 [PubMed - as supplied by publisher]

Crystallization and preliminary X-ray structural analysis of nucleoside triphosphate hydrolases from Neospora caninum and Toxoplasma gondii

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Nov 1;66(Pt 11):1445-8. Epub 2010 Oct 28.

Crystallization and preliminary X-ray structural analysis of nucleoside triphosphate hydrolases from Neospora caninum and Toxoplasma gondii

Matoba K, Shiba T, Takeuchi T, Sibley LD, Seiki M, Kikyo F, Horiuchi T, Asai T, Harada S.

Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto 606-8585, Japan.

Abstract
The nucleoside triphosphate hydrolases that are produced by Neospora caninum (NcNTPase) and Toxoplasma gondii (TgNTPase-I) have a different physiological function from the ubiquitous ecto-ATPases. The recombinant enzymes were crystallized at 293 K using polyethylene glycol 3350 as a precipitant and X-ray diffraction data sets were collected for NcNTPase (to 2.8 Å resolution) and TgNTPase-I (to 3.1 Å resolution) at 100 K using synchrotron radiation. The crystals of NcNTPase and TgNTPase-I belonged to the orthorhombic space group I222 (unit-cell parameters a = 93.6, b = 140.8, c = 301.1 Å) and the monoclinic space group P2(1) (unit-cell parameters a = 87.1, b = 123.5, c = 120.2 Å, β = 96.6°), respectively, with two NcNTPase (V(M) = 3.7 Å(3) Da(-1)) and four TgNTPase-I (V(M) = 2.7 Å(3) Da(-1)) molecules per asymmetric unit. SAD phasing trials using a data set (λ = 0.97904 Å) collected from a crystal of selenomethionylated NcNTPase gave an initial electron-density map of sufficient quality to build a molecular model of NcNTPase.

PMID: 21045291 [PubMed - in process]

An insertional trap for coditional gene expression in Toxoplasma gondii: identification of TAF250 as an essential gene

Mol Biochem Parasitol. 2010 Oct 27. [Epub ahead of print]

An insertional trap for coditional gene expression in Toxoplasma gondii: identification of TAF250 as an essential gene

Jammallo L, Eidell K, Davis PH, Dufort FJ, Cronin C, Thirugnanam S, Chiles TC, Roos DS, Gubbels MJ.

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

Abstract
Toxoplasmosis is characterized by fast lytic replication cycles leading to severe tissue lesions. Successful host cell invasion is essential for pathogenesis. The division cycle of Toxoplasma gondii is characterized by an unusual cell cycle progression and a distinct internal budding mechanism. To identify essential genes involved in the lytic cycle we devised an insertional gene trapping strategy using the Tet-transactivator system. In essence, a random, active promoter is displaced with a tetracycline regulatable promoter, which if in an essential gene, will result in a conditionally lethal phenotype upon tetracycline addition. We isolated eight mutants with growth defects, two of which displayed modest invasion defects, one of which had an additional cell cycle defect. The trapped loci were identified using expression microarrays, exploiting the tetracycline dependent expression of the trapped genes. In mutant 3.3H6 we identified TCP-1, a component of the chaperonin protein folding machinery under the control of the Tet promoter. However, this gene was not critical for growth of mutant 3.3H6. Subsequently, we identified a suppressor gene encoding a protein with a hypothetical function by guided cosmid complementation. In mutant 4.3B13, we identified TAF250, an RNA polymerase II complex component, as the trapped, essential gene. Furthermore, by mapping the plasmid insertion boundaries we identified multiple genomic rearrangements, which hint at a potential replication dependent DNA repair mechanism. Furthermore, these rearrangements provide an explanation for inconsistent locus rescue results observed by molecular biological approaches. Taken together, we have added an approach to identify and study essential genes in Toxoplasma.

A novel multifunctional oligonucleotide microarray for Toxoplasma gondii

BMC Genomics. 2010 Oct 25;11(1):603. [Epub ahead of print]

A novel multifunctional oligonucleotide microarray for Toxoplasma gondii

Bahl A, Davis PH, Behnke M, Dzierszinski F, Jagalur M, Chen F, Shanmugam D, White M, Kulp D, Roos DS.

Abstract
ABSTRACT:

BACKGROUND: Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a prohibitively expensive undertaking.

RESULTS: Taking advantage of available genome sequences and annotation for Toxoplasma gondii (the causative agent of opportunistic illness in immunocompromised individuals) and Plasmodium falciparum (a related parasite responsible for severe human malaria), we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for Toxoplasma, and single-nucleotide polymorphism (SNP)-based genotyping of both T. gondii and P. falciparum. Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis). The relatively small genomes of P. falciparum (23 Mb) and T. gondii (65 Mb), and the ability to isolate DNA from haploid stages, coupled with a careful analysis of signals from overlapping probes, permits a novel design enabling high confidence genotyping using only four probes per SNP. This strategy permits resolution of recombination points in the clonal progeny of sexual crosses, allowing detailed mapping of phenotypic traits. Recent sequencing of additional T. gondii isolates identifies >620K new SNPs, including >11K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating gene and transcript discovery, statistical analysis of expression profiles, etc. are also discussed.

CONCLUSIONS: In addition to providing an initial global view of the T. gondii transcriptome across major lineages and permitting detailed resolution of recombination points in a historical sexual cross, the multifunctional nature of this array also allowed opportunities to exploit probes for purposes beyond their intended use, enhancing analyses. This array is in widespread use by the T. gondii research community, and several aspects of the design strategy are likely to be useful for other pathogens.

PMID: 20974003 [PubMed - as supplied by publisher]

Generation of a Neutralizing Human Monoclonal Antibody Fab Fragment to SAG1 of Toxoplasma gondii Tachyzoites

Infect Immun. 2010 Oct 18. [Epub ahead of print]

Generation of a Neutralizing Human Monoclonal Antibody Fab Fragment to SAG1 of Toxoplasma gondii Tachyzoites

Fu YF, Feng M, Ohnishi K, Kimura T, Itoh J, Cheng XJ, Tachibana H.

Department of Medical Microbiology and Parasitology, Fudan University School of Medicine, Shanghai 200032, China; Department of Infectious Diseases, Tokyo Metropolitan Bokutoh General Hospital, Sumida-Ku, Tokyo 130-8575, Japan; Department of Infectious Diseases and Teaching and Research Support Center, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan.

Abstract
A combinatorial immunoglobulin gene library was constructed from lymphocytes in peripheral blood of a patient with toxoplasmosis and screened for production of human monoclonal antibody Fab fragments to recombinant SAG1 of Toxoplasma gondii. Two Fab clones, Tox203 and Tox1403, which consisted of a common heavy chain and different light chains, showed positive staining on the entire surface of tachyzoites in confocal microscopy. Sequence analysis of the heavy chain gene revealed that the closest germline V segments were VH3-23. The germline D segment was D1-7, and the closest germline J segment was JH4. In the light chain genes, the closest germline V segment was Vκ1-17 with the Jκ1 or Jκ4 segments. The dissociation constants of these Fab fragments with recombinant SAG1 were 3.09x10(-9) M for Tox203 and 2.01x10(-8) M for Tox1403, indicating that the affinity of Tox203 was 7 times higher than that of Tox1403. Preincubation of T. gondii tachyzoites with Tox203 significantly inhibited their attachment to cultured MDBK cells. Passive immunization of mice with Tox203 also significantly reduced mortality after challenge with T. gondii tachyzoites. This is the first report of bacterial expression of human monoclonal antibody Fab fragments to SAG1 of T. gondii. These results also demonstrate that human Fab fragments to SAG1 might be applicable for immunoprophylaxis of toxoplasmosis.

PMID: 20956568 [PubMed - as supplied by publisher]

Toxoplasma gondii primary infection in renal transplant recipients. Two case reports and literature review

Transpl Int. 2010 Oct 19. doi: 10.1111/j.1432-2277.2010.01173.x. [Epub ahead of print]

Toxoplasma gondii primary infection in renal transplant recipients. Two case reports and literature review
Martina MN, Cervera C, Esforzado N, Linares L, Torregrosa V, Sanclemente G, Hoyo I, Cofan F, Oppenheimer F, Miro JM, Campistol JM, Moreno A.

 Renal Transplant Unit, Hospital Clinic of Barcelona, IDIBAPS, University of Barcelona, Barcelona, Spain  Infectious Diseases Service, Hospital Clinic of Barcelona, IDIBAPS, University of Barcelona, Barcelona, Spain.

Abstract
Toxoplasmosis after solid organ transplantation is a complication associated with high morbidity and mortality. Universal prophylaxis with trimethoprim-sulfamethoxazole (TMP-SMX) is effective to prevent post-transplant toxoplasmosis. We report two cases of renal transplant recipients with negative antibodies against Toxoplasma gondii pretransplant who developed toxoplasmosis after TMP-SMX discontinuation. We have also performed a review of published cases of primary toxoplasmosis after renal transplantation. Of 20 cases reviewed, 11 were male and the mean age was 37.8 years (SD = 13.8). Donor serology for T. gondii was determined in 15 donors, two of them (13%) with negative immunoglobulin (Ig)G and four (27%) with positive IgG and IgM antibodies. Fever was present in 85% of primary toxoplasmosis and hematologic abnormalities were observed in 69% of the cases. Ten patients died (50%). All patients with fatal outcomes had clinical evidence of toxoplasmosis during the early post-transplant period (first 90 days), while no patient with late toxoplasmosis died (P = 0.003). Primary toxoplasmosis is associated with high mortality rates and TMP-SMX prophylaxis can delay the onset of symptoms resulting in an improvement of prognosis.

© 2010 The Authors. Transplant International © 2010 European Society for Organ Transplantation.
PMID: 20955469 [PubMed - as supplied by publisher]

Intracellular Protozoan Parasites of Humans: The Role of Molecular Chaperones in Development and Pathogenesis

Protein Pept Lett. 2010 Oct 18. [Epub ahead of print]

Intracellular Protozoan Parasites of Humans: The Role of Molecular Chaperones in Development and Pathogenesis
Shonhai A, Maier AG, Przyborski JM, Blatch GL.

Department of Biochemistry & Microbiology, University of Zululand, Kwadlangezwa, South Africa. G.Blatch@ru.ac.za.

Abstract
Certain kinetoplastid (Leishmania spp. and Tryapnosoma cruzi) and apicomplexan parasites (Plasmodium falciparum and Toxoplasma gondii) are capable of invading human cells as part of their pathology. These parasites appear to have evolved a relatively expanded or diverse complement of genes encoding molecular chaperones. The gene families encoding heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) chaperones show significant expansion and diversity (especially for Leishmania spp. and T. cruzi), and in particular the Hsp40 family appears to be an extreme example of phylogenetic radiation. In general, Hsp40 proteins act as co-chaperones of Hsp70 chaperones, forming protein folding pathways that integrate with Hsp90 to ensure proteostasis in the cell. It is tempting to speculate that the diverse environmental insults that these parasites endure have resulted in the evolutionary selection of a diverse and expanded chaperone network. Hsp90 is involved in development and growth of all of these intracellular parasites, and so far represents the strongest candidate as a target for chemotherapeutic interventions. While there have been some excellent studies on the molecular and cell biology of Hsp70 proteins, relatively little is known about the biological function of Hsp70-Hsp40 interactions in these intracellular parasites. This review focuses on intracellular protozoan parasites of humans, and provides a critique of the role of heat shock proteins in development and pathogenesis, especially the molecular chaperones Hsp90, Hsp70 and Hsp40.

PMID: 20955165 [PubMed - as supplied by publisher]

Concerted Action of Two Formins in Gliding Motility and Host Cell Invasion by Toxoplasma gondii.

PLoS Pathog. 2010 Oct 7;6(10). pii: e1001132.

Concerted Action of Two Formins in Gliding Motility and Host Cell Invasion by Toxoplasma gondii.

Daher W, Plattner F, Carlier MF, Soldati-Favre D.

Department of Microbiology and Molecular Medicine, CMU, University of Geneva, Geneva, Switzerland.

Abstract
The invasive forms of apicomplexan parasites share a conserved form of gliding motility that powers parasite migration across biological barriers, host cell invasion and egress from infected cells. Previous studies have established that the duration and direction of gliding motility are determined by actin polymerization; however, regulators of actin dynamics in apicomplexans remain poorly characterized. In the absence of a complete ARP2/3 complex, the formin homology 2 domain containing proteins and the accessory protein profilin are presumed to orchestrate actin polymerization during host cell invasion. Here, we have undertaken the biochemical and functional characterization of two Toxoplasma gondii formins and established that they act in concert as actin nucleators during invasion. The importance of TgFRM1 for parasite motility has been assessed by conditional gene disruption. The contribution of each formin individually and jointly was revealed by an approach based upon the expression of dominant mutants with modified FH2 domains impaired in actin binding but still able to dimerize with their respective endogenous formin. These mutated FH2 domains were fused to the ligand-controlled destabilization domain (DD-FKBP) to achieve conditional expression. This strategy proved unique in identifying the non-redundant and critical roles of both formins in invasion. These findings provide new insights into how controlled actin polymerization drives the directional movement required for productive penetration of parasites into host cells.

PMID: 20949068 [PubMed - in process]

Chronic infection with Toxoplasma gondii causes myenteric neuroplasticity of the jejunum in rats

Auton Neurosci. 2010 Oct 5. [Epub ahead of print]

Chronic infection with Toxoplasma gondii causes myenteric neuroplasticity of the jejunum in rats
Hermes-Uliana C, Pereira-Severi LS, Luerdes RB, Franco CL, da Silva AV, Araújo EJ, Sant'ana DD.

Programa de Pós-Graduação em Ciência Animal, Universidade Paranaense, (UNIPAR), Paraná, Brazil.

Abstract
Toxoplasma gondii is an aetiological agent of toxoplasmosis, which commonly causes diarrhoea in a number of species. This observation and the parasite's affinity for the nervous tissue support the theory that T. gondii infection may affect the myenteric neurons. The aim of this study was to evaluate the changes caused by T. gondii (genotype III) in the myenteric neurons of the jejunum in rats. Fifteen rats were distributed into three groups: control (CG), inoculated for 30days (G30) and inoculated for 90days (G90). Rats from the G30 and G90 groups received an oral inoculum with 500 oocysts from a genotype III (M7741) T. gondii strain. At 180days of age, all animals were anaesthetised and euthanised. Whole mounts were stained by using Giemsa (total population) and NADPH-diaphorase (nitrergic subpopulation) histochemistry. Maintenance of the width, length, area and neuronal density was observed; there was neuronal atrophy in the G30 group and a tendency to hypertrophy in the G90 group. Rats inoculated orally with sporulated oocysts did not show clinical illness or macroscopic or microscopic lesions, as do the majority of animal species. Therefore, infection was confirmed by a serum agglutination test; 30days of infection caused increased weight gain and atrophy of myenteric neurons. At 90days post-infection, weight gain became normal, and myenteric neurons hypertrophied.

PMID: 20932812 [PubMed - as supplied by publisher]

Comprehensive proteomic analysis of membrane proteins in toxoplasma gondii

Mol Cell Proteomics. 2010 Oct 10. [Epub ahead of print]

Comprehensive proteomic analysis of membrane proteins in toxoplasma gondii
Che FY, Madrid-Aliste C, Burd B, Zhang H, Nieves E, Kim K, Fiser A, Angeletti RH, Weiss LM.

Albert Einstein College of Medicine, United States.

Abstract
Toxoplasma gondii (T.gondii) is an obligate intracellular protozoan parasite that is important human and animal pathogen. Experimental information on T.gondii membrane proteins is limited, and the majority of gen predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T.gondii is important not only for understanding this parasite's invasion mechanism(s), but also for the discovery of potentail drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T.gondii, employing three proteomics strategies: 1D gel LC-MSMS annlysis (one dimensional gel electrophoresis liquid chromatography tandem mass spectrometry), biotin labeling in conjunction with 1D gel LC-MSMS analysis, and a novel strategy that combines Three Layer "Sandwich" gel Electrophoresis (TLSGE) with multidimensional protein identification technology (MudPIT). A total of 2, 241 T.gondii proteins with at least one predicted transmembrane segment were identified and grouped into 841 sequentially non-redundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T.gondii genmoe. A large portion (42%) of the identified T. gondii membrane proteins are hypothetical proteins. Furthermore, many of the membrane proteins validated by mass spectrometry are unique to T.gondii or to the Apicomplexa, providing a set of gene predictions ripe for experimental investigation, and potentially siutable targets for the development of therapeutic strategies.

PMID: 20935347 [PubMed - as supplied by publisher]

CNS-derived CCL21 is both sufficient to drive homeostatic CD4+ T cell proliferation and necessary for efficient CD4+ T cell migration

Brain Behav Immun. 2010 Sep 21. [Epub ahead of print]

CNS-derived CCL21 is both sufficient to drive homeostatic CD4+ T cell proliferation and necessary for efficient CD4+ T cell migration into the CNS parenchyma following Toxoplasma gondii infection

Ploix CC, Noor S, Crane J, Masek K, Carter W, Lo DD, Wilson EH, Carson MJ.

Roche Ltd., Basel, Switzerland.

Abstract
Injury, infection and autoimmune triggers increase CNS expression of the chemokine CCL21. Outside the CNS, CCL21 contributes to chronic inflammatory disease and autoimmunity by three mechanisms: recruitment of lymphocytes into injured or infected tissues, organization of inflammatory infiltrates into lymphoid-like structures and promotion of homeostatic CD4+ T-cell proliferation. To test if CCL21 plays the same role in CNS inflammation, we generated transgenic mice with astrocyte-driven expression of CCL21 (GFAP-CCL21 mice). Astrocyte-produced CCL21 was bioavailable and sufficient to support homeostatic CD4+ T-cell proliferation in cervical lymph nodes even in the absence of endogenous CCL19/CCL21. However, lymphocytes and glial-activation were not detected in the brains of uninfected GFAP-CCL21 mice, although CCL21 levels in GFAP-CCL21 brains were higher than levels expressed in inflamed Toxoplasma-infected nontransgenic brains. Following Toxoplasma infection, T-cell extravasation into submeningeal, perivascular and ventricular sites of infected CNS was not CCL21-dependent, occurring even in CCL19/CCL21-deficient mice. However, migration of extravasated CD4+, but not CD8+ T cells from extra-parenchymal CNS sites into the CNS parenchyma was CCL21-dependent. CD4+ T cells preferentially accumulated at perivascular, submeningeal and ventricular spaces in infected CCL21/CCL19-deficient mice. By contrast, greater numbers of CD4+ T cells infiltrated the parenchyma of infected GFAP-CCL21 mice than in wild-type or CCL19/CCL21-deficient mice. Together these data indicate that CCL21 expression within the CNS has the potential to contribute to T cell-mediated CNS pathology via: (a) homeostatic priming of CD4+ T-lymphocytes outside the CNS and (b) by facilitating CD4+ T-cell migration into parenchymal sites following pathogenic insults to the CNS.

PMID: 20868739 [PubMed - as supplied by publisher]

Topological journey of parasite-derived antigens for presentation by MHC class I molecules

Trends Immunol. 2010 Sep 22. [Epub ahead of print]

Topological journey of parasite-derived antigens for presentation by MHC class I molecules

Blanchard N, Shastri N.

Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA; Current address: Physiopathology Research Centre of Toulouse-Purpan/INSERM U563, Department of Immunology and Infectious diseases, CHU Purpan, BP3028, 31024 Toulouse Cedex, France.

Abstract
Within cells of their host, many bacteria and parasites inhabit specialized compartments, such as a modified phagosome for Mycobacterium tuberculosis or a parasitophorous vacuole for Toxoplasma gondii. These locations could exclude microbial material from entry into the MHC class I surveillance pathway. Remarkably, however, under these circumstances, cells can still signal the presence of invading pathogens to circulating CD8(+) T cells, which typically play a key role in protection against such intracellular organisms. Here, we review MHC I presentation pathways in various contexts, ranging from model antigens in non-infectious settings to pathogen-infected cells. We suggest that presentation of intracellular pathogens can be described as not just one, but several distinct pathways; perhaps because diverse pathogens have evolved different strategies to interact with host cells.

PMID: 20869317 [PubMed - as supplied by publisher]

{alpha}-tubulin Mutations Alter Oryzalin Affinity and Microtubule Assembly Properties to Confer Dinitroaniline Resistance

Eukaryot Cell. 2010 Sep 24. [Epub ahead of print]

{alpha}-tubulin Mutations Alter Oryzalin Affinity and Microtubule Assembly Properties to Confer Dinitroaniline Resistance

Lyons-Abbott S, Sackett DL, Wloga D, Gaertig J, Morgan RE, Werbovetz KA, Morrissette NS.

Department of Molecular Biology and Biochemistry, University of CA, Irvine 92617; Laboratory of Integrative and Medical Biophysics, Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA 20892; Department of Cellular Biology, University of Georgia, 30602; Division of Medicinal Chemistry & Pharmacognosy, Ohio State University, 43210.

Abstract
Plant and protozoan microtubules are selectively sensitive to dinitroanilines, which do not disrupt vertebrate or fungal microtubules. Tetrahymena thermophila is an abundant source of dinitroaniline-sensitive tubulin and we have modified the single T. thermophila α-tubulin gene to create strains that solely express mutant α-tubulin in functional dimers. Previous research identified multiple α-tubulin mutations that confer dinitroaniline resistance in the human parasite Toxoplasma gondii and when two of these mutations (L136F and I252L) were introduced into T. thermophila, they conferred resistance in these free-living ciliates. Purified tubulin heterodimers composed of L136F or I252L α-tubulin display decreased affinity for the dinitroaniline oryzalin relative to wild-type T. thermophila tubulin. Moreover, the L136F substitution dramatically reduces the critical concentration for microtubule assembly relative to the properties of wild-type T. thermophila tubulin. Our data provides additional support for the proposed dinitroaniline binding site on α-tubulin and validates the use of T. thermophila for expression of genetically homogeneous populations of mutant tubulins for biochemical characterization.

PMID: 20870876

Coordinated Progression through Two Subtranscriptomes Underlies the Tachyzoite Cycle of Toxoplasma gondii

PLoS One. 2010 Aug 26;5(8). pii: e12354.

Coordinated Progression through Two Subtranscriptomes Underlies the Tachyzoite Cycle of Toxoplasma gondii

Behnke MS, Wootton JC, Lehmann MM, Radke JB, Lucas O, Nawas J, Sibley LD, White MW.

Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana, United States of America.

Abstract
BACKGROUND: Apicomplexan parasites replicate by varied and unusual processes where the typically eukaryotic expansion of cellular components and chromosome cycle are coordinated with the biosynthesis of parasite-specific structures essential for transmission.

METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the global cell cycle transcriptome of the tachyzoite stage of Toxoplasma gondii. In dividing tachyzoites, more than a third of the mRNAs exhibit significant cyclical profiles whose timing correlates with biosynthetic events that unfold during daughter parasite formation. These 2,833 mRNAs have a bimodal organization with peak expression occurring in one of two transcriptional waves that are bounded by the transition into S phase and cell cycle exit following cytokinesis. The G1-subtranscriptome is enriched for genes required for basal biosynthetic and metabolic functions, similar to most eukaryotes, while the S/M-subtranscriptome is characterized by the uniquely apicomplexan requirements of parasite maturation, development of specialized organelles, and egress of infectious daughter cells. Two dozen AP2 transcription factors form a series through the tachyzoite cycle with successive sharp peaks of protein expression in the same timeframes as their mRNA patterns, indicating that the mechanisms responsible for the timing of protein delivery might be mediated by AP2 domains with different promoter recognition specificities.

CONCLUSION/SIGNIFICANCE: Underlying each of the major events in apicomplexan cell cycles, and many more subordinate actions, are dynamic changes in parasite gene expression. The mechanisms responsible for cyclical gene expression timing are likely crucial to the efficiency of parasite replication and may provide new avenues for interfering with parasite growth.

PMID: 20865045 [PubMed - in process]

Congenital infections with Trypanosoma cruzi or Toxoplasma gondii are associated with decreased serum interferon-γ and interleukin-18

Ann Trop Med Parasitol. 2010 Sep;104(6):485-92.

Congenital infections with Trypanosoma cruzi or Toxoplasma gondii are associated with decreased serum concentrations of interferon-γ and interleukin-18 but increased concentrations of interleukin-10

Mayer JP, Biancardi M, Altcheh J, Freilij H, Weinke T, Liesenfeld O.

Institut für Mikrobiologie und Hygiene, Charité Universitätsmedizin, Campus Benjamin Franklin, Hindenburgdamm 27, 12203 Berlin; Germany, Klinik für Kinderchirurgie, Helios Klinik Berlin-Buch, Schwanebecker Chaussee 50, 13125 Berlin, Germany.

Abstract
Little is known about the immune responses of newborns with congenital Chagas disease (CCD) or congenital toxoplasmosis (CT) but they probably differ to those seen in adults with Chagas disease or toxoplasmosis, leading to differences in pathology. The concentrations of interleukin-18 (IL-18), interferon-γ (IFN-γ) and interleukin 10 (IL-10) in the sera of infants with CCD or CT were determined and compared with those in the sera of uninfected controls (born to mothers who were seropositive or seronegative for Trypanosoma cruzi). The infants with CCD or CT were found to have lower IL-18 and IFN-γ concentrations but higher IL-10 concentrations than the uninfected controls. The IL-18 and IFN-γ concentrations were also significantly lower in the infants with CCD than in those with CT. Although the infants with symptomatic CT had significantly higher serum concentrations of IL-18 than those with asymptomatic infection with Toxoplasma, the infants with symptomatic CCD had similar serum concentrations of IL-18 to the infants with asymptomatic Tr. cruzi infection. Taken together, these results indicate that IL-10 contributes to the suppression of pro-inflammatory immune responses and therefore, perhaps, to clinically overt CCD and CT.

PMID: 20863437 [PubMed - in process]

UNC93B1 Mediates Host Resistance to Infection with Toxoplasma gondii

PLoS Pathog. 2010 Aug 26;6(8). pii: e1001071.

UNC93B1 Mediates Host Resistance to Infection with Toxoplasma gondii

Melo MB, Kasperkovitz P, Cerny A, Könen-Waisman S, Kurt-Jones EA, Lien E, Beutler B, Howard JC, Golenbock DT, Gazzinelli RT.

University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

Abstract
UNC93B1 associates with Toll-Like Receptor (TLR) 3, TLR7 and TLR9, mediating their translocation from the endoplasmic reticulum to the endolysosome, hence allowing proper activation by nucleic acid ligands. We found that the triple deficient '3d' mice, which lack functional UNC93B1, are hyper-susceptible to infection with Toxoplasma gondii. We established that while mounting a normal systemic pro-inflammatory response, i.e. producing abundant MCP-1, IL-6, TNFα and IFNγ, the 3d mice were unable to control parasite replication. Nevertheless, infection of reciprocal bone marrow chimeras between wild-type and 3d mice with T. gondii demonstrated a primary role of hemopoietic cell lineages in the enhanced susceptibility of UNC93B1 mutant mice. The protective role mediated by UNC93B1 to T. gondii infection was associated with impaired IL-12 responses and delayed IFNγ by spleen cells. Notably, in macrophages infected with T. gondii, UNC93B1 accumulates on the parasitophorous vacuole. Furthermore, upon in vitro infection the rate of tachyzoite replication was enhanced in non-activated macrophages carrying mutant UNC93B1 as compared to wild type gene. Strikingly, the role of UNC93B1 on intracellular parasite growth appears to be independent of TLR function. Altogether, our results reveal a critical role for UNC93B1 on induction of IL-12/IFNγ production as well as autonomous control of Toxoplasma replication by macrophages.

PMID: 20865117 [PubMed - in process]

Advances in imaging the innate and adaptive immune response to Toxoplasma gondii

Future Microbiol. 2010 Sep;5:1321-8.

Advances in imaging the innate and adaptive immune response to Toxoplasma gondii

John B, Weninger W, Hunter CA.

Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Abstract
Toxoplasma gondii is an intracellular protozoan parasite that infects a wide variety of warm-blooded hosts and can have devastating effects in the developing fetus as well as the immunocompromised host. An appreciation of how this organism interacts with the host immune system is crucial to understanding the pathogenesis of this disease. The last decade has been marked by the application of various imaging techniques, such as bioluminescent imaging as well as confocal and multiphoton microscopy to study toxoplasmosis. The ability to manipulate parasites to express fluorescent/bioluminescent markers or model antigens/enzymes combined with the development of reporter mice that allow the detection of distinct immune populations have been crucial to the success of many of these studies. These approaches have permitted the visualization of parasites and immune cells in real-time and provided new insights into the nature of host-pathogen interactions. This article highlights some of the advances in imaging techniques, their strengths and weaknesses, and how these techniques have impacted our understanding of the interaction between parasites and various immune populations during toxoplasmosis.

PMID: 20860479 [PubMed - in process]

Phosphorylation of eukaryotic initiation factor-2{alpha} promotes the extracellular survival of obligate intracellular parasite Toxoplasma gondii

Proc Natl Acad Sci U S A. 2010 Sep 20. [Epub ahead of print]

Phosphorylation of eukaryotic initiation factor-2{alpha} promotes the extracellular survival of obligate intracellular parasite Toxoplasma gondii

Joyce BR, Queener SF, Wek RC, Sullivan WJ Jr.

Departments of Pharmacology and Toxicology and Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202.

Abstract
While seeking a new host cell, obligate intracellular parasites, such as the protozoan Toxoplasma gondii, must be able to endure the stress of an extracellular environment. The mechanisms Toxoplasma use to remain viable while deprived of a host cell are not understood. We have previously shown that phosphorylation of Toxoplasma eukaryotic initiation factor-2α (TgIF2α) is a conserved response to stress. Here we report the characterization of Toxoplasma harboring a point mutation (S71A) in TgIF2α that prevents phosphorylation. Results show that TgIF2α phosphorylation is critical for parasite viability because the TgIF2α-S71A mutants are ill-equipped to cope with life outside the host cell. The TgIF2α-S71A mutants also showed a significant delay in producing acute toxoplasmosis in vivo. We conclude that the phosphorylation of TgIF2α plays a crucial role during the lytic cycle by ameliorating the stress of the extracellular environment while the parasite searches for a new host cell.

PMID: 20855600 [PubMed - as supplied by publisher]

The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1

PLoS One. 2010 Sep 14;5(9). pii: e12736.

The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1

Bakszt R, Wernimont A, Allali-Hassani A, Mok MW, Hills T, Hui R, Pizarro JC.

The Structural Genomics Consortium (SGC), University of Toronto, Toronto, Ontario, Canada.

Abstract
BACKGROUND: Pyruvate kinase (PK), which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population.

METHODOLOGY/PRINCIPAL FINDINGS: We have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain in the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers.

CONCLUSIONS: We hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two α-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery.

PMID: 20856875 [PubMed - as supplied by publisher]

Structure-Based Analysis of Toxoplasma gondii Profilin: A Parasite-Specific Motif is Required for Recognition by Toll-like Receptor 11

J Mol Biol. 2010 Sep 16. [Epub ahead of print]

Structure-Based Analysis of Toxoplasma gondii Profilin: A Parasite-Specific Motif is Required for Recognition by Toll-like Receptor 11

Kucera K, Koblansky AA, Saunders LP, Frederick KB, De La Cruz EM, Ghosh S, Modis Y.

Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA.

Abstract
Profilins promote actin polymerization by exchanging ADP for ATP on monomeric actin, and delivering ATP-actin to growing filament barbed ends. Apicomplexan protozoa like Toxoplasma gondii invade host cells using an actin-dependent gliding motility. Toll-like receptor 11 (TLR11) generates an innate immune response upon sensing T. gondii profilin (TgPRF). The crystal structure of TgPRF reveals a parasite-specific surface motif consisting of an acidic loop, followed by a long β-hairpin. A series of structure-based profilin mutants show that TLR11 recognition of the acidic loop is responsible for most of the IL-12 secretion response to TgPRF in peritoneal macrophages. Deletion of both the acidic loop and the β-hairpin completely abrogates IL-12 secretion. Insertion of the T. gondii acidic loop and β-hairpin into yeast profilin is sufficient to generate TLR11-dependent signaling. Substitution of the acidic loop in TgPRF with the homologous loop from the apicomplexan parasite C. parvum does not affect TLR11-dependent IL-12 secretion, while substitution with the acidic loop from P. falciparum results in reduced but significant IL-12 secretion. We conclude that the parasite-specific motif in TgPRF is the key molecular pattern recognized by TLR11. Unlike other profilins, TgPRF slows nucleotide exchange on monomeric rabbit actin, and binds rabbit actin weakly. The putative TgPRF actin-binding surface includes the β-hairpin, and diverges widely from the actin-binding surfaces of vertebrate profilins.

PMID: 20851125 [PubMed - as supplied by publisher]

A Novel Family of Toxoplasma IMC Proteins Displays a Hierarchical Organization and Functions in Coordinating Parasite Division

PLoS Pathog. 2010 Sep 9;6(9). pii: e1001094.

A Novel Family of Toxoplasma IMC Proteins Displays a Hierarchical Organization and Functions in Coordinating Parasite Division

Beck JR, Rodriguez-Fernandez IA, Cruz de Leon J, Huynh MH, Carruthers VB, Morrissette NS, Bradley PJ.

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

Abstract
Apicomplexans employ a peripheral membrane system called the inner membrane complex (IMC) for critical processes such as host cell invasion and daughter cell formation. We have identified a family of proteins that define novel sub-compartments of the Toxoplasma gondii IMC. These IMC Sub-compartment Proteins, ISP1, 2 and 3, are conserved throughout the Apicomplexa, but do not appear to be present outside the phylum. ISP1 localizes to the apical cap portion of the IMC, while ISP2 localizes to a central IMC region and ISP3 localizes to a central plus basal region of the complex. Targeting of all three ISPs is dependent upon N-terminal residues predicted for coordinated myristoylation and palmitoylation. Surprisingly, we show that disruption of ISP1 results in a dramatic relocalization of ISP2 and ISP3 to the apical cap. Although the N-terminal region of ISP1 is necessary and sufficient for apical cap targeting, exclusion of other family members requires the remaining C-terminal region of the protein. This gate-keeping function of ISP1 reveals an unprecedented mechanism of interactive and hierarchical targeting of proteins to establish these unique sub-compartments in the Toxoplasma IMC. Finally, we show that loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, indicating a role for the ISP proteins in coordinating this unique process of Toxoplasma replication.

PMID: 20844581 [PubMed - in process]

Toxoplasma gondii infection in first-episode and inpatient individuals with schizophrenia

Int J Infect Dis. 2010 Sep 13. [Epub ahead of print]

Toxoplasma gondii infection in first-episode and inpatient individuals with schizophrenia

Hamidinejat H, Ghorbanpoor M, Hosseini H, Alavi SM, Nabavi L, Jalali MH, Borojeni MP, Jafari H, Mohammadaligol S.

Department of Pathobiology, Shahid Chamran University, Ahvaz, Iran.

Abstract
BACKGROUND: A high seroprevalence of Toxoplasma gondii infection has been detected in psychiatric patients, particularly in schizophrenia cases.

METHODS: In the present study 98 patients suffering from schizophrenia (58 inpatients and 40 first-episode patients) and 96 control patients (50 healthy volunteers and 46 with a depressive disorder) were examined for the presence of both IgG and IgM antibodies against T. gondii by enzyme-linked immunosorbent assay (ELISA). We applied the Bradford Hill criteria to identify the weight of causal inference.

RESULTS: The positivity rate of anti-T. gondii IgG antibodies among individuals with schizophrenia (57.1%) was significantly higher than in healthy controls (29.2%). There were no associations between immune status ratio (ISR) values and the risk of schizophrenia. The weight of evidence approach using the Bradford Hill criteria revealed a 92% probability of a causal association.

CONCLUSION: Our results show that exposure to T. gondii may lead to schizophrenia.

PMID: 20843718 [PubMed - as supplied by publisher]

Apicomplexan parasite adhesins: novel strategies for targeting host cell carbohydrates

Curr Opin Struct Biol. 2010 Sep 13. [Epub ahead of print]

Apicomplexan parasite adhesins: novel strategies for targeting host cell carbohydrates

Boulanger MJ, Tonkin ML, Crawford J.

Department of Biochemistry and Microbiology, University of Victoria, PO Box 3055, STN CSC, Victoria, BC, Canada V8W 3P6.

Abstract
Apicomplexan parasites such as Plasmodium spp. (malaria) and Toxoplasma gondii (toxoplasmosis) are significant global pathogens of humans and animals. Unlike many intracellular bacterial and viral pathogens that rely on host cell uptake machinery to gain entry, apicomplexan parasites promote recognition, attachment and ultimately invasion of host cells through an orchestrated delivery of adhesins. While several of these adhesins are now known to target host cell glycans, only recently have atomic level insights been forthcoming. Here we review recent developments in defining detailed molecular blueprints used by these widespread pathogens to drive host cell adhesion and promote infectivity.

PMID: 20843678 [PubMed - as supplied by publisher]

A Toxoplasma MORN1 Null Mutant Undergoes Repeated Divisions but Is Defective in Basal Assembly, Apicoplast Division and Cytokinesis

PLoS One. 2010 Aug 19;5(8). pii: e12302.

A Toxoplasma MORN1 Null Mutant Undergoes Repeated Divisions but Is Defective in Basal Assembly, Apicoplast Division and Cytokinesis

Lorestani A, Sheiner L, Yang K, Robertson SD, Sahoo N, Brooks CF, Ferguson DJ, Striepen B, Gubbels MJ.

Department of Biology, Boston College, Chestnut Hill, Massachusetts, United States of America.

Abstract
The membrane occupation and recognition nexus protein 1 (MORN1) is highly conserved among apicomplexan parasites and is associated with several structures that have a role in cell division. Here we dissected the role of MORN1 using the relatively simple budding process of Toxoplasma gondii as a model. Ablation of MORN1 in a conditional null mutant resulted in pronounced defects suggesting a central role for MORN1 in apicoplast segregation and in daughter cell budding. Lack of MORN1 resulted in double-headed parasites. These Janus-headed parasites form two complete apical complexes but fail to assemble a basal complex. Moreover, these parasites were capable of undergoing several more budding rounds resulting in the formation of up to 16-headed parasites conjoined at the basal end. Despite this segregation defect, the mother's cytoskeleton was completely disassembled in every budding round. Overall this argues that successful completion of the budding is not required for cell cycle progression. None of the known basal complex components, including a set of recently identified inner membrane complex (IMC) proteins, localized correctly in these multi-headed parasites. These data suggest that MORN1 is essential for assembly of the basal complex, and that lack of the basal complex abolishes the contractile capacity assigned to the basal complex late in daughter formation. Consistent with this hypothesis we observe that MORN1 mutants fail to efficiently constrict and divide the apicoplast. We used the null background provided by the mutant to dissect the function of subdomains of the MORN1 protein. This demonstrated that deletion of a single MORN domain already prevented the function of MORN1 whereas a critical role for the short linker between MORN domains 6 and 7 was identified. In conclusion, MORN1 is required for basal complex assembly and loss of MORN1 results in defects in apicoplast division and daughter segregation.

PMID: 20808817 [PubMed - in process]

Identification of attractive drug targets in neglected-disease pathogens using an in silico approach

PLoS Negl Trop Dis. 2010 Aug 24;4(8). pii: e804.

Identification of attractive drug targets in neglected-disease pathogens using an in silico approach

Crowther GJ, Shanmugam D, Carmona SJ, Doyle MA, Hertz-Fowler C, Berriman M, Nwaka S, Ralph SA, Roos DS, Van Voorhis WC, Agüero F.

Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington, United States of America.

Abstract
BACKGROUND: The increased sequencing of pathogen genomes and the subsequent availability of genome-scale functional datasets are expected to guide the experimental work necessary for target-based drug discovery. However, a major bottleneck in this has been the difficulty of capturing and integrating relevant information in an easily accessible format for identifying and prioritizing potential targets. The open-access resource TDRtargets.org facilitates drug target prioritization for major tropical disease pathogens such as the mycobacteria Mycobacterium leprae and Mycobacterium tuberculosis; the kinetoplastid protozoans Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi; the apicomplexan protozoans Plasmodium falciparum, Plasmodium vivax, and Toxoplasma gondii; and the helminths Brugia malayi and Schistosoma mansoni.

METHODOLOGY/PRINCIPAL FINDINGS: Here we present strategies to prioritize pathogen proteins based on whether their properties meet criteria considered desirable in a drug target. These criteria are based upon both sequence-derived information (e.g., molecular mass) and functional data on expression, essentiality, phenotypes, metabolic pathways, assayability, and druggability. This approach also highlights the fact that data for many relevant criteria are lacking in less-studied pathogens (e.g., helminths), and we demonstrate how this can be partially overcome by mapping data from homologous genes in well-studied organisms. We also show how individual users can easily upload external datasets and integrate them with existing data in TDRtargets.org to generate highly customized ranked lists of potential targets.

CONCLUSIONS/SIGNIFICANCE: Using the datasets and the tools available in TDRtargets.org, we have generated illustrative lists of potential drug targets in seven tropical disease pathogens. While these lists are broadly consistent with the research community's current interest in certain specific proteins, and suggest novel target candidates that may merit further study, the lists can easily be modified in a user-specific manner, either by adjusting the weights for chosen criteria or by changing the criteria that are included.

PMID: 20808766 [PubMed - in process]

CXCL10 is required to maintain T cell populations and control parasite replication during chronic ocular toxoplasmosis

Invest Ophthalmol Vis Sci. 2010 Sep 1. [Epub ahead of print]

CXCL10 is required to maintain T cell populations and control parasite replication during chronic ocular toxoplasmosis

Norose K, Kikumura A, Luster AD, Hunter CA, Harris TH.

Infection and Host Defense, Chiba University, Chiba, Japan.

Abstract
PURPOSE. Toxoplasma gondii is a major cause of ocular disease, which can lead to permanent vision loss in humans. T cells are critically involved in parasite control, but little is known about the molecules that promote T cell trafficking and migration in the retina. Thus, the aim of this study was to image and dissect the T cell response during chronic toxoplasmic retinochoroiditis. METHODS. C57BL/6 mice were infected with the Me49 strain of T. gondii and T cells that infiltrated the eye were analyzed by flow cytometry and imaged using multi-photon microscopy. IFN-gamma, CXCL9, CXCL10, and CXCR3 mRNA levels were measured by real-time PCR. To investigate the role of CXCL10, mice were treated with anti-CXCL10 antibodies and histopathology and immunohistochemistry were performed to monitor changes in pathology, cellular infiltration, and parasite burden in the eye. RESULTS. Infection with T. gondii leads to the infiltration of highly-activated, motile T cells into the eye. These cells express CXCR3, are capable of producing IFN-gamma and TNF-alpha, and CD8+ T cells express granzyme B. The expression of CXCL9 and CXCL10 in the retina was significantly upregulated during chronic infection. Treatment of chronically infected mice with anti-CXCL10 antibodies led to decreases in the number of CD3+, CD4+, and CD8+ T cells and IFN-gamma mRNA expression in the retina and an increase in replicating parasites and ocular pathology. CONCLUSION. The maintenance of the T cell response and control of T. gondii in the eye during chronic infection is dependent on CXCL10.

PMID: 20811054 [PubMed - as supplied by publisher]

Nephromyces, a beneficial apicomplexan symbiont in marine animals

Proc Natl Acad Sci U S A. 2010 Aug 24. [Epub ahead of print]

Nephromyces, a beneficial apicomplexan symbiont in marine animals

Saffo MB, McCoy AM, Rieken C, Slamovits CH.

Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138-2902.

Abstract
With malaria parasites (Plasmodium spp.), Toxoplasma, and many other species of medical and veterinary importance its iconic representatives, the protistan phylum Apicomplexa has long been defined as a group composed entirely of parasites and pathogens. We present here a report of a beneficial apicomplexan: the mutualistic marine endosymbiont Nephromyces. For more than a century, the peculiar structural and developmental features of Nephromyces, and its unusual habitat, have thwarted characterization of the phylogenetic affinities of this eukaryotic microbe. Using short-subunit ribosomal DNA (SSU rDNA) sequences as key evidence, with sequence identity confirmed by fluorescence in situ hybridization (FISH), we show that Nephromyces, originally classified as a chytrid fungus, is actually an apicomplexan. Inferences from rDNA data are further supported by the several apicomplexan-like structural features in Nephromyces, including especially the strong resemblance of Nephromyces infective stages to apicomplexan sporozoites. The striking emergence of the mutualistic Nephromyces from a quintessentially parasitic clade accentuates the promise of this organism, and the three-partner symbiosis of which it is a part, as a model for probing the factors underlying the evolution of mutualism, pathogenicity, and infectious disease.

PMID: 20736348 [PubMed - as supplied by publisher]

Toxoplasma Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

J Vis Exp. 2010 Aug 12;(42). pii: 2091. doi: 10.3791/2091.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Tobin C, Pollard A, Knoll L.

Department of Medical Microbiology and Immunology, University of Wisconsin.

Abstract
Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO(2;) starvation, using medium with high a pH, or the addition of interferon gamma (IFNgamma). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO(2;) and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFNgamma and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO(2;) and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFNgamma and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

PMID: 20736916 [PubMed - in process]

Toxoplasma gondii: Infection natural congenital in cattle and an experimental inoculation of gestating cows with oocysts

Exp Parasitol. 2010 Aug 21. [Epub ahead of print]

Toxoplasma gondii: Infection natural congenital in cattle and an experimental inoculation of gestating cows with oocysts

Costa GH, da Costa AJ, Lopes WD, Bresciani KD, Dos Santos TR, Esper CR, Santana AE.

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

Abstract
Two studies, of a natural infection and an experimental infection, were performed in order to study congenital transmission of Toxoplasma gondii in cattle. In the first study, 50 fetuses were harvested from gestating cows that were eutanasied at a municipal slaughterhouse in Jaboticabal, São Paulo state, Brazil. In the second study, 11 gestating cows were divided into four groups for inoculation with T. gondii: GI consisted of 3 cows inoculated with 1.0 x 10(5) oocysts during their first trimester of gestation; GII consisted of three cows inoculated with 1.0 x 10(5) oocysts during their second trimester of gestation; GIII consisted of three cows inoculated with 1.0 x 10(5) oocysts during their last trimester of gestation; and GIV consisted of two control cows, one during its first and the other during its second trimester of gestation. In both studies, the presence of T. gondii was confirmed both indirectly by immunofluorescence assay (IFAT). In the natural infection experiment, 18% (9/50) of the gestating cows were confirmed to have specific antibodies (IFAT - 1:64) against T. gondii. The bioassay was able to diagnose the presence of T. gondii in the tissue samples from three calves. In the second experiment, the nine cows from groups I, II and III presented with specific antibodies (IFAT) against T. gondii. In contrast, T. gondii could not be detected by IFAT, histopathological examination or the bioassay in any of the nine calves born to cows experimentally infected with T. gondii oocysts. Based on the results from both studies, we conclude that congenital infection of Toxoplasma gondii in cattle, while infrequent, does occur naturally The pathogenicity of the strain of T. gondii may influence the likelihood of this route of transmission.

PMID: 20736009 [PubMed - as supplied by publisher]

Determination of diagnostic value of Toxoplasma gondii recombinant ROP2 and ROP4 antigens in mouse experimental model

Pol J Microbiol. 2010;59(2):137-41.

Determination of diagnostic value of Toxoplasma gondii recombinant ROP2 and ROP4 antigens in mouse experimental model

Gatkowska J, Dziadek B, Brzostek A, Dziadek J, Dzitko K, Długońska H.

Department of Immunoparasitology, University of Lódź, Poland. gatjus@biol.uni.lodz.pl

Abstract
The aim of this study was to test the potential diagnostic usefulness of recombinant Toxoplasma gondii rhoptry antigens, ROP2 and ROP4, with respect to toxoplasmosis detection and infection phase distinction in laboratory mouce by determining specific serum IgM and IgG antibodies with the use of indirect ELISA technique. The mice antibody response to ROP antigens was significantly higher in the IgM than in the IgG class with the peak on the turn of acute and latent infection, whereas the response to recombinant SAG1 antigen, used as control, revealed preferential synthesis of IgG antibodies with the highest absorbance values measured during latent toxoplasmosis.

PMID: 20734761 [PubMed - in process]

The binding of Toxoplasma gondii glycosylphosphatidylinositols to galectin-3 is required for their recognition by macrophages

J Biol Chem. 2010 Aug 20. [Epub ahead of print]

The binding of Toxoplasma gondii glycosylphosphatidylinositols to galectin-3 is required for their recognition by macrophages

Debierre-Grockiego F, Niehus S, Coddeville B, Elass E, Poirier F, Weingart R, Schmidt RR, Mazurier J, Guerardel Y, Schwarz RT.

UFR Sciences Pharmaceutiques, France;

Abstract
We showed that the production of tumor necrosis factor alpha by macrophages in response to Toxoplasma gondii glycosylphosphatidylinositols (GPIs) requires the expression of both Toll-Like Receptors TLR2 and TLR4, but not of their co-receptor CD14. Galectin-3 is a beta-galactoside-binding protein with immune-regulatory effects, which associates with TLR2. We demonstrate here by using the surface plasmon resonance method that the GPIs of T. gondii bind to human galectin-3 with strong affinity and in a dose-dependent manner. The use of a synthetic glycan and of the lipid moiety cleaved from the GPIs show that both parts are involved in the interaction with galectin-3. GPIs of T. gondii also bind to galectin-1 but with a lower affinity and only through the lipid moiety. At the cellular level, the production of tumor necrosis factor alpha induced by T. gondii GPIs in macrophages depends on the expression of galectin-3 but not of galectin-1. This study is the first identification of a galectin-3 ligand of T. gondii origin, and galectin-3 might be a co-receptor presenting the GPIs to the TLRs on macrophages.

PMID: 20729207 [PubMed - as supplied by publisher]

Modulation of immunity in mice with latent toxoplasmosis-the experimental support for the immunosuppression hypothesis

Parasitol Res. 2010 Aug 19. [Epub ahead of print]

Modulation of immunity in mice with latent toxoplasmosis-the experimental support for the immunosuppression hypothesis of Toxoplasma-induced changes in reproduction of mice and humans

Kaňková S, Holáň V, Zajícová A, Kodym P, Flegr J.

Department of Philosophy and History of Science, Faculty of Science, Charles University in Prague, Vinicná 7, 128 44, Prague 2, Czech Republic.

Abstract
The immunosuppression hypothesis suggests that the increased sex ratio in mice and women with latent toxoplasmosis, retarded embryonic growth in the early phases of pregnancy, prolonged pregnancy of Toxoplasma-infected women, and increased prevalence of toxoplasmosis in mothers of children with Down syndrome can be explained by the presumed immunosuppressive effects of latent toxoplasmosis. Here, we searched for indices of immunosuppression in mice experimentally infected with Toxoplasma gondii. Our results showed that mice in the early phase of latent infection exhibited temporarily increased production of interleukin (IL)-12 and decreased production of IL-10. In accordance with the immunosuppression hypothesis, the mice showed decreased production of IL-2 and nitric oxide and decreased proliferation reaction (synthesis of DNA) in the mixed lymphocyte culture in the early and also in the late phases of latent toxoplasmosis. Since about 30% of the world population are latently infected by T. gondii, the toxoplasmosis-associated immunosuppression might have serious public health consequences.

PMID: 20721578 [PubMed - as supplied by publisher]

Toxoplasma IgG and IgA, but not IgM, antibody titers increase in sera of immunocompetent mice in association with proliferation of tachyzoites

Microbes Infect. 2010 Aug 10. [Epub ahead of print]

Toxoplasma IgG and IgA, but not IgM, antibody titers increase in sera of immunocompetent mice in association with proliferation of tachyzoites in the brain during the chronic stage of infection
Singh J, Graniello C, Ni Y, Payne L, Sa Q, Hester J, Shelton BJ, Suzuki Y.

Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1410 Prices Fork Road, Blacksburg, VA 24061.

Abstract
Toxoplasma IgG and IgA, but not IgM, antibody titers were significantly higher in immunocompetent mice with cerebral proliferation of tachyzoites during the chronic stage of infection than those treated with sulfadiazine to inhibit the parasite growth. Their IgG and IgA antibody titers correlated significantly with the amounts of tachyzoite-specific SAG1 mRNA in their brains. In contrast, neither IgG, IgA, nor IgM antibody titers increased following two different doses of challenge infection in chronically infected mice. Increased antibody titers in IgG and IgA but not IgM may be a useful indicator suggesting an occurrence of cerebral tachyzoite growth in immunocompetent individuals chronically infected with T. gondii.

PMID: 20708090 [PubMed - as supplied by publisher]

Activity of the histone deacetylase inhibitor FR235222 on Toxoplasma gondii: inhibition of stage conversion of the parasite cyst form

Antimicrob Agents Chemother. 2010 Aug 16. [Epub ahead of print]

Activity of the histone deacetylase inhibitor FR235222 on Toxoplasma gondii: inhibition of stage conversion of the parasite cyst form and study of new derivative compounds

Maubon D, Bougdour A, Wong YS, Brenier-Pinchart MP, Curt A, Hakimi MA, Pelloux H.

Parasitology-Mycology Laboratory, Département des Agents Infectieux, Centre Hospitalier Universitaire, BP 217, 38043 Grenoble, cedex 09, France; UMR 5163, Laboratoire Adaptation et Pathogénie des Micro-organismes, ATIP+ group, CNRS-Université Joseph Fourier GRENOBLE 1, BP 170, F-38042 Grenoble cedex 9, France; Département de Pharmacochimie Moléculaire, Université de Grenoble, CNRS UMR 5063, CNRS ICMG FR 2607, Bâtiment E, 470 Rue de la Chimie, F-38 041 Grenoble cedex 9, France.

Abstract
Bradyzoite to tachyzoite conversion plays a role in pathogenesis of recrudescence of ocular toxoplasmosis and disease in immune compromised persons. The currently available medicines are ineffective on cysts and fail to prevent reactivation of latent toxoplasmosis. A previous paper showed that the histone deacetylase inhibitor FR235222 has a dramatic effect on tachyzoite growth and induces tachyzoite-to-bradyzoite conversion in vitro. This study shows that FR235222 can target in vitro converted cysts and bradyzoites. Moreover, the compound is active on ex vivo T. gondii cysts. Free bradyzoites isolated after lysis of the cell wall did not proliferate in vitro when the cyst was treated with FR235222. The results imply that this compound is able to cross the T. gondii cystic cell wall. Fluorescent labeling shows that the compound impairs the capacity of the bradyzoites to convert without damaging the cyst wall integrity. In vivo inoculation of formerly treated cysts fails to infect mice when these cysts were treated with FR235222. We used our structural knowledge of FR235222 and its target TgHDAC3 to synthesize new FR235222 derivative compounds. We identified two new molecules that are highly active against tachyzoites. They harbor a better selectivity index that is more suitable for a future in vivo approach in mice with chronic toxoplasmosis. These results identify FR235222 and its derivatives as new lead compounds in the range of therapeutics available in acute and chronic toxoplasmosis.

PMID: 20713670 [PubMed - as supplied by publisher]

Integrative Genomic Approaches Highlight a Family of Parasite-Specific Kinases that Regulate Host Responses

Cell Host Microbe. 2010 Aug 19;8(2):208-218.

Integrative Genomic Approaches Highlight a Family of Parasite-Specific Kinases that Regulate Host Responses

Peixoto L, Chen F, Harb OS, Davis PH, Beiting DP, Brownback CS, Ouloguem D, Roos DS.

Department of Biology and Penn Genome Frontiers Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.

Abstract
Apicomplexan parasites release factors via specialized secretory organelles (rhoptries, micronemes) that are thought to control host cell responses. In order to explore parasite-mediated modulation of host cell signaling pathways, we exploited a phylogenomic approach to characterize the Toxoplasma gondii kinome, defining a 44 member family of coccidian-specific secreted kinases, some of which have been previously implicated in virulence. Comparative genomic analysis suggests that "ROPK" genes are under positive selection, and expression profiling demonstrates that most are differentially expressed between strains and/or during differentiation. Integrating diverse genomic-scale analyses points to ROP38 as likely to be particularly important in parasite biology. Upregulating expression of this previously uncharacterized gene in transgenic parasites dramatically suppresses transcriptional responses in the infected cell. Specifically, parasite ROP38 downregulates host genes associated with MAPK signaling and the control of apoptosis and proliferation. These results highlight the value of integrative genomic approaches in prioritizing candidates for functional validation.

PMID: 20709297 [PubMed - as supplied by publisher]

Molecular characterization of a novel family of cysteine-rich proteins of Toxoplasma gondii and ultrastructural evidence of oocyst wall localization

Int J Parasitol. 2010 Aug 11. [Epub ahead of print]

Molecular characterization of a novel family of cysteine-rich proteins of Toxoplasma gondii and ultrastructural evidence of oocyst wall localization

Possenti A, Cherchi S, Bertuccini L, Pozio E, Dubey JP, Spano F.

Department of Infectious, Parasitic and Immunomediated Diseases, 299 - 00161 Rome, Italy.

Abstract
Among apicomplexan parasites, the coccidia and Cryptosporidium spp. are important pathogens of livestock and humans, and the environmentally resistant stage (oocyst) is essential for their transmission. Little is known of the chemical and molecular composition of the oocyst wall. Currently, the only parasite molecules shown to be involved in oocyst wall formation are the tyrosine-rich proteins gam56, gam82 and gam230 of Eimeria spp. and the cysteine-rich proteins COWP1 and COWP8 of Cryptosporidium parvum. In the present study, we searched the ToxoDB database for the presence of putative Toxoplasma gondii oocyst wall proteins (OWPs) and identified seven candidates, herein named TgOWP1 through TgOWP7, showing homology to the Cryptosporidium COWPs. We analysed a cDNA library from partially sporulated oocysts of T. gondii and cloned the full-length cDNAs encoding TgOWP1, TgOWP2 and TgOWP3, which consist of 499, 462 and 640 amino acids, respectively. The three proteins share 24% sequence identity with each other and a markedly similar overall structure, based on the presence of an N-terminal leader peptide followed by tandem duplications of a six-cysteine amino acid motif closely related to the Type I repeat of COWPs. Using antisera to recombinant TgOWP1, TgOWP2 and TgOWP3, we showed by Western blot that these molecules are expressed in T. gondii oocysts but are not detectable in tachyzoites. The solubilization of TgOWP1-3 strictly depended on the presence of reducing agents, consistent with a likely involvement of these proteins in multimeric complexes mediated by disulfide bridges. Immunofluorescence analysis allowed the localization of TgOWP1, TgOWP2 and TgOWP3 to the oocyst wall. Additionally, using immunoelectron microscopy and the 1G12 monoclonal antibody, TgOWP3 was specifically detected in the outer layer of the oocyst wall, thus representing the first validated molecular marker of this structure in T. gondii.

PMID: 20708619 [PubMed - as supplied by publisher]

A family of intermediate filament-like proteins is sequentially assembled into the cytoskeleton of Toxoplasma gondii

Cell Microbiol. 2010 Aug 2. [Epub ahead of print]

A family of intermediate filament-like proteins is sequentially assembled into the cytoskeleton of Toxoplasma gondii

Anderson-White BR, Ivey FD, Cheng K, Szatanek T, Lorestani A, Beckers CJ, Ferguson DJ, Sahoo N, Gubbels MJ.

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

Abstract
The intracellular protozoan parasite Toxoplasma gondii divides by a unique process of internal budding that involves the assembly of two daughter cells within the mother. The cytoskeleton of Toxoplasma, which is composed of microtubules associated with an inner membrane complex (IMC), has an important role in this process. The IMC, which is directly under the plasma membrane, contains a set of flattened membranous sacs lined on the cytoplasmic side by a network of filamentous proteins. This network contains a family of intermediate filament-like proteins or IMC proteins. In order to elucidate the division process, we have characterized a 14-member sub-family of Toxoplasma IMC proteins that share a repeat motif found in proteins associated with the cortical alveoli in all alveolates. By creating fluorescent protein fusion reporters for the family members we determined the spatio-temporal patterns of all 14 IMC proteins through tachyzoite development. This revealed several distinct distribution patterns and some provide the basis for novel structural models such as the assembly of certain family members into the basal complex. Furthermore we identified IMC15 as an early marker of budding and, lastly, the dynamic patterns observed throughout cytokinesis provide a timeline for daughter parasite development and division.

PMID: 20698859 [PubMed - as supplied by publisher]

Identification and Development of Novel Inhibitors of Toxoplasma gondii Enoyl Reductase

J Med Chem. 2010 Aug 10. [Epub ahead of print]

Identification and Development of Novel Inhibitors of Toxoplasma gondii Enoyl Reductase

Tipparaju SK, Muench SP, Mui EJ, Ruzheinikov SN, Lu JZ, Hutson SL, Kirisits MJ, Prigge ST, Roberts CW, Henriquez FL, Kozikowski AP, Rice DW, McLeod RL.

Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois.

Abstract
Toxoplasmosis causes significant morbidity and mortality, and yet available medicines are limited by toxicities and hypersensitivity. Because improved medicines are needed urgently, rational approaches were used to identify novel lead compounds effective against Toxoplasma gondii enoyl reductase (TgENR), a type II fatty acid synthase enzyme essential in parasites but not present in animals. Fifty-three compounds, including three classes that inhibit ENRs, were tested. Six compounds have antiparasite MIC(90)s < 6 muM without toxicity to host cells, three compounds have IC(90)s < 45 nM against recombinant TgENR, and two protect mice. To further understand the mode of inhibition, the cocrystal structure of one of the most promising candidate compounds in complex with TgENR has been determined to 2.7 A. The crystal structure reveals that the aliphatic side chain of compound 19 occupies, as predicted, space made available by replacement of a bulky hydrophobic residue in homologous bacterial ENRs by Ala in TgENR. This provides a paradigm, conceptual foundation, reagents, and lead compounds for future rational development and discovery of improved inhibitors of T. gondii.

PMID: 20698542 [PubMed - as supplied by publisher]

Genome-Wide Survey and Evolutionary Analysis of Trypsin Proteases in Apicomplexan Parasites

Genomics Proteomics Bioinformatics. 2010 Jun;8(2):103-112.

Genome-Wide Survey and Evolutionary Analysis of Trypsin Proteases in Apicomplexan Parasites

Arenas AF, Osorio-Méndez JF, Gutierrez AJ, Gomez-Marin JE.

Grupo de Parasitología Molecular (GEPAMOL), Centro de Investigaciones Biomédicas, Universidad del Quindío, Armenia, Colombia.

Abstract
Apicomplexa are an extremely diverse group of unicellular organisms that infect humans and other animals. Despite the great advances in combating infectious diseases over the past century, these parasites still have a tremendous social and economic burden on human societies, particularly in tropical and subtropical regions of the world. Proteases from apicomplexa have been characterized at the molecular and cellular levels, and central roles have been proposed for proteases in diverse processes. In this work, 16 new genes encoding for trypsin proteases are identified in 8 apicomplexan genomes by a genome-wide survey. Phylogenetic analysis suggests that these genes were gained through both intracellular gene transfer and vertical gene transfer. Identification, characterization and understanding of the evolutionary origin of protease-mediated processes are crucial to increase the knowledge and improve the strategies for the development of novel chemotherapeutic agents and vaccines. Copyright © 2010 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.

PMID: 20691395 [PubMed - as supplied by publisher]

Immunological response of sheep to injections of plasmids encoding Toxoplasma gondii SAG1 and ROP1 genes

Parasite Immunol. 2010 Sep;32(9-10):671-83.

Immunological response of sheep to injections of plasmids encoding Toxoplasma gondii SAG1 and ROP1 genes

Li B, Oledzka G, McFarlane RG, Spellerberg MB, Smith SM, Gelder FB, Kur J, Stankiewicz M.

Lincoln University - Agriculture and Life Sciences Division, Canterbury, New Zealand.

Abstract
Infection with the intracellular protozoan parasite Toxoplasma gondii (T. gondii) causes health problems to both humans and livestock and has a large economic impact worldwide. The immune response in sheep following infection with T. gondii was evaluated using six different combinations of plasmid DNA, recombinant antigen and adjuvant. Sheep were generally vaccinated twice by intramuscular injection with plasmid DNA containing gene sequences for either the surface antigen (SAG1) or the rhoptry protein (ROP1) of T. gondii. Two of the groups injected with plasmid DNA SAG1 were boosted with recombinant protein (SAG1). We investigated the efficacy of including oligodeoxynucleotides (ODN) that contain CG motifs (CpG) and the gene coding for ovine granulocyte-macrophage colony stimulating factor (GM-CSF) as potential adjuvants. Administration of the plasmid encoding the ROP1 gene significantly enhanced both IFN-gamma production from peripheral blood cells when cultured in vitro with Toxoplasma antigen, and ROP1-specific IgG1 and IgG2 antibody levels present in serum. However, injection with SAG1 did not stimulate IFN-gamma production. These results indicate the potential of ROP1, given as plasmid DNA, as a potential vaccine candidate to protect sheep against T. gondii infection.

PMID: 20691019 [PubMed - in process]

Identification of New Pathogens in the Intraocular Fluid of Patients With Uveitis

Am J Ophthalmol. 2010 Aug 4. [Epub ahead of print]

Identification of New Pathogens in the Intraocular Fluid of Patients With Uveitis

de Groot-Mijnes JD, de Visser L, Zuurveen S, Martinus RA, Völker R, Ten Dam-Van Loon NH, de Boer JH, Postma G, de Groot RJ, Van Loon AM, Rothova A.

Department of Virology, University Medical Center Utrecht, Utrecht, The Netherlands; Department of Ophthalmology, University Medical Center Utrecht, Utrecht, The Netherlands.

Abstract
PURPOSE: To determine infectious causes in patients with uveitis of unknown origin by intraocular fluids analysis. DESIGN: Case-control study. METHODS: Ocular fluids from 139 patients suspected of infectious uveitis, but negative for herpes simplex virus, varicella-zoster virus, cytomegalovirus, and Toxoplasma gondii by polymerase chain reaction and/or antibody analysis in intraocular fluids, were assessed for the presence of 18 viruses and 3 bacteria by real-time polymerase chain reaction (PCR). The ocular fluids from 48 patients with uveitis of known etiology or with cataract were included as controls. RESULTS: Positive PCR results were found for Epstein-Barr virus, for rubella virus, and for human herpesvirus 6 each in 1 patient and for human parechovirus in 4 patients. Of the human parechovirus-positive patients, 1 was immunocompromised and had panuveitis. The other 3 patients were immunocompetent and had anterior uveitis, all with corneal involvement. CONCLUSIONS: Human parechovirus might be associated with infectious (kerato)uveitis. Copyright © 2010 Elsevier Inc. All rights reserved.

PMID: 20691420 [PubMed - as supplied by publisher]

Transmission of Toxoplasmosis (Toxoplasma gondii) by Foods

Adv Food Nutr Res. 2010;60C:1-19.

Transmission of Toxoplasmosis (Toxoplasma gondii) by Foods

Pereira KS, Franco RM, Leal DA.

Departamento de Engenharia Bioquímica, Escola de Química, Centro de Tecnologia Bloco E - Sala 203, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.

Abstract
Protozoan foodborne diseases are generally underrecognized. Toxoplasma gondii is the causative agent of toxoplasmosis, one of the most prevalent parasitic infections to humans and domestic animals. The most likely source of T. gondii occurring through food is the consumption of raw or undercooked meat contaminated with tissue cysts. Sporulated T. gondii oocysts, from the feces of infected cats, present in the environment are a potential source of infection. The ingestion of water contaminated with oocysts and the eating of unwashed raw vegetables or fruits were identified as an important risk factor in most epidemiological studies. This review presents information and data to show the importance of T. gondii transmission by foods. Copyright © 2010 Elsevier Inc. All rights reserved.

PMID: 20691951 [PubMed - as supplied by publisher]

The Absence of IDO Upregulates Type I IFN Production, Resulting in Suppression of Viral Replication in the Retrovirus-Infected Mouse

J Immunol. 2010 Aug 6. [Epub ahead of print]

The Absence of IDO Upregulates Type I IFN Production, Resulting in Suppression of Viral Replication in the Retrovirus-Infected Mouse

Hoshi M, Saito K, Hara A, Taguchi A, Ohtaki H, Tanaka R, Fujigaki H, Osawa Y, Takemura M, Matsunami H, Ito H, Seishima M.

Department of Informative Clinical Medicine and.

Abstract
Indoleamine 2,3-dioxygenase, the l-tryptophan-degrading enzyme, plays a key role in the powerful immunomodulatory effects on several different types of cells. Because modulation of IDO activities after viral infection may have great impact on disease progression, we investigated the role of IDO following infection with LP-BM5 murine leukemia virus. We found suppressed BM5 provirus copies and increased type I IFNs in the spleen from IDO knockout (IDO(-/-)) and 1-methyl-d-l-tryptophan-treated mice compared with those from wild-type (WT) mice. Additionally, the number of plasmacytoid dendritic cells in IDO(-/-) mice was higher in the former than in the WT mice. In addition, neutralization of type I IFNs in IDO(-/-) mice resulted in an increase in LP-BM5 viral replication. Moreover, the survival rate of IDO(-/-) mice or 1-methyl-d-l-tryptophan-treated mice infected with LP-BM5 alone or with both Toxoplasma gondii and LP-BM5 was clearly greater than the survival rate of WT mice. To our knowledge, the present study is the first report to observe suppressed virus replication with upregulated type I IFN in IDO(-/-) mice, suggesting that modulation of the IDO pathway may be an effective strategy for treatment of virus infection.

PMID: 20693424 [PubMed - as supplied by publisher]

Comparative Analysis of Stage Specific Gene Regulation of Apicomplexan Parasites: Plasmodium falciparum and Toxoplasma gondii

Infect Disord Drug Targets. 2010 Aug 1;10(4):240-1.

Comparative Analysis of Stage Specific Gene Regulation of Apicomplexan Parasites: Plasmodium falciparum and Toxoplasma gondii

López-Estraño C.

Department of Biology, The University of Memphis, Memphis, TN 38152, USA. cestrano@memphis.edu.

PMID: 20687894 [PubMed - in process]

Biogenesis of the Inner Membrane Complex Is Dependent on Vesicular Transport by the Alveolate Specific GTPase Rab11B

PLoS Pathog. 2010 Jul 29;6(7):e1001029.

Biogenesis of the Inner Membrane Complex Is Dependent on Vesicular Transport by the Alveolate Specific GTPase Rab11B

Agop-Nersesian C, Egarter S, Langsley G, Foth BJ, Ferguson DJ, Meissner M.

Department of Infectiology, Parasitology, University Hospital Heidelberg, Heidelberg, Germany.

Abstract
Apicomplexan parasites belong to a recently recognised group of protozoa referred to as Alveolata. These protists contain membranous sacs (alveoli) beneath the plasma membrane, termed the Inner Membrane Complex (IMC) in the case of Apicomplexa. During parasite replication the IMC is formed de novo within the mother cell in a process described as internal budding. We hypothesized that an alveolate specific factor is involved in the specific transport of vesicles from the Golgi to the IMC and identified the small GTPase Rab11B as an alveolate specific Rab-GTPase that localises to the growing end of the IMC during replication of Toxoplasma gondii. Conditional interference with Rab11B function leads to a profound defect in IMC biogenesis, indicating that Rab11B is required for the transport of Golgi derived vesicles to the nascent IMC of the daughter cell. Curiously, a block in IMC biogenesis did not affect formation of sub-pellicular microtubules, indicating that IMC biogenesis and formation of sub-pellicular microtubules is not mechanistically linked. We propose a model where Rab11B specifically transports vesicles derived from the Golgi to the immature IMC of the growing daughter parasites.

PMID: 20686666 [PubMed - in process]

Transmission dynamics of Toxoplasma gondii along an urban-rural gradient

Theor Popul Biol. 2010 Jun 2. [Epub ahead of print]

Transmission dynamics of Toxoplasma gondii along an urban-rural gradient

Lélu M, Langlais M, Poulle ML, Gilot-Fromont E.

Université de Lyon, F-69000, Lyon; Université Lyon 1; CNRS, UMR5558, Laboratoire de Biométrie et Biologie Evolutive, F-69622, Villeurbanne, France; 2C2A-CERFE, 5 rue de la Héronniére, F-08240 Boult-aux-Bois, France; Université de Reims Champagne-Ardenne, Laboratoire de Parasitologie-Mycologie, EA 3800, UFR de Médecine, IFR 53, 51 rue Cognacq-Jay, F-51096 Reims, France.

Abstract
Recently, several authors proposed that the availability of intermediate hosts (IH) for definitive hosts (DH) may contribute to determine the dynamics and evolutionary ecology of parasites with facultative complex life cycles. The protozoa Toxoplasma gondii may be transmitted to DH either via predation of infected IH through a complex life cycle (CLC) or directly from contaminated environment through a simple life cycle (SLC). This parasite is also present in contrasting host density environments. We tested the hypothesis that the relative contributions of CLC and SLC along an urban-rural gradient depend on the IH supply. We built and analysed a deterministic model of T. gondii transmission cycle. SLC relative contribution is important only in urban-type environments, i.e., with low predation rate on IH. In contrast, the parasite is predominantly transmitted through CLC in suburban and rural environments. The association of the two cycles enables the parasite to spread in situations of low IH availability and low DH population size for which each cycle alone is insufficient. Copyright © 2010. Published by Elsevier Inc.

PMID: 20685358 [PubMed - as supplied by publisher]

Structure of the micronemal protein 2 (MIC2) A/I domain from Toxoplasma gondii

Protein Sci. 2010 Aug 3. [Epub ahead of print]

Structure of the micronemal protein 2 (MIC2) A/I domain from Toxoplasma gondii

Tonkin ML, Grujic O, Pearce M, Crawford J, Boulanger MJ.

Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia V8W 3P6.

Abstract
Toxoplasma gondii is a widespread zoonotic pathogen capable of causing serious disease in humans and animals. As an obligate intracellular parasite, T. gondii relies on the orchestrated secretion of proteins from its apical complex organelles including the multi-modular, transmembrane micronemal protein 2 (MIC2) that couples recognition of the host cell with cytoskeletal reorganization of the parasite to drive invasion. To probe the basis by which the von Willebrand Factor A (vWA) - Integrin like module of TgMIC2 engages the host cell, we solved the crystal structure of a truncated form of TgMIC2A/I (TgMIC2A/Ic) phased by iodide SIRAS and refined to a resolution of 2.05A. The TgMIC2A/Ic core is organized into a central twisted beta sheet flanked by alpha helices consistent with a canonical vWA fold. A restricted basic patch serves as the putative heparin binding site, but no heparin binding was detected in native gel shift assays. Furthermore, no metal was observed in the metal ion dependent adhesion site (MIDAS). Structural overlays with homologous A/I domains reveal a divergent organization of the MIDAS beta4-alpha4 loop in TgMIC2A/Ic, which is stabilized through the burial of Phe195 into a deep pocket formed by Gly185. Intriguingly, Gly185 appears be unique among A/I domains to TgMIC2A/I suggesting that the divergent loop conformation may also be unique to TgMIC2A/I. Though lacking the C-terminal extension, the TgMIC2A/Ic structure reported here is the first of an A/I domain from an apicomplexan parasite and provides valuable insight into defining the molecular recognition of host cells by these widespread pathogens.

PMID: 20684023 [PubMed - as supplied by publisher]

Toxoplasma gondii infection inhibits the mitochondrial apoptosis through induction of Bcl-2 and HSP70

Parasitol Res. 2010 Aug 3. [Epub ahead of print]

Toxoplasma gondii infection inhibits the mitochondrial apoptosis through induction of Bcl-2 and HSP70

Hwang IY, Quan JH, Ahn MH, Hassan Ahmed HA, Cha GH, Shin DW, Lee YH.

Department of Infection Biology, Research Institute for Medical Science, Chungnam National University School of Medicine, 6 Munhwa-dong, Jung-gu, Daejeon, 301-131, South Korea.

Abstract
Heat-shock protein 70 (HSP70) is highly expressed in Toxoplasma gondii-infected cells. However, the role of this protein is not well understood, especially during apoptosis. This study addresses the mechanism behind the antiapoptotic chaperone activity of HSP70 in Toxoplasma-infected host cells using a human macrophage cell line, THP-1 by Western blot, DNA fragmentation assay, immunoprecipitation, and a caspase-3/7 activity assay based on cleavage of the colorimetric substrate DEVD-pNA. Apoptosis induced by arsenic trioxide (As(2)O(3)) was inhibited in T. gondii-infected THP-1 cells, but not in uninfected cells. Without As(2)O(3) induction of apoptosis, T. gondii infection caused increased expression of Bcl-2 and HSP70, but not caspase-3. However, active form caspase-3 levels were lower in As(2)O(3)-treated infected cells as compared with As(2)O(3)-treated uninfected cells. Bcl-2 expression in As(2)O(3)-treated infected cells was similar to that in cells infected with T. gondii. Translocation of apoptosis-inducing factor (AIF) and release of cytochrome c from mitochondria were inhibited in As(2)O(3)-treated infected cells as compared with As(2)O(3)-treated uninfected cells. Increased parasite loads in Toxoplasma-infected macrophages caused higher HSP70 and Bcl-2 expression in whole-cell extracts and fractionated components, respectively. However, expression of AIF and cytochrome c was unaffected. Toxoplasma dose-dependently inhibited caspase-3 activation, thus revealing an anti-apoptotic parasite activity on cytochrome c-mediated caspase activation in subcellular components. In addition, immunoprecipitation analysis suggested that HSP70 is capable of binding to the pro-apoptotic factors AIF and Apaf-1, but not to cytochrome c or procaspase-9. Taken together, these data demonstrate that T. gondii infection inhibits mitochondrial apoptosis through overproduction of anti-apoptotic Bcl-2 as well as HSP70, which are increased parasite loads dependently.

PMID: 20680337 [PubMed - as supplied by publisher]

Toxoplasma gondii Protease TgSUB1 is Required for Cell Surface Processing of Micronemal Adhesive Complexes and Efficient Adhesion of Tachyzoites

Cell Microbiol. 2010 Jul 30. [Epub ahead of print]

Toxoplasma gondii Protease TgSUB1 is Required for Cell Surface Processing of Micronemal Adhesive Complexes and Efficient Adhesion of Tachyzoites

Lagal V, Binder EM, Huynh MH, Kafsack BF, Harris PK, Diez R, Chen D, Cole RN, Carruthers VB, Kim K.

Departments of Medicine and Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY 10461 USA.

Abstract
Abstract Host cell invasion by Toxoplasma gondii is critically dependent upon adhesive proteins secreted from the micronemes. Proteolytic trimming of microneme contents occurs rapidly after their secretion onto the parasite surface and is proposed to regulate adhesive complex activation to enhance binding to host cell receptors. However, the proteases responsible and their exact function are still unknown. In this report, we show that T. gondii tachyzoites lacking the microneme subtilisin protease TgSUB1 have a profound defect in surface processing of secreted microneme proteins. Notably parasites lack protease activity responsible for proteolytic trimming of MIC2, MIC4 and M2AP after release onto the parasite surface. Although complementation with full-length TgSUB1 restores processing, complementation of Deltasub1 parasites with TgSUB1 lacking the GPI anchor (Deltasub1::DeltaGPISUB1) only partially restores microneme protein processing. Loss of TgSUB1 decreases cell attachment and in vitro gliding efficiency leading to lower initial rates of invasion. Deltasub1 andDeltasub1::DeltaGPISUB1 parasites are also less virulent in mice. Thus TgSUB1 is involved in micronemal protein processing and regulation of adhesive properties of macromolecular adhesive complexes involved in host cell invasion.

PMID: 20678172 [PubMed - as supplied by publisher]

Synthesis and evaluation of oryzalin analogs against Toxoplasma

Bioorg Med Chem Lett. 2010 Jul 8. [Epub ahead of print]

Synthesis and evaluation of oryzalin analogs against Toxoplasma gondii

Endeshaw MM, Li C, Leon JD, Yao N, Latibeaudiere K, Premalatha K, Morrissette N, Werbovetz KA.

Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210-1291, USA.

Abstract
The synthesis and evaluation of 20 dinitroanilines and related compounds against the obligate intracellular parasite Toxoplasmagondii is reported. Using in vitro cultures of parasites in human fibroblasts, we determined that most of these compounds selectively disrupted Toxoplasma microtubules, and several displayed sub-micromolar potency against the parasite. The most potent compound was N(1),N(1)-dipropyl-2,6-dinitro-4-(trifluoromethyl)-1,3-benzenediamine (18b), which displayed an IC(50) value of 36nM against intracellular T. gondii. Based on these data and another recent report [Ma, C.; Tran, J.; Gu, F.; Ochoa, R.; Li, C.; Sept, D.; Werbovetz, K.; Morrissette, N. Antimicrob. AgentsChemother. 2010, 54, 1453], an antimitotic structure-activity relationship for dinitroanilines versus Toxoplasma is presented. Copyright © 2010 Elsevier Ltd. All rights reserved.

PMID: 20675138 [PubMed - as supplied by publisher]