Saturday, November 27, 2010

The antibiotic monensin causes cell cycle disruption of Toxoplasma gondii, mediated through the DNA repair enzyme TgMSH-1

Antimicrob Agents Chemother. 2010 Nov 22. [Epub ahead of print]

The antibiotic monensin causes cell cycle disruption of Toxoplasma gondii, mediated through the DNA repair enzyme TgMSH-1

Lavine MD, Arrizabalaga G.

Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Life Sciences South Room 142, Moscow, ID 83843, USA.

Abstract
Monensin is a polyether ionophore antibiotic that is widely used in the control of coccidia in animals. Despite its significance in veterinary medicine, little is known about its mode of action and potential mechanisms of resistance in coccidian parasites. Here we show that monensin causes accumulation of the coccidian Toxoplasma gondii at an apparent late S-phase cell cycle checkpoint. In addition, experiments utilizing a monensin-resistant T. gondii mutant show that this effect of monensin is dependent on the function of a mitochondrial homologue of the Mut-S DNA damage repair enzyme (TgMSH-1). Furthermore, the same TgMSH-1 dependent cell cycle disruption is observed with the anti-parasitic ionophore salinomycin and the DNA alkylating agent methyl nitrosourea. Our results suggest a novel mechanism for the mode of action of monensin and salynomicin on coccidial parasites, in which the drug activates a MSH-1 dependent cell cycle checkpoint by an unknown mechanism, ultimately leading to the death of the parasite. This model would indicate that cell cycle disruption is an important mediator of drug susceptibility and resistance to ionophoric antibiotics in coccidian parasites.

PMID: 21098240 [PubMed - as supplied by publisher]

Intracellular transport of Toxoplasma gondii through the blood-brain barrier

J Neuroimmunol. 2010 Nov 22. [Epub ahead of print]

Intracellular transport of Toxoplasma gondii through the blood-brain barrier

Lachenmaier SM, Deli MA, Meissner M, Liesenfeld O.

Institute of Microbiology and Hygiene, Charité-Medical School Berlin, Campus Benjamin Franklin, Berlin, Germany.

Abstract
Toxoplasma gondii establishes latent infection in the central nervous system of immunocompentent hosts. Toxoplasmic encephalitis is a life threatening reactivation of latent infection in the brain of immunocompromised patients. To further understand the mechanisms of entry into the brain of T. gondii we investigated host molecules and cells involved in the passage of the parasite through the blood-brain barrier. First, using microarrays brain endothelial cells were found to upregulate, among others, chemokines and adhesion molecules following infection with tachyzoites. Using flow cytometry we observed upregulated ICAM-1 expression on the surface of brain endothelial cells following infection; ICAM-1 expression was further increased after pre-incubation with IFN-γ. Compared to RH tachyzoites, ME49 tachyzoites induced a stronger upregulation of ICAM-1 and an earlier and stronger IL-6 and MCP-1 secretion by brain endothelial cells. Using an in vitro coculture model of the BBB (primary glia cells and brain endothelial cells) we found a stronger migration of infected antigen-presenting cells compared to lymphocytes (4.63% vs. 0.6% of all cells) across the BBB. Among all antigen-presenting cells CD11b(+)/CD11c(+) cells showed the highest infection rate, whereas the majority of infected cells that migrated through the blood-brain barrier were CD11b(+)/CD11c(-) cells. Infection of PBMCs with type I or type II Toxoplasma strains resulted in similar patterns of cell migration across the in vitro BBB model. In conclusion, these results suggest that T. gondii modulates gene expression of brain endothelial cells to promote its own migration through the blood-brain barrier in a 'Trojan horse' manner. Cells expressing CD11b either with or without CD11c are likely candidate cells for the intracellular transport of T. gondii across the BBB. T. gondii type I and type II strains induced similar migration patterns of antigen-presenting cells across the in vitro BBB.

Copyright © 2010 Elsevier B.V. All rights reserved.
PMID: 21106256 [PubMed - as supplied by publisher]

Protective immunity induced by Toxoplasma gondii rhoptry protein 16 (ROP16) against toxoplasmosis in mice

Clin Vaccine Immunol. 2010 Nov 24. [Epub ahead of print]

Protective immunity induced by Toxoplasma gondii rhoptry protein 16 (ROP16) against toxoplasmosis in mice

Yuan ZG, Zhang XX, He XH, Petersen E, Zhou DH, He Y, Lin RQ, Li XZ, Chen XL, Shi XR, Zhong XL, Zhang B, Zhu XQ.

State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, CAAS, Lanzhou, Gansu Province 730046, PR China; Department of Parasitology, College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong Province 510642, PR China; College of Agriculture, South China Agricultural University, 483 Wushan Street, Tianhe District, Guangzhou, Guangdong Province 510642, PR China; Department of Infectious Diseases, Clinical Institute, and Institute of Medical Microbiology and Immunology, Faculty of Health Sciences, Aarhus University, Aarhus, Denmark; College of Animal Science and Technology, Yunnan Agricultural University, Kunming, Yunnan Province 650201, PR China.

Abstract
Toxoplasma gondii can infect a large variety of domestic and wild animals and human beings, sometimes causing severe pathology. Rhoptries are involved in T. gondii invasion and host cell interaction, and have been implicated as important virulence factors. In this study, we constructed a DNA vaccine expressing rhoptry protein 16 (ROP16) of T. gondii, and evaluated the immune responses it induced in Kunming mice. The gene sequence encoding ROP16 was inserted into the eukaryotic expression vector pVAX I. We immunized Kunming mice intramuscularly. After immunization, we evaluated the immune response using lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged lethally. The results showed that mice immunized with pVAX-ROP16 developed a high level of specific antibody responses against T. gondii ROP16 expressed in Escherichia coli, a strong lymphoproliferative response, and significant levels of IFN-γ, IL-2, IL-4 and IL-10 production, compared with other mice immunized with either empty plasmid or phosphate-buffered saline, respectively. The results showed that pVAX-ROP16 induces significant humoral and cellular Th1 immune responses. After lethal challenge, the mice immunized with the pVAX-ROP16 showed a significantly (P values < 0.05) prolonged survival time (21.6 ± 9.9 days) compared with control mice which died within 7 days of challenge. Our data demonstrate, for the first time, that ROP16 triggered a strong humoral and cellular response against T. gondii, and that ROP16 is a promising vaccine candidate against toxoplasmosis, worth further development.

PMID: 21106780 [PubMed - as supplied by publisher]

NALP1 Influences Susceptibility to Human Congenital Toxoplasmosis, Pro-Inflammatory Cytokine Response and Fate of T. gondii-Infected Monocytic Cells

Infect Immun. 2010 Nov 22. [Epub ahead of print]

NALP1 Influences Susceptibility to Human Congenital Toxoplasmosis, Pro-Inflammatory Cytokine Response and Fate of T. gondii-Infected Monocytic Cells

Witola WH, Mui E, Hargrave A, Liu S, Hypolite M, Montpetit A, Cavailles P, Bisanz C, Cesbron-Delauw MF, Fournié GJ, McLeod R.

Departments of Surgery (Ophthalmology) and Pediatrics (Infectious Disease), The University of Chicago, Chicago, Illinois 60637, USA; Laboratoire Adaptation et Pathogénie des Micro-organismes, CNRS UMR 5163, Université Joseph Fourier GRENOBLE 1, Institut Jean Roget, BP 170, 38042 Grenoble cedex 9, France; Centre d'Innovation, Génome Québec, Montréal, Québec H3A 1A4, Canada; INSERM, U563, F-31000 Toulouse, France and University Toulouse III Paul Sabatier, F-31000 Toulouse, France.

Abstract
NALP1 is a member of the NOD-like receptor (NLR) family proteins that form inflammasomes. Upon cellular infection or stress, inflammasomes are activated, triggering maturation of pro-inflammatory cytokines and downstream cellular signaling mediated through the MyD88 adaptor. Toxoplasma gondii is an obligate intracellular parasite that stimulates production of high levels of pro-inflammatory cytokines important in innate immunity. Herein, susceptibility alleles for human congenital toxoplasmosis in NALP1 gene were identified. To investigate the role of the NALP1 inflammasome during infection with T. gondii, we genetically engineered a human monocytic cell line for NALP1 gene knockdown by RNA interference. NALP1 silencing attenuated progression of T. gondii infection, with accelerated host cell death and eventual cell disintegration. In line with this observation, up-regulation of pro-inflammatory cytokines IL-1β, IL-18 and IL-12 with T. gondii infection was not observed in monocytic cells with NALP1 knockdown. These findings suggest that the NALP1 inflammasome is critical for mediating innate immune responses to T. gondii infection and pathogenesis. Although there have been recent advances in understanding the potent activity of inflammasomes in directing innate immune responses to disease, to our knowledge, this is the first report on the crucial role of NALP1 inflammasome in the pathogenesis of T. gondii infections in humans.

PMID: 21098108 [PubMed - as supplied by publisher]

UNC93B1 is essential for TLR11 activation and IL-12 dependent host resistance to Toxoplasma gondii

J Biol Chem. 2010 Nov 19. [Epub ahead of print]

UNC93B1 is essential for TLR11 activation and IL-12 dependent host resistance to Toxoplasma gondii

Pifer R, Benson A, Sturge CR, Yarovinsky F.

University of Texas Southwestern Medical Center at Dallas, United States.

Abstract
Toll-like receptor (TLR) activation relies on biochemical recognition of microbial molecules and localization of the TLR within specific cellular compartments. Cell surface TLRs largely recognize bacterial membrane components, and intracellular TLRs are exclusively involved in sensing nucleic acids. Here we show that TLR11, an innate sensor for the Toxoplasma protein profilin, is an intracellular receptor that resides in the endoplasmic reticulum. The twelve membrane-spanning endoplasmic reticulum-resident protein UNC93B1 interacts directly with TLR11 and regulates the activation of dendritic cells (DC) in response to T. gondii profilin and parasitic infection in vivo. A deficiency in functional UNC93B1 protein abolished TLR11-dependent IL-12 secretion by DC, attenuated Th1 responses against T. gondii, and dramatically enhanced susceptibility to the parasite. Our results reveal that the association with UNC93B1 and the intracellular localization of TLRs is not a unique feature of nucleic acid-sensing TLRs but is also essential for TLR11-dependent recognition of T. gondii profilin and for host protection against this parasite.

PMID: 21097503 [PubMed - as supplied by publisher]

Towards an immunosense vaccine to prevent toxoplasmosis: Protective Toxoplasma gondii epitopes restricted by HLA-A*0201

Vaccine. 2010 Nov 20. [Epub ahead of print]

Towards an immunosense vaccine to prevent toxoplasmosis: Protective Toxoplasma gondii epitopes restricted by HLA-A*0201

Cong H, Mui EJ, Witola WH, Sidney J, Alexander J, Sette A, Maewal A, McLeod R.

Department of Surgery, The University of Chicago, 5841 S. Maryland Ave., MC 2114, Chicago, IL 60637, USA.

Abstract
The ideal vaccine to protect against toxoplasmosis in humans would include antigens that elicit a protective T helper cell type 1 immune response, and generate long-lived IFN-γ-producing CD8(+) T cells. Herein, we utilized a predictive algorithm to identify candidate HLA-A02 supertype epitopes from Toxoplasma gondii proteins. Thirteen peptides elicited production of IFN-γ from PBMC of HLA-A02 supertype persons seropositive for T. gondii infection but not from seronegative controls. These peptides displayed high-affinity binding to HLA-A02 proteins. Immunization of HLA-A*0201 transgenic mice with these pooled peptides, with a universal CD4(+) epitope peptide called PADRE, formulated with adjuvant GLA-SE, induced CD8(+) T cell IFN-γ production and protected against parasite challenge. Peptides identified in this study provide candidates for inclusion in immunosense epitope-based vaccines.

Copyright © 2010. Published by Elsevier Ltd.
PMID: 21095258 [PubMed - as supplied by publisher]

Evaluation of the immune response induced by DNA vaccine cocktail expressing complete SAG1 and ROP2 genes against toxoplasmosis

Vaccine. 2010 Nov 20. [Epub ahead of print]

Evaluation of the immune response induced by DNA vaccine cocktail expressing complete SAG1 and ROP2 genes against toxoplasmosis

Hoseinian Khosroshahi K, Ghaffarifar F, D'Souza S, Sharifi Z, Dalimi A.

Dept. Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

Abstract
Toxoplasma gondii, the pathogen of toxoplasmosis, can infect most mammals and birds. The high incidence and severe or lethal damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. We constructed a DNA cocktail, containing plasmids encoding the full-length SAG1 and ROP2 genes of T. gondii and evaluated its immune response and protective efficacy in comparison with single-gene vaccines and control groups. We immunized BALB/c mice intramuscularly three times. DNA cocktail elicited IgG and IFN-γ, TNF-α and IL-2 greater than single-gene plasmids and increased survival time against a lethal challenge with the highly virulent T. gondii RH strain. The current study shows that pc-SAG1+ pc-ROP2 as a cocktail DNA vaccine produces higher Th1 immune response than single-gene plasmids and cocktail DNA is effective to prime an enhanced and balanced specific immunity.

Copyright © 2010. Published by Elsevier Ltd.
PMID: 21095254 [PubMed - as supplied by publisher]

Segmental Meningomyelitis in 2 Cats Caused by Toxoplasma gondii

J Vet Intern Med. 2010 Nov 23. doi: 10.1111/j.1939-1676.2010.0635.x. [Epub ahead of print]

Segmental Meningomyelitis in 2 Cats Caused by Toxoplasma gondii

Alves L, Gorgas D, Vandevelde M, Gandini G, Henke D.

Department of Clinical Veterinary Medicine, Division of Clinical Neurology, Vetsuisse Faculty, University of Berne, Berne, Switzerland Division of Clinical Radiology, Vetsuisse Faculty, University of Berne, Berne, Switzerland Department of Veterinary Clinics, Faculty of Veterinary Medicine, University of Bologna, Bologna, Italy.

PMID: 21092003 [PubMed - as supplied by publisher]

Monday, November 22, 2010

Evaluation of three recombinant multi-antigenic vaccines composed of surface and secretory antigens of Toxoplasma gondii

Vaccine. 2010 Nov 15. [Epub ahead of print]

Evaluation of three recombinant multi-antigenic vaccines composed of surface and secretory antigens of Toxoplasma gondii in murine models of experimental toxoplasmosis

Dziadek B, Gatkowska J, Brzostek A, Dziadek J, Dzitko K, Grzybowski M, Dlugonska H.

Department of Immunoparasitology, University of Lodz, Lodz, Poland.

Abstract
The great clinical and economical impact of Toxoplasma gondii infections makes the development of an effective vaccine for controlling toxoplasmosis an extremely important aim. In the present study, we evaluate the protective and immunogenic properties of three recombinant subunit vaccines composed of rROP2+rGRA4+rSAG1, rROP2+rROP4+rGRA4 and rROP2+rROP4+rSAG1 proteins of T. gondii in an experimental toxoplasmosis model in the C3H/HeJ and C57BL/6 mouse strains. All three recombinant vaccines induced partial protection as measured by the reduction of brain cyst burden following challenge with five tissue cysts of the low virulence DX T. gondii strain. The level of protection was dependent on the antigen composition of the vaccine and the genetic background of the laboratory animals. The strongest protection against chronic toxoplasmosis was induced in both C3H/HeJ and C57BL/6 mice by the mixture of rhoptry proteins rROP2 and rROP4 combined with tachyzoite major protein SAG1. The average parasite burden in these groups of mice was reduced by 71% and 90%, respectively, compared to non-vaccinated mice. The observed protective effect was related to the vaccine-induced cellular and humoral immune responses, as measured by the antigen-induced release of the Th1 cytokines IFN-γ and IL-2, the antigen-stimulated proliferation of spleen cells of vaccinated animals in comparison to control animals and the development of systemic antigen-specific IgG1 and IgG2a (C3H/HeJ) or IgG2c (C57BL/6) antibodies. Our studies show that recombinant rROP2, rROP4, rGRA4 and rSAG1 antigens may be promising candidates for a subunit vaccine against toxoplasmosis. Additionally, we demonstrate that the ideal composition of vaccine antigens can be equally effective in mice with different genetic backgrounds and variable levels of innate resistance to toxoplasmosis, resulting in strong protection against T. gondii invasion.

Copyright © 2010. Published by Elsevier Ltd.
PMID: 21087690 [PubMed - as supplied by publisher]

Friday, November 19, 2010

New details on the fine structure of the rhoptry of Toxoplasma gondii

Microsc Res Tech. 2010 Nov 17. [Epub ahead of print]

New details on the fine structure of the rhoptry of Toxoplasma gondii

Lemgruber L, Lupetti P, De Souza W, Vommaro RC.

Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil.

Abstract
Rhoptries are organelles that have important, complex roles in Apicomplexa biology. During Toxoplasma gondii infection, these organelles take part in several essential and complex processes that include host cell entry and parasite development. Using different electron microscopy techniques, we characterized the fine morphology of the rhoptries of two of the most important life stages of T. gondii: the tachyzoite and the bradyzoite forms. The observed tachyzoite and bradyzoite rhoptries had delimited regions characterized by a dark and electron-dense neck, an amorphous and less electron-dense bulb, and a region of intermediate electron density, which connects the bulb to the neck. Metal replicas of frozen-fractured tachyzoites showed intramembranous particles of different densities and sizes on the fractured faces of rhoptry membranes. Both in tachyzoites and bradyzoites, the intramembranous particles were arranged in distinctive parallel arrays that decorated most part of these organelles. Tubulo-vesicular subcompartments and free particles within the rhoptry lumen were observed on freeze-fractured replicas. Cryo-fixed, deep-etched samples showed several pore-like structures localized in the bulb portion. No obvious evidence was found of a possible connection between rhoptries and micronemes. Microsc. Res. Tech., 2010. © 2010 Wiley-Liss, Inc.

PMID: 21086448 [PubMed - as supplied by publisher]

Tuesday, November 09, 2010

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]

Sunday, November 07, 2010

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]

Thursday, November 04, 2010

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.