Thursday, November 27, 2008

Apicomplexan cytoskeleton and motors: Key regulators in morphogenesis, cell division, transport and motility

Int J Parasitol. 2008 Nov 6. [Epub ahead of print]

Apicomplexan cytoskeleton and motors: Key regulators in morphogenesis, cell division, transport and motility

Santos JM, Lebrun M, Daher W, Soldati D, Dubremetz JF.

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

Protozoan parasites of the phylum Apicomplexa undergo a lytic cycle whereby a single zoite produced by the previous cycle has to encounter a host cell, invade it, multiply to differentiate into a new zoite generation and escape to resume a new cycle. At every step of this lytic cycle, the cytoskeleton and/or the gliding motility apparatus play a crucial role and recent results have elucidated aspects of these processes, especially in terms of the molecular characterization and interaction of the increasing number of partners involved, and the signalling mechanisms implicated. The present review aims to summarize the most recent findings in the field.

PMID: 19028497 [PubMed - as supplied by publisher]

The temperature-sensitive mutants of Toxoplasma gondii and ocular toxoplasmosis

Vaccine. 2008 Nov 18. [Epub ahead of print]

The temperature-sensitive mutants of Toxoplasma gondii and ocular toxoplasmosis

Lu F, Huang S, Kasper LH.

Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510080, PR China.

The risk of blindness caused by ocular toxoplasmosis supports efforts to improve our understanding for control of this disease. In this study, the involvement of CD8(+), CD4(+), B cell, and IL-10 gene in the immune response of primary ocular infection with the temperature-sensitive mutant (ts-4) of the RH Toxoplasma gondii strain, and in the protective immunity of ocular ts-4 vaccination and challenge with RH strain was investigated in murine models utilizing inbred C57BL/6 mice-deficient in CD4(+), CD8(+), B cells (muMT), or IL-10 gene. Compared to naive mice, all WT and mutant mice had different degree of ocular pathological changes after ts-4 ocular infection, in which both CD8 KO and IL-10 KO mice showed the most severe ocular lesions. Immunized by ts-4 intracameral (i.c.) inoculation, all mutant mice had partially decreased vaccine-induced resistance associated with increased ocular parasite burdens after RH strain challenge. A significant increase of the percentages of B cells and CD8(+) T cells in the draining lymph nodes were observed in WT and IL-10 KO mice after either infection or challenge. The levels of specific anti-toxoplasma IgG in both eye fluid and serum from all the mice were significantly increased after ts-4 i.c. immunization, except muMT mice. These results suggest that the avirulent ts-4 of T. gondii inoculated intracamerally can induce both ocular pathology and ocular protective immunity; CD4(+), CD8(+), B cell, and IL-10 gene are all necessary to the vaccine-induced resistance to ocular challenge by virulent RH strain, in which CD8(+) T cells are the most important component.

PMID: 19026704 [PubMed - as supplied by publisher]

Host cell autophagy is induced by Toxoplasma gondii and contributes to parasite growth

J Biol Chem. 2008 Nov 21. [Epub ahead of print]

Host cell autophagy is induced by Toxoplasma gondii and contributes to parasite growth

Wang Y, Weiss LM, Orlofsky A.

Pathology, Albert Einstein College of Medicine, Bronx, NY 10461.

Autophagy has been shown to contribute to defense against intracellular bacteria and parasites. In comparison, the ability of such pathogens to manipulate host cell autophagy to their advantage has not been examined. Here we present evidence that infection by Toxoplasma gondii, an intracellular protozoan parasite, induces host cell autophagy in both HeLa cells and primary fibroblasts, via a mechanism dependent on host Atg5 but independent of host mTOR suppression. Infection led to the conversion of LC3 to the autophagosome-associated form LC3-II, to the accumulation of LC3-containing vesicles near the parasitophorous vacuole, and to the relocalization towards the vacuole of structures labeled by the phosphatidylinositol-3-phosphate indicator, YFP-2xFYVE. The autophagy regulator beclin 1 was concentrated in the vicinity of the parasitophorous vacuole in infected cells. Inhibitor studies indicated that parasite-induced autophagy is dependent on calcium signaling and on abscisic acid. At physiologically relevant amino acid levels parasite growth became defective in Atg5-deficient cells, indicating a role for host cell autophagy in parasite recovery of host cell nutrients. A flow cytometric analysis of cell size as a function of parasite content revealed that autophagy-dependent parasite growth correlates with autophagy-dependent consumption of host cell mass that is dependent on parasite progression. These findings indicate a new role for autophagy as a pathway by which parasites may effectively compete with the host cell for limiting anabolic resources.

PMID: 19028680 [PubMed - as supplied by publisher]

Central nervous system infections in immunocompromised patients - update on diagnostics and therapy

Leuk Lymphoma. 2008 Nov 21:1-13. [Epub ahead of print]

Central nervous system infections in immunocompromised patients - update on diagnostics and therapy

Schmidt-Hieber M Md, Zweigner J, Uharek L, Blau IW, Thiel E.

Medizinische Klinik III, Charite Universitatsmedizin Berlin, Berlin, Germany.

Infections of the central nervous system (CNS) are increasingly reported in patients with malignancies. Heavily immunocompromised patients like those after allogeneic stem cell transplantation (SCT) or previous T cell depleting treatment regimens (e.g. with fludarabine or alemtuzumab) are at highest risk for cerebral infections. The spectrum of causative organisms may vary greatly, depending on the underlying malignancy, its treatment and various other factors. Toxoplasma gondii and fungi are the leading causative organisms in patients after allogeneic SCT, but also viruses such as herpes simplex virus or JC virus may be detected in these patients. Definitive diagnosis of cerebral infection still remains a high challenge, although diagnostics have improved by the wide availability of imaging techniques and polymerase chain reaction in recent years. Novel therapeutic options are arising, particularly for fungal CNS infections. Here, we summarise aspects on epidemiology, clinical symptoms and prognosis of CNS infections in patients with malignancies. Additionally, we give an overview on the diagnostics and management of cerebral infections in these patients including evidence evaluation of efficacy of treatment.

PMID: 19031169 [PubMed - as supplied by publisher]

Congenital Toxoplasmosis and Reinfection during Pregnancy

J Infect Dis. 2008 Nov 25. [Epub ahead of print]

Congenital Toxoplasmosis and Reinfection during Pregnancy: Case Report, Strain Characterization, Experimental Model of Reinfection, and Review

Elbez-Rubinstein A, Ajzenberg D, Dardé ML, Cohen R, Dumètre A, Yera H, Gondon E, Janaud JC, Thulliez P.

1Service de Néonatologie and 2Laboratoire de Bactériologie, Hôpital Intercommunal de Créteil, Créteil, 3Centre National de Référence Toxoplasmose/Toxoplasma Biological Resource Center, Centre Hospitalier-Universitaire Dupuytren, and 4Laboratoire de Parasitologie-Mycologie, EA 3174-NETEC, Faculté de Médecine, Université de Limoges, Limoges, and 5Institut de Puériculture de Paris, Paris, France.

We present a case of disseminated congenital toxoplasmosis in a newborn born to a mother who had been immunized against toxoplasmosis before conception. The mother was reinfected, likely by ingestion of imported raw horse meat during pregnancy. This clinical presentation is exceptional in France and raised the possibility of infection by a highly virulent Toxoplasma strain. The strain responsible was isolated from the peripheral blood of the newborn, and when genotyped with microsatellite markers, it exhibited an atypical genotype, one which is very uncommon in Europe but had been described in South America. We tested the hypothesis of a reinfection with a different genotype by using an experimental mouse model, which confirmed that acquired immunity against European Toxoplasma strains may not protect against reinfection by atypical strains acquired during travel outside Europe or by eating imported meat.

PMID: 19032062 [PubMed - as supplied by publisher]

Toxoplasma gondii infection blocks the development of allergic airway inflammation in BALB/c mice

Clin Exp Immunol. 2008 Nov 20. [Epub ahead of print]

Toxoplasma gondii infection blocks the development of allergic airway inflammation in BALB/c mice

Fenoy I, Giovannoni M, Batalla E, Martin V, Frank FM, Piazzon I, Goldman A.

Centro de Estudios en Salud y Medio Ambiente (CESyMA), Escuela de Ciencia y Tecnología, Universidad Nacional de San Martín, Argentina.

Summary There is a link between increased allergy and a reduction of some infections in western countries. Epidemiological data also show that respiratory allergy is less frequent in people exposed to orofaecal and foodborne microbes such as Toxoplasma gondii. Infection with T. gondii induces a strong cell-mediated immunity with a highly polarized T helper type 1 (Th1) response in early stages of infection. Using a well-known murine model of allergic lung inflammation, we sought to investigate whether T. gondii infection could modulate the susceptibility to develop respiratory allergies. Both acute and chronic infection with T. gondii before allergic sensitization resulted in a diminished allergic inflammation, as shown by a decrease in bronchoalveolar lavage (BAL) eosinophilia, mononuclear and eosinophil cell infiltration around airways and vessels and goblet cell hyperplasia. Low allergen-specific immunoglobulin (Ig)E and IgG1 and high levels of allergen-specific IgG2a serum antibodies were detected. A decreased interleukin (IL)-4 and IL-5 production by lymph node cells was observed, while no antigen-specific interferon-gamma increase was detected. Higher levels of the regulatory cytokine IL-10 were found in BAL from infected mice. These results show that both acute and chronic parasite infection substantially blocked development of airway inflammation in adult BALB/c mice. Our results support the hypothesis that T. gondii infection contributes to protection against allergy in humans.

PMID: 19032550 [PubMed - as supplied by publisher]

Friday, November 21, 2008

Prevention of toxoplasmosis in transplant patients

Clin Microbiol Infect. 2008 Nov 15. [Epub ahead of print]

Prevention of toxoplasmosis in transplant patients

Derouin F, Pelloux H; on behalf of the of the ESCMID Study Group on Clinical Parasitology.

Laboratory of Parasitology and Mycology, University Paris and Hôpital Saint-Louis, Assistance Publique-Hôpitaux de Paris, Paris, France.

Toxoplasmosis is a life-threatening opportunistic infection that affects haematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) recipients. Its incidence in these patients is closely related to the prevalence of toxoplasmosis in the general population, which is high in Europe. In SOT recipients, toxoplasmosis results mainly from transmission of the parasite with the transplanted organ from a Toxoplasma-seropositive donor to a Toxoplasma-seronegative recipient. This risk is high in cases of transplantation of organs that are recognized sites of encystation of the parasite, e.g. the heart, and is markedly lower in other SOT recipients. Clinical symptoms usually occur within the first 3 months after transplantation, sometimes as early as 2 weeks post transplant, and involve febrile myocarditis, encephalitis or pneumonitis. In HSCT recipients, the major risk of toxoplasmosis results from the reactivation of a pre-transplant latent infection in seropositive recipients. The median point of disease onset is estimated at 2 months post transplant, with <10% of cases occurring before 30 days and 15-20% later than day 100. Toxoplasmosis usually manifests as encephalitis or pneumonitis, and frequently disseminates with multiple organ involvement. Diagnosis of toxoplasmosis is based on the demonstration of parasites or parasitic DNA in blood, bone marrow, cerebrospinal fluid, bronchoalveolar lavage fluid or biopsy specimens, and serological tests do not often contribute to the diagnosis. For prevention of toxoplasmosis, serological screening of donors and recipients before transplantation allows the identification of patients at higher risk of toxoplasmosis, i.e. seropositive HSCT recipients and mismatched (seropositive donor/seronegative recipients) SOT recipients. Preventing toxoplasmosis disease in those patients presently relies on prophylaxis via prescription of co-trimoxazole.

PMID: 19018809 [PubMed - as supplied by publisher]

Toxoplasmosis infection in a kidney-transplant patient

NDT Plus. 2008 Dec;1(6):429-432. Epub 2008 Oct 18

Unusual presentation of primary toxoplasmosis infection in a kidney-transplant patient complicated by an acute left-ventricular failure

Hébraud B, Kamar N, Borde JS, Bessières MH, Galinier M, Rostaing L.

Department of Nephrology, Dialysis and Multiorgan Transplantation , University Hospital , CHU Rangueil.

Although primary toxoplasmosis is a rare event following kidney transplantation, it can be life threatening. This report describes this complication. The patient presented with high-grade fever, haemolytic anaemia and haemophagocytic-syndrome-related pancytopaenia. Toxoplasma gondii diagnosis was ascertained by blood and bone-marrow PCR assays. After 6 weeks with Clindamycin plus pyrimethamine therapies and despite negativation of T. gondii blood PCR assay, the patient developed left-ventricular failure. After adding sulfamethoxazole/ trimethoprim, ramipril, digoxine, bisoprolol and spironolactone, he progressively recovered. Anti-T. gondii therapy was continued for 6 months. Four years later he received a third kidney allograft: at that time anti-T. gondii antibodies had become negative. The outcome was uneventful despite immunosuppression but with inclusion of sulfamethoxazole/trimethoprim prophylaxis. More than 3 years after the third kidney transplantation the patient has had no toxoplasmosis reactivation. This case report highlights that T. gondii can be the cause of myocarditis in a renal transplant recipient.

PMID: 19020669 [PubMed]

Thursday, November 20, 2008

The histone methylase KMTox interacts with the redox-sensor peroxiredoxin-1 and targets genes involved in Toxoplasma gondii antioxidant defences

Mol Microbiol. 2008 Nov 5. [Epub ahead of print]

The histone methylase KMTox interacts with the redox-sensor peroxiredoxin-1 and targets genes involved in Toxoplasma gondii antioxidant defences

Sautel CF, Ortet P, Saksouk N, Kieffer S, Garin J, Bastien O, Hakimi MA.

Laboratoire Adaptation et Pathogénie des Micro-organismes, Université Joseph Fourier GRENOBLE 1, BP 170, F-38042 Grenoble cedex 9, France.

Summary The ability of living cells to alter their gene expression patterns in response to environmental changes is essential for viability. Oxidative stress represents a common threat for all aerobic life. In normally growing cells, in which hydrogen peroxide generation is transient or pulsed, the antioxidant systems efficiently control its concentration. Intracellular parasites must also protect themselves against the oxidative burst imposed by the host. In this work, we have investigated the role of KMTox, a new histone lysine methyltransferase, in the obligate intracellular parasite Toxoplasma gondii. KMTox is a nuclear protein that holds a High Mobility Group domain, which is thought to recognize bent DNA. The enzyme methylates both histones H4 and H2A in vitro with a great preference for the substrate in reduced conditions. Importantly, KMTox interacts specifically with the typical 2-cys peroxiredoxin-1 and the binding is to some extent enhanced upon oxidation. It appears that the cellular functions that are primarily regulated by the KMTox are antioxidant defences and maintenance of cellular homeostasis. KMTox may regulate gene expression in T. gondii by providing the rapid re-arrangement of chromatin domains and by interacting with the redox-sensor TgPrx1 contribute to establish the antioxidant 'firewall' in T. gondii.

PMID: 19017266 [PubMed - as supplied by publisher]

Wednesday, November 19, 2008

The Proinflammatory Cytokine Induced IRG1 Protein Associates with Mitochondria

J Interferon Cytokine Res. 2008 Nov 17. [Epub ahead of print]

The Proinflammatory Cytokine Induced IRG1 Protein Associates with Mitochondria

Degrandi D, Hoffmann R, Beuter-Gunia C, Pfeffer K.

Institute of Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University of Duesseldorf, Germany.

Interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF) are essential cytokines for successful clearance of microbial infections. Activation of macrophages by synergistic effects of these cytokines leads to induction of antimicrobial effector systems like reactive oxygen and reactive nitrogen intermediates. Strikingly, IFN-gammaR(-/-) and TNFRp55(-/-) mice are considerably more susceptible to infections than inducible nitric oxide synthase(-/-) and p47phox(-/-) mice. Thus we applied transcriptome-profiling studies to identify genes synergistically upregulated by IFN-gamma and TNF in macrophages which are potentially involved in the defense against intracellular pathogens. From a total of 234 regulated genes we found 35 genes that were upregulated by combined effects of IFN-gamma and TNF and were at least 2-fold induced. The majority of these genes are involved in signal transduction and transcriptional regulation. However, we found several genes were poorly characterized with regard to immunological functions. As a prototypic TNF- and IFN-gamma-coregulated gene we characterized the expression and the subcellular localization of immunoresponsive gene 1 (IRG1) in murine macrophages. IRG1 is highly upregulated in murine ANA-1 macrophages by several proinflammatory cytokines and Toll-like receptor (TLR) agonists, as well as in spleen and lung of Listeria monocytogenes or Toxoplasma gondii infected mice, respectively. Furthermore, this study identifies 35 genes that constitute the IFN-gamma/TNF-triggered effector program in innate immunity.

PMID: 19014335 [PubMed - as supplied by publisher]

Friday, November 14, 2008

Role of an Ancestral D-bifunctional Protein Containing Two Sterol-carrier Protein-2 Domains in Lipid Uptake and Trafficking

Mol Biol Cell. 2008 Nov 12. [Epub ahead of print]

Role of an Ancestral D-bifunctional Protein Containing Two Sterol-carrier Protein-2 Domains in Lipid Uptake and Trafficking in Toxoplasma

Lige B, Jayabalasingham B, Zhang H, Pypaert M, Coppens I.

Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Public Health, Baltimore, MD 21205; Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520.

Monitoring Editor: Howard Riezman The inability to synthesize cholesterol is universal among protozoa. The intracellular pathogen Toxoplasma depends on host lipoprotein-derived cholesterol to replicate in mammalian cells. Mechanisms of cholesterol trafficking in this parasite must be important for delivery to proper organelles. We characterized a unique D-bifunctional protein variant expressed by Toxoplasma consisting of one N-terminal D-3-hydroxyacyl-CoA dehydrogenase domain fused to two tandem sterol carrier protein-2 (SCP-2) domains. This multidomain protein undergoes multiple cleavage steps to release free SCP-2. The most C-terminal SCP-2 carries a PTS1 that directs the protein to vesicles before processing. Abrogation of this signal results in SCP-2 accumulation in the cytoplasm. Cholesterol specifically binds to parasite SCP-2 but with 10-fold lower affinity than phosphatidylcholine. In mammalian cells and Toxoplasma, the two parasite SCP-2 domains promote the circulation of various lipids between organelles and to the surface. Compared with wild-type parasites, TgHAD-2SCP-2-transfected parasites replicate faster and show enhanced uptake of cholesterol and oleate, which are incorporated into neutral lipids that accumulate at the basal end of Toxoplasma. This work provides the first evidence that the lipid transfer capability of an ancestral eukaryotic SCP-2 domain can influence the lipid metabolism of an intracellular pathogen to promote its multiplication in mammalian cells.

PMID: 19005217 [PubMed - as supplied by publisher]

Thursday, November 13, 2008

Stanley Medical Research Institute: T. gondii and schizophrenia

To: All researchers interested in Toxoplasma gondii

The Stanley Medical Research Institute (SMRI) is interested in the possible etiological relationship between T. gondii and schizophrenia, and we are supporting multiple research projects on this relationship. We have therefore created a section on our website summarizing past and present research that may be relevant; this can be accessed at (click on Laboratory of Developmental Neurovirology, then on Toxoplasmosis–Schizophrenia Research, then on the section you wish to view). We are applying for permission to make many of the most important papers available in PDF format. We welcome corrections and suggestions for this section and will keep it updated.

Periodically, SMRI will issue requests for proposals (RFPs) for specific research projects; such RFPs will be advertised in this section of the SMRI website. Investigators are free to contact either Dr. Torrey or Dr. Yolken regarding specific questions or ideas in this area.

E. Fuller Torrey, M.D.

Robert H. Yolken, M.D.

Post-treatment assessment of acute Toxoplasma infection during pregnancy

J Obstet Gynaecol. 2008 Aug;28(6):593-5

Post-treatment assessment of acute Toxoplasma infection during pregnancy

Habib FA.

Department of Obstetric and Gynecology, Faculty of Medicine, Taibah University, Saudi Arabia.

Serological immune profile in cases of Toxoplasma infection is heterogeneous, and responses may be delayed or fail to be represented: this makes it an unreliable method for diagnosis and/or treatment follow-up. Therefore, the present study relied on a sensitive and specific molecular procedure (nested polymerase chain reaction, PCR), using the whole blood sample to establish the diagnosis of acute maternal toxoplasmosis in 27 pregnant women. All of them received the recommended dose of Spiramycin. Only 19 returned for follow-up visits and completed their pregnancies to full term. The achievement of the treatment regimen was evaluated according to the results of PCR amplification of T. gondii DNA at the end of the treatment course. Patients who continued to have positive PCR results were given another treatment course. After treatment with a single course of Spiramycin, 11(57.9%) patients retained T. gondii DNA in their peripheral blood and in eight (42.1%) patients, T. gondii DNA was absent by PCR: four (21.01%) patients received up to three courses of treatment.

PMID: 19003652 [PubMed - in process]

Tpl2 kinase regulates T cell interferon-{gamma} production and host resistance

J Exp Med. 2008 Nov 10. [Epub ahead of print]

Tpl2 kinase regulates T cell interferon-{gamma} production and host resistance to Toxoplasma gondii

Watford WT, Hissong BD, Durant LR, Yamane H, Muul LM, Kanno Y, Tato CM, Ramos HL, Berger AE, Mielke L, Pesu M, Solomon B, Frucht DM, Paul WE, Sher A, Jankovic D, Tsichlis PN, O'Shea JJ.

Lymphocyte Cell Biology Section, Molecular Immunology and Inflammation Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892.

Tpl2 (Tumor progression locus 2), also known as Cot/MAP3K8, is a hematopoietically expressed serine-threonine kinase. Tpl2 is known to have critical functions in innate immunity in regulating tumor necrosis factor-alpha, Toll-like receptor, and G protein-coupled receptor signaling; however, our understanding of its physiological role in T cells is limited. We investigated the potential roles of Tpl2 in T cells and found that it was induced by interleukin-12 in human and mouse T cells in a Stat4-dependent manner. Deficiency of Tpl2 was associated with impaired interferon (IFN)-gamma production. Accordingly, Tpl2(-/-) mice had impaired host defense against Toxoplasma gondii with reduced parasite clearance and decreased IFN-gamma production. Furthermore, reconstitution of Rag2(-/-) mice with Tpl2-deficient T cells followed by T. gondii infection recapitulated the IFN-gamma defect seen in the Tpl2-deficient mice, confirming a T cell-intrinsic defect. CD4(+) T cells isolated from Tpl2(-/-) mice showed poor induction of T-bet and failure to up-regulate Stat4 protein, which is associated with impaired TCR-dependent extracellular signal-regulated kinase activation. These data underscore the role of Tpl2 as a regulator of T helper cell lineage decisions and demonstrate that Tpl2 has an important functional role in the regulation of Th1 responses.

PMID: 19001140 [PubMed - as supplied by publisher]

Intracellular survival of apicomplexan parasites and host cell modification

Int J Parasitol. 2008 Oct 25. [Epub ahead of print]

Intracellular survival of apicomplexan parasites and host cell modification

Lüder CG, Stanway RR, Chaussepied M, Langsley G, Heussler VT.

Institute for Medical Microbiology, Georg-August-University Göttingen, Kreuzbergring 57, 37075 Göttingen, Germany.

The intracellular stages of apicomplexan parasites are known to extensively modify their host cells to ensure their own survival. Recently, considerable progress has been made in understanding the molecular details of these parasite-dependent effects for Plasmodium-, Toxoplasma- and Theileria-infected cells. We have begun to understand how Plasmodium liver stage parasites protect their host hepatocytes from apoptosis during parasite development and how they induce an ordered cell death at the end of the liver stage. Toxoplasma parasites are also known to regulate host cell survival pathways and it has been convincingly demonstrated that they block host cell major histocompatibility complex (MHC)-dependent antigen presentation of parasite epitopes to avoid cell-mediated immune responses. Theileria parasites are the masters of host cell modulation because their presence 'immortalises' the infected cell. It is now accepted that multiple pathways are activated to induce Theileria-dependent host cell transformation. Although it is now known that similar host cell pathways are affected by the different parasites, the outcome for the infected cell varies considerably. Improved imaging techniques and new methods to control expression of parasite and host cell proteins will help us to analyse the molecular details of parasite-dependent host cell modifications.

PMID: 19000910 [PubMed - as supplied by publisher]

Tuesday, November 11, 2008

Targeting the transcriptional and translational machinery of the endosymbiotic organelle in apicomplexans

Curr Drug Targets. 2008 Nov;9(11):948-56.

Targeting the transcriptional and translational machinery of the endosymbiotic organelle in apicomplexans

Fleige T, Soldati-Favre D.

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

Apicomplexans are obligate intracellular parasites causing devastating disease in both humans and livestock. Nearly all apicomplexans, with the exception of Cryptosporidium, contain two endosymbiontic organelles carrying their own DNA; the mitochondrion and the plastid-like organelle called the apicoplast. The apicoplast is an attractive drug target as it harbors not only metabolic pathways not found in the host cell, but it is also dependent on its ancient transcriptional and translational machinery. These parasites rely on the plastid, and inhibition of its function or loss of this organelle leads to immediate or delayed death. Replication of plastidic DNA shows differences between the members of this phylum. In Plasmodium parasites, two forms of replication are observed - unidirectional single-stranded replication and a rolling circle mechanism - whereas in Toxoplasma gondii only the rolling circle is found. Targeting enzymes involved in DNA-replication leads to a delayed death of the parasite. Most of the genes in the apicoplast genome encode elements of their own transcriptional and translational machinery, and they are highly similar to those found in bacteria. Several anti-bacterials which target this machinery are also active against apicomplexan parasites and inhibition leads mostly to the delayed death phenomenon.

PMID: 18991607 [PubMed - in process]

Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II

Int J Parasitol. 2008 Oct 21. [Epub ahead of print]

Genetic identification of essential indels and domains in carbamoyl phosphate synthetase II of Toxoplasma gondii.

Fox BA, Ristuccia JG, Bzik DJ.

Department of Microbiology and Immunology, Dartmouth Medical School, 1 Medical Center Drive, 652E Borwell Building, Lebanon, NH 03756, USA.

New treatments need to be developed for the significant human diseases of toxoplasmosis and malaria to circumvent problems with current treatments and drug resistance. Apicomplexan parasites causing these lethal diseases are deficient in pyrimidine salvage, suggesting that selective inhibition of de novo pyrimidine biosynthesis can lead to a severe loss of uridine 5'-monophosphate (UMP) and thymidine 5'-monophosphate (dTMP) pools, thereby inhibiting parasite RNA and DNA synthesis. Disruption of Toxoplasma gondii carbamoyl phosphate synthetase II (CPSII) induces a severe uracil auxotrophy with no detectable parasite replication in vitro and complete attenuation of virulence in mice. Here we show that a CPSII cDNA minigene efficiently complements the uracil auxotrophy of CPSII-deficient mutants, restoring parasite growth and virulence. Our complementation assays reveal that engineered mutations within, or proximal to, the catalytic triad of the N-terminal glutamine amidotransferase (GATase) domain inactivate the complementation activity of T. gondii CPSII and demonstrate a critical dependence on the apicomplexan CPSII GATase domain in vivo. Surprisingly, indels present within the T. gondii CPSII GATase domain as well as the C-terminal allosteric regulatory domain are found to be essential. In addition, several mutations directed at residues implicated in allosteric regulation in Escherichia coli CPS either abolish or markedly suppress complementation and further define the functional importance of the allosteric regulatory region. Collectively, these findings identify novel features of T. gondii CPSII as potential parasite-selective targets for drug development.

PMID: 18992249 [PubMed - as supplied by publisher]

Toxoplasma gondii: Uptake and survival of oocysts in free-living amoebae

Exp Parasitol. 2008 Oct 18. [Epub ahead of print]

Toxoplasma gondii: Uptake and survival of oocysts in free-living amoebae

Winiecka-Krusnell J, Dellacasa-Lindberg I, Dubey JP, Barragan A.

Department of Parasitology, Mycology and Environmental Microbiology, Swedish Institute for Infectious Disease Control, 171 82 Solna, Sweden.

Waterborne transmission of the oocyst stage of Toxoplasma gondii can cause outbreaks of clinical toxoplasmosis in humans and infection of marine mammals. In water-related environments and soil, free-living amoebae are considered potential carriers of various pathogens, but knowledge on interactions with parasitic protozoa remains elusive. In the present study, we assessed whether the free-living Acanthamoebacastellanii, due to its phagocytic activity, can interact with T. gondii oocysts. We report that amoebae can internalize T. gondii oocysts by active uptake. Intracellular oocysts in amoebae rarely underwent phagocytic lysis, retained viability and established infection in mice. Interaction of T. gondii with amoebae did not reduce the infectivity and pathogenicity of oocysts even after prolonged co-cultivation. Our results show that uptake of oocysts by A. castellanii does not restrain the transmission of T. gondii in a murine infection model.

PMID: 18992742 [PubMed - as supplied by publisher]

Digest this! A role for autophagy in controlling pathogens

Cell Host Microbe. 2008 Nov 13;4(5):413-4

Digest this! A role for autophagy in controlling pathogens

Whitmarsh RJ, Hunter CA.

Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 380 South University Avenue, Philadelphia, PA 19104, USA.

Autophagy provides a mechanism for cells to conserve nutrients, but was recently associated with immunity to intracellular pathogens. Here, Zhao et al. (2008) present direct in vivo evidence that autophagy is linked to macrophage control of Toxoplasma gondii and Listeria monocytogenes and highlights that this process intersects with cytokine-mediated antimicrobial responses.

PMID: 18996340 [PubMed - in process]

Autophagosome-independent essential function for the autophagy protein atg5 in cellular immunity to intracellular pathogens

Cell Host Microbe. 2008 Nov 13;4(5):458-69

Autophagosome-independent essential function for the autophagy protein atg5 in cellular immunity to intracellular pathogens

Zhao Z, Fux B, Goodwin M, Dunay IR, Strong D, Miller BC, Cadwell K, Delgado MA, Ponpuak M, Green KG, Schmidt RE, Mizushima N, Deretic V, Sibley LD, Virgin HW.

Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.

The physiologic importance of autophagy proteins for control of mammalian bacterial and parasitic infection in vivo is unknown. Using mice with granulocyte- and macrophage-specific deletion of the essential autophagy protein Atg5, we show that Atg5 is required for in vivo resistance to the intracellular pathogens Listeria monocytogenes and Toxoplasma gondii. In primary macrophages, Atg5 was required for interferongamma (IFN-gamma)/LPS-induced damage to the T. gondii parasitophorous vacuole membrane and parasite clearance. While we did not detect classical hallmarks of autophagy, such as autophagosomes enveloping T. gondii, Atg5 was required for recruitment of IFN-gamma-inducible p47 GTPase IIGP1 (Irga6) to the vacuole membrane, an event that mediates IFN-gamma-mediated clearance of T. gondii. This work shows that Atg5 expression in phagocytic cells is essential for cellular immunity to intracellular pathogens in vivo, and that an autophagy protein can participate in immunity and intracellular killing of pathogens via autophagosome-independent processes such as GTPase trafficking.

PMID: 18996346 [PubMed - in process]

Proteomes and transcriptomes of Apicomplexa - Where's the message?

Int J Parasitol. 2008 Nov 1. [Epub ahead of print]

Proteomes and transcriptomes of Apicomplexa - Where's the message?

Wastling JM, Xia D, Sohal A, Chaussepied M, Pain A, Langsley G.

Department of Pre-Clinical Veterinary Science, Faculty of Veterinary Science, University of Liverpool, Liverpool L69 7ZJ, UK.

The Apicomplexa now have some of the most comprehensive and integrated proteome datasets of all pathogenic micro-organisms. Coverage is currently at a level where these data can be used to help predict the potential biological function of proteins in these parasites, without having to defer to measurement of mRNA levels. Transcriptomic data for the Apicomplexa (microarrays, expressed sequence tag (EST) collections, serial analysis of gene expression (SAGE) and massively parallel signature sequencing (MPSS) tags) are also copious, enabling us to investigate the extent to which global mRNA levels correlate with proteomic data. Here, we present a proteomic and transcriptomic perspective of gene expression in key apicomplexan parasites, including Plasmodium spp., Toxoplasma gondii, Cryptosporidium parvum, Neospora caninum and Theileria spp., and discuss the alternative views of gene expression that they provide. Although proteomic evidence does not detect every protein for every gene, many examples of readily detected proteins whose corresponding genes display little or no detectable transcription, are detected across the Apicomplexa. These examples are not easily explained by the "guilt by association", or "stock and go" hypotheses of gene transcription. With the advent of ultra-high-throughput sequencing technologies there will be a quantum shift in transcriptional analysis which, combined with improving quantitative proteome datasets, will provide a core component of a systems-wide approach to studying the Apicomplexa.

PMID: 18996390 [PubMed - as supplied by publisher]

Statins inhibit Toxoplasma gondii multiplication in macrophages in vitro

Int J Antimicrob Agents. 2008 Nov 7. [Epub ahead of print]

Statins inhibit Toxoplasma gondii multiplication in macrophages in vitro

Cortez E, Stumbo AC, Oliveira M, Barbosa HS, Carvalho L.

Laboratório Cultura de Células, Departamento de Histologia e Embriologia, IBRAG, Universidade do Estado do Rio de Janeiro (UERJ), Av. Prof. Manoel de Abreu 444, 3 degrees andar, 20550-170, Rio de Janeiro, RJ, Brazil.

Publication Types:

PMID: 18996682 [PubMed - as supplied by publisher]

Saturday, November 08, 2008

Artemisinin derivatives inhibit Toxoplasma gondii in vitro at multiple steps in the lytic cycle

J Antimicrob Chemother. 2008 Nov 6. [Epub ahead of print]

Artemisinin derivatives inhibit Toxoplasma gondii in vitro at multiple steps in the lytic cycle

D'Angelo JG, Bordón C, Posner GH, Yolken R, Jones-Brando L.

Department of Chemistry and The Malaria Research Institute, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.

Objectives We sought to improve upon the usefulness of artemisinins as anti-Toxoplasma agents by synthesizing new unsaturated, carba derivatives and then testing them for in vitro efficacy against three steps of the lytic cycle of Toxoplasma gondii tachyzoites. Methods Novel derivatives of ART were synthesized and then tested for in vitro antiparasitic activity using T. gondii tachyzoites constitutively expressing beta-galactosidase and human fibroblast host cells. Compounds were evaluated for parasite growth inhibition and cytotoxicity, inhibition of replication and inhibition of parasite invasion of host cells. Results Five of the seven new derivatives, 3a-c, 3e and 3f, effectively inhibited T. gondii growth (IC(50) = 1.0-4.4 microM); however, only three of these proved to be relatively non-cytotoxic (TD(50) >/= 200 microM). The same five derivatives also inhibited tachyzoite replication, and attachment to and invasion of host cells as effectively as or better than the parent compound ART. In addition, one of the derivatives incapable of inhibiting growth, deoxy-3a, was found to inhibit parasite invasion. Conclusions These new artemisinin derivatives have the ability to inhibit multiple steps of T. gondii's lytic cycle. Synthetic unsaturated, carba derivatives of ART have potential as therapeutic agents for the prevention and treatment of toxoplasmosis in humans.

PMID: 18988681 [PubMed - as supplied by publisher]

Quantitative real-time polymerase chain reaction for the accurate detection of Toxoplasma gondii in amniotic fluid

Diagn Microbiol Infect Dis. 2008 Nov 5. [Epub ahead of print]

Quantitative real-time polymerase chain reaction for the accurate detection of Toxoplasma gondii in amniotic fluid

Kasper DC, Sadeghi K, Prusa AR, Reischer GH, Kratochwill K, Förster-Waldl E, Gerstl N, Hayde M, Pollak A, Herkner KR.

Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna 1090, Austria; Research Core Unit of Pediatric Biochemistry and Analytics, Vienna 1090, Austria.

Infection with Toxoplasma gondii during pregnancy is often asymptomatic and may cause severe fetal damage. A quantitative TaqMan minor groove binder real-time polymerase chain reaction (PCR) assay was developed for the specific and sensitive detection of the previously described 529-bp repeat element occurring up to 200 to 300 times in T. gondii genome. The qualitative and quantitative detection limits determined were 6 and 20 marker copies (1/30 to 1/50 of 1 parasite) per PCR, respectively. In addition to standard PCR cycling conditions, 3 different fast PCR protocols were evaluated to minimize run time. A higher variability but no loss of specificity was observed. For the evaluation of clinical applicability, a total of 135 amniotic fluid samples were analyzed targeting both 529-bp and B1 gene. The sensitivity and specificity were 88.0% and 100.0% for B1, and 100.0% and 98.2% for 529-bp PCR assay (positive predictive value and negative predictive value: 100.0% and 97.4%, and 92.6% and 100.0%, respectively). Our results demonstrated an increased sensitivity of the 529-bp PCR assay even in a faster protocol.

PMID: 18990529 [PubMed - as supplied by publisher]

Friday, November 07, 2008

Toxoplasma actively remodels the microtubule network in host cells

Microbes Infect. 2008 Oct 17. [Epub ahead of print]

Toxoplasma gondii actively remodels the microtubule network in host cells

Walker ME, Hjort EE, Smith SS, Tripathi A, Hornick JE, Hinchcliffe EH, Archer W, Hager KM.

Department of Biological Sciences, Center for Global Health and Infectious Disease, 216 Galvin Life Sciences Building, University of Notre Dame, Notre Dame, IN 46556-0369, USA.

Toxoplasma gondii infection triggers host microtubule rearrangement and organelle recruitment around the parasite vacuole. Factors affecting initial stages of microtubule remodeling are unknown. To illuminate the mechanism, we tested the hypothesis that the parasite actively remodels host microtubules. Utilizing heat-killed parasites and time-lapse analysis, we determined microtubule rearrangement requires living parasites and is time dependent. We discovered a novel aster of microtubules (MTs) associates with the vacuole within 1h of infection. This aster lacks the concentrated foci of gamma (gamma)-tubulin normally associated with MT nucleation sites. Unexpectedly, vacuole enlargement does not correlate with an increase in MT staining around the vacuole. We conclude microtubule remodeling does not result from steric constraints. Using nocodazole washout studies, we demonstrate the vacuole nucleates host microtubule growth in-vivo via gamma-tubulin-associated sites. Moreover, superinfected host cells display multiple gamma-tubulin foci. Microtubule dynamics are critical for cell cycle control in uninfected cells. Using non-confluent monolayers, we show host cells commonly fail to finish cytokinesis resulting in larger, multinucleated cells. Our data suggest intimate interactions between T. gondii and host microtubules result in suppression of cell division and/or cause a mitotic defect, thus providing a larger space for parasite duplication.

PMID: 18983931 [PubMed - as supplied by publisher]

Molecular characterization and expression analysis of a P-glycoprotein homologue in Toxoplasma

Mol Biochem Parasitol. 2008 Oct 17. [Epub ahead of print]

Molecular characterization and expression analysis of a P-glycoprotein homologue in Toxoplasma gondii

Schmid A, Sauvage V, Escotte-Binet S, Aubert D, Terryn C, Garnotel R, Villena I.

Laboratoire de Parasitologie-Mycologie, EA 3800, IFR 53, UFR Médecine, Université de Reims Champagne-Ardenne, 51 rue Cognacq-Jay, 51095 Reims cedex, France.

ATP-binding cassette (ABC) transporters represent an important family of membrane proteins involved in drug resistance and other biological activities. The present study reports on the characterization of a P-glycoprotein (Pgp), TgABC.B1, in the protozoan parasite Toxoplasma gondii. The protein encoded by the TgABC.B1 gene displays the typical (TMD-NBD)(2) structural organization of the "full" ABC transporter and shows significant identity and similarity with two apicomplexan Pgps; Pgh1 in Plasmodium falciparum and CpABC3 in Cryptosporidium parvum. The TgABC.B1 gene is a single copy gene transcribed into a full-length mRNA of 4.3kb and expressed as a protein of approximately 150kDa, which cellular localization revealed a membrane-associated labelling in tachyzoites. The TgABC.B1 gene is constitutively expressed in the three major T. gondii genotypes but demonstrated a higher expression in virulent type I, at both transcriptional and translational levels. Further characterization of this Pgp-like protein will increase our knowledge of the membrane transport system in this parasite and could result in the identification of a new therapeutic target in Toxoplasma.

PMID: 18984013 [PubMed - as supplied by publisher]

Molecular approaches to diversity of populations of apicomplexan parasites

Int J Parasitol. 2008 Oct 21. [Epub ahead of print]

Molecular approaches to diversity of populations of apicomplexan parasites

Beck HP, Blake D, Dardé ML, Felger I, Pedraza-Díaz S, Regidor-Cerrillo J, Gómez-Bautista M, Ortega-Mora LM, Putignani L, Shiels B, Tait A, Weir W.

Swiss Tropical Institute, Socinstrasse 57, CH 4002 Basel, Switzerland.

Apicomplexan parasites include many parasites of importance either for livestock or as causative agents of human diseases. The importance of these parasites has been recognised by the European Commission and resulted in support of the COST (Cooperation in Science and Technology) Action 857 'Apicomplexan Biology in the Post-Genomic Era'. In this review we discuss the current understanding in 'Biodiversity and Population Genetics' of the major apicomplexan parasites, namely the Eimeria spp., Cryptosporidium spp., Toxoplasma gondii, Neosporacaninum, Theileria spp. and Plasmodium spp. During the past decade molecular tools for characterizing and monitoring parasite populations have been firmly established as an integral part of field studies and intervention trials. Analyses have been conducted for most apicomplexan pathogens to describe the extent of genetic diversity, infection dynamics or population structure. The underlying key question for all parasites is to understand how genetic diversity influences epidemiology and pathogenicity and its implication in therapeutic and vaccination strategies as well as disease control. Similarities in the basic biology and disease or transmission patterns among this order of parasites promote multifaceted discussions and comparison of epidemiological approaches and methodological tools. This fosters mutual learning and has the potential for cross-fertilisation of ideas and technical approaches.

PMID: 18983997 [PubMed - as supplied by publisher]

A phylogenetic perspective of developmental gene expression in the Apicomplexa

Int J Parasitol. 2008 Oct 21. [Epub ahead of print]

New eukaryotic systematics: A phylogenetic perspective of developmental gene expression in the Apicomplexa

Gissot M, Kim K, Schaap D, Ajioka JW.

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

The phylum Apicomplexa consists of obligate intracellular protistan parasites, some of which are responsible for global disease causing serious morbidity and mortality in humans and animals. Understanding the mechanisms of gene expression that drive the cellular changes required to complete their life cycles will be critical in combating infection and disease. Plasmodium spp. and Toxoplasma gondii have served as good models for growth and development in the Apicomplexa. Elucidating developmental gene expression relies on comparisons with known mechanisms and their DNA, RNA and protein components. Transcriptional profiling across asexual development suggests a model where a cascade of gene expression results in a "just-in-time" production process that makes products only when needed. Some mechanisms that control transcription such as chromatin/histone modification are highly conserved in the phylum compared with the traditional model organisms, yeast, worms, flies and mammals. Studies exploiting this phenomenon show great potential for both investigating the effects of chromatin structure on developmental gene expression, and helping to identify genes that are expressed in a stage-specific manner. Transcription factors and their cognate cis-acting binding sites have been difficult to identify. This may be because the DNA binding motifs that have evolved to act as transcription factors in the Apicomplexa, e.g. the AP2 family, may be more like plants than the traditional model organisms. A new eukaryotic phylogenetic model comprised of six super-groups divides the traditional model organisms, plants and the Apicomplexa into separate super-groups. This phylogenetic model helps explain why basic functions such as transcriptional regulation appear be a composite of mechanisms in the Apicomplexa compared with what is known from other eukaryotes.

PMID: 18983845 [PubMed - as supplied by publisher]

Wednesday, November 05, 2008

The Dual Origin of Toxoplasma gondii N-Glycans

Biochemistry. 2008 Nov 1. [Epub ahead of print]

The Dual Origin of Toxoplasma gondii N-Glycans

Garénaux E, Shams-Eldin H, Chirat F, Bieker U, Schmidt J, Michalski JC, Cacan R, Guérardel Y, Schwarz RT.

Unite de Glycobiologie Structurale et Fonctionnelle, UMR 8576 CNRS, Universite des Sciences et Technologies de Lille, 59655 Villeneuve d'Ascq cedex, France, and Institut fur Virologie, AG Parasitologie, Philipps-Universitat Marburg, Hans-Meerwein-Strasse 2, 35043 Marburg, Germany

N-Linked glycosylation is the most frequent modification of secreted proteins in eukaryotic cells that plays a crucial role in protein folding and trafficking. Mature N-glycans are sequentially processed in the endoplasmic reticulum and Golgi apparatus through a pathway highly conserved in most eukaryotic organisms. Here, we demonstrate that the obligate intracellular protozoan parasite Toxoplasma gondii independently transfers endogenous truncated as well as host-derived N-glycans onto its own proteins. Therefore, we propose that the apicomplexan parasite scavenges N-glycosylation intermediates from the host cells to compensate for the rapid evolution of its biosynthetic pathway, which is primarily devoted to modification of proteins with glycosylphosphatidylinositols rather than N-glycans.

PMID: 18975916 [PubMed - as supplied by publisher]

Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens

Nat Immunol. 2008 Nov 2. [Epub ahead of print]

Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens

El Kasmi KC, Qualls JE, Pesce JT, Smith AM, Thompson RW, Henao-Tamayo M, Basaraba RJ, König T, Schleicher U, Koo MS, Kaplan G, Fitzgerald KA, Tuomanen EI, Orme IM, Kanneganti TD, Bogdan C, Wynn TA, Murray PJ.

[1] Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 38015, USA. [2] Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee 38015, USA. [3] Present address: Department of Pediatrics, Division of Gastroenterology and Hepatology, University of Colorado at Denver and Health Sciences Center, Anschutz Medical Campus, Colorado 80045, USA. [4] These authors contributed equally to this work.

Toll-like receptor (TLR) signaling in macrophages is required for antipathogen responses, including the biosynthesis of nitric oxide from arginine, and is essential for immunity to Mycobacterium tuberculosis, Toxoplasma gondii and other intracellular pathogens. Here we report a 'loophole' in the TLR pathway that is advantageous to these pathogens. Intracellular pathogens induced expression of the arginine hydrolytic enzyme arginase 1 (Arg1) in mouse macrophages through the TLR pathway. In contrast to diseases dominated by T helper type 2 responses in which Arg1 expression is greatly increased by interleukin 4 and 13 signaling through the transcription factor STAT6, TLR-mediated Arg1 induction was independent of the STAT6 pathway. Specific elimination of Arg1 in macrophages favored host survival during T. gondii infection and decreased lung bacterial load during tuberculosis infection.

PMID: 18978793 [PubMed - as supplied by publisher]

Protozoa in respiratory pathology: a review

Eur Respir J. 2008 Nov;32(5):1354-70.

Protozoa in respiratory pathology: a review

Martínez-Girón R, Esteban JG, Ribas A, Doganci L.

Protozoal Respiratory Pathology Research Unit, Fundación INCLINICA, Anatomical Pathology Service, Hospital Universitario Central de Asturias, Oviedo, Spain.

Among the micro-organisms that may affect the respiratory apparatus are the protozoa. The diseases they may give rise to constitute a relatively uncommon group of respiratory ailments with, in the majority of cases, an underlying clinical situation corresponding to states of suppressed immunity (AIDS, transplants, malign haemopathies, corticotherapy, etc.). Other factors, such as visits to endemic areas and immigration, also have to be taken into account. In view of the probable increase in the number of cases and the appearance of new emerging diseases, it is the intention of the present work to review the publications available, in different fields of medicine, that refer to the principal kinds of protozoa (Entamoeba, Acanthamoeba, Balamuthia, Leishmania, Trypanosoma, Trichomonas, Lophomonas, Cryptosporidium, Cyclospora, Toxoplasma, Plasmodium, Babesia, Encephalitozoon, Enterocytozoon and Balantidium) and, at the same time, detail and comment on the latest findings on this subject.

PMID: 18978136 [PubMed - in process]

Toxoplasma gondii: A simple high-throughput assay for drug screening in vitro

Exp Parasitol. 2008 Oct 17. [Epub ahead of print]

Toxoplasma gondii: A simple high-throughput assay for drug screening in vitro

Jin C, Kaewintajuk K, Jiang J, Jeong W, Kamata M, Kim HS, Wataya Y, Park H.

Department of Infection Biology, Zoonosis Research Center, Wonkwang University School of Medicine, 344-2, Shinyong-dong, Iksan, Chonbuk 570-749, Republic of Korea.

Toxoplasma gondii is the etiologic agent of toxoplasmosis. Although the combination of sulfadiazine and pyrimethamine is used as therapy for this disease, these drugs can have serious side effects and its use is limited in pregnancy. Therefore there is a need for new anti-T. gondii drugs in the clinic. Some systems for T. gondii drug screening have been described, but these have limitations and can be difficult. In order to solve these problems, we established a system to screen drugs in vitro that involved using cell viability methods to calculate drug selectivities, which are Trypan blue, [3-(4,5-dimethylthiazol-zyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliuzolium, inner salt] (MTS) method and lactate dehydrogenase (LDH) assay. These assays were simple to establish and perform. The IC(50) values calculated from the morphological assay were not significantly different from the EC(50) values calculated using the other three methods. In particular, the results of the morphological assay showed a distinct association with the MTS assay (R=0.9841). These assays could be used for a wide range of applications in the screening of new drugs and may provide an alternative to the techniques currently used to screen for candidate anti-T. gondii compounds in vitro. In this study, we also tested many compounds and identified some that had a good anti-T. gondii effect in vitro based on the MTS assay. This simple and fast system allowed us to determine which compounds to investigate further using in vivo experiments.

PMID: 18977350 [PubMed - as supplied by publisher]

Sunday, November 02, 2008

In Silico Identification of Specialized Secretory-Organelle Proteins in Apicomplexan Parasites and In Vivo Validation

PLoS ONE. 2008;3(10):e3611. Epub 2008 Oct 31

In Silico Identification of Specialized Secretory-Organelle Proteins in Apicomplexan Parasites and In Vivo Validation in Toxoplasma gondii

Chen Z, Harb OS, Roos DS.

Department of Biology, Penn Genomic Frontiers Institute, and the Graduate Program in Genomics and Computational Biology, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

Apicomplexan parasites, including the human pathogens Toxoplasma gondii and Plasmodium falciparum, employ specialized secretory organelles (micronemes, rhoptries, dense granules) to invade and survive within host cells. Because molecules secreted from these organelles function at the host/parasite interface, their identification is important for understanding invasion mechanisms, and central to the development of therapeutic strategies. Using a computational approach based on predicted functional domains, we have identified more than 600 candidate secretory organelle proteins in twelve apicomplexan parasites. Expression in transgenic T. gondii of eight proteins identified in silico confirms that all enter into the secretory pathway, and seven target to apical organelles associated with invasion. An in silico approach intended to identify possible host interacting proteins yields a dataset enriched in secretory/transmembrane proteins, including most of the antigens known to be engaged by apicomplexan parasites during infection. These domain pattern and projected interactome approaches significantly expand the repertoire of proteins that may be involved in host parasite interactions.

PMID: 18974850 [PubMed - in process]