Sunday, March 30, 2008

Cationic amino Acid transporter-2 regulates immunity by modulating arginase activity

PLoS Pathog. 2008 Mar 14;4(3):e1000023

Cationic amino Acid transporter-2 regulates immunity by modulating arginase activity

Thompson RW, Pesce JT, Ramalingam T, Wilson MS, White S, Cheever AW, Ricklefs SM, Porcella SF, Li L, Ellies LG, Wynn TA.

Immunopathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

Cationic amino acid transporters (CAT) are important regulators of NOS2 and ARG1 activity because they regulate L-arginine availability. However, their role in the development of Th1/Th2 effector functions following infection has not been investigated. Here we dissect the function of CAT2 by studying two infectious disease models characterized by the development of polarized Th1 or Th2-type responses. We show that CAT2(-/-) mice are significantly more susceptible to the Th1-inducing pathogen Toxoplasma gondii. Although T. gondii infected CAT2(-/-) mice developed stronger IFN-gamma responses, nitric oxide (NO) production was significantly impaired, which contributed to their enhanced susceptibility. In contrast, CAT2(-/-) mice infected with the Th2-inducing pathogen Schistosoma mansoni displayed no change in susceptibility to infection, although they succumbed to schistosomiasis at an accelerated rate. Granuloma formation and fibrosis, pathological features regulated by Th2 cytokines, were also exacerbated even though their Th2 response was reduced. Finally, while IL-13 blockade was highly efficacious in wild-type mice, the development of fibrosis in CAT2(-/-) mice was largely IL-13-independent. Instead, the exacerbated pathology was associated with increased arginase activity in fibroblasts and alternatively activated macrophages, both in vitro and in vivo. Thus, by controlling NOS2 and arginase activity, CAT2 functions as a potent regulator of immunity.

PMID: 18369473 [PubMed - in process]

Itraconazole affects Toxo endodyogeny

FEMS Microbiol Lett. 2008 Mar 26 [Epub ahead of print]

Itraconazole affects Toxoplasma gondii endodyogeny

Martins-Duarte ED, de Souza W, Vommaro RC.

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

The antifungal agent itraconazole is an effective drug against systemic mycoses inhibiting cytochrome P-450-mediated ergosterol synthesis, essential for fungal survival. In this work, we show the activity of this azole as a potential agent against Toxoplasma gondii, the causative agent of toxoplasmosis. Monolayers of LLC-MK2 epithelial cells infected with tachyzoites of RH strain were incubated with different concentrations of itraconazole for 24 and 48 h. The IC(50) values obtained were 114.0 and 53.6 nM for 24 and 48 h, respectively. Transmission electron microscopy (TEM) analysis of itraconazole-treated intracellular tachyzoites showed endoplasmic reticulum and nuclear envelope swelling. The drug also caused rupture of the parasite's surface membrane and affected the parasite's division by endodyogeny. This observation was confirmed both by fluorescence microscopy of cells labeled with diamidino-2-phenylindole and by three-dimensional reconstruction of serial thin sections analyzed by TEM. The treatment with itraconazole led to the formation of a mass of daughter cells, suggesting the interruption of the scission process during the parasite's cell division.

PMID: 18371067 [PubMed - as supplied by publisher]

Acute immune-mediated thrombocytopenic purpura related to Toxo infection

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

Acute immune-mediated thrombocytopenic purpura related to Toxoplasma gondii infection

Tuon FF, Higashino HR, Amato VS, Nicodemo AC.

Department of Infectious and Parasitic Diseases, Medical School, University of São Paulo, São Paulo, SP, Brazil.

Publication Types:

Wednesday, March 26, 2008

Alveolins, a New Family of Cortical Proteins that Define the Protist Infrakingdom Alveolata

Mol Biol Evol. 2008 Mar 21 [Epub ahead of print]

Alveolins, a New Family of Cortical Proteins that Define the Protist Infrakingdom Alveolata

Gould SB, Tham WH, Cowman AF, McFadden GI, Waller RF.

School of Botany, University of Melbourne, VIC 3010, Australia.

Alveolates are a recently recognised group of unicellular eukaryotes that unites disparate protists including apicomplexan parasites (which cause malaria and toxoplasmosis), dinoflagellate algae (which cause red tides and are symbionts in many corals) and the ciliates (which are microscopic predators and common rumen symbionts). Gene sequence trees provide robust support for the alveolate alliance but beyond the common presence of membranous sacs (alveoli) subtending the plasma membrane the group has no unifying morphological feature. We describe a family of proteins, alveolins, associated with these membranous sacs in apicomplexa, dinoflagellates and ciliates. Alveolins contain numerous simple peptide repeats and are encoded by multi gene families. We generated antibodies against a peptide motif common to all alveolins and identified a range of apparently abundant proteins in apicomplexans, dinoflagellates and ciliates. Immunolocalization reveals that alveolins are associated exclusively with the cortical regions of apicomplexa, dinoflagellates and ciliates where the alveolar sacs occur. Alveolins are the first molecular nexus between the unifying structures that defines this eukaryotic group. They provide an excellent opportunity to explore the exceptional compartment that was apparently the key to a remarkable diversification of unique protists that occupy a wide array of lifestyle niches.

PMID: 18359944 [PubMed - as supplied by publisher]

Host cells participate in the in vitro effects of new-generation diamidine-analogues

Antimicrob Agents Chemother. 2008 Mar 24 [Epub ahead of print]

Host cells participate in the in vitro effects of new-generation diamidine-analogues against tachyzoites of the intracellular apicomplexan parasites Neospora caninum and Toxoplasma gondii

Leepin A, Stüdli A, Brun R, Stephens CE, Boykin DW, Hemphill A.

Institute of Parasitology, University of Berne, Länggass-Strasse 122, CH-3012 Berne, Switzerland; Swiss Tropical Institute, Antiparasite Chemotherapy, Socinstrasse 57, CH-4002 Basel, Switzerland; Department of Chemistry, Georgia State University, PO Box 4098, Atlanta, Georgia 30302-4098, USA.

The in vitro effects of 19 dicationic diamidine-derivatives against the proliferative tachyzoite stages of the apicomplexan parasites Neospora caninum and Toxoplasma gondii were investigated. Four compounds (DB811, DB786, DB750 and DB766) with similar structural properties exhibited profound inhibition of tachyzoite proliferation. The lowest IC50 values were noted for DB786 (Neospora-IC50 = 0.21microM; Toxoplasma-IC50 = 0.22microM) and DB750 (Neospora-IC50 = 0.23microM; Toxoplasma-IC50 = 0.16microM), with complete proliferation inhibition at 1.7microM for both drugs in both species. DB750 and DB786 were chosen for further studies. Electron microscopy of N. caninum-infected HFF cultures revealed distinct alterations and damage of parasite ultrastructure upon drug treatment, while host cells remained unaffected. To exert true parasiticidal efficacy against N. caninum, a treatment duration of 3 h at 1.7microM was sufficient for DB750, while a longer treatment period (24 h) was necessary for DB786. Pretreatment of tachyzoites for 1 h prior to host cell exposure had no effect on infectivity. However, pretreatment of uninfected host cells had a significant adverse effect on N. caninum proliferation: exposure of HFF to 1,7microM DB750 for 6, 12 and 24 h, respectively, followed by infection with N. caninum tachyzoites and subsequent culture in the absence of DB750, resulted in a significantly delayed parasite proliferation. This suggests that either these compounds or respective active metabolites were still present after removal of the drugs, or that drug treatments reversably impaired some functional activities in HFF, which were essential for parasite proliferation and/or survival.

PMID: 18362190 [PubMed - as supplied by publisher]

Sunday, March 23, 2008

Effects of ozone and ultraviolet radiation treatments on the infectivity of Toxo oocysts

Vet Parasitol. 2008 Feb 13 [Epub ahead of print]

Effects of ozone and ultraviolet radiation treatments on the infectivity of Toxoplasma gondii oocysts

Dumètre A, Le Bras C, Baffet M, Meneceur P, Dubey JP, Derouin F, Duguet JP, Joyeux M, Moulin L.

Centre de recherche d’expertise et de contrôle des eaux de Paris, 144 Avenue Paul Vaillant-Couturier, 75014 Paris, France.

Clinical toxoplasmosis in humans has been epidemiologically linked to the consumption of drinking water contaminated by Toxoplasma gondii oocysts. We evaluated killing of T. gondii oocysts after ultraviolet (UV) or ozone treatments by bioassay in mice and/or cell culture. A 4-log inactivation of the oocyst/sporozoite infectivity was obtained for UV fluences >20mJcm(-2). In contrast, oocysts were not inactivated by ozone with an exposure (Ct) up to 9.4mgminl(-1) in water at 20 degrees C. In conclusion, UV treatment can be an effective disinfection method to inactivate T. gondii oocysts in drinking water, but ozone did not show promise in this research.

PMID: 18355965 [PubMed - as supplied by publisher]

Friday, March 21, 2008

Intrinsic disorder in pathogenic and non-pathogenic microbes

Mol Biosyst. 2008 Apr;4(4):328-40. Epub 2008 Feb 21

Intrinsic disorder in pathogenic and non-pathogenic microbes: discovering and analyzing the unfoldomes of early-branching eukaryotes

Mohan A, Sullivan WJ Jr, Radivojac P, Dunker AK, Uversky VN

School of Informatics, Indiana University, Bloomington, IN 47401, USA.

Parasitic protozoal infections have long been known to cause profound degrees of sickness and death in humans as well as animal populations. Despite the increase in the number of annotated genomes available for a large variety of protozoa, a great deal more has yet to be learned about them, from their fundamental physiology to mechanisms invoked during host-pathogen interactions. Most of these genomes share a common feature, namely a high prevalence of low complexity regions in their predicted proteins, which is believed to contribute to the uniqueness of the individual species within this diverse group of early-branching eukaryotes. In the case of Plasmodium species, which cause malaria, such regions have also been reported to hamper the identification of homologues, thus making functional genomics exceptionally challenging. One of the better accepted theories accounting for the high number of low complexity regions is the presence of intrinsic disorder in these microbes. In this study we compare the degree of disordered proteins that are predicted to be expressed in many such ancient eukaryotic cells. Our findings indicate an unusual bias in the amino acids comprising protozoal proteomes, and show that intrinsic disorder is remarkably abundant among their predicted proteins. Additionally, the intrinsically disordered regions tend to be considerably longer in the early-branching eukaryotes. An analysis of a Plasmodium falciparum interactome indicates that protein-protein interactions may be at least one function of the intrinsic disorder. This study provides a bioinfomatics basis for the discovery and analysis of unfoldomes (the complement of intrinsically disordered proteins in a given proteome) of early-branching eukaryotes. It also provides new insights into the evolution of intrinsic disorder in the context of adapting to a parasitic lifestyle and lays the foundation for further work on the subject.

PMID: 18354786 [PubMed - in process]

Thursday, March 20, 2008

Identification of trafficking determinants for polytopic rhomboid proteases

Traffic. 2008 Mar 10 [Epub ahead of print]

Identification of trafficking determinants for polytopic rhomboid proteases in Toxoplasma gondii

Sheiner L, Dowse TJ, Soldati-Favre D.

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

Rhomboids constitute a family of polytopic serine proteases conserved throughout evolution. The obligate intracellular parasite Toxoplasma gondii possesses six genes coding for rhomboid-like proteases that are targeted to distinct subcellular compartments: TgROM1 localizes to regulated secretory organelles, micronemes, TgROM2 is present in the Golgi, while TgROM4 and TgROM5 are found in the pellicle of the parasite. The targeting mechanism/s of rhomboid proteins is an aspect that has not yet been assessed. The existence of TgROM family members localized to different sub-cellular compartments provides a convenient system to study their sorting mechanisms in a genetically tractable organism that possesses an elaborate secretory pathway and conserved trafficking machineries. In this study, we experimentally established the topology of TgROM1 and TgROM4 at the plasma membrane and applied domain-exchange and site-directed mutagenesis approaches to identify critical sorting determinants on the N-terminal cytosolic domains of TgROM2 and TgROM1 that confer their Golgi and post-Golgi localizations, respectively.

PMID: 18346213 [PubMed - as supplied by publisher]

Cluster of four surface antigen genes specifically expressed in bradyzoites, SAG2CDXY, plays an important role in persistence

Infect Immun. 2008 Mar 17 [Epub ahead of print]

A cluster of four surface antigen genes specifically expressed in bradyzoites, SAG2CDXY, plays an important role in Toxoplasma gondii persistence

Saeij JP, Arrizabalaga G, Boothroyd JC.

Stanford University School of Medicine, Department of Microbiology and Immunology, Stanford, CA 94305.

Toxoplasma gondii is one of the most successful protozoan parasites of warm-blooded animals. Key to its ability to establish a chronic infection in immunocompetent animals is thought to be stage-specific expression of its surface molecules. The rapidly dividing tachyzoite stage displays a different subset of the family of surface antigen-1 (SAG1)-related-sequences (SRS) compared to the encysted bradyzoite stage. It is possible that this switch is necessary to protect the bradyzoites against an immune response raised against the tachyzoite stage. Alternatively, it might be that bradyzoite SRSs evolved to facilitate invasion of different cell types, such as found in the brain, where cysts develop, or small intestine where bradyzoites must enter after oral infection. Here, we studied the function of a cluster of four tandem genes encoding bradyzoite SRSs called SAG2C, -D, -X and -Y. Using bioluminescent imaging (BLI) of mice infected with parasites expressing firefly luciferase (FLUC) driven by the SAG2D promoter, we show stage conversion for the first time in living animals. A truncated version of the SAG2D promoter (SAG2Dmin) gave efficient expression of FLUC in both tachyzoites and bradyzoites indicating that the bradyzoite specificity of the complete SAG2D promoter is likely due to element(s) that normally suppress expression in tachyzoites. Comparing mice infected with wild-type or mutants where the SAG2CDXY cluster of genes has been deleted (DeltaSAG2CDXY), we demonstrate that whereas DeltaSAG2CDXY parasites are less capable of maintaining a chronic infection in the brain, they do not show a defect in oral infectivity.

PMID: 18347037 [PubMed - as supplied by publisher]

Tuesday, March 18, 2008

The role of MHC haplotypes H2d/H2b in mouse resistance/susceptibility to cyst formation is influenced by the strain

An Acad Bras Cienc. 2008 Mar;80(1):85-99.

The role of MHC haplotypes H2d/H2b in mouse resistance/susceptibility to cyst formation is influenced by the lineage of infective Toxoplasma gondii strain

Resende MG, Fux B, Caetano BC, Mendes EA, Silva NM, Ferreira AM, Melo MN, Vitor RW, Gazzinelli RT.

Laboratório de Imunopatologia, Instituto René Rachou, Belo Horizonte, MG, 30190-002, Brasil.

Toxoplasma gondii strains displaying the Type I/III genotype are associated with acquired ocular toxoplasmosis in humans. Here, we used a mice model to characterize some immunological mechanisms involved in host resistance to infection with such strains. We have chosen the Type I/III strains D8, G2 and P-Br, which cause a chronic infection in mice that resembles human toxoplamosis. Mice deficient of molecules MyD88, IFN-gamma, and IL-12 were susceptible to all three parasite strains. This finding indicates the importance of innate mechanisms in controlling infection. On the other hand, MHC haplotype did not influenced resistance/susceptibility; since mice lineages displaying a same genetic background but different MHC haplotypes (H2b or H2d) developed similar mortality and cyst numbers after infection with those strains. In contrast, the C57BL/6 genetic background, and not MHC haplotype, was critical for development of intestinal inflammation caused by any of the studied strains. Finally, regarding effector mechanisms, weobserved that B and CD8+ T lymphocytes controlled survival,whereas the inducible nitric oxide synthase influenced cyst numbers in brains of mice infected with Type I/III strains. These findings are relevant to further understanding of the immunologic mechanisms involved in host protection and pathogenesis during infection with T. gondii.

PMID: 18345378 [PubMed - in process]

Serum and aqueous humour cytokine response and histopathological alterations during ocular Toxo infection in C57BL/6 mice

Micron. 2008 Feb 14 [Epub ahead of print]

Serum and aqueous humour cytokine response and histopathological alterations during ocular Toxoplasma gondii infection in C57BL/6 mice

Calabrese KS, Tedesco RC, Zaverucha do Valle T, Barbosa HS.

Laboratório de Imunomodulação, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Av. Brasil 4365, CEP 21040-900 Rio de Janeiro, Brazil.

Toxoplasma gondii, an obligate intracellular protozoan parasite, infects most species of warm-blooded animals, and in humans it causes toxoplasmosis. Healthy people that become infected rarely present clinical symptoms because the immune system prevents the parasite from causing illness. Congenital toxoplasmosis may result in abortion, hydrocephalus, as well as neurological and ocular disease (most frequently retinochoroiditis) of the newborn. In immunocompromised patients, reactivation of latent disease can cause encephalitis. Cell-mediated immunity to T. gondii antigens involves innate acute inflammatory responses and antigen-specific adaptive immunity. Considering the complexity of the immunological events triggered during toxoplasmosis, systemic and local responses were evaluated by cytokine measurements. Aqueous humour and serum were obtained from non-infected and T. gondii Me-49 strain infected C57BL/6 mice for cytokine quantification. Histopathological analyses were made with eyes enucleated from mice after 30 days of infection. ELISA assays showed an increase of IFN-gamma levels both in serum and aqueous humour of infected mice in opposition to a decrease in IL-10 levels. On the other hand, TGF-beta was high, whereas IL-12 and TNF-alpha were present in small levels in both groups. We also detected higher levels of IL-4 and IL-6 in aqueous humour than in serum of infected mice when compared to the control group. MIP-2 presented no significant differences between the two groups. Fas and Fas-L were also present in similar levels in serum of non-infected and infected mice, but both chemokines were increased in the aqueous humour of infected mice. Histopathological analysis of infected mice showed inflammatory infiltrates around blood vessels and alteration of the outer photoreceptor segments, on the external and inner nuclear layer. Parasites were observed in 82% of eyes, inside the blood vessels associated with inflammatory infiltrate. Edema, characterized by the increase of interstitial spaces between the FTR, forming lacunae was also noted. These alterations take the form of projections (retino-vitreal), characteristic of retinochoroiditis. In conclusion, T. gondii infection of C57BL/6 mice revealed that cytokine patterns alone do not assure susceptibility or resistance against infection, thus reinforcing the notion that it is necessary more than cytokine dosage to determine Th1 or Th2 profile in this model.

PMID: 18343675 [PubMed - as supplied by publisher]

Analysis of Recurrence Patterns Associated with Toxoplasmic Retinochoroiditis

Am J Ophthalmol. 2008 Mar 14 [Epub ahead of print]

Analysis of Recurrence Patterns Associated with Toxoplasmic Retinochoroiditis

Holland GN, Crespi CM, Loon NT, Charonis AC, Yu F, Bosch-Driessen LH, Rothova A.

Ocular Inflammatory Disease Center, Jules Stein Eye Institute and the Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, California.

PURPOSE: Toxoplasmic retinochoroiditis is thought to recur randomly. We sought to determine whether there is, instead, a longitudinal pattern of recurrences and to identify risk factors for recurrence. DESIGN: Longitudinal cohort study. METHODS: We collected the following data for 143 patients with toxoplasmic retinochoroiditis in The Netherlands: gender, first affected eye, age at first episode, mode of Toxoplasma gondii infection (congenital vs postnatal), treatment history, and presence of retinal scars at initial examination. For each episode, we determined age, duration since first episode, and interval since previous episode. We estimated the relationship between disease-free interval after an episode and recurrence risk. The influence of host and disease factors on recurrence risk was analyzed using Cox regression with frailty modeling for correlated intrapatient recurrence times. We performed a Monte Carlo test for occurrence of clusters after prolonged disease-free intervals. RESULTS: Follow-up ranged from 0.3 to 41 years (323 episodes in first-affected eyes). Recurrence risk was highest immediately after an episode, then decreased with increasing disease-free intervals, a pattern consistent with clustering. Relative risk (RR) of recurrence declined 72% (RR, 0.28; 95% confidence interval [CI], 0.22 to 0.36; P < .001) with each 10-year interval since first episode, and declined 15% (RR, 0.85; 95% CI, 0.71 to 1.01; P = .06) for each 10-year increase in age at first episode. Patients more than 40 years of age were at higher risk of recurrence than younger patients (RR, 1.74; 95% CI, 1.06 to 2.86; P = .03). Clusters of episodes occurred after prolonged disease-free intervals. CONCLUSIONS: Toxoplasmic retinochoroiditis occurs in clusters over time. Recurrence risk is influenced by patient age and duration of infection.

PMID: 18343351 [PubMed - as supplied by publisher]

Friday, March 14, 2008

Localisation of gluconeogenesis and tricarboxylic acid (TCA)-cycle enzymes and first functional analysis of the TCA cycle in Toxo

Int J Parasitol. 2008 Feb 13 [Epub ahead of print]

Localisation of gluconeogenesis and tricarboxylic acid (TCA)-cycle enzymes and first functional analysis of the TCA cycle in Toxoplasma gondii

Fleige T, Pfaff N, Gross U, Bohne W.

Institute of Medical Microbiology, University of Göttingen, Kreuzbergring 57, Göttingen D-37075, Germany.

The apicomplexan parasite Toxoplasma gondii displays some unusual localisations of carbohydrate converting enzymes, which is due to the presence of a vestigial, non-photosynthetic plastid, referred to as the apicoplast. It was recently demonstrated that the single pyruvate dehydrogenase complex (PDH) in T. gondii is exclusively localised inside the apicoplast but absent in the mitochondrion. This raises the question about expression, localisation and function of enzymes for the tricarboxylic acid (TCA)-cycle, which normally depends on PDH generated acetyl-CoA. Based on the expression and localisation of epitope-tagged fusion proteins, we show that all analysed TCA cycle enzymes are localised in the mitochondrion, including both isoforms of malate dehydrogenase. The absence of a cytosolic malate dehydrogenase suggests that a typical malate-aspartate shuttle for transfer of reduction equivalents is missing in T. gondii. We also localised various enzymes which catalyse the irreversible steps in gluconeogenesis to a cellular compartment and examined mRNA expression levels for gluconeogenesis and TCA cycle genes between tachyzoites and in vitro bradyzoites. In order to get functional information on the TCA cycle for the parasite energy metabolism, we created a conditional knock-out mutant for the succinyl-CoA synthetase. Disruption of the sixth step in the TCA cycle should leave the biosynthetic parts of the cycle intact, but prevent FADH(2) production. The succinyl-CoA synthetase depletion mutant displayed a 30% reduction in growth rate, which could be restored by supplementation with 2muM succinate in the tissue culture medium. The mitochondrial membrane potential in these parasites was found to be unaltered. The lack of a more severe phenotype suggests that a functional TCA cycle is not essential for T. gondii replication and for maintenance of the mitochondrial membrane potential.

PMID: 18336823 [PubMed - as supplied by publisher]

Thursday, March 13, 2008

Congenital toxoplasmosis infection in an infant born to an HIV-1-infected mother

Braz J Infect Dis. 2007 Dec;11(6):610-1.

Congenital toxoplasmosis infection in an infant born to an HIV-1-infected mother

Cruz ML, Cardoso CA, Saavedra MC, Santos ED, Melino T.

Infectious Diseases Department, Hospital dos Servidores do Estado, Rio de Janeiro, RJ, Brazil.

We report the occurrence of congenital toxoplasmosis in an infant born to an HIV infected mother who had high anti-toxoplasma IgG and negative IgM at nine weeks of gestation. We briefly review available literature and discuss the possible mechanisms of transmission of congenital toxoplasmosis among HIV infected pregnant women.

PMID: 18327477 [PubMed - in process]

Host cell manipulation by the human pathogen Toxoplasma

Cell Mol Life Sci. 2008 Mar 10 [Epub ahead of print]

Host cell manipulation by the human pathogen Toxoplasma gondii

Laliberté J, Carruthers VB.

Department of Microbiology and Immunology, University of Michigan Medical School, 1150 West Medical Center Drive, 5751 Medical Science Building II, Ann Arbor, Michigan, 48109-0620, USA,

Toxoplasma gondii is an obligate intracellular parasite that can infect virtually any nucleated cell. During invasion Toxoplasma creates the parasitophorous vacuole, a subcellular compartment that acts as an interface between the parasite and host, and serves as a platform for modulation of host cell functions that support parasite replication and infection. Spatial reorganization of host organelles and cytoskeleton around the parasitophorous vacuole are observed following entry, and recent evidence suggests this interior redecorating promotes parasite nutrient acquisition. New findings also reveal that Toxoplasma manipulates host signaling pathways by deploying parasite kinases and a phosphatase, including at least two that infiltrate the host nucleus. Toxoplasma infection additionally controls several cellular pathways to establish an anti-apoptotic environment, and subverts immune cells as a conduit for dissemination. In this review we discuss these recent developments in understanding how Toxoplasma achieves widespread success as a human and animal parasite by manipulating its host.

PMID: 18327664 [PubMed - as supplied by publisher]

Formin Regulates Actin Polymerization and Localizes to the Parasite-Erythrocyte Moving Junction during Invasion

Cell Host Microbe. 2008 Mar 13;3(3):188-98.

A Malaria Parasite Formin Regulates Actin Polymerization and Localizes to the Parasite-Erythrocyte Moving Junction during Invasion

Baum J, Tonkin CJ, Paul AS, Rug M, Smith BJ, Gould SB, Richard D, Pollard TD, Cowman AF.

Infection and Immunity Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia.

Malaria parasites invade host cells using actin-based motility, a process requiring parasite actin filament nucleation and polymerization. Malaria and other apicomplexan parasites lack Arp2/3 complex, an actin nucleator widely conserved across eukaryotes, but do express formins, another type of actin nucleator. Here, we demonstrate that one of two malaria parasite formins, Plasmodium falciparum formin 1 (PfFormin 1), and its ortholog in the related parasite Toxoplasma gondii, follows the moving tight junction between the invading parasite and the host cell, which is the predicted site of the actomyosin motor that powers motility. Furthermore, in vitro, the PfFormin1 actin-binding formin homology 2 domain is a potent nucleator, stimulating actin polymerization and, like other formins, localizing to the barbed end during filament elongation. These findings support a conserved molecular mechanism underlying apicomplexan parasite motility and, given the essential role that actin plays in cell invasion, highlight formins as important determinants of malaria parasite pathogenicity.

PMID: 18329618 [PubMed - in process]

Protein Trafficking inside Toxoplasma gondii

Traffic. 2008 Mar 4 [Epub ahead of print]

Protein Trafficking inside Toxoplasma gondii

Sheiner L, Soldati-Favre D.

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

The accurate targeting of proteins to their final destination is an essential process in all living cells. Apicomplexans are obligate intracellular protozoan parasites that possess a compartmental organization similar to that of free-living eukaryotes but can be viewed as professional secretory cells. Establishment of parasitism involves the sequential secretion from highly specialized secretory organelles, including micronemes, rhoptries and dense granules. Additionally, apicomplexans harbor a tubular mitochondrion, a nonphotosynthetic plastid organelle termed the apicoplast, acidocalcisomes and an elaborated inner membrane complex composed of flattened membrane cisternae that are derived from the secretory pathway. Given the multitude of destinations both inside and outside the parasite, the endoplasmic reticulum/Golgi of the apicomplexans constitutes one of the most busy roads intersections in eukaryotic traffic.

PMID: 18331382 [PubMed - as supplied by publisher]

Protective effect of a multiantigenic DNA vaccine against Toxo with co-delivery of IL-12 in mice

Parasite Immunol. 2008 Mar 3 [Epub ahead of print]

Protective effect of a multiantigenic DNA vaccine against Toxoplasma gondii with co-delivery of IL-12 in mice

Cui YL, He SY, Xue MF, Zhang J, Wang HX, Yao Y.

Department of Parasitology, School of Medicine, Shandong University, Shandong, China.

In this study, we constructed a multiantigenic DNA vaccine, pSAG1-ROP2-SAG2 and examined its effect with co-delivery of a plasmid encoding IL-12 (pIL-12) as an adjuvant in BALB/c mice against Toxoplasma gondii. After a lethal challenge of T. gondii RH strain, survival of the mice immunized with this pSAG1-ROP2-SAG2 vaccine was significantly prolonged in comparison to the control groups. Furthermore, the protection was significantly augmented by pIL-12 co-delivery. As demonstrated by lymphocyte proliferation assay, cytokine and antibody level determinations, the humoral and Th1-type cellular responses elicited by this multiantigenic DNA vaccine were significantly stronger than those elicited by double-antigenic, or single-antigenic DNA vaccines. Our data suggest that multiantigenic DNA vaccine with pIL-12 co-delivery is a very effective approach in the protection against T. gondii.

PMID: 18331395 [PubMed - as supplied by publisher]

Monday, March 10, 2008

Novel GDP-dependent pyruvate kinase isozyme

J Biol Chem. 2008 Mar 6 [Epub ahead of print]

A novel GDP-dependent pyruvate kinase isozyme from Toxoplasma gondii localizes to both the apicoplast and the mitochondrion

Saito T, Nishi M, Lim MI, Wu B, Maeda T, Hashimoto H, Takeuchi T, Roos DS, Asai T.

Department of Tropical Medicine and Parasitology, Keio University School of Medicine, Tokyo 1608582.

We previously reported a cytosolic pyruvate kinase (EC from Toxoplasma gondii (TgPyKI) that differs from most eukaryotic pyruvate kinases in being regulated by glucose 6-phosphate rather than fructose 1,6-diphosphate. Another putative pyruvate kinase (TgPyKII) was identified from parasite genome, which exhibits 32% amino acid sequence identity to TgPyKI and retains pyruvate kinase signature motifs and amino acids essential for substrate binding and catalysis. While TgPyKI is most closely related to plant/algal enzymes, phylogenetic analysis suggests a proteobacterial origin for TgPyKII. Enzymatic characterization of recombinant TgPyKII shows a high pH optimum at 8.5, and a preference for GDP as a phosphate recipient. Catalytic activity is independent of K+, and no allosteric or regulatory effects were observed in the presence of fructose 1,6-diphosphate, fructose 2,6-diphosphate, glucose 6-phosphate, ribose 5-phosphate, AMP or ATP. Unlike TgPyKI, native TgPyKII activity was exclusively associated with the membranous fraction of a T. gondii tachyzoite lysate. TgPyKII possesses a long N-terminal extension containing five putative start codons before the conserved region and localizes to both apicoplast and mitochondrion by immunofluorescence assay using native antibody and fluorescent protein fusion to the N-terminal extension. Further deletional and site-directed mutagenesis suggest that a translate from 1st methionine (Met) is responsible for the localization to the apicoplast whereas one from 3rd Met is for the mitochondrion. This is the first report of a potential mitochondrial pyruvate kinase in any system.

PMID: 18326043 [PubMed - as supplied by publisher]

Thursday, March 06, 2008

National seroprevalence of Toxo in India

J Parasitol. 2007 Dec;93(6):1520-1.

National seroprevalence of Toxoplasma gondii in India

Dhumne M, Sengupta C, Kadival G, Rathinaswamy A, Velumani A

Thyrocare Technologies Ltd., #37, 1/D, TTC, MIDC, Turbhe, Navi Mumbai, India.

This article reports the first national serological prevalence of Toxoplasma gondii in India. In total, 23,094 serum samples were tested for T. gondii IgG and IgM antibodies with the use of a solid-phase immunocapture ELISA. Antibodies (IgG) were found in 24.3%; IgM antibodies were detected in 2% of the samples. The lowest seroprevalences were in the northern parts of India, with the highest in the south. These data probably reflect the effects of significantly drier conditions and, therefore, a negative impact on the survivability of T. gondii oocysts.

Hsp20 is a stripe-arranged chaperone like protein associated with the outer leaflet of the inner membrane complex

Biol Cell. 2008 Mar 3 [Epub ahead of print]

Toxoplasma gondii Hsp20 is a stripe-arranged chaperone like protein associated with the outer leaflet of the inner membrane complex

de Miguel N, Lebrun M, Heaslip A, Hu K, Beckers CJ, Matrajt M, Dubremetz JF, Angel SO.

Background information: Toxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. T. gondii has 5 small heat shock (sHsp) proteins located in different subcellular compartments. Among them, Hsp20 showed to be localized at the periphery of the parasite body. sHsps are widespread, constituting the most poorly conserved family of molecular chaperones. The presence of sHsps in membrane structures is unusual. Results: The localization of Hsp20 was further analyzed using high resolution fluorescent light microscopy as well as electron microscopy, which revealed that Hsp20 is associated to the outer surface of the inner membrane complex (IMC), in a set of discontinuous stripes following the same spiraling trajectories as the subpellicular microtubules. Detergent extraction profile of Hsp20 was similar to that of GAP45, a glideosome protein associated to inner membrane complex (IMC), but different from that of IMC1 protein. Although we were unable to detect interacting protein partners of Hsp20 either in normal or stressed tachyzoites, an interaction of Hsp20 with phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5 biphosphate phospholipids could be observed. Conclusions: Hsp20 showed to be associated to a specialized membranous structure of the parasite, the IMC. This discontinuous striped-arrangement is unique in T. gondii, indicating that the topology of the outer leaflet of the IMC is not homogeneous.

PMID: 18315523 [PubMed - as supplied by publisher]

Novel Triazine JPC-2067-B Inhibits Toxo In Vitro and In Vivo

PLoS Negl Trop Dis. 2008 Mar 5;2(3):e190.

Novel Triazine JPC-2067-B Inhibits Toxoplasma gondii In Vitro and In Vivo

Mui EJ, Schiehser GA, Milhous WK, Hsu H, Roberts CW, Kirisits M, Muench S, Rice D, Dubey JP, Fowble JW, Rathod PK, Queener SF, Liu SR, Jacobus DP, McLeod R.

Department of Ophthalmology and Visual Science, University of Chicago, Chicago, Illinois, United States of America.

BACKGROUND AND METHODOLOGY: Toxoplasma gondii causes substantial morbidity, mortality, and costs for healthcare in the developed and developing world. Current medicines are not well tolerated and cause hypersensitivity reactions. The dihydrotriazine JPC-2067-B (4, 6-diamino-1, 2-dihydro-2, 2-dimethyl-1-(3'(2-chloro-, 4-trifluoromethoxyphenoxy)propyloxy)-1, 3, 5-triazine), which inhibits dihydrofolate reductase (DHFR), is highly effective against Plasmodium falciparum, Plasmodium vivax, and apicomplexans related to T. gondii. JPC-2067-B is the primary metabolite of the orally active biguanide JPC-2056 1-(3'-(2-chloro-4-trifluoromethoxyphenyloxy)propyl oxy)- 5-isopropylbiguanide, which is being advanced to clinical trials for malaria. Efficacy of the prodrug JPC-2056 and the active metabolite JPC-2067-B against T. gondii and T. gondii DHFR as well as toxicity toward mammalian cells were tested. PRINCIPAL FINDINGS AND CONCLUSIONS: Herein, we found that JPC-2067-B is highly effective against T. gondii. We demonstrate that JPC-2067-B inhibits T. gondii growth in culture (IC50 20 nM), inhibits the purified enzyme (IC50 6.5 nM), is more efficacious than pyrimethamine, and is cidal in vitro. JPC-2067-B administered parenterally and the orally administered pro-drug (JPC-2056) are also effective against T. gondii tachyzoites in vivo. A molecular model of T. gondii DHFR-TS complexed with JPC-2067-B was developed. We found that the three main parasite clonal types and isolates from South and Central America, the United States, Canada, China, and Sri Lanka have the same amino acid sequences preserving key binding sites for the triazine. SIGNIFICANCE: JPC-2056/JPC-2067-B have potential to be more effective and possibly less toxic treatments for toxoplasmosis than currently available medicines.

PMID: 18320016 [PubMed - in process]

Apicomplexa in mammalian cells: trafficking to the parasitophorous vacuole

Traffic. 2008 Feb 24 [Epub ahead of print]

Apicomplexa in mammalian cells: trafficking to the parasitophorous vacuole

Cesbron-Delauw MF, Gendrin C, Travier L, Ruffiot P, Mercier C.

Laboratoire Adaptation et Pathogénie des Microorganismes, CNRS UMR 5163, Université Joseph Fourier Grenoble 1, BP 170, 38042 Grenoble cedex 9, France.

Most Apicomplexa reside and multiply in the cytoplasm of their host cell, within a parasitophorous vacuole (PV) originating from both parasite and host cell components. Trafficking of parasite-encoded proteins destined to membrane compartments beyond the confine of the parasite plasma membrane is a process that offers a rich territory to explore novel mechanisms of protein-membrane interactions. Here, we focus on the parasitophorous vacuoles formed by the asexual stages of two pathogens of medical importance, Plasmodium and Toxoplasma. We compare the PV of both parasites, with a particular emphasis on their evolutionary divergent compartmentalization within the host cell. We also discuss the existence of peculiar export mechanisms and/or sorting determinants that are potentially involved in the post-secretory targeting of parasite proteins to the PV sub-compartments.

PMID: 18315533 [PubMed - as supplied by publisher]

Influence of low-density lipoprotein (LDL) receptor on lipid composition, inflammation and parasitism

Microbes Infect. 2007 Dec 8 [Epub ahead of print]

Influence of low-density lipoprotein (LDL) receptor on lipid composition, inflammation and parasitism during Toxoplasma gondii infection

Portugal LR, Fernandes LR, Pietra Pedroso VS, Santiago HC, Gazzinelli RT, Alvarez-Leite JI.

Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil.

Intracellular replication of Toxoplasma gondii requires cholesterol uptake by host cell low-density lipoprotein receptor (LDLr), a critical element in atherosclerosis. We evaluated host parasitism, inflammatory responses and development of atherosclerosis in LDLr knockout (LDLr(-/-)) and their controls C57BL/6 mice infected with T. gondii. Our results show that T. gondii cysts were reduced in LDLr(-/-) mice when compared to C57BL/6 mice. However, in presence of hypercholesterolemic diet, parasite growth in LDLr(-/-) mice was similar to that seen in infected C57BL/6 mice. In presence of a hypercholesterolemic diet, T. gondii infection leads to a 60% reduction of serum triacylglycerol, total and atherogenic lipoprotein cholesterol. When aortic valve lesion was analyzed, infected mice showed a reduction of atherosclerotic lesion area as well as CD36 expression. MCP-1, SRA-I, SRA-II, ICAM-1 and VCAM-1 mRNA expression was kept similar between infected and control groups. Thus, despite the intense inflammatory process, the drastic reduction in serum lipids seems to limit the development of atherosclerosis in LDLr(-/-) mice infected with T. gondii. In conclusion, our results indicate that T. gondii employs host LDLr to acquire cholesterol and favor its growth. However, in the presence of hypercholesterolemia, T. gondii parasites are able to acquire cholesterol-rich lipoproteins through an alternative host receptor, and overcome LDLr deficiency, favoring host parasitism and impairing lipid loading of foam cells.

PMID: 18316222 [PubMed - as supplied by publisher]

Microneme protein 8 - a new essential invasion factor

J Cell Sci. 2008 Mar 4 [Epub ahead of print]

Microneme protein 8 - a new essential invasion factor in Toxoplasma gondii

Kessler H, Herm-Götz A, Hegge S, Rauch M, Soldati-Favre D, Frischknecht F, Meissner M.

Apicomplexan parasites rely on sequential secretion of specialised secretory organelles for the invasion of the host cell. First, micronemes release their content upon contact with the host cell. Second, rhoptries are discharged, leading to the formation of a tight interaction (moving junction) with the host cell, through which the parasite invades. The functional characterisation of several micronemal proteins in Toxoplasma gondii suggests the occurrence of a stepwise process. Here, we show that the micronemal protein MIC8 of T. gondii is essential for the parasite to invade the host cell. When MIC8 is not present, a block in invasion is caused by the incapability of the parasite to form a moving junction with the host cell. We furthermore demonstrate that the cytosolic domain is crucial for the function of MIC8 and can not be functionally complemented by any other micronemal protein characterised so far, suggesting that MIC8 represents a novel, functionally distinct invasion factor in this apicomplexan parasite.

PMID: 18319299 [PubMed - as supplied by publisher]

Wednesday, March 05, 2008

Deploying parasite profilin on a mission of invasion and danger

Cell Host Microbe. 2008 Feb 14;3(2):61-3.

Deploying parasite profilin on a mission of invasion and danger

Denkers EY, Striepen B

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.

Interleukin-12 (IL-12) is important in inducing Th1 responses during infection with microbial pathogens such as the protozoan Toxoplasma gondii. In this issue of Cell Host & Microbe, Plattner and colleagues describe an engineered Toxoplasma strain that lacks profilin, an actin-binding molecule previously implicated in Toll-like receptor-11-dependent IL-12 induction and now shown to be important in parasite motility and host cell invasion.

Publication Types:

PMID: 18312839 [PubMed - in process]

Toxoplasma profilin is essential for host cell invasion and TLR11-dependent induction of an interleukin-12 response

Cell Host Microbe. 2008 Feb 14;3(2):77-87

Toxoplasma profilin is essential for host cell invasion and TLR11-dependent induction of an interleukin-12 response

Plattner F, Yarovinsky F, Romero S, Didry D, Carlier MF, Sher A, Soldati-Favre D

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

Apicomplexan parasites exhibit actin-dependent gliding motility that is essential for migration across biological barriers and host cell invasion. Profilins are key contributors to actin polymerization, and the parasite Toxoplasma gondii possesses a profilin-like protein that is recognized by Toll-like receptor TLR11 in the host innate immune system. Here, we show by conditional disruption of the corresponding gene that T.gondii profilin, while not required for intracellular growth, is indispensable for gliding motility, host cell invasion, active egress from host cells, and virulence in mice. Furthermore, parasites lacking profilin are unable to induce TLR11-dependent production in vitro and in vivo of the defensive host cytokine interleukin-12. Thus, profilin is an essential element of two aspects of T. gondii infection. Like bacterial flagellin, profilin plays a role in motility while serving as a microbial ligand recognized by the host innate immune system.

Publication Types:
Research Support, N.I.H., Intramural
Research Support, Non-U.S. Gov't

PMID: 18312842 [PubMed - in process]

The microneme rhomboid protease TgROM1 is required for efficient intracellular growth of Toxoplasma

Eukaryot Cell. 2008 Feb 29 [Epub ahead of print]

The microneme rhomboid protease TgROM1 is required for efficient intracellular growth of Toxoplasma gondii

Brossier F, Starnes GL, Beatty WL, Sibley LD

Department of Molecular Microbiology, Washington University School of Medicine, 660. S Euclid Avenue, Saint Louis, MO, 63110-1093.

Rhomboids are serine proteases that cleave their substrates within the transmembrane domain. Toxoplasma gondii contains six rhomboids that are expressed in different life cycle stages and localized to different cellular compartments. Toxoplasma rhomboid protein 1 (TgROM1) has previously been shown to be active in vitro and the orthologue in Plasmodium falciparum processes the essential microneme protein AMA1 in a heterologous system. We investigated the role of TgROM1 to determine its role in during in vitro growth of T. gondii. TgROM1 was localized in the secretory pathway of the parasite including the Golgi and micronemes, which contain adhesive proteins involved in invasion of host cells. However, unlike other micronemal proteins, TgROM1 was not released onto the parasite surface during cell invasion, suggesting it does not play a critical role in cell invasion. Suppression of TgROM1 using the tetracycline-regulatable system revealed that ROM1 deficient parasites were out-competed by wide type T. gondii. ROM1 deficient parasites showed only modest decrease in invasion but replicated more slowly than wild type cells. Collectively, these results indicate that ROM1 is required for efficient intracellular growth by T. gondii.

PMID: 18310358 [PubMed - as supplied by publisher]

MORN1 has a conserved role in asexual and sexual development across the Apicomplexa

Eukaryot Cell. 2008 Feb 29 [Epub ahead of print]

MORN1 has a conserved role in asexual and sexual development across the Apicomplexa

Ferguson DJ, Sahoo N, Pinches RA, Bumstead JM, Tomley FM, Gubbels MJ

Nuffield Department of Pathology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK; Department of Biology, Boston College, Chestnut Hill, MA, USA; Nuffield Department of Medicine, Molecular Parasitology, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, UK; Division of Microbiology, Institute for Animal Health, Compton, Newbury, Berkshire, RG20 7NN, UK.

The gene encoding the membrane occupation and recognition nexus protein, MORN1, is conserved across the Apicomplexa. In Toxoplasma gondii MORN1 is associated with the spindle poles, the anterior and posterior rings of the inner membrane complex (IMC). The present study examines the localization of MORN1 during the coccidian development of T. gondii and three Eimeria species (in the definitive host) and erythrocytic schizogony of Plasmodium falciparum. During asexual proliferation MORN1 is associated with the posterior ring of the IMCs of the multiple daughters forming during T. gondii endopolygeny and schizogony in Eimeria and P. falciparum. Furthermore, P. falciparum MORN1 protein expression peaked in late schizogony. These data fit a model with a conserved role for MORN1 during IMC assembly in all variations of asexual development. An important new observation is reactivity of MORN1 antibody with certain sexual stages in T. gondii and Eimeria species. Here MORN1 is organized as a ring-like structure where the microgametes bud from the microgametocyte while in mature microgametes it is present near the flagellar basal bodies and mitochondrion. These observations suggest a conserved role for MORN1 across the Apicomplexa involving both asexual and sexual development.

PMID: 18310354 [PubMed - as supplied by publisher]

Monday, March 03, 2008

T cell expression of MyD88 is required for resistance to Toxoplasma

Proc Natl Acad Sci U S A. 2008 Feb 28 [Epub ahead of print]

T cell expression of MyD88 is required for resistance to Toxoplasma gondii

Larosa DF, Stumhofer JS, Gelman AE, Rahman AH, Taylor DK, Hunter CA, Turka LA.

Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104.

Resistance to Toxoplasma gondii depends on dendritic cells to recognize this pathogen and secrete IL-12, in turn promoting IFN-gamma production from responding T cells. The adaptor protein, myeloid differentiation primary-response gene 88 (MyD88), is important for most Toll-like receptor (TLR) signaling, as well as IL-1R/IL-18R signals. There is considerable evidence that MyD88 is required for the innate sensing of T. gondii and IL-12 responses. Although Myd88(-/-) mice challenged with T. gondii have defective IL-12 and Th1 effector responses and succumb to disease, administration of IL-12 to Myd88(-/-) mice partially restores the Th1 response and yet fails to prolong survival. This finding suggested that MyD88 may mediate signals within T cells important for resistance to this pathogen. To evaluate the role of MyD88 in T cells under noncompetitive conditions, bone marrow chimeras were generated, in which the T cells lacked MyD88, but MyD88-dependent innate immune responses were intact. Upon challenge with T. gondii, these chimeric mice were more susceptible to disease, developing severe toxoplasmic encephalitis and succumbing within 30 days. Splenocytes and brain mononuclear cells isolated from infected chimeric mice produced less IFN-gamma when cultured with a T. gondii-derived antigen. The increase in susceptibility observed was independent of signals via the IL-1R and IL-18R, suggesting a role for TLRs in MyD88-mediated T cell responses to T. gondii. These observations show that, in addition to a role for MyD88 in innate responses, T cell expression of MyD88 is necessary for prolonged resistance to a pathogen.

PMID: 18308927 [PubMed - as supplied by publisher]