Saturday, October 29, 2011

Separation and purification of Toxoplasma gondii tachyzoites from in vitro and in vivo culture systems

Exp Parasitol. 2011 Oct 15. [Epub ahead of print]

Separation and purification of Toxoplasma gondii tachyzoites from in vitro and in vivo culture systems.

Wu L, Chen SX, Jiang XG, Fu XL, Shen YJ, Cao JP.

SourceSchool of Medical Science and Laboratory Medicine, Jiangsu University, 212013 Zhenjiang, People's Republic of China.

In this study, we evaluated four methods to separate and purify Toxoplasma gondii tachyzoites from in vivo and in vitro culture systems, including trypsin digestion, purification with a 3-μm filter, CF-11 cellulose purification, and Percoll purification. Our results indicate that both purification with a 3-μm filter and CF11 cellulose purification methods remove leukocytes or HeLa cells, and can therefore be used as candidate methods for the purification of in vivo and in vitro culture products. Trypsin digestion had a high tachyzoite recovery rate, but 22.35% of leukocytes and 69.64% of HeLa cells remained in the purified products. Percoll solution [30% (v/v)] also had a high tachyzoite recovery rate, but 3.44% of leukocytes and 61.61% of HeLa cells remained in the purified products. The 40% Percoll solution was also a candidate method for purifying tachyzoites from in vivo culture products, with a 65.45% tachyzoite recovery rate and without leukocytes.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID:22033076[PubMed - as supplied by publisher]

Molecular cloning and characterization of Mitogen-activated protein kinase 2 in Toxoplasma gondii

Cell Cycle. 2011 Oct 31;10(20). [Epub ahead of print]

Molecular cloning and characterization of Mitogen-activated protein kinase 2 in Toxoplasma gondii.

Huang H, Ma YF, Bao Y, Lee H, Lisanti MP, Tanowitz HB, Weiss LM.

SourceDepartment of Pathology; Albert Einstein College of Medicine; Bronx, NY USA.

Toxoplasma gondii is an obligate intracellular protozoan that is both a human and animal pathogen. This Apicomplexan causes significant morbidity and mortality in immune competent and immune compromised hosts. In humans, the most common manifestations of T. gondii infections are chorioretinitis in congenital infection and encephalitis in immune compromised patients such as patients with advanced AIDS. Mitogen-activated protein kinase (MAPK) pathways are major signal transduction systems by which eukaryotic cells convert environmental cues to intracellular events such as proliferation and differentiation. We have identified a T. gondii homologue of the MAPK family that we have called TgMAPK2. Sequence analyses demonstrated TgMAPK2 has homology with lower eukaryotic ERK2, but has significant differences from mammalian ERK2. TgMAPK2 has an open reading frame of 2037 bp, 678 amino acids, and its molecular weight is 73.1 kDa. It contains the typical twelve sub-domains of a MAPK and has a TDY motif in the dual phosphorylation and activation subdomains. This suggests that TgMAPK2 may play an important role in stress response. Recombinant TgMAPK2 was catalytically active and was not inhibited by a human ERK2 inhibitor, FR180204. A partial TgMAPK2 lacking the ATP binding motifs, GxGxxGxV, was successfully regulated by a ligand-controlled destabilization domain (ddFKBP) expression vector system in T. gondii. Since TgMAPK2 is significantly different from its mammalian counterpart it may be useful as a drug target. This work establishes a foundation for further study for this unique kinase.

PMID:22030559[PubMed - as supplied by publisher]

Friday, October 28, 2011

Toxoplasma gondii: Determination of the onset of chronic infection in mice and the in vitro reactivation of brain cysts

Exp Parasitol. 2011 Oct 18. [Epub ahead of print]

Toxoplasma gondii: Determination of the onset of chronic infection in mice and the in vitro reactivation of brain cysts.

Kit CW, Wah MJ, Ambu S, Segarra I.

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

Toxoplasma gondii is an intra-cellular parasite that infects humans through vertical and horizontal transmission. The cysts remain dormant in the brain of infected humans and can reactivate in immunocompromised hosts resulting in acute toxoplasmic encephalitis which may be fatal. We determined the onset and progression of brain cysts generation in a mouse model following acute toxoplasmosis as well as the ability of brain cysts to reactivate in vitro. Male Balb/c mice, (uninfected control group, n=10) were infected orally (study group, n=50) with 1000 tachyzoites of T. gondii (ME49 strain) and euthanized at 1, 2, 4, 8 and 16weeks post infection. Brain tissue was harvested, homogenized, stained and the number of brain cysts counted. Aliquots of brain homogenate with cysts were cultured in vitro with confluent Vero cells and the number of cysts and tachyzoites counted after 1week. Brain cysts but not tachyzoites were detected at week 2 post infection and reached a plateau by week 4. In vitro Vero cells culture showed similar pattern for cysts and tachyzoites and reactivation of cyst in vitro was not influenced by the age of the brain cysts.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID:22027550[PubMed - as supplied by publisher]

Thursday, October 27, 2011

The apicoplast: a red alga in human parasites

Essays Biochem. 2011 Oct 24;51:111-25.

The apicoplast: a red alga in human parasites.

Striepen B.

SourceCenter for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, 500 D.W. Brooks Drive, Athens, GA 30602, U.S.A.

Surprisingly, some of the world's most dangerous parasites appear to have had a benign photosynthetic past in the ocean. The phylum Apicomplexa includes the causative agents of malaria and a number of additional human and animal diseases. These diseases threaten the life and health of hundreds of millions each year and pose a tremendous challenge to public health. Recent findings suggest that Apicomplexa share their ancestry with diatoms and kelps, and that a key event in their evolution was the acquisition of a red algal endosymbiont. A remnant of this endosymbiont is still present today, albeit reduced to a small chloroplast-like organelle, the apicoplast. In the present chapter, I introduce the remarkably complex biology of this organelle. The apicoplast is bounded by four membranes, and these membranes trace their ancestry to three different organisms. Intriguingly, this divergent ancestry is still reflected in their molecular makeup and function. We also pursue the raison d'être of the apicoplast. Why did Apicomplexa retain a chloroplast when they abandoned photosynthesis for a life as obligate parasites? The answer to this question appears to lie in the profound metabolic dependence of the parasite on its endosymbiont. This dependence may prove to be a liability to the parasite. As humans lack chloroplasts, the apicoplast has become one of the prime targets for the development of parasite-specific drugs.

PMID:22023445[PubMed - in process]

The Phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii Reveal Unusual Adaptations Within and Beyond the Parasites' Boundaries

Cell Host Microbe. 2011 Oct 4;10(4):410-9.

The Phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii Reveal Unusual Adaptations Within and Beyond the Parasites' Boundaries.

Treeck M, Sanders JL, Elias JE, Boothroyd JC.

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

Plasmodium falciparum and Toxoplasma gondii are obligate intracellular apicomplexan parasites that rapidly invade and extensively modify host cells. Protein phosphorylation is one mechanism by which these parasites can control such processes. Here we present a phosphoproteome analysis of peptides enriched from schizont stage P. falciparum and T. gondii tachyzoites that are either "intracellular" or purified away from host material. Using liquid chromatography-tandem mass spectrometry, we identified over 5,000 and 10,000 previously unknown phosphorylation sites in P. falciparum and T. gondii, respectively, revealing that protein phosphorylation is an extensively used regulation mechanism both within and beyond parasite boundaries. Unexpectedly, both parasites have phosphorylated tyrosines, and P. falciparum has unusual phosphorylation motifs that are apparently shaped by its A:T-rich genome. This data set provides important information on the role of phosphorylation in the host-pathogen interaction and clues to the evolutionary forces operating on protein phosphorylation motifs in both parasites.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID:22018241[PubMed - in process]

Toxoplasmosis: diagnosis, treatment, and prevention in congenitally exposed infants

J Pediatr Health Care. 2011 Nov;25(6):355-64. Epub 2010 Jun 12.

Toxoplasmosis: diagnosis, treatment, and prevention in congenitally exposed infants.

Kaye A.

Toxoplasmosis is a rare disease caused by the obligate intracellular protozoan parasite, Toxoplasma gondii. Most persons with toxoplasmosis in the United States are asymptomatic, but if a woman is infected during pregnancy, the parasite can cross the placenta and cause congenital toxoplasmosis in the fetus. The severity of congenital toxoplasmosis depends on when in the pregnancy the mother is exposed, but it can cause ocular and central nervous system disease as well as lead to growth failure and hearing and vision abnormalities. Congenital toxoplasmosis is treated with a combination of pyrimethamine, sulfadiazine, and leucovorin. It is important for pediatric nurse practitioners to be aware of the clinical presentation and treatment of congenital toxoplasmosis.

Copyright © 2011 National Association of Pediatric Nurse Practitioners. Published by Mosby, Inc. All rights reserved.

PMID:22018426[PubMed - in process]

Toxoplasma gondii: Identification and immune response against a group of proteins involved in cellular invasion

Exp Parasitol. 2011 Oct 12. [Epub ahead of print]

Toxoplasma gondii: Identification and immune response against a group of proteins involved in cellular invasion.

Azzouz S, Maache M, Osuna A, Lawton P, Pétavy AF.

SourceDepartment of Parasitology and Medical Mycology, Université de Lyon, Université Claude Bernard-Lyon I, ISPB, Faculté de Pharmacie, 8 Avenue Rockefeller, 69373 Lyon Cedex 08, France.

Toxoplasma gondii is an ubiquitous intracellular parasite, causative agent of toxoplasmosis, and a worldwide zoonosis for which an effective vaccine is needed. A group of proteins secreted by tachyzoites during host-cell invasion was isolated from the interaction medium. It induced the permeability of the cells as assessed by alpha-sarcin and consequently facilitated the entry of the parasite into the cells. SDS-PAGE of the purified proteins showed a pattern of four proteins of 67, 42, 32 and 27kDa. MRC-5 cells incubated with the total protein and the different electroeluted bands endured a high cellular death in presence of alpha-sarcin. BALb/C mice immunized with the group of proteins had a mixed Th1/Th2 response and were protected upon challenge with the parasites.

Copyright © 2011. Published by Elsevier Inc.

PMID:22019410[PubMed - as supplied by publisher]

Identification of tissue cyst wall components by transcriptome analysis of in vivo and in vitro Toxoplasma bradyzoites

Eukaryot Cell. 2011 Oct 21. [Epub ahead of print]

Identification of tissue cyst wall components by transcriptome analysis of in vivo and in vitro Toxoplasma bradyzoites.

Buchholz KR, Fritz HM, Chen X, Durbin-Johnson B, Rocke DM, Ferguson DJ, Conrad PA, Boothroyd JC.

SourceDepartment of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, 94305, USA.

The Toxoplasma gondii bradyzoite is essential to establish persistent infection yet little is known about what factors this developmental form secretes to establish the cyst or interact with its host cell. To identify candidate bradyzoite-secreted effectors, the transcriptomes of in vitro tachyzoites 2 days post-infection, in vitro bradyzoites 4 days post-infection, and in vivo bradyzoites 21 days post-infection were interrogated by microarray, and the program SignalP was used to identify signal peptides indicating secretion. One-hundred-and-two putative bradyzoite-secreted effectors were identified with this approach. Two candidates, bradyzoite pseudokinase 1 (BPK1) and microneme adhesive repeat (MAR)-domain containing protein 4 (MCP4), were chosen for further investigation and confirmed to be induced and secreted by bradyzoites in vitro and in vivo. Thus, we report the first analysis of the transcriptomes of in vitro and in vivo bradyzoites and identify two new protein components of the Toxoplasma tissue cyst wall.

PMID:22021236[PubMed - as supplied by publisher]

SPM1 Stabilizes Subpellicular Microtubules in Toxoplasma gondii

Eukaryot Cell. 2011 Oct 21. [Epub ahead of print]

SPM1 Stabilizes Subpellicular Microtubules in Toxoplasma gondii.

Tran JQ, Li C, Chyan A, Chung L, Morrissette NS.

SourceDepartment of Molecular Biology and Biochemistry, University of California, Irvine, Irvine CA 92697.

We have identified two novel proteins that co-localize with the subpellicular microtubules in the protozoan parasite Toxoplasma gondii and named these proteins SPM1 and SPM2. These proteins have basic isoelectric points and both have homologs in other apicomplexan parasites. SPM1 contains six tandem copies of a 32 amino acid repeat whereas SPM2 lacks defined protein signatures. Alignment of Toxoplasma SPM2 with apparent Plasmodium SPM2 homologs indicates that the greatest degree of conservation lies in the carboxy-terminal half of the protein. Analysis of Plasmodium homologs of SPM1 indicates that while the central 32 amino acid repeats have expanded to different degrees (seven, eight, nine, twelve or thirteen repeats), the amino and carboxy-terminal regions remain conserved. In contrast, although the Cryptosporidium SPM1 homolog has a conserved carboxy-tail, the five repeats are considerably diverged, and it has a smaller amino-terminal domain. SPM1 is localized along the full length of the subpellicular microtubules but does not associate with the conoid or spindle microtubules. SPM2 has a restricted localization along the middle region of the subpellicular microtubules. Domain deletion analysis indicates that four or more copies of the SPM1 repeat are required for localization to microtubules, and the amino-terminal 63 residues of SPM2 are required for localization to the subpellicular microtubules. Gene deletion studies indicate that neither SPM1 nor SPM2 is essential for tachyzoite viability. However, loss of SPM1 decreases overall parasite fitness and eliminates the stability of subpellicular microtubules to detergent extraction.

PMID:22021240[PubMed - as supplied by publisher]

Unrecognized Ingestion of Toxoplasma gondii Oocysts Leads to Congenital Toxoplasmosis and Causes Epidemics in North America

Clin Infect Dis. 2011 Oct 21. [Epub ahead of print]

Unrecognized Ingestion of Toxoplasma gondii Oocysts Leads to Congenital Toxoplasmosis and Causes Epidemics in North America.

Boyer K, Hill D, Mui E, Wroblewski K, Karrison T, Dubey JP, Sautter M, Noble AG, Withers S, Swisher C, Heydemann P, Hosten T, Babiarz J, Lee D, Meier P, McLeod R; other members of the Toxoplasmosis Study Group.
SourceDepartment of Pediatrics.

Background. Congenital toxoplasmosis presents as severe, life-altering disease in North America. If mothers of infants with congenital toxoplasmosis could be identified by risks, it would provide strong support for educating pregnant women about risks, to eliminate this disease. Conversely, if not all risks are identifiable, undetectable risks are suggested. A new test detecting antibodies to sporozoites demonstrated that oocysts were the predominant source of Toxoplasma gondii infection in 4 North American epidemics and in mothers of children in the National Collaborative Chicago-based Congenital Toxoplasmosis Study (NCCCTS). This novel test offered the opportunity to determine whether risk factors or demographic characteristics could identify mothers infected with oocysts.Methods. Acutely infected mothers and their congenitally infected infants were evaluated, including in-person interviews concerning risks and evaluation of perinatal maternal serum samples.Results. Fifty-nine (78%) of 76 mothers of congenitally infected infants in NCCCTS had primary infection with oocysts. Only 49% of these mothers identified significant risk factors for sporozoite acquisition. Socioeconomic status, hometown size, maternal clinical presentations, and ethnicity were not reliable predictors.Conclusions. Undetected contamination of food and water by oocysts frequently causes human infections in North America. Risks are often unrecognized by those infected. Demographic characteristics did not identify oocyst infections. Thus, although education programs describing hygienic measures may be beneficial, they will not suffice to prevent the suffering and economic consequences associated with congenital toxoplasmosis. Only a vaccine or implementation of systematic serologic testing of pregnant women and newborns, followed by treatment, will prevent most congenital toxoplasmosis in North America.

PMID:22021924[PubMed - as supplied by publisher]

Identification of intracellular and plasma membrane calcium channel homologues in pathogenic parasites

PLoS One. 2011;6(10):e26218. Epub 2011 Oct 14.

Identification of intracellular and plasma membrane calcium channel homologues in pathogenic parasites.

Prole DL, Taylor CW.

SourceDepartment of Pharmacology, University of Cambridge, Cambridge, United Kingdom.

Ca(2+) channels regulate many crucial processes within cells and their abnormal activity can be damaging to cell survival, suggesting that they might represent attractive therapeutic targets in pathogenic organisms. Parasitic diseases such as malaria, leishmaniasis, trypanosomiasis and schistosomiasis are responsible for millions of deaths each year worldwide. The genomes of many pathogenic parasites have recently been sequenced, opening the way for rational design of targeted therapies. We analyzed genomes of pathogenic protozoan parasites as well as the genome of Schistosoma mansoni, and show the existence within them of genes encoding homologues of mammalian intracellular Ca(2+) release channels: inositol 1,4,5-trisphosphate receptors (IP(3)Rs), ryanodine receptors (RyRs), two-pore Ca(2+) channels (TPCs) and intracellular transient receptor potential (Trp) channels. The genomes of Trypanosoma, Leishmania and S. mansoni parasites encode IP(3)R/RyR and Trp channel homologues, and that of S. mansoni additionally encodes a TPC homologue. In contrast, apicomplexan parasites lack genes encoding IP(3)R/RyR homologues and possess only genes encoding TPC and Trp channel homologues (Toxoplasma gondii) or Trp channel homologues alone. The genomes of parasites also encode homologues of mammalian Ca(2+)influx channels, including voltage-gated Ca(2+) channels and plasma membrane Trp channels. The genome of S. mansoni also encodes Orai Ca(2+) channel and STIM Ca(2+) sensor homologues, suggesting that store-operated Ca(2+) entry may occur in this parasite. Many anti-parasitic agents alter parasite Ca(2+) homeostasis and some are known modulators of mammalian Ca(2+) channels, suggesting that parasite Ca(2+) channel homologues might be the targets of some current anti-parasitic drugs. Differences between human and parasite Ca(2+) channels suggest that pathogen-specific targeting of these channels may be an attractive therapeutic prospect.

PMID:22022573[PubMed - in process] PMCID: PMC3194816

Strain-dependent host transcriptional responses to toxoplasma infection are largely conserved in Mammalian and avian hosts

PLoS One. 2011;6(10):e26369. Epub 2011 Oct 13.

Strain-dependent host transcriptional responses to toxoplasma infection are largely conserved in Mammalian and avian hosts.

Ong YC, Boyle JP, Boothroyd JC.

SourceStanford University, Department of Microbiology and Immunology, Stanford, California, United States of America.

Toxoplasma gondii has a remarkable ability to infect an enormous variety of mammalian and avian species. Given this, it is surprising that three strains (Types I/II/III) account for the majority of isolates from Europe/North America. The selective pressures that have driven the emergence of these particular strains, however, remain enigmatic. We hypothesized that strain selection might be partially driven by adaptation of strains for mammalian versus avian hosts. To test this, we examine in vitro, strain-dependent host responses in fibroblasts of a representative avian host, the chicken (Gallus gallus). Using gene expression profiling of infected chicken embryonic fibroblasts and pathway analysis to assess host response, we show here that chicken cells respond with distinct transcriptional profiles upon infection with Type II versus III strains that are reminiscent of profiles observed in mammalian cells. To identify the parasite drivers of these differences, chicken fibroblasts were infected with individual F1 progeny of a Type II x III cross and host gene expression was assessed for each by microarray. QTL mapping of transcriptional differences suggested, and deletion strains confirmed, that, as in mammalian cells, the polymorphic rhoptry kinase ROP16 is the major driver of strain-specific responses. We originally hypothesized that comparing avian versus mammalian host response might reveal an inversion in parasite strain-dependent phenotypes; specifically, for polymorphic effectors like ROP16, we hypothesized that the allele with most activity in mammalian cells might be less active in avian cells. Instead, we found that activity of ROP16 alleles appears to be conserved across host species; moreover, additional parasite loci that were previously mapped for strain-specific effects on mammalian response showed similar strain-specific effects in chicken cells. These results indicate that if different hosts select for different parasite genotypes, the selection operates downstream of the signaling occurring during the beginning of the host's immune response.

PMID:22022607[PubMed - in process] PMCID: PMC3192797

Calcium storage and function in apicomplexan parasites

Essays Biochem. 2011 Oct 24;51:97-110.

Calcium storage and function in apicomplexan parasites.

Moreno SN, Ayong L, Pace DA.

SourceCenter for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, GA 30602, U.S.A.

Calcium is relevant for several vital functions in apicomplexan parasites, including host cell invasion, parasite motility and differentiation. The ER (endoplasmic reticulum) and calcium-rich acidocalcisomes have been identified as major calcium stores. Other potential calcium-storage organelles include the Golgi, the mitochondrion, the apicoplast and the recently described plant-like vacuole in Toxoplasma gondii. Compared with most eukaryotic systems, apicomplexan parasites contain a reduced number of calcium-related genes, a vast majority of which remain uncharacterized. Several Ca2+-ATPases have been described in apicomplexans, several of which are annotated in the different genomes. There is experimental evidence for an IP3 (inositol 1,4,5-trisphosphate)-dependent calcium response in Plasmodium spp. and T. gondii, although no IP3 or ryanodine receptors have been identified. Genes encoding potential calcium channels are present in T. gondi, but not in Plasmodium spp. and Cryptosporidium spp. Effector calcium-binding proteins including calmodulins and CDPK (calcium-dependent protein kinase) genes mainly found in plants have also been described. The characterized CDPKs were found to play important roles in protein secretion, host cell invasion and parasite differentiation. Taken together, the available information on calcium storage and function in apicomplexans, although fragmented, suggest the existence of unique calcium-mediated pathways in these parasites. An in-depth functional characterization of the apicomplexan calcium-related genes could lead to the identification of novel therapeutic targets, and will improve our understanding of the role of calcium in parasite development and virulence.

PMID:22023444[PubMed - in process]

Cell invasion and strain dependent induction of suppressor of cytokine signaling-1 by Toxoplasma gondii

Immunobiology. 2011 Aug 27. [Epub ahead of print]

Cell invasion and strain dependent induction of suppressor of cytokine signaling-1 by Toxoplasma gondii.

Stutz A, Kessler H, Kaschel ME, Meissner M, Dalpke AH.

SourceDept. of Infectious Diseases - Medical Microbiology and Hygiene, University Heidelberg, 69120 Heidelberg, Germany.

Toxoplasma gondii is an intracellular parasite that has to cope with the microbicidal actions of IFNγ. Previously we reported that parasite-mediated induction of suppressor of cytokine signaling protein 1 (SOCS1) contributes to inhibition of IFNγ signaling. However, the signaling requirements remained elusive. We now show that induction of SOCS1 and inhibition of nitric oxide production by IFNγ was independent of stimulation of Toll-like receptors. Instead, infection by T. gondii resulted in induction of egr transcription factors which have been reported to regulate SOCS expression. Indeed, induction of egr2 as well as SOCS1 was dependent on p38 MAP kinase and blockade of egr inhibited SOCS1 expression. Moreover, we found that Mic8, a previously identified invasion factor of T. gondii, was necessary for SOCS1 regulation and escape of IFNγ mediated nitric oxide secretion within macrophages. Surprisingly, when further analyzing Mic8 deficient parasites we noted that inhibition of IFNγ mediated up-regulation of MHC-class II and ICAM1 molecules was independent of cell invasion. Furthermore, these inhibitory effects were equally observed in type I and II strains of T. gondii and were dependent on excreted and secreted antigens. In contrast, only the virulent RH type I strain additionally induced SOCS1 and efficiently inhibited nitric oxide secretion by IFNγ. The results show that T. gondii makes use of two different mechanisms to escape from IFNγ activity with one mode being strain dependent and relying on active cell invasion and SOCS1 induction.

Copyright © 2011 Elsevier GmbH. All rights reserved.

PMID:22015046[PubMed - as supplied by publisher]

Potential immunomodulatory effects of latent toxoplasmosis in humans

BMC Infect Dis. 2011 Oct 18;11(1):274. [Epub ahead of print]

Potential immunomodulatory effects of latent toxoplasmosis in humans.

Flegr J, Striz I.


BACKGROUND: About 30% of the population worldwide are infected with the protozoan parasite Toxoplasma gondii. Latent toxoplasmosis has many specific behavioral and physiological effects on the human organism. Modified reactivity of the immune system has been suggested to play a key role in many of these effects. For example, the immunosuppression hypothesis explains the higher probability of the birth of male offspring observed in Toxoplasma-positive humans and mice by the protection of the (more immunogenic) male embryos against abortion.

METHODS: Here we searched for indices of immunosuppression in Toxoplasma-positive subjects by comparing clinical records of immunology outpatients.

RESULTS: Our cohort study showed that the male patients with latent toxoplasmosis had decreased and the Toxoplasma-positive women had increased leukocyte, NK-cell and monocyte counts in comparison with controls. The B-cell counts were reduced in both Toxoplasma-positive men and women. The difference between Toxoplasma-positive and Toxoplasma-negative subjects diminished with the decline of the specific Toxoplasma antibody titre (a proxy for the length of infection), which is consistent with the observed decreasing strength of the effect of latent toxoplasmosis on human reproduction. The prevalence of toxoplasmosis in 128 male patients was unusually low (10.9%) which contrasted with normal prevalence in 312 female patients (23.7%) and in general population Prague (20-30%).

CONCLUSIONS: Latent toxoplasmosis has immunomodulatory effects in human and probably protects men against some classes of immunopathological diseases. The main limitation of the present study was the absence of the data on the immunoreactivity of immune cells subpopulations. Therefore further studies are needed to search for indices of immunosuppression in human using more specific markers.

PMID:22008411[PubMed - as supplied by publisher]

Characterization of Toxoplasma gondii 5' UTR with Encyclopedic TSS Information

J Parasitol. 2011 Oct 19. [Epub ahead of print]

Characterization of Toxoplasma gondii 5' UTR with Encyclopedic TSS Information.

Yamagishi J, Watanabe J, Goo YK, Masatani T, Suzuki Y, Xuan X.

SourceObihiro University of Agriculture and Veterynary Medicine, Professor, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterynary Medicine.

Abstract The 5' UTR is widely involved in gene expression via post-transcriptional regulation. However, the detail profile of the 5' UTR for Toxoplasma gondii has not demonstrated yet. To investigate the issue, we compared the predicted ORFs and transcription start sites (TSSs) of T. gondii obtained by TSS-seq, a method which enables to analyze encyclopedic TSSs with next-generation sequencers. As a result, it was demonstrated that the mode length of the 5' UTR distributes between 120 and 140 nts when a subset of genes with predicted signal peptides was examined. On the contrary, when genes without the signal peptide were examined, the length was extended to around 600 nts (nucleotides). Because additional information on the predicted signal peptide endows increased reliability to the 5' end estimation of each ORF, we believe that the former value was more reliable as a representative 5' UTR length of T. gondii and that the discrepancy indicates that current predictions of the 5' end of the ORF were less accurate and considerably more discordant with the natural status.

PMID:22010783[PubMed - as supplied by publisher]

A new alternative in vitro method for quantification of Toxoplasma gondii infectivity

J Parasitol. 2011 Oct 19. [Epub ahead of print]

A new alternative in vitro method for quantification of Toxoplasma gondii infectivity.

Useo R, Husson F, De Coninck J, Khaldi S, Gervais P.

Sourcea AgroSup Dijon, Université de Bourgogne, AgroSup Dijon, Université de Bourgogne.

Abstract An in vitro method to determine the infectious potency of an unknown suspension of the protozoan parasite Toxoplasma gondii based on kinetics of host cells lysis was developed. Mic1-3KO a mutant strain of Toxoplasma gondii RH tachyzoïtes were inoculated in 25-cm² flasks containing a 90% confluent monolayer of Human Foreskin fibroblast. Lysis kinetics was monitored for infection ratios ranging from 1:10⁶ to 1:10¹ and the threshold value for parasite egress was defined at 10⁶¹. Results allowed to build a calibration curve relating the initial infection ratios to the time needed to reach 10⁶¹. Finally, the method was validated using of a known mixture of dead and live parasites. This method was found to estimate with accuracy the initial ratio of infection of an unknown parasites suspension. This easy-to-use method is reproducible and application to any Toxoplasma gondii tachyzoïtes RH strain, genetically modified or not. This method is suitable for testing promising candidates for an effective live vaccine.

PMID:22010815[PubMed - as supplied by publisher]

In vitro interactions of Toxoplasma gondii tachyzoites with di-cationic compounds

Parasitology. 2011 Oct 19:1-13. [Epub ahead of print]

The adaptive potential of a survival artist: characterization of the in vitro interactions of Toxoplasma gondii tachyzoites with di-cationic compounds in human fibroblast cell cultures.

Kropf C, Debache K, Rampa C, Barna F, Schorer M, Stephens CE, Ismail MA, Boykin DW, Hemphill A.

SourceInstitute of Parasitology, Vetsuisse Faculty, University of Berne, Switzerland.

SUMMARYThe impact of di-cationic pentamidine-analogues against Toxoplama gondii (Rh- and Me49-background) was investigated. The 72 h-growth assays showed that the arylimidamide DB750 inhibited the proliferation of tachyzoites of T. gondii Rh and T. gondii Me49 with an IC50 of 0·11 and 0·13 μm, respectively. Pre-incubation of fibroblast monolayers with 1 μm DB750 for 12 h and subsequent culture in the absence of the drug also resulted in a pronounced inhibiton of parasite proliferation. However, upon 5-6 days of drug exposure, T. gondii tachyzoites adapted to the compound and resumed proliferation up to a concentration of 1·2 μm. Out of a set of 32 di-cationic compounds screened for in vitro activity against T. gondii, the arylimidamide DB745, exhibiting an IC50 of 0·03 μm and favourable selective toxicity was chosen for further studies. DB745 also inhibited the proliferation of DB750-adapted T. gondii (IC50=0·07 μm). In contrast to DB750, DB745 also had a profound negative impact on extracellular non-adapted T. gondii tachyzoites, but not on DB750-adapted T. gondii. Adaptation of T. gondii to DB745 (up to a concentration of 0·46 μm) was much more difficult to achieve and feasible only over a period of 110 days. In cultures infected with DB750-adapted T. gondii seemingly intact parasites could occasionally be detected by TEM. This illustrates the astonishing capacity of T. gondii tachyzoites to adapt to environmental changes, at least under in vitro conditions, and suggests that DB745 could be an interesting drug candidate for further assessments in appropriate in vivo models.

PMID:22011664[PubMed - as supplied by publisher]

Intrinsic expression of Nod2 in CD4(+) T lymphocytes is not necessary for the development of cell-mediated immunity and host resistance to Toxoplasma

Eur J Immunol. 2011 Oct 17. doi: 10.1002/eji.201141876. [Epub ahead of print]

Intrinsic expression of Nod2 in CD4(+) T lymphocytes is not necessary for the development of cell-mediated immunity and host resistance to Toxoplasma gondii.

Caetano BC, Biswas A, Lima-Junior DS, Benevides L, Mineo TW, Horta CV, Lee KH, Silva JS, Gazzinelli RT, Zamboni DS, Kobayashi KS.

SourceDepartment of Medicine, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA.

Nod2 belongs to the NLR family of proteins and senses bacterial cell wall components to initiate innate immune responses against various pathogens. Recently it has been reported that T cell-intrinsic expression of Nod2 promotes host defense against Toxoplasma gondii infection by inducing type 1 immunity. Here we present results that demonstrate that Nod2 does not play a role in the defense against T. gondii infection. Nod2-deficient mice were fully capable of inducing Th1 immune responses and did not show enhanced susceptibility to infection. Upon T-cell receptor stimulation in vitro, Nod2-deficient CD4(+) T cells showed normal activation, IL-2 production, proliferation and Th1/2 differentiation. Nod2 mRNA and protein were expressed in CD4(+) T and CD8(+) T cells at substantial levels. Therefore Nod2, although expressed in CD4(+) T cells, does not have an intrinsic function in T-cell activation and differentiation.

Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PMID:22002196[PubMed - as supplied by publisher]

An agent-based model for the transmission dynamics of Toxoplasma gondii

J Theor Biol. 2011 Oct 12;293C:15-26. [Epub ahead of print]

An agent-based model for the transmission dynamics of Toxoplasma gondii.

Jiang W, Sullivan AM, Su C, Zhao X.

SourceDepartment of Mechanical, Aerospace and Biomedical Engineering, University of Tennessee, Knoxville, TN 37996-2030, United States; School of Civil Engineering and Mechanics, Huazhong University of Science & Technology, Wuhan 430074, China.

Toxoplasma gondii (T. gondii) is a unicellular protozoan that infects up to one-third of the world's human population. Numerous studies revealed that a latent infection of T. gondii can cause life-threatening encephalitis in immunocompromised people and also has significant effects on the behavior of healthy people and animals. However, the overall transmission of T. gondii has not been well understood although many factors affecting this process have been found out by different biologists separately. Here we synthesize what is currently known about the natural history of T. gondii by developing a prototype agent-based model to mimic the transmission process of T. gondii in a farm system. The present model takes into account the complete life cycle of T. gondii, which includes the transitions of the parasite from cats to environment through feces, from contaminated environment to mice through oocysts, from mice to cats through tissue cysts, from environment to cats through oocysts as well as the vertical transmission among mice. Although the current model does not explicitly include humans and other end-receivers, the effect of the transition to end-receivers is estimated by a developed infection risk index. The current model can also be extended to include human activities and thus be used to investigate the influences of human management on disease control. Simulation results reveal that most cats are infected through preying on infected mice while mice are infected through vertical transmission more often than through infection with oocysts, which clearly suggests the important role of mice during the transmission of T. gondii. Furthermore, our simulation results show that decreasing the number of mice on a farm can lead to the eradication of the disease and thus can lower the infection risk of other intermediate hosts on the farm. In addition, with the assumption that the relation between virulence and transmission satisfies a normal function, we show that intermediate virulent lineages (type II) can sustain the disease most efficiently, which can qualitatively agree with the fact that the evolution of the parasite favors intermediate virulence. The effects of other related factors on transmission, including the latent period and imprudent behavior of mice, and prevention strategies are also studied based on the present model.

Copyright © 2011 Elsevier Ltd. All rights reserved.

PMID:22004993[PubMed - as supplied by publisher]

Interferon-gamma- and perforin-mediated immune responses for resistance against Toxoplasma gondii in the brain

Expert Rev Mol Med. 2011 Oct 4;13:e31.

Interferon-gamma- and perforin-mediated immune responses for resistance against Toxoplasma gondii in the brain.

Suzuki Y, Sa Q, Gehman M, Ochiai E.

SourceDepartment of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY, USA.

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes various diseases, including lymphadenitis, congenital infection of fetuses and life-threatening toxoplasmic encephalitis in immunocompromised individuals. Interferon-gamma (IFN-γ)-mediated immune responses are essential for controlling tachyzoite proliferation during both acute acquired infection and reactivation of infection in the brain. Both CD4+ and CD8+ T cells produce this cytokine in response to infection, although the latter has more potent protective activity. IFN-γ can activate microglia, astrocytes and macrophages, and these activated cells control the proliferation of tachyzoites using different molecules, depending on cell type and host species. IFN-γ also has a crucial role in the recruitment of T cells into the brain after infection by inducing expression of the adhesion molecule VCAM-1 on cerebrovascular endothelial cells, and chemokines such as CXCL9, CXCL10 and CCL5. A recent study showed that CD8+ T cells are able to remove T. gondii cysts, which represent the stage of the parasite in chronic infection, from the brain through their perforin-mediated activity. Thus, the resistance to cerebral infection with T. gondii requires a coordinated network using both IFN-γ- and perforin-mediated immune responses. Elucidating how these two protective mechanisms function and collaborate in the brain against T. gondii will be crucial in developing a new method to prevent and eradicate this parasitic infection.

PMID:22005272[PubMed - in process]

A patatin-like protein protects Toxoplasma gondii from degradation in a nitric oxide dependent manner

Infect Immun. 2011 Oct 17. [Epub ahead of print]

A patatin-like protein protects Toxoplasma gondii from degradation in a nitric oxide dependent manner

Tobin CM, Knoll LJ

SourceDepartment of Medical Microbiology and Immunology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706.

Toxoplasma gondii is an obligate intracellular parasite that uses immune cells to disseminate throughout its host. T. gondii can persist and even slowly replicate in activated host macrophages by reducing the antimicrobial effects of molecules such as nitric oxide (NO). A T. gondii patatin-like protein called TgPL1 was previously shown to be important for survival in activated macrophages. Here we show that a T. gondii mutant with a deletion of the TgPL1 gene (ΔTgPL1) is degraded in activated macrophages. This degradation phenotype is abolished by the removal of NO by the use of an iNOS inhibitor or iNOS deficient macrophages. Exogenous addition of NO to macrophages results in reduced parasite growth, but not degradation of ΔTgPL1 parasites. These results suggest that NO is necessary but not sufficient for the degradation of ΔTgPL1 parasites in activated macrophages. While some patatin-like proteins have phospolipase A(2) (PLA(2)) activity, recombinant TgPL1 purified from E. coli does not have phospholipase activity. This result was not surprising as TgPL1 contains a G to S change at the predicted catalytic serine residue. An epitope tagged version of TgPL1 partially co-localizing with a dense granule protein in the parasitophorous vacuole space. These results may suggest that TgPL1 moves to the parasitophorous vacuole to protect parasites from nitric oxide by an undetermined mechanism.

PMID:22006568[PubMed - as supplied by publisher]

Saturday, October 15, 2011

Evolutionarily divergent, unstable filamentous actin is essential for gliding motility in apicomplexan parasites

PLoS Pathog. 2011 Oct;7(10):e1002280. Epub 2011 Oct 6

Evolutionarily divergent, unstable filamentous actin is essential for gliding motility in apicomplexan parasites

Skillman KM, Diraviyam K, Khan A, Tang K, Sept D, Sibley LD

SourceDepartment of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri.

Apicomplexan parasites rely on a novel form of actin-based motility called gliding, which depends on parasite actin polymerization, to migrate through their hosts and invade cells. However, parasite actins are divergent both in sequence and function and only form short, unstable filaments in contrast to the stability of conventional actin filaments. The molecular basis for parasite actin filament instability and its relationship to gliding motility remain unresolved. We demonstrate that recombinant Toxoplasma (TgACTI) and Plasmodium (PfACTI and PfACTII) actins polymerized into very short filaments in vitro but were induced to form long, stable filaments by addition of equimolar levels of phalloidin. Parasite actins contain a conserved phalloidin-binding site as determined by molecular modeling and computational docking, yet vary in several residues that are predicted to impact filament stability. In particular, two residues were identified that form intermolecular contacts between different protomers in conventional actin filaments and these residues showed non-conservative differences in apicomplexan parasites. Substitution of divergent residues found in TgACTI with those from mammalian actin resulted in formation of longer, more stable filaments in vitro. Expression of these stabilized actins in T. gondii increased sensitivity to the actin-stabilizing compound jasplakinolide and disrupted normal gliding motility in the absence of treatment. These results identify the molecular basis for short, dynamic filaments in apicomplexan parasites and demonstrate that inherent instability of parasite actin filaments is a critical adaptation for gliding motility.

PMID:21998582[PubMed - in process]

Friday, October 14, 2011

Opposing Biological Functions of Tryptophan Catabolizing Enzymes During Intracellular Infection

J Infect Dis. 2011 Oct 11. [Epub ahead of print]

Opposing Biological Functions of Tryptophan Catabolizing Enzymes During Intracellular Infection

Divanovic S, Sawtell NM, Trompette A, Warning JI, Dias A, Cooper AM, Yap GS, Arditi M, Shimada K, Duhadaway JB, Prendergast GC, Basaraba RJ, Mellor AL, Munn DH, Aliberti J, Karp CL

SourceDivision of Molecular Immunology.

Recent studies have underscored physiological and pathophysiological roles for the tryptophan-degrading enzyme indolamine 2,3-dioxygenase (IDO) in immune counterregulation. However, IDO was first recognized as an antimicrobial effector, restricting tryptophan availability to Toxoplasma gondii and other pathogens in vitro. The biological relevance of these findings came under question when infectious phenotypes were not forthcoming in IDO-deficient mice. The recent discovery of an IDO homolog, IDO-2, suggested that the issue deserved reexamination. IDO inhibition during murine toxoplasmosis led to 100% mortality, with increased parasite burdens and no evident effects on the immune response. Similar studies revealed a counterregulatory role for IDO during leishmaniasis (restraining effector immune responses and parasite clearance), and no evident role for IDO in herpes simplex virus type 1 (HSV-1) infection. Thus, IDO plays biologically important roles in the host response to diverse intracellular infections, but the dominant nature of this role-antimicrobial or immunoregulatory-is pathogen-specific.

PMID:21990421[PubMed - as supplied by publisher]

The organization of the wall filaments and characterization of the matrix structures of Toxoplasma gondii cyst form

Cell Microbiol. 2011 Sep 8. doi: 10.1111/j.1462-5822.2011.01681.x. [Epub ahead of print]

The organization of the wall filaments and characterization of the matrix structures of Toxoplasma gondii cyst form

Lemgruber L, Lupetti P, Martins-Duarte ES, De Souza W, Vommaro RC

SourceLaboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Rio de Janeiro, Brazil. Parasitology - Department of Infectious Diseases, University of Heidelberg Medical School, Heidelberg, Germany. Department of Evolutionary Biology, University of Siena, Siena, Italy. Instituto Nacional de Metrologia e Qualidade Industrial - Inmetro, Rio de Janeiro, Brazil.

The encystation process is a key step in Toxoplasma gondii life cycle, allowing the parasite to escape from the host immune system and the transmission among the hosts. A detailed characterization of the formation and structure of the cyst stage is essential for a better knowledge of toxoplasmosis. Here we isolated cysts from mice brains and analysed the cyst wall structure and cyst matrix organization using different electron microscopy techniques. Images obtained showed that the cyst wall presented a filamentous aspect, with circular openings on its surface. The filaments were organized in two layers: a compact one, facing the exterior of the whole cyst and a more loosen one, facing the matrix. Within the cyst wall, we observed tubules and a large number of vesicles. The cyst matrix presented vesicles of different sizes and tubules, which were organized in a network connecting the bradyzoites to each other and to the cyst wall. Large vesicles, with a granular material in their lumen of glycidic nature were observed. Similar vesicles were also found associated with the posterior pole of the bradyzoites and in proximity to the cyst wall.

© 2011 Blackwell Publishing Ltd.

PMID:21899696[PubMed - as supplied by publisher]

Wednesday, October 12, 2011

Immunological responses induced by a DNA vaccine expressing RON4 and by immunogenic recombinant protein RON4 failed to protect mice against chronic to

Vaccine. 2011 Oct 4. [Epub ahead of print]

Immunological responses induced by a DNA vaccine expressing RON4 and by immunogenic recombinant protein RON4 failed to protect mice against chronic toxoplasmosis

Rashid I, Hedhli D, Moiré N, Pierre J, Debierre-Grockiego F, Dimier-Poisson I, Mévélec MN

SourceUniversité François Rabelais, INRA, UMR 0483 Université-INRA d'Immunologie Parasitaire, Vaccinologie et Biothérapie anti-infectieuse, IFR136 Agents Transmissibles et Infectiologie, UFR des Sciences Pharmaceutiques, 31 Avenue Monge, 37200 Tours, France.

The development of an effective vaccine against Toxoplasma gondii infection is an important issue due to the seriousness of the related public health problems, and the economic importance of this parasitic disease worldwide. Rhoptry neck proteins (RONs) are components of the moving junction macromolecular complex formed during invasion. The aim of this study was to evaluate the vaccine potential of RON4 using two vaccination strategies: DNA vaccination by the intramuscular route, and recombinant protein vaccination by the nasal route. We produced recombinant RON4 protein (RON4S2) using the Schneider insect cells expression system, and validated its antigenicity and immunogenicity. We also constructed optimized plasmids encoding full length RON4 (pRON4), or only the N-terminal (pNRON4), or the C-terminal part (pCRON4) of RON4. CBA/J mice immunized with pRON4, pNRON4 or pCRON4 plus a plasmid encoding the granulocyte-macrophage-colony-stimulating factor showed high IgG titers against rRON4S2. Mice immunized by the nasal route with rRON4S2 plus cholera toxin exhibited low levels of anti-RON4S2 IgG antibodies, and no intestinal IgA antibodies specific to RON4 were detected. Both DNA and protein vaccination generated a mixed Th1/Th2 response polarized towards the IgG1 antibody isotype. Both DNA and protein vaccination primed CD4+ T cells in vivo. In addition to the production of IFN-γ, and IL-2, Il-10 and IL-5 were also produced by the spleen cells of the immunized mice stimulated with RON4S2, suggesting that a mixed Th1/Th2 type immune response occurred in all the immunized groups. No cytokine was detectable in stimulated mesenteric lymph nodes from mice immunized by the nasal route. Immune responses were induced by both DNA and protein vaccination, but failed to protect the mice against a subsequent oral challenge with T. gondii cysts. In conclusion, strategies designed to enhance the immunogenicity and to redirect the cellular response towards a Th1 type response against RON4 could lead to more encouraging results.

Copyright © 2011. Published by Elsevier Ltd.

PMID:21983362[PubMed - as supplied by publisher]

The role of melatonin in parasite biology

Mol Biochem Parasitol. 2011 Oct 1. [Epub ahead of print]

The role of melatonin in parasite biology

Bagnaresi P, Nakabashi M, Thomas AP, Reiter RJ, Garcia CR

SourceDepartamento de Biofísica, Universidade Federal de São Paulo, São Paulo, Brazil.

Regarded as the circadian hormone in mammals, melatonin is a highly conserved molecule, present in nearly all species. In this review, we discuss the role of this indolamine and its precursors in the cell biology of parasites and the role of the molecule in the physiology of the host. In Plasmodium, melatonin can modulate intracellular concentrations of calcium and cAMP, which in turn can regulate kinase activity and cell cycle. In Trypanosoma infections, modulation of the immune system by melatonin is extremely important in controlling the parasite population. Melatonin also contributes to the inflammatory response to Toxoplasma gondii infection. Thus, there are a number of unique adaptations involving intricate connections between melatonin and the biology of the parasite-host relationship.

Copyright © 2011. Published by Elsevier B.V.

PMID:21982826[PubMed - as supplied by publisher]

Saturday, October 08, 2011

Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex

PLoS Pathog. 2011 Sep;7(9):e1002222. Epub 2011 Sep 29

Quantitative in vivo Analyses Reveal Calcium-dependent Phosphorylation Sites and Identifies a Novel Component of the Toxoplasma Invasion Motor Complex

Nebl T, Prieto JH, Kapp E, Smith BJ, Williams MJ, Yates JR 3rd, Cowman AF, Tonkin CJ.
SourceThe Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia.

Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca(2+)-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of (32)[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca(2+)-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component.

PMID:21980283[PubMed - in process]

Friday, October 07, 2011

MIF Participates in Toxoplasma gondii-Induced Pathology Following Oral Infection

PLoS One. 2011;6(9):e25259. Epub 2011 Sep 22

MIF Participates in Toxoplasma gondii-Induced Pathology Following Oral Infection

Cavalcanti MG, Mesquita JS, Madi K, Feijó DF, Assunção-Miranda I, Souza HS, Bozza MT

SourceDepartamento de Imunologia, Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is essential for controlling parasite burden and survival in a model of systemic Toxoplasma gondii infection. Peroral T. gondii infection induces small intestine necrosis and death in susceptible hosts, and in many aspects resembles inflammatory bowel disease (IBD). Considering the critical role of MIF in the pathogenesis of IBD, we hypothesized that MIF participates in the inflammatory response induced by oral infection with T. gondii.

METHODOLOGY/PRINCIPAL FINDINGS: Mif deficient (Mif(-)/(-)) and wild-type mice in the C57Bl/6 background were orally infected with T. gondii strain ME49. Mif(-)/(-) mice had reduced lethality, ileal inflammation and tissue damage despite of an increased intestinal parasite load compared to wt mice. Lack of MIF caused a reduction of TNF-α, IL-12, IFN-γ and IL-23 and an increased expression of IL-22 in ileal mucosa. Moreover, suppressed pro-inflammatory responses at the ileal mucosa observed in Mif(-)/(-) mice was not due to upregulation of IL-4, IL-10 or TGF-β. MIF also affected the expression of matrix metalloproteinase-9 (MMP-9) but not MMP-2 in the intestine of infected mice. Signs of systemic inflammation including the increased concentrations of inflammatory cytokines in the plasma and liver damage were less pronounced in Mif(-)/(-) mice compared to wild-type mice.

CONCLUSION/SIGNIFICANCE: In conclusion, our data suggested that in susceptible hosts MIF controls T. gondii infection with the cost of increasing local and systemic inflammation, tissue damage and death.

PMID:21977228[PubMed - in process]

Review of the series "disease of the year 2011: toxoplasmosis" pathophysiology of toxoplasmosis

Ocul Immunol Inflamm. 2011 Oct;19(5):297-306

Review of the series "disease of the year 2011: toxoplasmosis" pathophysiology of toxoplasmosis

Subauste CS, Ajzenberg D, Kijlstra A.
SourceDivision of Infectious Diseases and HIV Medicine, Department of Medicine, Case Western Reserve University School of Medicine , Cleveland, Ohio , USA ; Department of Ophthalmology and Visual Sciences, Case Western Reserve University School of Medicine , Cleveland, Ohio , USA ; and Department of Pathology, Case Western Reserve University School of Medicine , Cleveland, Ohio , USA.

Toxoplasma gondii is a major cause of chronic parasitic infection in the world. This protozoan can cause retino-choroiditis in newborns and in adults, both immunocompetent and immunodeficient. This disease tends to be recurrent and can lead to severe visual impairment. The authors review current knowledge on the role of parasite genetics in influencing susceptibility to ocular toxoplasmosis and on the immuno-pathogenesis of this disease.

PMID:21970661[PubMed - in process]

Wednesday, October 05, 2011

Nuclear actin-related protein is required for chromosome segregation in Toxoplasma gondii

Mol Biochem Parasitol. 2011 Sep 22. [Epub ahead of print]

Nuclear actin-related protein is required for chromosome segregation in Toxoplasma gondii

Suvorova ES, Lehmann MM, Kratzer S, White MW

SourceDepartment of Molecular Medicine, University of South Florida, Tampa, FL 33612, United States; Department of Global Health, University of South Florida, Tampa, FL 33612, United States.

Apicomplexa parasites use complex cell cycles to replicate that are not well understood mechanistically. We have established a robust forward genetic strategy to identify the essential components of parasite cell division. Here we describe a novel temperature sensitive Toxoplasma strain, mutant 13-20C2, which growth arrests due to a defect in mitosis. The primary phenotype is the mis-segregation of duplicated chromosomes with chromosome loss during nuclear division. This defect is conditional-lethal with respect to temperature, although relatively mild in regard to the preservation of the major microtubule organizing centers. Despite severe DNA loss many of the physical structures associated with daughter budding and the assembly of invasion structures formed and operated normally at the non-permissive temperature before completely arresting. These results suggest there are coordinating mechanisms that govern the timing of these events in the parasite cell cycle. The defect in mutant 13-20C2 was mapped by genetic complementation to Toxoplasma chromosome III and to a specific mutation in the gene encoding an ortholog of nuclear actin-related protein 4. A change in a conserved isoleucine to threonine in the helical structure of this nuclear actin related protein leads to protein instability and cellular mis-localization at the higher temperature. Given the age of this protist family, the results indicate a key role for nuclear actin-related proteins in chromosome segregation was established very early in the evolution of eukaryotes.

Copyright © 2011 Elsevier B.V. All rights reserved.

PMID:21963440[PubMed - as supplied by publisher]

The Cryptosporidium parvum Kinome

BMC Genomics. 2011 Sep 30;12(1):478. [Epub ahead of print]

The Cryptosporidium parvum Kinome

Artz JD, Wernimont AK, Allali-Hassani A, Zhao Y, Amani M, Lin YH, Senisterra G, Wasney GA, Fedorov O, King O, Roos A, Lunin VV, Qiu W, Finerty P Jr, Hutchinson A, Chau I, von Delft F, Mackenzie F, Lew J, Kozieradzki I, Vedadi M, Schapira M, Zhang C, Shokat K, Heightman T, Hui R.


BACKGROUND: Hundreds of millions of people are infected with cryptosporidiosis annually, with immunocompromised individuals suffering debilitating symptoms and children in socioeconomically challenged regions at risk of repeated infections. There is currently no effective drug available. In order to facilitate the pursuit of anti-cryptosporidiosis targets and compounds, our study spans the classification of the Cryptosporidium parvum kinome and the structural and biochemical characterization of representatives from the CDPK family and a MAP kinase.

RESULTS: The C. parvum kinome comprises over 70 members, some of which may be promising drug targets. These C. parvum protein kinases include members in the AGC, Atypical, CaMK, CK1, CMGC, and TKL groups; however, almost 35 % could only be classified as OPK (other protein kinases). In addition, about 25 % of the kinases identified did not have any known orthologues outside of Cryptosporidium spp. Comparison of specific kinases with their Plasmodium falciparum and Toxoplasma gondii orthologues revealed some distinct characteristics within the C. parvum kinome, including potential targets and opportunities for drug design. Structural and biochemical analysis of 4 representatives of the CaMK group and a MAP kinase confirms features that may be exploited in inhibitor design. Indeed, screening CpCDPK1 against a library of kinase inhibitors yielded a set of the pyrazolopyrimidine derivatives (PP1-derivatives) with IC50 values of <10 nM. The binding of a PP1-derivative is further described by an inhibitor-bound crystal structure of CpCDPK1. In addition, structural analysis of CpCDPK4 identified an unprecedented Zn-finger within the CDPK kinase domain that may have implications for its regulation.

CONCLUSIONS: Identification and comparison of the C. parvum protein kinases against other parasitic kinases shows how orthologue- and family-based research can be used to facilitate characterization of promising drug targets and the search for new drugs.

PMID:21962082[PubMed - as supplied by publisher] Free full text

Monday, October 03, 2011

The neurotropic parasite toxoplasma gondii increases dopamine metabolism

PLoS One. 2011;6(9):e23866. Epub 2011 Sep 21

The neurotropic parasite toxoplasma gondii increases dopamine metabolism

Prandovszky E, Gaskell E, Martin H, Dubey JP, Webster JP, McConkey GA.

SourceInstitute of Integrative and Comparative Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.

The highly prevalent parasite Toxoplasma gondii manipulates its host's behavior. In infected rodents, the behavioral changes increase the likelihood that the parasite will be transmitted back to its definitive cat host, an essential step in completion of the parasite's life cycle. The mechanism(s) responsible for behavioral changes in the host is unknown but two lines of published evidence suggest that the parasite alters neurotransmitter signal transduction: the disruption of the parasite-induced behavioral changes with medications used to treat psychiatric disease (specifically dopamine antagonists) and identification of a tyrosine hydroxylase encoded in the parasite genome. In this study, infection of mammalian dopaminergic cells with T. gondii enhanced the levels of K+-induced release of dopamine several-fold, with a direct correlation between the number of infected cells and the quantity of dopamine released. Immunostaining brain sections of infected mice with dopamine antibody showed intense staining of encysted parasites. Based on these analyses, T. gondii orchestrates a significant increase in dopamine metabolism in neural cells. Tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, was also found in intracellular tissue cysts in brain tissue with antibodies specific for the parasite-encoded tyrosine hydroxylase. These observations provide a mechanism for parasite-induced behavioral changes. The observed effects on dopamine metabolism could also be relevant in interpreting reports of psychobehavioral changes in toxoplasmosis-infected humans.

PMID:21957440[PubMed - in process]

Severe Congenital Toxoplasmosis in the United States: Clinical and Serologic Findings in Untreated Infants

Pediatr Infect Dis J. 2011 Sep 27. [Epub ahead of print]

Severe Congenital Toxoplasmosis in the United States: Clinical and Serologic Findings in Untreated Infants

Olariu TR, Remington JS, McLeod R, Alam A, Montoya JG.

SourceFrom the *Toxoplasma Serology Laboratory, Palo Alto Medical Foundation, Palo Alto, CA; †Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA; ‡Department of Medicine, University of Chicago School of Medicine, Chicago, IL; §Toxoplasmosis Center, University of Chicago Medical Center, Chicago, IL; and ¶Department of Pediatrics, University of Chicago School of Medicine, Chicago, IL.

BACKGROUND: Congenital toxoplasmosis can cause significant neurologic manifestations and other untoward sequelae.

METHODS: The Palo Alto Medical Foundation Toxoplasma Serology Laboratory database was searched for data on infants 0 to 180 days old, in whom congenital toxoplasmosis had been confirmed and who had been tested for Toxoplasma gondii-specific immunoglobulin G (IgG), IgM, and IgA antibodies, between 1991 and 2005. Their clinical findings were confirmed at the National Collaborative Chicago-based Congenital Toxoplasmosis Study center. We reviewed available clinical data and laboratory profiles of 164 infants with congenital toxoplasmosis whose mothers had not been treated for the parasite during gestation.

RESULTS: One or more severe clinical manifestations of congenital toxoplasmosis were reported in 84% of the infants and included eye disease (92.2%), brain calcifications (79.6%), and hydrocephalus (67.7%). In 61.6% of the infants, eye disease, brain calcifications, and hydrocephalus were present concurrently. T. gondii-specific IgM, IgA, and IgE antibodies were demonstrable in 86.6%, 77.4%, and 40.2% of the infants, respectively. Testing for IgM and IgA antibodies increased the sensitivity of making the diagnosis of congenital toxoplasmosis to 93% compared with testing for IgM or IgA individually. IgM and IgA antibodies were still present in 43.9% of infants diagnosed between 1 and 6 months of life.

CONCLUSIONS: Our study reveals that severe clinical signs of congenital toxoplasmosis including hydrocephalus, eye disease, or intracranial calcifications occurred in 85% infants whose sera were referred to our reference Toxoplasma Serology Laboratory during a period of 15 years. Laboratory tests, including serologic and polymerase chain reaction tests, were critical for diagnosis in the infants. Our results contrast remarkably with those of European investigators who rarely observe severe clinical signs in infants with congenital toxoplasmosis.

PMID:21956696[PubMed - as supplied by publisher]