Saturday, April 27, 2013

Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole

Mol Biol Cell. 2013 Apr 24. [Epub ahead of print]

Toxoplasma gondii salvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole

Romano JD, Sonda S, Bergbower E, Smith ME, Coppens I

Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD 21205, USA Institute of Parasitology, University of Zurich, CH-8057, Switzerland

The obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and, upon entry, forms its own membrane-bound compartment, named the parasitophorous vacuole (PV). Within the PV, the parasite replicates and scavenges nutrients, including lipids, from host organelles. Although T. gondii can synthesize sphingolipids de novo, it also scavenges these lipids from the host Golgi. How the parasite obtains sphingolipids from the Golgi remains unclear as the PV avoids fusion with host organelles. In this study, we have explored the host Golgi-PV interaction and evaluated the importance of host-derived sphingolipids for parasite growth. We demonstrate that the PV preferentially localizes near the host Golgi early during an infection and remains closely associated with this organelle throughout infection. The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous mini-stacks, which surround the PV, and hijacks host Golgi-derived vesicles within the PV. These vesicles, marked with Rab14, Rab30 or Rab43, colocalize with host-derived sphingolipids in the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite's growth. Thus, our results reveal that T. gondii relies on host-derived sphingolipids for its development and scavenges these lipids via Golgi-derived vesicles.

PMID: 23615442 [PubMed - as supplied by publisher]

Wednesday, April 24, 2013

Host Cell Subversion by Toxoplasma GRA16

Cell Host Microbe. 2013 Apr 17;13(4):489-500. doi: 10.1016/j.chom.2013.03.002.

Host Cell Subversion by Toxoplasma GRA16, an Exported Dense Granule Protein that Targets the Host Cell Nucleus and Alters Gene Expression

Bougdour A, Durandau E, Brenier-Pinchart MP, Ortet P, Barakat M, Kieffer S, Curt-Varesano A, Curt-Bertini RL, Bastien O, Coute Y, Pelloux H, Hakimi MA

UMR 5163, LAPM, Centre National de la Recherche Scientifique, 38041 Grenoble, France; Université Joseph Fourier, 38000 Grenoble, France. Electronic address:

After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function.

PMID: 23601110 [PubMed - in process]

Lateral gene transfer of Family A DNA polymerases between thermophilic viruses, Aquificae, and Apicomplexa

Mol Biol Evol. 2013 Apr 22. [Epub ahead of print]

Lateral gene transfer of Family A DNA polymerases between thermophilic viruses, Aquificae, and Apicomplexa

Schoenfeld TW, Murugapiran S, Dodsworth JA, Floyd S, Lodes M, Mead DA, Hedlund BP.

Lucigen, 2905 Parmenter Street, Middleton, Wisconsin 53562.

Bioinformatics and functional screens identified a group of Family A-type DNA Polymerase (polA) genes encoded by viruses inhabiting circumneutral and alkaline hot springs in Yellowstone National Park and the U.S. Great Basin. The proteins encoded by these viral polA genes (PolAs) shared no significant sequence similarity with any known viral proteins, but were remarkably similar to PolAs encoded by two of three families of the bacterial phylum Aquificae and by several apicoplast-targeted PolA-like proteins found in the eukaryotic phylum Apicomplexa, which includes the obligate parasites Plasmodium, Babesia, and Toxoplasma. The viral gene products share signature elements previously associated only with Aquificae and Apicomplexa PolA-like proteins and were similar to proteins encoded by prophage elements of a variety of otherwise unrelated Bacteria, each of which additionally encoded a prototypical bacterial PolA. Unique among known viral DNA polymerases, the viral PolA proteins of this study share with the Apicomplexa proteins large amino-terminal domains with putative helicase/primase elements but low primary sequence similarity. The genomic context and distribution, phylogeny, and biochemistry of these PolA proteins suggest that thermophilic viruses transferred polA genes to the Apicomplexa, likely through secondary endosymbiosis of a virus-infected proto-apicoplast, and to the common ancestor of two of three Aquificae families, where they displaced the orthologous cellular polA gene. Based on biochemical activity, gene structure, and sequence similarity we speculate that the xenologous viral-type polA genes may have functions associated with diversity-generating recombination in both Bacteria and Apicomplexa.

 PMID: 23608703 [PubMed - as supplied by publisher]

Saturday, April 20, 2013

Upregulated expression of Tim-3 involved in the process of toxoplasmic encephalitis in mouse model

Parasitol Res. 2013 Apr 18. [Epub ahead of print]

Upregulated expression of Tim-3 involved in the process of toxoplasmic encephalitis in mouse model

Wu B, Huang B, Chen Y, Li S, Yan J, Zheng H, Huang S, Shen J, Lun ZR, Wang Y, Kasper LH, Lu F.

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

Toxoplasma gondii can establish chronic infection and is characterized by the formation of tissue cysts in the brain. The cysts may remain throughout the life of the host but can reactivate and cause life-threatening toxoplasmic encephalitis (TE) in immunocompromised patients. T cell-mediated immune responses are essential for preventing the reactivation of chronic infection of T. gondii in the brain. The immunoinhibitory receptor T cell immunoglobulin and mucin domain (Tim)-1 and Tim-3 are expressed on terminally differentiated T helper (Th) 2 and Th1 cells, respectively, participating in the regulation of Th immune response. However, there is no report concerning the role of Tim genes in TE. In this study, Kunming outbred mice were infected with Prugniaud (Pru), a type II strain of T. gondii by oral gavage. Compared with the uninfected controls, there were mild brain inflammations at 3 weeks postinfection (p.i.), moderate brain inflammations at 5 weeks p.i., and aggravated brain inflammations and necrosis at 7 and 9 weeks p.i. The expressions of tachyzoite stage-specific genes in brains were consistent with the severity of brain histopathology of TE at 5 and 7 weeks p.i., while the expressions of bradyzoite stage-specific genes in brains were significantly increased at 7 and 9 weeks p.i. Using quantitative real-time PCR detection and immunohistochemistry staining, our results showed that the expressions of Tim-3 were significantly upregulated in both brains and spleens at 5 weeks p.i. and in spleens at 9 weeks p.i., which showed the similar dynamic tendency as that of interferon-γ expressions in both brains and spleens at the same times. In contrast, the Th2-specific marker Tim-1 expressions were significantly downregulated in both brains and spleens at 3 weeks p.i. and upregulated in both brains and spleens at 7 and 9 weeks p.i., which showed the similar dynamic tendency as that of interleukin-4 expressions in both brains and spleens at the same time. Our data indicate that Tim-3 may involve in the process of TE in mice infected with T. gondii Pru strain.

PMID: 23595213 [PubMed - as supplied by publisher]

Thursday, April 18, 2013

Toxoplasmosis-Associated Difference in Intelligence and Personality in Men Depends on Their Rhesus Blood Group but Not ABO Blood Group

PLoS One. 2013 Apr 10;8(4):e61272. Print 2013.

Toxoplasmosis-Associated Difference in Intelligence and Personality in Men Depends on Their Rhesus Blood Group but Not ABO Blood Group

Flegr J, Preiss M, Klose J.

Department of Biology, Faculty of Science, Charles University in Prague, Prague, Czech Republic.

The parasite Toxoplasma gondii influences the behaviour of infected animals and probably also personality of infected humans. Subjects with a Rhesus-positive blood group are protected against certain behavioural effects associated with Toxoplasma infection, including the deterioration of reaction times and personality factor shift.
Here, we searched for differences in the toxoplasmosis-associated effects between RhD-positive and RhD-negative subjects by testing 502 soldiers with two personality tests and two intelligence tests. The infected subjects expressed lower levels of all potentially pathognomic factors measured with the N-70 questionnaire and in neurasthenia measured with NEO-PI-R. The RhD-positive, Toxoplasma-infected subjects expressed lower while RhD-negative, Toxoplasma-infected subjects expressed higher intelligence than their Toxoplasma-free peers. The observed Toxoplasma-associated differences were always larger in RhD-negative than in RhD-positive subjects.
RhD phenotype plays an important role in the strength and direction of association between latent toxoplasmosis and not only psychomotor performance, but also personality and intelligence.

 PMID: 23593448 [PubMed - as supplied by publisher]

Wednesday, April 17, 2013

Hammondia hammondi, an avirulent relative of Toxoplasma gondii, has functional orthologs of known T. gondii virulence genes

Proc Natl Acad Sci U S A. 2013 Apr 15. [Epub ahead of print]

Hammondia hammondi, an avirulent relative of Toxoplasma gondii, has functional orthologs of known T. gondii virulence genes

Walzer KA, Adomako-Ankomah Y, Dam RA, Herrmann DC, Schares G, Dubey JP, Boyle JP.

Department of Biological Sciences, Dietrich School of Arts and Sciences, University of Pittsburgh, Pittsburgh, PA 15260.

Toxoplasma gondii is a ubiquitous protozoan parasite capable of infecting all warm-blooded animals, including humans. Its closest extant relative, Hammondia hammondi, has never been found to infect humans and, in contrast to T. gondii, is highly attenuated in mice. To better understand the genetic bases for these phenotypic differences, we sequenced the genome of a H. hammondi isolate (HhCatGer041) and found the genomic synteny between H. hammondi and T. gondii to be >95%. We used this genome to determine the H. hammondi primary sequence of two major T. gondii mouse virulence genes, TgROP5 and TgROP18. When we expressed these genes in T. gondii, we found that H. hammondi orthologs of TgROP5 and TgROP18 were functional. Similar to T. gondii, the HhROP5 locus is expanded, and two distinct HhROP5 paralogs increased the virulence of a T. gondii TgROP5 knockout strain. We also identified a 107 base pair promoter region, absent only in type III TgROP18, which is necessary for TgROP18 expression. This result indicates that the ROP18 promoter was active in the most recent common ancestor of these two species and that it was subsequently inactivated in progenitors of the type III lineage. Overall, these data suggest that the virulence differences between these species are not solely due to the functionality of these key virulence factors. This study provides evidence that other mechanisms, such as differences in gene expression or the lack of currently uncharacterized virulence factors, may underlie the phenotypic differences between these species.

PMID: 23589877 [PubMed - as supplied by publisher]

Changes in the proteomic profiles of mouse brain after infection with cyst-forming Toxoplasma gondii

Parasit Vectors. 2013 Apr 12;6(1):96. [Epub ahead of print]

Changes in the proteomic profiles of mouse brain after infection with cyst-forming Toxoplasma gondii

Zhou DH, Zhao FR, Huang SY, Xu MJ, Song HQ, Su C, Zhu XQ.

Toxoplasma gondii is an opportunistic pathogenic protozoan parasite, which infects approximately one third of the human population worldwide, causing opportunistic zoonotic toxoplasmosis. The predilection of T. gondii for the central nervous system (CNS) causes behavioral disorders and fatal necrotizing encephalitis and thus constitutes a major threat especially to AIDS patients.
In the present study, we explored the proteomic profiles of brain tissues of the specific pathogen-free (SPF) Kunming mice at 7 d, 14 d and 21 d after infection with cysts of the Toxoplasma gondii Prugniaud (PRU) strain (Genotype II), by two-dimensional gel electrophoresis (2-DE) combined with MALDI-TOF/TOF tandem mass spectrometry (MS/MS).
A total of 60 differentially expressed protein spots were selected. Fifty-six spots were successfully identified, which corresponded to 45 proteins of the mouse. Functional analysis using a Gene Ontology database showed that these proteins were mainly involved in metabolism, cell structure, signal transduction and immune responses, and will be beneficial for the understanding of molecular mechanisms of T. gondii pathogenesis.
This study identified some mouse brain proteins involved in the response with cyst-forming T. gondii PRU strain. These results provided an insight into the responsive relationship between T. gondii and the host brain tissues, which will shed light on our understanding of the mechanisms of pathogenesis in toxoplasmic encephalitis, and facilitate the discovery of new methods of diagnosis, prevention, control and treatment of toxoplasmic encephalopathy.

 PMID: 23587304 [PubMed - as supplied by publisher]

Tuesday, April 16, 2013

TgGRA23, a novel Toxoplasma gondii dense granule protein associated with the parasitophorous vacuole membrane and intravacuolar network

Parasitol Int. 2013 Apr 10. pii: S1383-5769(13)00051-2. doi: 10.1016/j.parint.2013.04.003. [Epub ahead of print]

TgGRA23, a novel Toxoplasma gondii dense granule protein associated with the parasitophorous vacuole membrane and intravacuolar network

Masatani T, Matsuo T, Tanaka T, Terkawi MA, Lee EG, Goo YK, Aboge GO, Yamagishi J, Hayashi K, Kameyama K, Shinuo C, Nishikawa Y, Xuan X.

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan

Toxoplasma gondii is an intracellular protozoan parasite, which relies on a specialized compartment, the parasitophorous vacuole (PV), to survive within host cells. Dense granules within the parasite release a large variety of proteins to maintain the integrity of the vacuole structure. Here, we identified a novel dense granule protein in T. gondii, TgGRA23, which is a homolog of the Sarcocystis muris dense granule protein, SmDG32. Recombinant TgGRA23 (rTgGRA23) expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein was used to raise antisera in mice and rabbits. Immunoblotting showed that antisera from the immunized mice and rabbits reacted with parasite lysates to yield a 21-kDa native protein. In addition, immuno-electron microscopic examination showed that TgGRA23 resides in the dense granules, PV membrane and intravacuolar network of the parasite. To confirm the precise subcellular localization of TgGRA23 in T. gondii, an immunofluorescent antibody test was performed using dense granule markers. Notably, TgGRA23 co-localized with other dense granule proteins including TgGRA4 and TgGRA7, in the extracellular-stage parasites. Biochemical experiments indicated that TgGRA23 is insoluble and may form an electrostatic complex that is resistant to non-ionic detergents. Furthermore, specific antibodies to TgGRA23 were detected during the chronic stage of Toxoplasma infection in mice. Our results suggest that TgGRA23 is an as yet unknown member of the T. gondii dense granule proteins, and that it may be involved in remodeling or maintenance of the PV.

PMID: 23583316 [PubMed - as supplied by publisher]

Analysis of Structures and Epitopes of Surface Antigen Glycoproteins Expressed in Bradyzoites of Toxoplasma gondii

Biomed Res Int. 2013;2013:165342. doi: 10.1155/2013/165342. Epub 2013 Mar 21.

Analysis of Structures and Epitopes of Surface Antigen Glycoproteins Expressed in Bradyzoites of Toxoplasma gondii

Cong H, Zhang M, Zhang Q, Gong J, Cong H, Xin Q, He S.

Department of Human Parasitology, School of Medicine, Shandong University, No. 44 Wenhuaxi Road, Jinan, Shandong 250012, China.

Toxoplasma gondii is a protozoan parasite capable of infecting humans and animals. Surface antigen glycoproteins, SAG2C, -2D, -2X, and -2Y, are expressed on the surface of bradyzoites. These antigens have been shown to protect bradyzoites against immune responses during chronic infections. We studied structures of SAG2C, -2D, -2X, and -2Y proteins using bioinformatics methods. The protein sequence alignment was performed by T-Coffee method. Secondary structural and functional domains were predicted using software PSIPRED v3.0 and SMART software, and 3D models of proteins were constructed and compared using the I-TASSER server, VMD, and SWISS-spdbv. Our results showed that SAG2C, -2D, -2X, and -2Y are highly homologous proteins. They share the same conserved peptides and HLA-I restricted epitopes. The similarity in structure and domains indicated putative common functions that might stimulate similar immune response in hosts. The conserved peptides and HLA-restricted epitopes could provide important insights on vaccine study and the diagnosis of this disease.

PMID: 23586017 [PubMed - in process]

Thursday, April 11, 2013

ApiAP2 transcription factor restricts development of the Toxoplasma tissue cyst

Proc Natl Acad Sci U S A. 2013 Apr 9. [Epub ahead of print]

ApiAP2 transcription factor restricts development of the Toxoplasma tissue cyst

Radke JB, Lucas O, De Silva EK, Ma Y, Sullivan WJ Jr, Weiss LM, Llinas M, White MW.

Departments of Molecular Medicine and Global Health, University of South Florida, Tampa, FL 33612.

Cellular differentiation leading to formation of the bradyzoite tissue cyst stage is the underlying cause of chronic toxoplasmosis. Consequently, mechanisms responsible for controlling development in the Toxoplasma intermediate life cycle have long been sought. Here, we identified 15 Toxoplasma mRNAs induced in early bradyzoite development that encode proteins with apicomplexan AP2 (ApiAP2) DNA binding domains. Of these 15 mRNAs, the AP2IX-9 mRNA demonstrated the largest expression increase during alkaline-induced differentiation. At the protein level, we found that AP2IX-9 was restricted to the early bradyzoite nucleus and is repressed in tachyzoites and in mature bradyzoites from 30-d infected animals. Conditional overexpression of AP2IX-9 significantly reduced tissue cyst formation and conferred alkaline pH-resistant growth, whereas disruption of the AP2IX-9 gene increased tissue cyst formation, indicating AP2IX-9 operates as a repressor of bradyzoite development. Consistent with a role as a repressor, AP2IX-9 specifically inhibited the expression of bradyzoite mRNAs, including the canonical bradyzoite marker, bradyzoite antigen 1 (BAG1). Using protein binding microarrays, we established the AP2 domain of AP2IX-9 binds a CAGTGT DNA sequence motif and is capable of binding cis-regulatory elements controlling the BAG1 and bradyzoite-specific nucleoside triphosphatase (B-NTPase) promoters. The effect of AP2IX-9 on BAG1 expression was direct because this factor inhibits expression of a firefly luciferase reporter under the control of the BAG1 promoter in vivo, and epitope-tagged AP2IX-9 can be immunoprecipitated with the BAG1 promoter in parasite chromatin. Altogether, these results indicate AP2IX-9 restricts Toxoplasma commitment to develop the mature bradyzoite tissue cyst.

PMID: 23572590 [PubMed - as supplied by publisher]

Molecular cloning, sequencing, and biological characterization of GRA4 gene of Toxoplasma gondii

Parasitol Res. 2013 Apr 10. [Epub ahead of print]

Molecular cloning, sequencing, and biological characterization of GRA4 gene of Toxoplasma gondii

Ram H, Rao JR, Tewari AK, Banerjee PS, Sharma AK.

Division of Parasitology, Indian Veterinary Research Institute, Izatnagar, 243 122, Uttar Pradesh, India,

In the present study, GRA4 (dense granule antigen) gene of Toxoplasma gondii was cloned, sequenced, and biologically characterized. The nucleotide sequence data obtained were analyzed and submitted in GenBank database (accession no. EU660037). Analysis of nucleotide sequence of GRA4 gene revealed 99.2 % homology with the published sequence (accession no. M76432). The gene segment (open reading frame) of 1,054 bp was further amplified and re-cloned in expression vector pET-32a. The recombinant protein obtained following the expression in prokaryotic system had a molecular mass of approx. 50 kDa and showed good immunoreactivity with T. gondii sera collected from infected goats. The immunization study of the recombinant protein performed in laboratory mice and live challenge with T. gondii revealed a high level of IgG response against the tachyzoite lysate antigen (TLA) by an indirect ELISA. Protection against T. gondii challenge infection was not evident in immunized mice except for the prolongation of survival period by 2 days. Humoral immune response profile revealed initially a high level of IgG antibody, but at 1 week post-challenge, a sudden drop in the level of the antibody was appreciable. Cytokine profiling by enzyme-linked immunosorbent spot method revealed relatively high level of IFN-γ production by the rodent spleen cells followed by IL-10 and IL-4. Increase in IFN-γ production by spleen cells of immunized mice following TLA stimulation suggested direct correlation to the up-regulated Th1 cells. However, the present immunization trial failed to show any positive relationship with the protection of mice following T. gondii challenge infection.

PMID: 23572047 [PubMed - as supplied by publisher]

Wednesday, April 10, 2013

The 12th International Workshops on Opportunistic Protists (IWOP-12)

J Eukaryot Microbiol. 2013 Mar 4. doi: 10.1111/jeu.12034. [Epub ahead of print]

The 12th International Workshops on Opportunistic Protists (IWOP-12)

Weiss LM, Cushion MT, Didier E, Xiao L, Marciano-Cabral F, Sinai AP, Matos O, Calderon EJ, Kaneshiro ES

Departments of Medicine and Pathology, Albert Einstein College of Medicine, Bronx, New York, USA.

The 12th International Workshops on Opportunistic Protists (IWOP-12) was held in August 2012 in Tarrytown, New York. The objectives of the IWOP meetings are to: (1) serve as a forum for exchange of new information among active researchers concerning the basic biology, molecular genetics, immunology, biochemistry, pathogenesis, drug development, therapy, and epidemiology of these immunodeficiency-associated pathogenic eukaryotic microorganisms that are seen in patients with AIDS and (2) foster the entry of new and young investigators into these underserved research areas. The IWOP meeting focuses on opportunistic protists, e.g. the free-living amoebae, Pneumocystis, Cryptosporidium, Toxoplasma, the Microsporidia, and kinetoplastid flagellates. This conference represents the major conference that brings together research groups working on these opportunistic pathogens. Slow but steady progress is being achieved on understanding the biology of these pathogenic organisms, their involvement in disease causation in both immune-deficient and immune-competent hosts, and is providing critical insights into these emerging and reemerging pathogens. This IWOP meeting demonstrated the importance of newly developed genomic level information for many of these pathogens and how analysis of such large data sets is providing key insights into the basic biology of these organisms. A great concern is the loss of scientific expertise and diversity in the research community due to the ongoing decline in research funding. This loss of researchers is due to the small size of many of these research communities and a lack of appreciation by the larger scientific community concerning the state of art and challenges faced by researchers working on these organisms.

PMID: 23560871 [PubMed - as supplied by publisher]

Tuesday, April 09, 2013

Evidence of tRNA cleavage in apicomplexan parasites

Mol Biochem Parasitol. 2013 Apr 1. pii: S0166-6851(13)00036-4. doi: 10.1016/j.molbiopara.2013.03.003. [Epub ahead of print]

Evidence of tRNA cleavage in apicomplexan parasites: half-tRNAs as new potential regulatory molecules of Toxoplasma gondii and Plasmodium berghei

Galizi R, Spano F, Giubilei MA, Capuccini B, Magini A, Urbanelli L, Ogawa T, Dubey JP, Spaccapelo R, Emiliani C, Di Cristina M.

Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy.

Several lines of evidence demonstrated that organisms ranging from bacteria to higher animals possess a regulated endonucleolytic cleavage pathway producing half-tRNA fragments. In the present study, we investigated the occurrence of this phenomenon in two distantly-related apicomplexan parasites, Toxoplasma gondii, the agent of toxoplasmosis, and the rodent malaria parasite Plasmodium berghei. A low-scale molecular characterization of the small RNA fraction of T. gondii revealed the endonucleolytic processing of 10 distinct tRNA species, with cleavage in the anticodon loop and upstream of the 3'-terminal CCA sequence yielding 5'- or 3'-end half-tRNAs. T. gondii and P. berghei exhibited variable rates of tRNA cleavage upon egress from host cells and in response to stage differentiation, amino acid starvation and heat-shock. Moreover, avirulent isolates of T. gondii and attenuated P. berghei parasites showed a higher rate of tRNA cleavage than virulent strains. Interestingly, half-tRNA production was significantly higher in the metabolically quiescent bradyzoite and sporozoite stages of T. gondii, compared to the fast-growing tachyzoite. Collectively, our findings shed light for the first time on the occurrence of tRNA cleavage in apicomplexan parasites and suggest a relationship between half-tRNA production and growth rate in this important group of organisms.

PMID: 23557708 [PubMed - as supplied by publisher]

Toxoplasma found in ancient Egyptian mummies

J Appl Genet. 2013 Apr 4. [Epub ahead of print]

First insights into the metagenome of Egyptian mummies using next-generation sequencing

Khairat R, Ball M, Chang CC, Bianucci R, Nerlich AG, Trautmann M, Ismail S, Shanab GM, Karim AM, Gad YZ, Pusch CM.

Institute of Human Genetics, University of Tübingen, Wilhelmstraße 27, 72074, Tübingen, Germany

We applied, for the first time, next-generation sequencing (NGS) technology on Egyptian mummies. Seven NGS datasets obtained from five randomly selected Third Intermediate to Graeco-Roman Egyptian mummies (806 BC-124AD) and two unearthed pre-contact Bolivian lowland skeletons were generated and characterised. The datasets were contrasted to three recently published NGS datasets obtained from cold-climate regions, i.e. the Saqqaq, the Denisova hominid and the Alpine Iceman. Analysis was done using one million reads of each newly generated or published dataset. Blastn and megablast results were analysed using MEGAN software. Distinct NGS results were replicated by specific and sensitive polymerase chain reaction (PCR) protocols in ancient DNA dedicated laboratories. Here, we provide unambiguous identification of authentic DNA in Egyptian mummies. The NGS datasets showed variable contents of endogenous DNA harboured in tissues. Three of five mummies displayed a human DNA proportion comparable to the human read count of the Saqqaq permafrost-preserved specimen. Furthermore, a metagenomic signature unique to mummies was displayed. By applying a "bacterial fingerprint", discrimination among mummies and other remains from warm areas outside Egypt was possible. Due to the absence of an adequate environment monitoring, a bacterial bloom was identified when analysing different biopsies from the same mummies taken after a lapse of time of 1.5 years. Plant kingdom representation in all mummy datasets was unique and could be partially associated with their use in embalming materials. Finally, NGS data showed the presence of Plasmodium falciparum and Toxoplasma gondii DNA sequences, indicating malaria and toxoplasmosis in these mummies. We demonstrate that endogenous ancient DNA can be extracted from mummies and serve as a proper template for the NGS technique, thus, opening new pathways of investigation for future genome sequencing of ancient Egyptian individuals.

 PMID: 23553074 [PubMed - as supplied by publisher]

Thursday, April 04, 2013

Toxoplasma rhoptry kinase ROP16 promotes host resistance to oral infection and intestinal inflammation only in the context of the dense granule protein GRA15

Infect Immun. 2013 Apr 1. [Epub ahead of print]

Toxoplasma rhoptry kinase ROP16 promotes host resistance to oral infection and intestinal inflammation only in the context of the dense granule protein GRA15

Jensen KD, Hu K, Whitmarsh RJ, Hassan MA, Julien L, Lu D, Chen L, Hunter CA, Saeij JP.

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Toxoplasma gondii transmission between intermediate hosts is dependent on the ingestion of walled cysts formed during the chronic phase of infection. Immediately following consumption, the parasite must ensure survival of the host by preventing adverse inflammatory responses and/or by limiting its own replication. Since the Toxoplasma secreted effectors rhoptry 16 kinase (ROP16) and dense granule 15 (GRA15) activate the JAK-STAT3/6 and NF-κB signaling pathways, respectively, we explored whether a particular combination of these effectors impacted intestinal inflammation and parasite survival in vivo. Here we report that expression of the STAT-activating version of ROP16 in the type II strain (II+ROP16I) promotes host resistance to oral infection only in the context of endogenous GRA15 expression. Protection was characterized by lower intestinal parasite burden and dampened inflammation. Host resistance to the II+ROP16I strain occurred independently of STAT6, and the T cell co-inhibitory receptors B7-DC and B7-H1, two receptors that are upregulated by ROP16. In addition, co-expression of ROP16 and GRA15 enhanced parasite susceptibility within TNFα/IFNγ-stimulated macrophages in a STAT3/6 independent manner. Transcriptional profiling of infected STAT3- and STAT6-deficient macrophages and parasitized Peyer's patches from II+ROP16I orally challenged mice suggested that ROP16 activated STAT5 to modulate host gene expression. Consistent with this supposition, the ROP16 kinase induced the sustained phosphorylation and nuclear localization of STAT5 in Toxoplasma infected cells. In summary, only the combined expression of both GRA15 and ROP16 promoted host resistance to acute oral infection, and Toxoplasma may possibly target the STAT5 signaling pathway to generate protective immunity in the gut.

PMID: 23545295 [PubMed - as supplied by publisher]

Determination of stage interconversion in vitro and in vivo by construction of transgenic Toxoplasma gondii that stably express stage-specific fluorescent proteins

Exp Parasitol. 2013 Mar 29. pii: S0014-4894(13)00085-4. doi: 10.1016/j.exppara.2013.03.015. [Epub ahead of print]

Determination of stage interconversion in vitro and in vivo by construction of transgenic Toxoplasma gondii that stably express stage-specific fluorescent proteins
Zhang H, Zhang Y, Cao J, Zhou Y, Wang N, Zhou J.

Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.

Detection of Toxoplasma gondii conversion from the tachyzoite stage to the bradyzoite stage in living brain tissue is difficult because the parasites are small and conversion and reactivation of the parasites are transient events. To better understand the mechanisms of T. gondii stage conversion between tachyzoites and bradyzoites, and to recognize stage conversion in an intermediate host, we constructed a transgenic cyst-forming strain (PLK) of T. gondii. The parasites stably expressed enhanced green fluorescence protein (EGFP) in the tachyzoite stage and red fluorescence protein (RFP) in the bradyzoite stage, under the control of the SAG1 and BAG1 promoters, respectively. The resulting transgenic parasite was designated as PLK/Bi. The PLK/Bi zoites expressed only green fluorescence in the tachyzoite stage and only red fluorescence in the bradyzoite stage in vitro and in vivo. Fluorescence analyses showed that recombinant GFP and RFP were located to the intracellular vacuolar spaces. In addition, an analysis of growth and culture conditions of transgenic T. gondii was performed in vitro and the virulence was evaluated in vivo. Our data suggested that the stage-specific fluorescence expression by PLK/Bi may be rationally designed for in vitro and in vivo studies on stage conversion and reactivation of T. gondii.

PMID: 23545429 [PubMed - as supplied by publisher]

Tuesday, April 02, 2013

"Becoming Karl": One family’s struggle with toxoplasmosis

“Becoming Karl: A brain-injured baby's journey to recovery” is a wonderful book written by Norma Delgarno about her son, Karl. In 1966, Karl was born with severe complications stemming from toxoplasmosis. Toxoplasmosis is caused by a single-celled parasite (Toxoplasma gondii) that can cross the placenta if a woman becomes infected for the first time during her pregnancy. The prognosis for Karl was poor and doctors claimed that he would be incapable of learning, requiring constant care the rest of his life.

With vibrant prose, Norma shares her journey of what it was like to raise a child born with such a devastating infection and brain injury. With unwavering love and determination, Norma tirelessly worked against the odds to give her son as close to a “normal” life as possible. Her writing is often poetic and humorous, making the book all the more engaging. This is a moving and inspirational documentary that anyone would enjoy, especially those affected by toxoplasmosis or other neurological disease.

The book is available through Amazon in paperback or Kindle editions.

More information about the author

Karl’s art

TgMAPK1 is a Toxoplasma gondii MAP kinase that hijacks host MKK3 signals to regulate virulence and interferon-γ-mediated nitric oxide production

Exp Parasitol. 2013 Mar 26. pii: S0014-4894(13)00086-6. doi: 10.1016/j.exppara.2013.03.016. [Epub ahead of print]

TgMAPK1 is a Toxoplasma gondii MAP kinase that hijacks host MKK3 signals to regulate virulence and interferon-γ-mediated nitric oxide production

Brumlik MJ, Pandeswara S, Ludwig SM, Jeansonne DP, Lacey MR, Murthy K, Daniel BJ, Wang RF, Thibodeaux SR, Church KM, Hurez V, Kious MJ, Zhang B, Alagbala A, Xia X, Curiel TJ.

The Cancer Therapy and Research Center/Adult Cancer Program, and the Institute for Drug Development, STRF/8403 Floyd Curl Drive MS 8252/University of Texas Health Science Center, San Antonio, TX 78229, USA. Electronic address:

The parasite Toxoplasma gondii controls tissue-specific nitric oxide (NO), thereby augmenting virulence and immunopathology through poorly-understood mechanisms. We now identify TgMAPK1, a Toxoplasma mitogen-activated protein kinase (MAPK), as a virulence factor regulating tissue-specific parasite burden by manipulating host interferon (IFN)-γ-mediated inducible nitric oxide synthase (iNOS). Toxoplasma with reduced TgMAPK1 expression (TgMAPK1lo) demonstrated that TgMAPK1 facilitates IFN-γ-driven p38 MAPK activation, reducing IFN-γ-generated NO in an MKK3-dependent manner, blunting IFN-γ-mediated parasite control. TgMAPK1lo infection in wild type mice produced >ten-fold lower parasite burden versus control parasites with normal TgMAPK1 expression (TgMAPK1con). Reduced parasite burdens persisted in IFN-γ KO mice, but equalized in normally iNOS-replete organs from iNOS KO mice. Parasite MAPKs are far less studied than other parasite kinases, but deserve additional attention as targets for immunotherapy and drug discovery.

PMID: 23541881 [PubMed - as supplied by publisher]