Wednesday, December 31, 2008

Induction of mitotic S-phase of host and neighboring cells by Toxoplasma gondii enhances parasite invasion

Mol Biochem Parasitol. 2008 Dec 6. [Epub ahead of print]

Induction of mitotic S-phase of host and neighboring cells by Toxoplasma gondii enhances parasite invasion

Lavine MD, Arrizabalaga G.

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

The intracellular parasite Toxoplasma gondii extensively modifies its host cell so as to efficiently grow and divide. Among these cellular changes, T. gondii alters the cell cycle of host cells it has invaded. We found that T. gondii affects the cell cycle of not only the cells it directly invades, but neighboring cells as well. Both direct invasion by T. gondii and exposure to filtered medium from cultures of T. gondii-infected cells (conditioned medium) caused normally quiescent fibroblasts to enter S-phase. T. gondii has been shown to attach to and invade S-phase host cells more readily, and we found that conditioned medium increased the rate of invasion of T. gondii into new host cells. Thus it appears that T. gondii directly releases, or induces parasitized host cells to release, a factor that induces neighboring cells to enter S-phase, allowing more rapid invasion by extracellular T. gondii and providing a possible selective advantage for the parasite.

PMID: 19111577 [PubMed - as supplied by publisher]

The cathepsin L of Toxoplasma gondii (TgCPL) and its endogenous macromolecular inhibitor, toxostatin

Mol Biochem Parasitol. 2008 Dec 6. [Epub ahead of print]

The cathepsin L of Toxoplasma gondii (TgCPL) and its endogenous macromolecular inhibitor, toxostatin

Huang R, Que X, Hirata K, Brinen LS, Lee JH, Hansell E, Engel J, Sajid M, Reed S.

Department of Medicine, University of California, San Diego, CA 92103, United States.

Toxoplasma gondii is an obligate intracellular parasite of all vertebrates, including man. Successful invasion and replication requires the synchronized release of parasite proteins, many of which require proteolytic processing. Unlike most parasites, T. gondii has a limited number of Clan CA, family C1 cysteine proteinases with one cathepsin B (TgCPB), one cathepsin L (TgCPL) and three cathepsin Cs (TgCPC1, 2, 3). Previously, we characterized toxopain, the only cathepsin B enzyme, which localizes to the rhoptry organelle. Two cathepsin Cs are trafficked through dense granules to the parasitophorous vacuole where they degrade peptides. We now report the cloning, expression, and modeling of the sole cathepsin L gene and the identification of two new endogenous inhibitors. TgCPL differs from human cathepsin L with a pH optimum of 6.5 and its substrate preference for leucine (vs. phenylalanine) in the P2 position. This distinct preference is explained by homology modeling, which reveals a non-canonical aspartic acid (Asp 216) at the base of the predicted active site S2 pocket, which limits substrate access. To further our understanding of the regulation of cathepsins in T. gondii, we identified two genes encoding endogenous cysteine proteinase inhibitors (ICPs or toxostatins), which are active against both TgCPB and TgCPL in the nanomolar range. Over expression of toxostatin-1 significantly decreased overall cysteine proteinase activity in parasite lysates, but had no detectable effect on invasion or intracellular multiplication. These findings provide important insights into the proteolytic cascades of T. gondii and their endogenous control.

PMID: 19111576 [PubMed - as supplied by publisher]

Cell Type-Specific Analysis of mRNA Synthesis and Decay In Vivo with Uracil Phosphoribosyltransferase and 4-thiouracil

Methods Enzymol. 2008;448:379-406

Chapter 19 Cell Type-Specific Analysis of mRNA Synthesis and Decay In Vivo with Uracil Phosphoribosyltransferase and 4-thiouracil

Cleary MD.

Microarray-based analysis of mRNA expression has provided a genome-wide understanding of the genes and pathways involved in many biological processes. However, two limitations are often associated with traditional microarray experiments. First, standard methods of microarray analysis measure mRNA abundance, not mRNA synthesis or mRNA decay, and, therefore, do not provide any information regarding the mechanisms regulating transcript levels. Second, microarrays are often performed with mRNA from a mixed population of cells, and data for a specific cell-type of interest can be difficult to obtain. This chapter describes a method, referred to here as "4TU-tagging," which can be used to overcome these limitations. 4TU-Tagging uses cell type-specific expression of the uracil phosphoribosyltransferase gene of Toxoplasma gondii and the uracil analog 4-thiouracil (4TU) to selectively tag and purify RNA. Pulse-labeling of newly synthesized RNA with 4TU followed by a "chase" with unmodified uracil allows in vivo measurements of mRNA synthesis and decay in specific cells. Experimental design considerations for applying 4TU-tagging to different systems and protocols for cell type-specific RNA tagging, purification, and microarray analysis are covered in this chapter.

PMID: 19111186 [PubMed - in process]

Protection against Toxoplasma gondii brain cyst formation in mice

Vet Parasitol. 2008 Nov 25. [Epub ahead of print]

Protection against Toxoplasma gondii brain cyst formation in mice immunized with Toxoplasma gondii cytoskeleton proteins and Lactobacillus casei as adjuvant

Martínez-Gómez F, García-González LF, Mondragón-Flores R, Bautista-Garfias CR.

Departamento de Parasitología ENCB-IPN, Mexico.

The aim of the study was to evaluate the protection generated in mice against Toxoplasma gondii brain cyst burden by vaccination with T. gondii cytoskeleton proteins using Lactobacillus casei as adjuvant. One hundred and sixty-eight NIH mice were randomly allocated into eight groups of 21 mice each. Animals were immunized as follows: in group 1 with Toxoplasma lysate antigen (TLA) in Freund's modified adjuvant, containing L. casei (FMA), in group 2 with Toxoplasma cytoskeleton proteins (TCPs) in FMA, in group 3 with FMA, in group 4 with phosphate buffered saline (PBS), in group 5 with L. casei dead by heath (Lc), in group 6 with Freund's complete adjuvant (FCA), in group 7 with TLA in FCA, and in group 8 with TCP in FCA. Mean brain cyst burden (+/-S.E.M.) was assessed in mice 8 weeks after challenge with T. gondii Me49 strain (20 cysts per mouse). The percentages of reduction in cyst burden per brain (P<0.01) as compared with the group 4 (control: mean 3181+/-97.5) were 77.25% (724+/-98) in group 1, 88.02% (381+/-97.5) in group 2, 38.92% (1943+/-130.3) in group 3, 44.31% (1771.4+/-102) in group 5, 59.28% (1295.2+/-99.1) in group 7 and 55.69% (1409.5+/-89.9) in group 8. In order of importance, the best protection was obtained in groups 2, 1, 7, 8, 5 and 3. Noticeably the mice inoculated with L. casei alone showed a significant reduction in T. gondii brain cysts (P<0.01), while those animals treated with FCA alone did not. Additionally, IgM anti-T. gondii antibody levels, as determined by ELISA 2 weeks after challenge, were highest in group 2 (P<0.01) than in the other seven groups. Results suggest that T. gondii cytoskeleton proteins with L. casei as adjuvant constitute a good anti-toxoplasmosis vaccine candidate.

PMID: 19111993 [PubMed - as supplied by publisher]

Saturday, December 27, 2008

Toxoplasma gondii Prevents Chromatin Remodeling Initiated by TLR-Triggered Macrophage Activation

J Immunol. 2009 Jan 1;182(1):489-497

Toxoplasma gondii Prevents Chromatin Remodeling Initiated by TLR-Triggered Macrophage Activation

Leng J, Butcher BA, Egan CE, Abi Abdallah DS, Denkers EY.

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

Macrophages infected with the opportunistic protozoan Toxoplasma gondii are unable to up-regulate many proinflammatory cytokine genes, including TNF (TNF-alpha), upon stimulation with LPS and other TLR ligands. In this study, we examined the influence of T. gondii on transcription factors associated with TNF-alpha transcription, as well as phosphorylation and acetylation of histone H3 at distal and proximal regions of the TNF-alpha promoter. During LPS stimulation, we found that Toxoplasma blocks nuclear accumulation of transcription factor c-Jun, but not that of cAMP response element-binding protein or NF-kappaB. However, chromatin immunoprecipitation studies revealed that binding of all of these transcription factors to the TNF promoter was decreased by T. gondii infection. Furthermore, the parasite blocked LPS-induced Ser(10) phosphorylation and Lys(9)/Lys(14) acetylation of histone H3 molecules associated with distal and proximal regions of the TNF-alpha promoter. Our results show that Toxoplasma inhibits TNF-alpha transcription by interfering with chromatin remodeling events required for transcriptional activation at the TNF promoter, revealing a new mechanism by which a eukaryotic pathogen incapacitates proinflammatory cytokine production during infection.

PMID: 19109180 [PubMed - as supplied by publisher]

Wednesday, December 24, 2008

Primary skeletal muscle cells trigger spontaneous Toxoplasma gondii tachyzoite-to-bradyzoite conversion at higher rates than fibroblasts

Int J Med Microbiol. 2008 Dec 17. [Epub ahead of print]

Primary skeletal muscle cells trigger spontaneous Toxoplasma gondii tachyzoite-to-bradyzoite conversion at higher rates than fibroblasts

Ferreira-da-Silva MD, Takács AC, Barbosa HS, Gross U, Lüder CG.

Institute for Medical Microbiology, Georg-August-University, Kreuzbergring 57, 37075 Göttingen, Germany; Laboratory of Structural Biology, Oswaldo Cruz Institut, Oswaldo Cruz Foundation, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil.

Toxoplasma gondii is one of the most common eukaryotic parasites and an important opportunistic pathogen of humans. The interconversion from proliferative tachyzoites into quiescent encysted bradyzoites plays pivotal roles in the lifelong persistence of T. gondii in its host and the pathogenesis of toxoplasmosis. Stage conversion and persistence in skeletal muscle tissue may be particularly important for the food-borne transmission of T. gondii to humans via raw or undercooked meat products. Here, we have followed the transition of tachyzoites to bradyzoites after infection of skeletal muscle cells (SkMC). Primary murine myoblasts were differentiated to multinucleated syncytial myotubes that displayed regular contractions in vitro and expressed myogenic markers Myf5 and MyoD. Tachyzoites of T. gondii invaded SkMC within 4h of infection and started to replicate within 24h of infection. Remarkably, intracellular tachyzoites readily differentiated to bradyzoites in SkMC without the need of exogenous stress factors. Double immunofluorescence labelling revealed significantly higher percentages of bradyzoite-containing vacuoles in SkMC than in murine fibroblasts at 24h until 6 days after infection. Furthermore, transcript levels of bradyzoite-specific ENO1 but not tachyzoite-specific ENO2 strongly increased in T. gondii-infected SkMC until 6 days of infection. These findings indicate that the commitment of T. gondii to differentiate to bradyzoites in SkMC does not require exogenous stress factors but could be rather regulated by cell-type specific factors.

PMID: 19097936 [PubMed - as supplied by publisher]

Sunday, December 21, 2008

Rapid Membrane Disruption by a Perforin-Like Protein Facilitates Parasite Exit from Host Cells

Science. 2008 Dec 18. [Epub ahead of print]

Rapid Membrane Disruption by a Perforin-Like Protein Facilitates Parasite Exit from Host Cells

Kafsack BF, Pena JD, Coppens I, Ravindran S, Boothroyd JC, Carruthers VB.

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.; Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, USA.

Perforin-like proteins are expressed by many bacterial and protozoan pathogens, yet little is known about their function or mode of action. Here, we describe TgPLP1, a secreted perforin-like protein of the intracellular protozoan pathogen Toxoplasma gondii that displays structural features necessary for pore-formation. Following intracellular growth, TgPLP1-deficient parasites failed to exit normally, resulting in entrapment within host cells. We show that this defect is due to an inability to permeabilize rapidly the parasitophorous vacuole membrane and host plasma membrane during exit. TgPLP1 ablation had little effect on growth in culture, but resulted in a >5-order of magnitude reduction of acute virulence in mice. Perforin-like proteins from other intracellular pathogens may play a similar role in microbial egress and virulence.

PMID: 19095897 [PubMed - as supplied by publisher]

Thursday, December 18, 2008

Modulation of Caspase Activation by Toxoplasma gondii

Methods Mol Biol. 2009;470:275-88

Modulation of Caspase Activation by Toxoplasma gondii

Hippe D, Gais A, Gross U, Lüder CG.

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

Apoptosis plays crucial roles for the outcome of infection with various infectious agents. The host's apoptotic program may be modulated after infection in order to combat the pathogen or to restrict the immune response. In addition, distinct microorganisms alter the apoptotic program of the host in order to meet the requirements for their further distribution. The activation of caspases (i.e., cysteine proteases with specificity for aspartic acid residues) preludes the disassembly of the cell in response to apoptosis-inducing stimuli. This depends on the proteolytic cleavage of inactive proforms into catalytically active subunits. Analyses of the proteolysis and the enzymatic activity of caspases therefore represent valuable tools to study apoptotic programs during infection. The apicomplexan parasite Toxoplasma gondii interferes with the caspase cascade of its host cell in order to facilitate intracellular survival. The modulation of caspase activation by T. gondii is determined by SDS-PAGE and immunoblotting with caspase-specific antibodies. Furthermore, the impact of the parasite on caspase activity is fluorimetrically determined by measuring the cleavage of caspase-specific substrate analogues.

PMID: 19089389 [PubMed - in process]

Diagnosis of Toxoplasmosis after Allogeneic Stem Cell Transplantation

Clin Infect Dis. 2008 Dec 11. [Epub ahead of print]

Diagnosis of Toxoplasmosis after Allogeneic Stem Cell Transplantation: Results of DNA Detection and Serological Techniques

Fricker-Hidalgo H, Bulabois CE, Brenier-Pinchart MP, Hamidfar R, Garban F, Brion JP, Timsit JF, Cahn JY, Pelloux H.

1Parasitologie-Mycologie, Pôle Biologie, 2Hématologie, Pôle Cancérologie, and 3Réanimation Médicale and 4Maladie Infectieuse, Pôle Médecine Aigüe et Communautaire, Centre Hospitalier Universitaire, Grenoble, France.

Background. @nbsp; The biological diagnosis of toxoplasmosis after allogeneic hematopoietic stem cell transplantation (HSCT) is based on the detection of Toxoplasma gondii DNA in blood specimens or other samples. Serological testing is used mainly to define the immunity status of the patient before HSCT. The aim of our study was to examine the performance of polymerase chain reaction (PCR) and serological techniques in the diagnosis of toxoplasmosis after HSCT. Methods. @nbsp; Seventy patients underwent allogeneic HSCT from September 2004 through September 2006. DNA was detected by PCR, and immunoglobulin G and immunoglobulin M were detected by enzyme-linked immunosorbent assay. Results. @nbsp; The results of immunoglobulin G detection before allogeneic HSCT were positive in 40 (57.1%) of the patients and negative in 30 (42.9%). After HSCT, 57 patients (81.4%) had test results that were negative for immunoglobulin M and had negative results of DNA detection, without toxoplasmosis infection. Four patients (5.7%) had at least 4 samples with positive PCR results and/or test results positive for immunoglobulin M against T. gondii; toxoplasmosis was then confirmed by clinical symptoms. Nine patients (12.9%) with positive PCR results and 1 or 2 samples with test results negative for immunoglobulin M were considered to have asymptomatic T. gondii infection. Reactivation of latent infection was the cause of toxoplasmosis in 3 of the 4 patients, and toxoplasmosis occurred as a primary infection in 1 patient. The detection of specific anti-T. gondii immunoglobulin M was the only biological evidence of toxoplasmosis in 2 patients, and samples were positive for immunoglobulin M before PCR was performed in 1 patient. Conclusions. @nbsp; Thus, after HSCT, all patients were at risk for toxoplasmosis; all patients who receive HSCTs should be followed up with biological testing that combines PCR and serological techniques.

PMID: 19072243 [PubMed - as supplied by publisher]

Thursday, December 11, 2008

Toxoplasma gondii: Reproductive parameters in experimentally infected male rats

Exp Parasitol. 2008 Nov 27. [Epub ahead of print]

Toxoplasma gondii: Reproductive parameters in experimentally infected male rats

Terpsidis KI, Papazahariadou MG, Taitzoglou IA, Papaioannou NG, Georgiadis MP, Theodoridis IT.

Laboratory of Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine, Aristotle University, Thessaloniki, P.O. Box 377, Agia Paraskevi, Thermi 57001, Greece.

Toxoplasma gondii is a protozoan parasite that is globally widespread and infects man and animals. With the aim of studying the influence of toxoplasmosis on male reproductive parameters, we investigated sperm motility, concentration and morphology of male rats experimentally infected by T. gondii. The GT F1 strain of T. gondii tissue cysts were fed at a dose of 5x10(3) tissue cysts per rat by oral gavage in an experimental group of 42 healthy adult male Wistar rats, while 42 male rats were used as controls. On days 10, 20, 30, 40, 50 and 60 post-inoculation (p.i.) 7 rats from each group were anesthetized. The body weight of each animal was recorded, then epididymis and testes were immediately removed, weighed and semen evaluation was undertaken. Weight of the right epididymis was significantly decreased on day 30 p.i., sperm motility was significantly decreased on days 10, 20, 30, 40, 50 and 60 p.i. and sperm concentration was significantly decreased on days 10, 30, 40 and 60 p.i. A marked increase of sperm abnormalities was noticed on days 30 and 40 p.i. No pathological lesions were detected either in the pituitary gland or the testes. In this study it was found that toxoplasmosis can affect main reproductive parameters in male rats, which are the most predictive of their fertilizing capacity.

PMID: 19063884 [PubMed - as supplied by publisher]

Computational Analysis and Experimental Validation of Gene Predictions in Toxoplasma gondii

PLoS ONE. 2008;3(12):e3899. Epub 2008 Dec 9

Computational Analysis and Experimental Validation of Gene Predictions in Toxoplasma gondii

Dybas JM, Madrid-Aliste CJ, Che FY, Nieves E, Rykunov D, Angeletti RH, Weiss LM, Kim K, Fiser A.

Biodefense Proteomics Research Center, Albert Einstein College of Medicine, Bronx, New York, United States of America.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan that infects 20 to 90% of the population. It can cause both acute and chronic infections, many of which are asymptomatic, and, in immunocompromized hosts, can cause fatal infection due to reactivation from an asymptomatic chronic infection. An essential step towards understanding molecular mechanisms controlling transitions between the various life stages and identifying candidate drug targets is to accurately characterize the T. gondii proteome. METHODOLOGY/PRINCIPAL FINDINGS: We have explored the proteome of T. gondii tachyzoites with high throughput proteomics experiments and by comparison to publicly available cDNA sequence data. Mass spectrometry analysis validated 2,477 gene coding regions with 6,438 possible alternative gene predictions; approximately one third of the T. gondii proteome. The proteomics survey identified 609 proteins that are unique to Toxoplasma as compared to any known species including other Apicomplexan. Computational analysis identified 787 cases of possible gene duplication events and located at least 6,089 gene coding regions. Commonly used gene prediction algorithms produce very disparate sets of protein sequences, with pairwise overlaps ranging from 1.4% to 12%. Through this experimental and computational exercise we benchmarked gene prediction methods and observed false negative rates of 31 to 43%. CONCLUSIONS/SIGNIFICANCE: This study not only provides the largest proteomics exploration of the T. gondii proteome, but illustrates how high throughput proteomics experiments can elucidate correct gene structures in genomes.

PMID: 19065262 [PubMed - in process]
PMCID: PMC2587701

Regulation of toll-like receptors in intestinal epithelial cells by stress and Toxoplasma gondii infection

Parasite Immunol. 2008 Nov-Dec;30(11-12):563-76

Regulation of toll-like receptors in intestinal epithelial cells by stress and Toxoplasma gondii infection

Gopal R, Birdsell D, Monroy FP.

Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011, USA.

Intestinal epithelial cells (IECs) form a barrier between invading microorganisms and the underlying host tissues. IECs express toll-like receptors (TLRs) that recognize specific molecular signatures on microbes, which activate intracellular signalling pathways leading to production of proinflammatory cytokines and chemokines. Stress hormones play an important role in modulation of proinflammatory cytokines and down-regulation of immune responses. Here we demonstrated that expression levels of TLR-2, TLR-4, TLR-9 and TLR-11 were significantly increased in mouse IECs following infection with Toxoplasma gondii on day 8 postinfection. In contrast, expression of TLRs was significantly decreased in infected mice subjected to cold water stress (CWS + INF). Expression of TLR-9 and TLR-11 in the mouse MODE-K cell line was significantly increased after infection. Expression of TLR-9 and TLR-11 in cells exposed to norepinephrine (NE) and parasites was significantly decreased when compared to cells exposed to parasites only. A significant increase was observed in SIGIRR, a negative regulator of TLRs in the CWS + INF group when compared to the INF group. Stress components were able to decrease expression levels of TLRs in IECs, decrease parasite load, and increase expression of a negative regulator thereby ameliorating intestinal inflammatory responses commonly observed during per oral T. gondii infection in C57BL/6 mice.

Publication Types:
Research Support, N.I.H., Extramural

PMID: 19067837 [PubMed - in process]

Apoptotic pulsed dendritic cells induce a protective immune response against Toxoplasma

Parasite Immunol. 2008 Nov-Dec;30(11-12):620-9

Apoptotic pulsed dendritic cells induce a protective immune response against Toxoplasma gondii

Bertaux L, Mevelec MN, Dion S, Suraud V, Gregoire M, Berthon P, Dimier-Poisson I.

Université François Rabelais; INRA; UMR 0483 Université-INRA d'Immunologie Parasitaire et Vaccinologie, Biothérapies anti-infectieuses, IFR 136 Agents Transmissibles en Infectiologie, UFR des Sciences Pharmaceutiques, Montpellier, France.

Infection with the intracellular protozoan parasite Toxoplasma gondii may cause severe sequelae in foetuses and life-threatening neuropathy in immunocompromised patients. We recently reported that vaccination with T. gondii-pulsed dendritic cells induces protective humoral and cellular immune responses against this intracellular pathogen in CBA/J mice. We assessed the feasibility of using a nonlive vaccine, by inducing the apoptosis of T. gondii-pulsed dendritic cells before injecting them into mice. Apoptosis was induced by culturing cells to confluence. We investigated whether these apoptotic T. gondii-pulsed dendritic cells elicited an immune response in vivo. Some studies have shown that immunization with apoptotic cells leads to the activation of innate and adaptive immune mechanisms. Our results are consistent with apoptotic cells having immunomodulatory properties in a model of parasite infection. We showed that the adoptive transfer of T. gondii-pulsed apoptotic dendritic cells elicited humoral and cellular Toxoplasma-specific immune responses with a Th1/Th2 profile, and conferred specific protection. The protective immune response induced was independent of inducible HSP70 production by apoptotic dendritic cells.

Publication Types:
Research Support, Non-U.S. Gov't

PMID: 19067843 [PubMed - in process]

Tuesday, December 09, 2008

ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

Biochem Biophys Res Commun. 2008 Dec 2. [Epub ahead of print]

ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

da Silva CV, da Silva EA, Cruz MC, Chavrier P, Isberg R, Mortara RA.

Instituto de Ciências Biomédicas, Universidade Federal de Uberlândia; Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, Rua Botucatu, 862, 60 andar, 04023-062 Sao Paulo, SP, Brazil.

Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP(2) and PIP(3) to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.

PMID: 19061866 [PubMed - as supplied by publisher]

Surface properties of Toxoplasma gondii oocysts and surrogate microspheres

Appl Environ Microbiol. 2008 Dec 5. [Epub ahead of print]

Surface properties of Toxoplasma gondii oocysts and surrogate microspheres

Shapiro K, Largier J, Mazet JA, Bernt W, Ell JR, Melli AC, Conrad PA.

Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis CA 95616; Department of Environmental Science and Policy, University of California, Davis CA 95616; Wildlife Health Center, School of Veterinary Medicine, University of California, Davis. CA 95616; Particle Characterization Laboratories, Novato CA 94945; Department of Chemistry, University of California, Davis CA 95616.

Physical properties that govern waterborne transmission of Toxoplasma gondii oocysts from land to sea were evaluated and compared to properties of carboxylated microspheres that could serve as surrogates for T. gondii oocysts in transport and water treatment studies. Electrophoretic mobilities of T. gondii oocysts, lightly carboxylated Dragon Green microspheres, and heavily carboxylated Glacial Blue microspheres were determined in ultrapure water, artificial fresh water with and without dissolved organic carbon, artificial estuarine water, and artificial sea water. The surface wettabilities of oocysts and microspheres were determined using a water contact angle approach. Toxoplasma gondii oocysts and microspheres were negatively charged in fresh water solutions, but their charge was neutralized in estuarine and sea waters. Oocysts, Glacial Blue microspheres, and unwashed Dragon Green microspheres had a low contact angle indicating that they were hydrophilic; however, once washed, Dragon Green microspheres became markedly hydrophobic. The hydrophilic nature and negative charge of T. gondii oocysts in fresh water could facilitate widespread contamination of waterways. The loss of charge observed in saline waters may lead to flocculation and subsequent accumulation of T. gondii oocysts in locations where fresh and marine waters mix, indicating a high risk of exposure to humans and wildlife with this zoonotic pathogen in estuarine habitats. While microspheres did not have identical surface properties as T. gondii, similar properties shared between each microsphere type and oocysts suggest that their joint application in transport and fate studies could provide a range of transport potentials in which oocysts are likely to behave.

PMID: 19060174 [PubMed - as supplied by publisher]

Thursday, December 04, 2008


Eukaryot Cell. 2008 Dec 1. [Epub ahead of print]


Gilk SD, Gaskins E, Ward GE, Beckers CJ

Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC 27599; and Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05404.

Toxoplasma gondii motility is powered by the myosin XIV motor complex which consists of myosin XIV heavy chain (MyoA), myosin light chain (MLC1), GAP45 and GAP50, the membrane anchor of the complex. MyoA, MLC1, and GAP45 are initially assembled into a soluble complex, which then associates with GAP50, an integral membrane protein of the parasite inner membrane complex. While all proteins in the myosin XIV motor complex are essential for parasite survival, the specific role of GAP45 remains unclear. We demonstrate here that final assembly of the motor complex is controlled by phosphorylation of GAP45. This protein is phosphorylated on multiple residues and, using mass spectroscopy, we have identified two of these, Ser(163) and Ser(167). The importance of these phosphorylation events was determined by mutation of Ser(163) and Ser(167) to Glu and Ala residues to mimic phosphorylated and non-phosphorylated residues, respectively. Mutation of Ser(163) and Ser(167) to either Ala or Glu residues does not affect targeting of GAP45 to the inner membrane complex or its association with MyoA and MLC1. Mutation of Ser(163) and Ser(167) into Ala residues also does not affect assembly of the mutant GAP45 into the myosin motor complex. Mutation of Ser(163) and Ser(167) to Glu residues, however, prevents association of the MyoA-MLC1-GAP45 complex with GAP50. These observations indicate that phosphorylation of Ser(163) and Ser(167) in GAP45 controls the final step in assembly of the myosin XIV motor complex.

PMID: 19047362 [PubMed - as supplied by publisher]

Recognition of Toxoplasma gondii by TLR11 Prevents Parasite-Induced Immunopathology

J Immunol. 2008 Dec 15;181(12):8478-84

Recognition of Toxoplasma gondii by TLR11 Prevents Parasite-Induced Immunopathology

Yarovinsky F, Hieny S, Sher A.

Department of Immunology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390.

TLRs are thought to play a critical role in self/non-self discrimination by sensing microbial infections and initiating both innate and adaptive immunity. In this study, we demonstrate that in the absence of TLR11, a major TLR involved in recognition of Toxoplasma gondii, infection with this protozoan parasite induces an abnormal immunopathological response consisting of pancreatic tissue destruction, fat necrosis, and systemic elevations in inflammatory reactants. We further show that this immunopathology is the result of non-TLR dependent activation of IFN-gamma secretion by NK cells in response to the infection. These findings reveal that in addition to triggering host resistance to infection, TLR recognition can be critical for the prevention of pathogen-induced immune destruction of self tissue.

PMID: 19050265 [PubMed - in process]

Dendritic Cells Expressing Plasmacytoid Marker PDCA-1 Are Trojan Horses during Toxoplasma Infection

J Immunol. 2008 Dec 15;181(12):8485-91

Dendritic Cells Expressing Plasmacytoid Marker PDCA-1 Are Trojan Horses during Toxoplasma gondii Infection

Bierly AL, Shufesky WJ, Sukhumavasi W, Morelli AE, Denkers EY.

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

Plasmacytoid dendritic cells (pDCs) play a key role in the innate immune response to viral infection, due largely to their ability to produce large quantities of type I IFNs. These cells are also notable for their ability to differentiate into conventional dendritic cells after appropriate stimulation. Here, we show that a splenic population of murine CD11c(+) cells expressing pDC markers Gr-1, B220, and PDCA-1 is preferentially parasitized after infection with the virulent RH strain of Toxoplasma gondii. Although these markers are closely associated with pDCs, the population we identified was unusual because the cells express CD11b and higher than expected levels of CD11c. By adoptive transfer of CD45.1-positive cells into CD45.2 congenic mice, we show that CD11c(+)Gr-1(+) cells migrate from the peritoneal cavity to the spleen. During infection, these cells accumulate in the marginal zone region. Recruitment of infected CD11c(+)Gr-1(+) cells to the spleen is partially dependent upon signaling through chemokine receptor CCR2. Intracellular cytokine staining demonstrates that infected, but not noninfected, splenic CD11c(+)Gr-1(+) dendritic cells are suppressed in their ability to respond to ex vivo TLR stimulation. We hypothesize that Toxoplasma exploits pDCs as Trojan horses, targeting them for early infection, suppressing their cytokine effector function, and using them for dissemination within the host.

PMID: 19050266 [PubMed - in process]