Wednesday, April 30, 2008

Comparison of cholera toxin A2/B and murine interleukin-12 as adjuvants of Toxoplasma multi-antigenic SAG1-ROP2 DNA vaccine

Exp Parasitol. 2008 Mar 23 [Epub ahead of print]

Comparison of cholera toxin A2/B and murine interleukin-12 as adjuvants of Toxoplasma multi-antigenic SAG1-ROP2 DNA vaccine

Xue M, He S, Zhang J, Cui Y, Yao Y, Wang H.

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

Toxoplasmosis can lead to severe pathology in both humans and animals. However, an effective vaccine for humans has not been successfully developed. In this study, we used multi-antigenic SAG1-ROP2 as a DNA vaccine and cholera toxin A2/B subunit and murine interleukin-12 to compare their effectiveness as genetic adjuvants. Bagg albino/c (BAL/c) mice were immunized intramuscularly with pcDNA3.1-SAG1-ROP2 alone (control group), or pcDNA3.1-SAG1-ROP2 with co-administration of pCTA2/B or pIL-12, respectively. After immunization, the effectiveness of these two adjuvants were compared using lymphocyte proliferation assay, cytokine and antibody measurements. The group co-administered pIL-12 elicited stronger humoral and Th1-type cellular immune responses than those immunized with pcDNA3.1-SAG1-ROP2 alone, while in the group co-administered pCTA2/B there was no obvious enhancement of immunity. When challenged with Toxoplasma gondii RH strain, mice immunized with pIL-12 co-administration had significantly higher survival rates, whereas there was no notable augmentation of immunity in pCTA2/B group. Therefore, since pIL-12 significantly enhanced the antigenicity of multi-antigenic DNA vaccine, this suggests that IL-12 is a better and more effective adjuvant than CTA2/B in this situation.

PMID: 18442818 [PubMed - as supplied by publisher]

Characterization of a nitric oxide synthase-like protein

Exp Parasitol. 2008 Mar 23 [Epub ahead of print]

Toxoplasma gondii: Molecular cloning and characterization of a nitric oxide synthase-like protein

Gutierrez-Escobar AJ, Arenas AF, Villoria-Guerrero Y, Padilla-Londoño JM, Gómez-Marin JE.

Grupo de Parasitología Molecular (GEPAMOL), Centro de Investigaciones Biomédicas, Facultad de Ciencias de la Salud, Universidad del Quindío, Armenia, Colombia.

Toxoplasma gondii has a nitrite production and a putative nitric oxide synthase (NOS) motif genomic sequence. In order to demonstrate that this sequence is functional and could be involved in the metabolism of l-arginine derivatives, we constructed a baculovirus carrying the previously identified Toxoplasma NOS-like DNA sequence. The recombinant protein was expressed into insect Sf9 cells and his activity was tested in serial microplate colorimetric assays. The protein produced 21nmol/min/ml nitrites per microgram of protein and followed Michaelis-Menten kinetics, with a K(m) for l-arginine of 2.3mM. Furthermore, the optimal pH, temperature and incubation time for the recombinant Toxoplasma NOS-like protein were established. Toxoplasma NOS runs as a band of 11.6kDa on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results indicate that the recombinant protein derived from the putative genomic sequence, at the chromosome 1b of T. gondii, is able to produce nitrites from l-arginine as substrate.

PMID: 18439581 [PubMed - as supplied by publisher]

Revision of the positive predictive value of IgM anti-Toxoplasma antibodies as an index of recent infection

New Microbiol. 2008 Jan;31(1):105-11.

Revision of the positive predictive value of IgM anti-Toxoplasma antibodies as an index of recent infection

De Paschale M, Agrappi C, Belvisi L, Cagnin D, Cerulli T, Clerici P, Mirri P, Manco MT, Cavallari S, Viganò EF.

Microbiology Unit, Hospital of Legnano, Milan, Italy.

The severity of congenital Toxoplasma gondii infection underlines the need for a precise diagnosis of acute infection during pregnancy. The search for specific IgM has been widely used for this purpose, but their possible early disappearance or persistence over time limits their meaning. In order to estimate the positive predictive value of anti-Toxoplasma IgM testing, we made an epidemiological analysis of the presence of anti-Toxoplasma IgG and IgM using ELISA in 4786 subjects attending the Hospital of Legnano in 2004-2005: 1360 seen for a clinical check-up and 3426 pregnant women for serological screening. In relation to IgG avidity, the positive predictive value of IgM was 45.98% (95% CI: 35.51-56.45) as a whole: this increased to 83.87% (95% CI: 70.92-96.82) in the patients with a highly positive test for IgM, but decreased to 9.52% (95% CI: 0.00-22.07) in pregnant women with a weakly positive test for IgM. Our results indicate that a highly positive IgM value in patients can be a good index of recent infection, but its poor predictive value in pregnant women underlines the need for additional tests with a follow-up if necessary.

PMID: 18437848 [PubMed - in process]

Friday, April 25, 2008

The transcription of bradyzoite genes in Toxo is controlled by autonomous promoter elements

Mol Microbiol. 2008 Apr 21 [Epub ahead of print]

The transcription of bradyzoite genes in Toxoplasma gondii is controlled by autonomous promoter elements

Behnke MS, Radke JB, Smith AT, Sullivan WJ Jr, White MW.

Department of Veterinary Molecular Biology, Montana State University Bozeman, MT 59717, USA.

Experimental evidence suggests that apicomplexan parasites possess bipartite promoters with basal and regulated cis-elements similar to other eukaryotes. Employing a dual luciferase model adapted for recombinational cloning and use in Toxoplasma gondii, we show that genomic regions flanking 16 parasite genes, which encompass examples of constitutive and tachyzoite- and bradyzoite-specific genes, are able to reproduce the appropriate developmental stage expression in a transient luciferase assay. Mapping of cis-acting elements in several bradyzoite promoters led to the identification of short sequence spans that are involved in control of bradyzoite gene expression in multiple strains and under different bradyzoite induction conditions. Promoters that regulate the heat shock protein BAG1 and a novel bradyzoite-specific NTPase during bradyzoite development were fine mapped to a 6-8 bp resolution and these minimal cis-elements were capable of converting a constitutive promoter to one that is induced by bradyzoite conditions. Gel-shift experiments show that mapped cis-elements are bound by parasite protein factors with the appropriate functional sequence specificity. These studies are the first to identify the minimal sequence elements that are required and sufficient for bradyzoite gene expression and to show that bradyzoite promoters are maintained in a "poised" chromatin state throughout the intermediate host life cycle in low passage strains. Together, these data demonstrate that conventional eukaryotic promoter mechanisms work with epigenetic processes to regulate developmental gene expression during tissue cyst formation.

PMID: 18433450 [PubMed - as supplied by publisher]

Evolutionary Origin of the Protozoan Parasites Histone-like Proteins (HU)

In Silico Biol. 2008;8(1):15-20

Evolutionary Origin of the Protozoan Parasites Histone-like Proteins (HU)

Arenas AF, Escobar AJ, Gómez-Marin JE.

Grupo de Parasitología Molecular (GEPAMOL), Centro de Investigaciones Biomédicas, Facultad de Ciencias de la Salud, Universidad del Quindío, Armenia, Colombia.

The histone-like proteins (HU) belong to a family of DNA architectural proteins that stabilize nucleoprotein complexes. We found a putative HU protein (TgGlmHMM_3045) in Toxoplasma gondii genome that was homologous to the bacterial HU protein. This putative sequence was located in the scaffold TGG_995361 of the chromosome 10. The sequence included the prokaryotic bacterial histone-like domain, KFGSLGlRRRGERVARNPRT (ID number PS00045). HU protein sequences were also found in Plasmodium falciparum, Neospora caninum, Theileria parva and Theileria annulata. We found that the homology of the putative HU protein in Apicomplexa was greater with bacterial histone-like proteins than with eukaryotic histone proteins. The phylogenetic tree indicated that the putative HU protein genes were acquired in Apicomplexa by means of a secondary endosymbiotic event from red algae and later they were transfered from the apicoplast organelle to the nuclear genome.

PMID: 18430986 [PubMed - in process]

Wednesday, April 23, 2008

IL-12 Signaling Drives CD8+ T Cell IFN-{gamma} Production and Differentiation of KLRG1+ Effector Subpopulations During Infection

J Immunol. 2008 May 1;180(9):5935-45.

IL-12 Signaling Drives CD8+ T Cell IFN-{gamma} Production and Differentiation of KLRG1+ Effector Subpopulations during Toxoplasma gondii Infection

Wilson DC, Matthews S, Yap GS.

Department of Molecular Microbiology and Immunology, Brown University, Providence, RI 02912.

IFN-gamma-producing CD8(+) T lymphocytes are essential effector cells that mediate protective immunity during murine toxoplasmosis, and yet their effector development remains poorly characterized. Vaccination with the carbamoyl phosphate synthase (CPS) mutant strain of Toxoplasma gondii was used to examine the CD8(+) T cell response in the peritoneal effector site. Four CTL subpopulations with varying effector potentials were defined based on the expression of effector molecules and the cell surface activation markers CD62L and killer cell lectin-like receptor G1 (KLRG1). Further phenotypic analysis revealed that the acquisition of KLRG1 among effector subpopulations correlated with the down-regulation of both IL-7R and CD27, suggesting that KLRG1 marks dominant, end-stage effector cells. Using gene-targeted mice, we tested the in vivo requirements of key IL-12 signaling components for effector CTL differentiation. Contrary to established models of viral and bacterial infection, CD8(+) T cell-intrinsic IL-12 signaling was required for the generation of IFN-gamma-producing CTLs in response to T. gondii. Importantly, the development of the KLRG1(+) effector subpopulations, but not the memory precursor-containing KLRG1(-) effector subset, was critically reliant on IL-12. Furthermore, IL-12 signaling-dependent T-bet expression was also found to be important for differentiation of KLRG1(+) effectors. Our results underscore a vital role for IL-12 in not only the induction of IFN-gamma expression but also in the development of heterogeneous subpopulations of effector CD8(+) T cells generated in response to the intracellular parasite T. gondii.

PMID: 18424713 [PubMed - in process]

Plasmacytoid Dendritic Cells Are Activated by Toxo to Present Antigen and Produce Cytokines

J Immunol. 2008 May 1;180(9):6229-36.

Plasmacytoid Dendritic Cells Are Activated by Toxoplasma gondii to Present Antigen and Produce Cytokines

Pepper M, Dzierszinski F, Wilson E, Tait E, Fang Q, Yarovinsky F, Laufer TM, Roos D, Hunter CA.

Department of Pathobiology.

Infection with the parasite Toxoplasma gondii leads to the induction of a Th1-type response dominated by IFN-gamma production and control of this pathogen. Cells of the innate immune system are essential in initiating this response both through the production of IL-12 as well as the presentation of parasite-derived Ags to MHC-restricted T cells. Although dendritic cells (DCs) have been implicated in these events, the contribution of individual DC populations remains unclear. Therefore, multiparameter flow cytometry was used to identify and characterize subsets of murine DCs during acute toxoplasmosis. This approach confirmed that infection leads to the expansion and activation of conventional DC (cDC) subsets. Unexpectedly, however, this analysis further revealed that plasmacytoid DCs are also expanded and that these cells up-regulate MHC class II and costimulatory molecules associated with their acquired ability to prime naive CD4(+) T cells. Furthermore, T. gondii-activated plasmacytoid DCs produce high levels of IL-12 and both plasmacytoid DC maturation and cytokine production are dependent on TLR11. Together these studies suggest that pDCs are a prominent DC subset involved in the initial stages of T. gondii infection, presenting parasite Ags and producing cytokines that are important for controlling infection.

PMID: 18424745 [PubMed - in process]

Tuesday, April 22, 2008

Translation regulation by eIF2 kinases in the development of latent cysts in Toxo

J Biol Chem. 2008 Apr 16 [Epub ahead of print]

Translation regulation by eIF2 kinases in the development of latent cysts in Toxoplasma gondii

Narasimhan J, Joyce BR, Naguleswaran A, Smith AT, Livingston MR, Dixon SE, Coppens I, Wek RC, Sullivan WJ Jr.

Pharmacology & Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202.

A key problem in the treatment of numerous pathogenic eukaryotes centers on their development into latent forms during stress. For example, the opportunistic protist Toxoplasma gondii converts to latent cysts (bradyzoites) responsible for recrudescence of disease. We report that Toxoplasma eukaryotic initiation factor-2 alpha (TgIF2alpha) is phosphorylated during stress, and establish that protozoan parasites utilize translation control to modulate gene expression during development. Importantly, TgIF2alpha remains phosphorylated in bradyzoites, explaining how these cells maintain their quiescent state. Further, we have characterized novel eIF2 kinases: one in the endoplasmic reticulum (ER) and a likely regulator of the unfolded protein response (TgIF2K-A), and another that is a probable responder to cytoplasmic stresses (TgIF2K-B). Significantly, our data suggest that 1) the regulation of protein translation through eIF2 kinases is associated with development, 2) eIF2alpha phosphorylation is employed by cells to maintain a latent state, and 3) ER and cytoplasmic stress responses evolved in eukaryotic cells prior to the early-diverging Apicomplexa. Given its importance to pathogenesis, eIF2 kinase-mediated stress responses may provide opportunities for novel therapeutics.

PMID: 18420584 [PubMed - as supplied by publisher]

Friday, April 18, 2008

Toxoplasma infection in horses. Giddyup.

Parassitologia. 2007 Jun;49(1-2):7-15.

Toxoplasma gondii infection in horses. A review

Tassi P.

Dipartimento di Sanità e Benessere degli Animali, Università degli Studi di Bari, Italy.

This review updates those written by Dubey and Beattie in 1988 (1988a) and by Tenter et al in 2000, on pathological and epidemiological aspects of Toxoplasma infection in horses. Under natural conditions, seroprevalence may variate from 0% up to 90%. This wide variation may be due to the sensitivity of the serological methods, to the age of animals, to the geographical area, and even to the hygienic condition of the farms and farm management. With few exceptions, horses are considered one of the less sensitive specie to the pathogenic effect of Toxoplasma gondii. In fact, neither under experimental nor under natural condition a genuine pathologic picture related to the toxoplasmic infection has been described. In one occasion the organism has been isolated from an eye condition and in others a connection between a higher frequency of unspecified pathological conditions and a positive response to serological test for Toxoplasma has been speculated. Diaplacental transmission and the following abortion have been only occasionally reported, and at least in one case in a quite trustworthy way, therefore it must be considered possible, though rare. Although infection of humans due to the consumption of horse meat has never been reported, the existence of a possible risk arouses by the demonstration of the presence of parasite stages in either naturally or experimentally infected horses, which resulted to be infective for mice and/or cats.

PMID: 18412038 [PubMed - in process]

Thursday, April 17, 2008

Organellar dynamics during the cell cycle of Toxoplasma

J Cell Sci. 2008 Apr 14 [Epub ahead of print]

Organellar dynamics during the cell cycle of Toxoplasma gondii

Nishi M, Hu K, Murray JM, Roos DS.

The protozoan phylum Apicomplexa encompasses approximately 5000 species of obligate intracellular parasites, including those responsible for malaria and toxoplasmosis. Rather than dividing by binary fission, apicomplexans use a remarkable mechanism for replication, assembling daughters de novo within the cytoplasm. Here, we exploit time-lapse microscopy of fluorescent markers targeted to various subcellular structures in Toxoplasma gondii tachyzoites to determine how these unicellular eukaryotes efficiently package a complete set of organelles, maintaining the highly polarized organization necessary for host cell invasion and pathogenesis. Golgi division and elongation of the apicoplast are among the first morphologically observable events, associated with an unusual pattern of centriolar migration. Daughter parasites are assembled on cytoskeletal scaffolding, whose growth proceeds from the apical end, first encapsulating the divided Golgi. Further extension of the cytoskeletal scaffold results in partitioning of the apicoplast, nucleus, endoplasmic reticulum, and finally the mitochondrion, which enters the developing daughters rapidly, but only very late during the division cycle. The specialized secretory organelles (micronemes and rhoptries) form de novo. This distinctive pattern of replication - in which organellar segregation spans approximately 75% of the cell cycle, completely encompassing S phase - suggests an unusual mechanism of cell cycle regulation.

PMID: 18411248 [PubMed - as supplied by publisher]

Fas/CD95-mediated apoptosis of type II cells is blocked by Toxo

Infect Immun. 2008 Apr 14 [Epub ahead of print]

Fas/CD95-mediated apoptosis of type II cells is blocked by Toxoplasma gondii primarily via interference with the mitochondrial amplification loop

Hippe D, Lytovchenko O, Schmitz I, Lüder CG.

Institute for Medical Microbiology, Georg-August-University, Kreuzbergring 57, 37075 Göttingen, Germany; and Institute for Molecular Medicine, Heinrich-Heine-University, Universitätsstrasse 1, 40225 Düsseldorf, Germany.

The intracellular protozoan Toxoplasma gondii induces persistent infections in various hosts and is an important opportunistic pathogen of humans with immature or deficient immune responses. The ability to survive intracellularly largely depends on the blocking of different pro-apoptotic signalling cascades of its host cell. Fas/CD95 triggers an apoptotic cascade that is crucial for immunity and the outcome of infectious diseases. Here we have determined the mechanism by which T. gondii counteracts death receptor-mediated cell death in type II cells that transduce Fas/CD95 ligation via caspase 8-mediated activation of the mitochondrial amplification loop. The results showed that infection with T. gondii significantly reduced Fas/CD95-triggered apoptosis in HeLa cells by inhibiting the activities of initiator caspases 8 and 9 and effector caspase 3/7. Parasitic infection dose-dependently diminished cleavage of caspase 8, the BH3-only protein Bid and the downstream caspases 9 and 3. Importantly, interference with Fas/CD95-triggered caspase 8 and caspase 3/7 activities after parasitic infection was largely dependent on the presence of caspase 9. Within the mitochondrial amplification loop, T. gondii significantly inhibited the Fas/CD95-triggered release of cytochrome c into the host cell cytosol. These results indicate that T. gondii inhibits Fas/CD95-mediated apoptosis in type II cells primarily by decreasing the apoptogenic function of mitochondria.

PMID: 18411295 [PubMed - as supplied by publisher]

Wednesday, April 16, 2008

Acute acquired toxoplasmosis presenting as polymyositis and chorioretinitis in immunocompetent patient

Joint Bone Spine. 2008 Apr 9 [Epub ahead of print]

Acute acquired toxoplasmosis presenting as polymyositis and chorioretinitis in immunocompetent patient

Hassene A, Vital A, Anghel A, Guez S, Series C.

Department of Internal Medicine, Service de Médecine Interne, CHU Pellegrin, 33076 Bordeaux, France.

The parasite Toxoplasma gondii mainly encysts in brain, retina, myocardium, and skeletal muscle. It has been implicated in the genesis of inflammatory myopathies for years, but the parasite usually cannot be detected in the muscle. It is established, however, that toxoplasmosis can cause myositis either by recent infection or by reactivation. The case of a non-HIV patient who developed an acute polymyositis upon infection by T. gondii is reported. We suggest that all patients with polymyositis should have serological tests for toxoplasmosis as a part of their initial evaluation and early trial of antiprotozoal therapy in case of positive findings.

PMID: 18406191 [PubMed - as supplied by publisher]

Regulation of innate immunity by suppressor of cytokine signaling (SOCS) proteins

Immunobiology. 2008;213(3-4):225-35. Epub 2007 Nov 28.

Regulation of innate immunity by suppressor of cytokine signaling (SOCS) proteins

Dalpke A, Heeg K, Bartz H, Baetz A.

Department of Hygiene and Medical Microbiology, Institute of Hygiene, University of Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany.

Innate immunity represents the first line of defense against invading pathogens. Toll-like receptors (TLRs) are important for activation of innate immunity. Moreover, cytokines mediate communication of cells and are necessary to mount an appropriately regulated immune response. However, activation of innate immunity has to be tightly controlled to avoid overshooting immune reactions. Suppressor of cytokine signaling (SOCS) proteins have been identified as inducible feedback inhibitors of cytokine receptors and have been shown to be of crucial importance for the limitation of inflammatory responses. In this review, we describe the role of SOCS proteins in macrophages and dendritic cells (DCs). Based on our own findings, we show that SOCS proteins are directly induced by stimulation of TLRs. However, SOCS proteins do not interfere with direct TLR signaling, but avoid overshooting activation by regulating paracrine IFN-beta signaling. In addition, SOCS proteins in macrophages and DCs regulate the sensitivity towards IFN-gamma and GM-CSF, thereby modulating anti-microbial activity of macrophages and differentiation of DCs. We discuss that SOCS induction can also be used by microbes to evade immune defense, and this is exemplified by the parasite Toxoplasma gondii which induces SOCS1 to inhibit IFN-gamma-mediated macrophage activation. Taken together, the findings indicate that SOCS proteins play an important role in the balanced activation of innate immunity during infectious encounter.

PMID: 18406369 [PubMed - in process]

Programmed Cell Death 5: A secreted molecule that exerts a pro-apoptotic effect on hosts

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

Programmed Cell Death 5 from Toxoplasma gondii: A secreted molecule that exerts a pro-apoptotic effect on host cells

Bannai H, Nishikawa Y, Matsuo T, Kawase O, Watanabe J, Sugimoto C, Xuan X.

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

Although parasite-infected host cells become resistant to apoptosis, uninfected bystander cells undergo apoptosis during Toxoplasma gondii infection. The Programmed Cell Death 5 (TgPDCD5) gene, a homologue of the human apoptosis-related molecule, was cloned from a T. gondii full-length cDNA database and subsequently characterized. The native TgPDCD5 was located in the cytosol and also detected in the secreted fraction. Immuno-electron microscopic analysis showed TgPDCD5 was primarily located close to the rhoptries or vesicle-like structures near the surface membrane of the parasite. Studies using recombinant TgPDCD5 (rTgPDCD5) demonstrated that host cells internalize the molecule in a heparan sulfate proteoglycan-binding motif-dependent manner. Furthermore, the addition of rTgPDCD5 to culture medium resulted in the enhancement of host-cell apoptosis triggered by etoposide in macrophage cell line J774A.1 and leukemic cell line HL-60 cells. Additionally, rTgPDCD5 induced apoptosis in J774A.1 cells in the presence of IFN-gamma. This report is the first to identify a parasitic molecule of T. gondii that has a pro-apoptotic effect on host cells.

PMID: 18406478 [PubMed - as supplied by publisher]

Novel actin-related protein is associated with daughter cell formation

Eukaryot Cell. 2008 Apr 11 [Epub ahead of print]

A novel actin-related protein is associated with daughter cell formation in Toxoplasma

Gordon JL, Beatty WL, Sibley LD.

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.

Cell division in Toxoplasma gondii occurs by an unusual budding mechanism termed endodyogeny, during which twin daughters are formed within the body of the mother cell. Cytokinesis begins with the coordinated assembly of the inner membrane complex (IMC), which surrounds the growing daughter cells. The IMC is compiled of both flattened membrane cisternae and subpellicular filaments composed of articulin-like proteins attached to underlying singlet microtubules. While proteins that comprise the elongating IMC have been described, little is known about its initial formation. Using Toxoplasma as a model system, we demonstrate that actin-like protein 1 (ALP1) is partially redistributed to the IMC at early stages in its formation. Immuno-EM localized ALP1 to a discrete region of the nuclear envelope, on transport vesicles, and on the nascent IMC of the daughter cells, prior to the arrival of proteins such as IMC-1. Over-expression of ALP1 under control of a strong constitutive promoter disrupted formation of the daughter cell IMC, leading to delayed growth and defects in nuclear and apicoplast segregation. Collectively, these data suggest that ALP1 participates in formation of daughter cell membranes during cell division in apicomplexan parasites.

PMID: 18408052 [PubMed - as supplied by publisher]

Sunday, April 13, 2008

Analyses of murine GBP homology clusters based on in silico, in vitro and in vivo studies

BMC Genomics. 2008 Apr 10;9(1):158 [Epub ahead of print]

Analyses of murine GBP homology clusters based on in silico, in vitro and in vivo studies

Kresse A, Konermann C, Degrandi D, Beuter-Gunia C, Wuerthner J, Pfeffer K, Beer S.

ABSTRACT: BACKGROUND: The interactions between pathogens and hosts lead to a massive upregulation of antimicrobial host effector molecules. Among these, the 65 kDa guanylate binding proteins (GBPs) are interesting candidates as intricate components of the host effector molecule repertoire. Members of the GBP family are highly conserved in vertebrates. Previous reports indicate an antiviral activity of human GBP1 (hGBP1) and murine GBP2 (mGBP2). We recently demonstrated that distinct murine GBP (mGBP) family members are highly upregulated upon Toxoplasma gondii infection and localize around the intracellular protozoa T. gondii. Moreover, we characterised five new mGBP family members within the murine 65 kDa GBP family. RESULTS: Here, we identified a new mGBP locus named mGbp11. Based on bacterial artificial chromosome (BAC), expressed sequence tag (EST), and RT-PCR analyses this study provides a detailed insight into the genomic localization and organization of the mGBPs. These analyses revealed a 166-kb spanning region on chromosome 3 harboring five transcribed mGBPs (mGbp1, mGbp2, mGbp3, mGbp5, and mGbp7) and one pseudogene (pseudomGbp1), as well as a 332-kb spanning region on chromosome 5 consisting of six transcribed mGBPs (mGbp4, mGbp6, mGbp8, mGbp9, mGbp10, and mGbp11), and one pseudogene (pseudomgbp2). Besides the strikingly high homology of 65% to 98% within the coding sequences, the mGBPs on chromosome 5 cluster also exhibit a highly homologous exon-intron structure whereas the mGBP on chromosome 3 reveals a more divergent exon-intron structure. CONCLUSIONS: This study details the comprehensive genomic organization of mGBPs and suggests that a continuously changing microbial environment has exerted evolutionary pressure on this gene family leading to multiple gene amplifications.

PMID: 18402675 [PubMed - as supplied by publisher]

Evaluation of a rapid ELISA technique for detection of circulating antigens of Toxoplasma

Microbiol Immunol. 2008;52(3):180-7.

Evaluation of a rapid ELISA technique for detection of circulating antigens of Toxoplasma gondii

Chen R, Lu S, Lou D, Lin A, Zeng X, Ding Z, Wen L, Ohta N, Wang J, Fu C.

Institute of Parasitic Diseases, Zhejiang Academy of Medical Sciences, and College of Life Sciences, Zhejiang University, Hangzhou, China.

To evaluate a modified rapid ELISA method for detecting CAg during Toxoplasma gondii infection, we analyzed the specificity and sensitivity of the ELISA method by using experimental Toxoplasma infection in rabbits and also tested this method in human samples including 5428 serum, 548 cerebrospinal fluid and two breast milk samples. We prepared PcAb, and used it for rapid one-step sandwich ELISA testing in which an incubation time in the regular ELISA procedure was omitted. This method detected CAg at the concentration of 31.2 ng/mL, and no cross-reaction was found with antigens of protozoa (Cryptosporidium parvum, Plasmodium falciparum), trematode (Schistosoma japonicum, Paragonimus sp.) and nematode (Brugia malayi, Ancylostoma duodenale, Ascaris lumbricoides and Trichinella spiralis). CAg was detected in rabbit serum 3 days after infection, and optical density values reached a peak 9 approximately 13 days after infection, then declined gradually. Among human serum samples, the positive rate of CAg was 2.11% in cerebral paralysis patients, whereas it was 0.22% or 0.71% in patients without neurological symptoms or in uncomplicated pregnant women. The difference among these three groups was statistically significant (P < 0.05). The positive rate of cerebrospinal fluid samples from cerebral paralysis patients was 10.58%. There is a statistically significant difference between the positive rates of meat-packing workers and blood donors (P < 0.01). In the retrospective analysis, CAg was detected in accordance with the onset of clinical symptoms, suggesting that CAg could reflect the clinical course in humans. Together with these results, CAg detected in the modified rapid sandwich ELISA could be a sensitive marker for acute and active infection of T. gondii.

PMID: 18402600 [PubMed - in process]

Saturday, April 12, 2008

Comparison of immunoblotting, Goldmann-Witmer coefficient and real-time PCR on aqueous humor for diagnosis of ocular toxoplasmosis

J Clin Microbiol. 2008 Apr 9 [Epub ahead of print]

Comparison of immunoblotting, Goldmann-Witmer coefficient and real-time PCR on aqueous humor for diagnosis of ocular toxoplasmosis

Fekkar A, Bodaghi B, Touafek F, Le Hoang P, Mazier D, Paris L.

AP-HP, Groupe hospitalier Pitié-Salpêtrière, Service Parasitologie Mycologie, Paris, F-75013 France; Université Pierre et Marie Curie-Paris 6, UMR S511 Paris, F-75013 France; INSERM, U511, Paris, F-75013 France; AP-HP, Groupe hospitalier Pitié-Salpêtrière, Service Ophtalmologie Paris, F-75013 France.

We compared three biological methods for the diagnosis of ocular toxoplasmosis (OT). Paired aqueous humor and serum samples from 34 patients with OT and from 76 patients with other ocular disorders were analysed with three methods: immunoblotting or western blot (WB), calculation of the Goldmann-Witmer coefficient (GWC), and polymerase chain reaction (PCR). WB and GWC each revealed intraocular production of specific anti-Toxoplasma immunoglobulin G (IgG) in 81% of samples (30 of 37). PCR detected toxoplasmic DNA in 38% of samples (13 of 34). Nine of the 13 PCR-positive patients were immunocompetent. Combining the techniques significantly improved the diagnostic sensitivity, to 92% with the GWC-WB combination, 90% with the WB-PCR combination, and 93% with the GWC-PCR combination. Combination of all three techniques improved the sensitivity to 97%.

PMID: 18400917 [PubMed - as supplied by publisher]

Thursday, April 10, 2008

Protection against colonization of the brain and muscles in a rat model

Exp Parasitol. 2008 Mar 7 [Epub ahead of print]

Toxoplasma gondii: Protection against colonization of the brain and muscles in a rat model

Freyre A, Falcón J, Mendez J, Gonzalez M.

Laboratorio de Toxoplasmosis, Departamento de Parasitologı´a, Universidad de la Republica, Facultad de Veterinaria, Alberto Lasplaces 1550, Montevideo 11600, Uruguay.

The objective was to test immune protection against the formation of Toxoplasma gondii tissue cysts in rats. It has been previously shown that 50 T. gondii tissue cysts of strain Me49 are not pathogenic for CF-1 mice, whereas 1 T. gondii tissue cyst of strain M-7741, can be lethal for mice 11-13 days after subcutaneous or oral administration. In the present study, ten rats were fed T. gondii oocysts of strain Me49 and after a further 30 days they were each orally challenged with T. gondii oocysts of strain M-7741. Thirty days after this, they were euthanased and brain and muscle samples inoculated subcutaneously or orally dosed, respectively, to mice for bioassay. None of the mice died, whereas all the mice that were inoculated with brain homogenates or were fed muscle samples from four non-immunized rats that had been inoculated with T. gondii oocysts of strain M-7741, died. These results encourage further research towards achieving vaccinal protection against the formation of T. gondii tissue cysts in meat animals and people.

PMID: 18397785 [PubMed - as supplied by publisher]

Wednesday, April 09, 2008

Molecular signals in the trafficking of MIC3 to the micronemes

Eukaryot Cell. 2008 Apr 4 [Epub ahead of print]

Molecular signals in the trafficking of the Toxoplasma gondii protein MIC3 to the micronemes

El Hajj H, Papoin J, Cérède O, Garcia-Réguet N, Soête M, Dubremetz JF, Lebrun M.

UMR 5235 CNRS, Université de Montpellier 2, CP 107, Place Eugène Bataillon, 34090 Montpellier, France; FRE 2377 CNRS, Institut de Biologie de Lille, 1 rue du professeur Calmette, 59021 Lille, France; UMR Université-INRA d'Immunologie Parasitaires, Faculté des Sciences Pharmaceutiques et Biologiques, 31 Avenue Monge, 37200 Tours, France.

The protozoan parasite Toxoplasma gondii is equipped with a sophisticated secretory apparatus including three distinct exocytic organelles named micronemes, rhoptries and dense granules. We have dissected the requirements for targeting the microneme protein MIC3, a key component of T. gondii infection. We have shown that MIC3 is processed in a post-Golgi compartment, and that the MIC3 pro-peptide and epidermal growth factor (EGF) modules contain microneme targeting information. The minimal requirement for microneme delivery is defined by the pro-peptide plus any one of the three EGF domains. We have demonstrated that the cleavage of the pro-peptide, the dimerization of MIC3 and the chitin-binding-like sequence, which are crucial for host cell binding and virulence, are dispensable for proper targeting. Finally, we have shown that part of MIC3 is withheld in the secretory pathway in a cell cycle-dependant manner.

PMID: 18390648 [PubMed - as supplied by publisher]

Inhibition of poly(ADP-ribose) polymerase is dispensable for parasite-mediated blockade of host cell apoptosis and intracellular parasite replication

Microbes Infect. 2007 Dec 28 [Epub ahead of print]

Transient inhibition of poly(ADP-ribose) polymerase expression and activity by Toxoplasma gondii is dispensable for parasite-mediated blockade of host cell apoptosis and intracellular parasite replication

Gais A, Beinert N, Gross U, Lüder CG.

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

Poly(ADP-ribose) polymerase-1 (PARP-1; EC is an abundant nuclear protein that is involved in DNA repair, cell cycle control, programmed cell death and transcriptional regulation. It also plays critical roles in the pathogenesis of inflammatory disorders. Here we have performed a detailed analysis of the interplay between the apicomplexan parasite Toxoplasma gondii and host cell PARP and its consequences for the host-parasite relationship. Our results have shown that T. gondii significantly decreased PARP expression in its host cells within 10min of infection but that the amount of PARP normalized during prolonged infection. Importantly, down-regulation of PARP expression after infection abrogated the ADP-ribosylation of acceptor proteins in response to oxidative stress. Overexpression of PARP in RAW264.7 cells revealed that elevated amounts of PARP neither affected host cell invasion nor intracellular development of T. gondii in non-stimulated or IFN-gamma/LPS-stimulated monocytes/macrophages. Furthermore, measurements of the activities of effector caspases 3 and 7 indicated that the blockade of host cell apoptosis by T. gondii occurs independently of the inhibition of PARP after infection. These findings suggest that the prominent decrease of host cell PARP and poly(ADP-ribos)ylation after parasitic infection do not affect the intracellular development of T. gondii in vitro.

PMID: 18396434 [PubMed - as supplied by publisher]

Wednesday, April 02, 2008

Primary culture of skeletal muscle cells as a model for studies of Toxo cystogenesis

J Parasitol. 2008 Feb;94(1):72-83.

Primary culture of skeletal muscle cells as a model for studies of Toxoplasma gondii cystogenesis

Guimarães EV, de Carvalho L, Barbosa HS.

Laboratório de Biologia Estrutural, Instituto Oswaldo Cruz - Fundação Oswaldo Cruz, Av. Brasil 4365, 21040-900 Rio de Janeiro, RJ, Brazil.

Toxoplasma gondii is a protozoan pathogen of birds and mammals, including humans. The infective stage, the bradyzoite, lives within cysts, which occur predominantly in cells of the central nervous system and skeletal and cardiac muscles, characterizing the chronic phase of toxoplasmosis. In the present study, we employed for the first time primary mouse culture of skeletal muscle cells (SkMC) infected with bradyzoites, as a cellular model for cystogenesis. The interconversion of bradyzoite and tachyzoite was analyzed by immunofluorescence using 2 stage-specific antibodies, i.e., anti-bradyzoite (anti-BAG1) and anti-tachyzoite (anti-SAG1). After 24 hr of interaction only bradyzoites were multiplying, as revealed by anti-BAG1 incubation; interconversion to tachyzoites was not observed. After 48 hr of infection, 2 types of vacuoles were seen, i.e., BAG1+ and SAG1+, indicating the presence of bradyzoites as well as their interconversion to tachyzoites. After 96 hr of infection, BAG1+ vacuoles presented a higher number of parasites when compared to 48 hr, indicating multiplication of bradyzoites without interconversion. Using ultrastructural analysis, bradyzoites were found to adhere to the cell membranes via both the apical and posterior regions or were associated with SkMC membrane expansions. During bradyzoite invasion of SkMC, migration of the rough endoplasmic reticulum (RER) profiles to the parasite invasion site was observed. Later, RER profiles were localized between the mitochondria and parasitophorous vacuole membrane (PVM) that contained the parasite. After 31 days of parasite-host cell infection, RER profiles and mitochondria were not observed in association with the cyst wall. Alterations of the PVM, including increased thickness and electrondensity gain on its inner membrane face, were observed 48 hr after infection. Cystogenesis was complete 96 hr after infection, resulting in the formation of the cyst wall, which displayed numerous membrane invaginations. In addition, an electron-dense granular region enriched with vesicles and tubules was present, as well as numerous intracystic bradyzoites. These results show that the in vitro T. gondii model and SkMC are potential tools for both the study of cystogenesis using molecular approaches and the drug screening action on tissue cysts and bradyzoites.

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

PMID: 18372624 [PubMed - in process]

Antibodies in cold stressed mice recognize a surface protein in tachyzoites

J Parasitol. 2008 Feb;94(1):114-8.

Antibodies in cold stressed mice recognize a surface protein in Toxoplasma gondii tachyzoites

Thompson EG, Aviles HO, Monroy FP.

Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona 87011, USA.

Physical or psychological stressors are known to have significant consequences for immune function and the outcome of disease in human and animal models. In mice, cold water stress (CWS) has been shown to delay control of acute infection and reactivation of latent infections. Increased levels of parasite-specific IgG and IgM antibodies are observed when CWS is applied in the chronic phase. The present study examined the effects of a physical stressor, CWS, on tachyzoites antigens of Toxoplasma gondii, with particular emphasis on a low molecular weight antigen, 5 kDa, which seems to be recognized by antibodies from mice subjected to CWS in the chronic phase. This antigen is not recognized by antibodies from infected mice not subjected to CWS. Sera obtained from stressed and infected (CWS + INF) mice subjected to CWS during the chronic phase (CWS + INF + CWS) were used to harvest anti-5-kDa antibodies for immunolocalization studies. Tachyzoite lysate preparations were electrophoretically separated and transferred to nitrocellulose membranes. Strips of nitrocellulose containing tachyzoite antigens in the 4-10-kDa range were used to select for anti-5-kDa antibodies. Harvested anti-5-kDa localized this antigen on the surface of tachyzoites. This antigen was not present in bradyzoite preparations. Treatment with phosphatidylinositol-specific phospholipase C showed this antigen was not anchored to the cell membrane through glycosyl-phosphatidylinositol. Strong antibody responses in stressed animals during the chronic phase are associated with parasite reactivation. The 5-kDa antigen constitutes a unique immunogenic component of T. gondii, with significant diagnostic potential for identifying reactivation of latent infections.

Toll-like receptor-4-mediated macrophage activation is differentially regulated by progesterone via the glucocorticoid and progesterone receptors

Immunology. 2008 Mar 28 [Epub ahead of print]

Toll-like receptor-4-mediated macrophage activation is differentially regulated by progesterone via the glucocorticoid and progesterone receptors

Jones LA, Anthony JP, Henriquez FL, Lyons RE, Nickdel MB, Carter KC, Alexander J, Roberts CW.

Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK.

Macrophage function has been demonstrated to be subject to modulation by progesterone. However, as this steroid hormone can act through the glucocorticoid receptor as well as the progesterone receptor, the mechanism of action has not been precisely characterized. To determine the mode of action, we compared the ability of progesterone, norgestrel (a synthetic progesterone-receptor-specific agonist) and dexamethasone (a synthetic glucocorticoid receptor agonist) to modulate macrophage function following stimulation of the Toll-like receptor-4 (TLR-4) ligand lipopolysaccharide (LPS). The results demonstrate that following stimulation of TLR-4 with LPS and cotreatment with either progesterone or dexamethasone, but not norgestrel, there is a significant reduction in nitric oxide (NO) production, indicating that this progesterone-mediated effect is through ligation of the glucocorticoid receptor. In contrast, LPS-induced interleukin-12 (IL-12) production could be downregulated by all three steroids, indicating that ligation by progesterone of either the glucocorticoid or the progesterone receptors or both could mediate this effect. While progesterone downmodulated NO-mediated killing of Leishmania donovani by activated macrophages in vitro, most probably via the glucocorticoid receptor, it had little effect on Toxoplasma gondii growth in these cells. This would suggest that progesterone-mediated increased susceptibility to T. gondii during pregnancy is more likely to be related to the ability of the hormone to downregulate IL-12 production and a type-1 response utilizing the progesterone as well as the glucocorticoid receptors.

PMID: 18373668 [PubMed - as supplied by publisher]

Patterns and role of diversifying selection in the evolution of SAG5 locus

Parasitol Res. 2008 Apr 1 [Epub ahead of print]

Patterns and role of diversifying selection in the evolution of Toxoplasma gondii SAG5 locus

Elsheikha HM, Zhao X.

Division of Veterinary Medicine, The School of Veterinary Medicine and Science, The University of Nottingham, College Road, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK,

The higher intergenotypic polymorphism of the surface antigen genes 5 (SAG5)A, SAG5C, and SAG5E in Toxoplasma gondii was proposed to be the outcome of positive selection pressure favoring variation within these loci. However, the exact nature and magnitude of this selection is not completely known. To address this issue, the amino acids on which natural selection may operate were identified by comparing the ratios of nonsynonymous and synonymous substitutions (p (N/) p (S)) of homologous DNA sequences in strains belonging to the three major genotypes of T. gondii. Both positive and negative selections were detected and are likely to have contributed to shaping the patterns of nucleotide substitution and polymorphism in SAG5 genes. Several sites identified in SAG5 loci as likely to be under positive selection suggesting that diversifying selection may have promoted divergence in these genes. Also, it was noted that the SAG5 genetic loci contain many areas that exhibit signs of purifying selection; some of these areas might be the attractive candidates for drug targets. Phylogenetic analysis using the neighbor-joining and maximum parsimony methods grouped the SAG5 sequences of T. gondii strains into three distinct statistically well-supported evolutionary lineages. These findings carry important implications for human and veterinary toxoplasmosis epidemiology and may provide important insights into the pathways through which virulence has evolved in T. gondii.

PMID: 18379821 [PubMed - as supplied by publisher]