Tuesday, June 26, 2007

Applications of bioluminescence imaging to the study of infectious diseases

Cell Microbiol. 2007 Jun 24; [Epub ahead of print]

Applications of bioluminescence imaging to the study of infectious diseases

Hutchens M, Luker GD.

Immunology Program, University of Michigan Medical School, Ann Arbor, MI, USA.

Bioluminescence imaging (BLI) has emerged as a powerful new method to analyse infectious diseases in animal models. BLI offers real-time monitoring of spatial and temporal progression of infection in the same animal, as opposed to euthanizing a cohort of animals and quantifying colony or plaque forming units at multiple time points. Pathogens or mice are engineered to express genetically encoded luciferase enzymes from bacteria, insects, or the sea pansy. The seminal study showing the feasibility of detecting microbially generated luminescence within a living mouse was published by Contag and colleagues in 1995, using Salmonella typhimurium transformed with the lux operon from Photorhabdus luminescens. Following this, they and others performed many studies of infection by bioluminescent Gram-negative and Gram-positive bacteria. Viruses can also be engineered to encode luciferase. Our laboratory has used bioluminescent reporter viruses to follow HSV and vaccinia pathogenesis; others have used an alphavirus or novirhabdovirus. Recently, even eukaryotic parasites Plasmodium, Leishmania and Toxoplasma have been transformed with luciferase and yielded unique insights into their in vivo behaviour. We expect that both the range of organisms and the molecular events able to be studied by BLI will continue to expand, yielding important insights into mechanisms of pathogenesis.

PMID: 17587328 [PubMed - as supplied by publisher]

High-level bacterial expression and purification of apicomplexan micronemal proteins for structural studies

Protein Pept Lett. 2007;14(5):411-5.

High-level bacterial expression and purification of apicomplexan micronemal proteins for structural studies

Saouros S, Blumenschein TM, Sawmynaden K, Marchant J, Koutroukides T, Liu B, Simpson P, Carpenter EP, Matthews SJ.

Department of Biological Sciences, Division of Molecular Biosciences, Centre for Structural Biology, Imperial College London, South Kensington, London SW7 2AZ, UK. s.j.matthews@imperial.ac.uk.

The cysteine-rich N-terminal domain of the micronemal adhesive protein MIC1 (MIC1-NT) from Toxoplasma gondii was cloned, expressed in Escherichia coli and purified. MIC1-NT is amenable to structural studies as shown by preliminary NMR and X-ray analysis. Positive results with two further micronemal proteins indicate that our strategy has wider application.

PMID: 17584164 [PubMed - in process]

Immune response induced by recombinant Mycobacterium bovis BCG expressing ROP2 gene

Parasitol Int. 2007 May 10; [Epub ahead of print]

Immune response induced by recombinant Mycobacterium bovis BCG expressing ROP2 gene of Toxoplasma gondii

Wang H, Liu Q, Liu K, Zhong W, Gao S, Jiang L, An N.

Shandong Institute of Parasitic Diseases, Jining, China.

Toxoplasma gondii is an obligate intracellular parasite, capable of infecting a variety of mammals and birds. Development of vaccine against T. gondii would be of great medical and veterinary value. In this study, the DNA sequence encoding ROP2 from T. gondii was cloned into the muticopy mycobacterial expression vector, pMV262, under the control of the Bacillus Calmette-Guerin (BCG) hsp60 promoter, and electroporated into BCG. Following selection of kanamycin, the recombinant BCG/pMV262-ROP2 was constructed and the expression of ROP2 was confirmed by Western blotting. The BALB/c mice inoculated with the BCG/pMV262-ROP2 developed specific immune responses against ROP2 protein, and there was an obvious delay in the mortality curve than the control (P<0.05). These results indicated that M. bovis BCG is an adequate vector to express and present antigens of T. gondii, and it may be used to further study the induction of protective immunity in other animals.

PMID: 17587637 [PubMed - as supplied by publisher]

Thursday, June 21, 2007

Neospora inhibits host cell apoptosis in the absence of NF-{kappa}B activation

Infect Immun. 2007 Jun 18; [Epub ahead of print]

The apicomplexan pathogen Neospora caninum inhibits host cell apoptosis in the absence of discernible NF-{kappa}B activation

Herman RK, Molestina RE, Sinai AP, Howe DK

Department of Veterinary Science and Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY 40546.

Neospora caninum, a causative agent of bovine abortions, is an apicomplexan parasite that is closely related to the human pathogen Toxoplasma gondii. Since a number of intracellular parasites, including T. gondii, have been shown to modulate host cell apoptosis, the present study was conducted to establish whether N. caninum is similarly capable of subverting apoptotic pathways in its host cells. Our results indicated that death receptor-mediated apoptosis is repressed during N. caninum infection, and the data further showed that the executioner caspase, caspase 3, does not become activated in the infected cells. Surprisingly, nuclear translocation of the NF-kappaB subunit p65 was not detected in N. caninum infected cells, although this host transcription factor has been shown to upregulate pro-survival genes in cells infected with T. gondii. Consistent with these findings, the distinct accumulation of phosphorylated IkappaB that is seen at the parasitophorous vacuole membrane (PVM) of T. gondii was not apparent on the N. caninum PVM. Although a putative IkappaB kinase activity was detected in N. caninum extracts, thereby implying that this parasite is capable of modulating NF-kappaB translocation into the host cell nucleus, the data collectively suggest that a profound and sustained activation of the NF-kappaB pathway is not central to the ability of N. caninum to prevent apoptosis of their host cells.

PMID: 17576757 [PubMed - as supplied by publisher]

NK Cells Enhance Dendritic Cell Response against Parasite Antigens via NKG2D Pathway

J Immunol. 2007 Jul 1;179(1):590-6.

NK Cells Enhance Dendritic Cell Response against Parasite Antigens via NKG2D Pathway

Guan H, Moretto M, Bzik DJ, Gigley J, Khan IA.

Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA 70112.

Recent studies have shown that NK-dendritic cell (DC) interaction plays an important role in the induction of immune response against tumors and certain viruses. Although the effect of this interaction is bidirectional, the mechanism or molecules involved in this cross-talk have not been identified. In this study, we report that coculture with NK cells causes several fold increase in IL-12 production by Toxoplasma gondii lysate Ag-pulsed DC. This interaction also leads to stronger priming of Ag-specific CD8(+) T cell response by these cells. In vitro blockade of NKG2D, a molecule present on human and murine NK cells, neutralizes the NK cell-induced up-regulation of DC response. Moreover, treatment of infected animals with Ab to NKG2D receptor compromises the development of Ag-specific CD8(+) T cell immunity and reduces their ability to clear parasites. These studies emphasize the critical role played by NKG2D in the NK-DC interaction, which apparently is important for the generation of robust CD8(+) T cell immunity against intracellular pathogens. To the best of our knowledge, this is the first work that describes in vivo importance of NKG2D during natural infection.

PMID: 17579080 [PubMed - in process]

Tuesday, June 19, 2007

Molecules targeting the purine salvage pathway in Apicomplexan parasites

Trends Parasitol. 2007 Jun 14; [Epub ahead of print]

Molecules targeting the purine salvage pathway in Apicomplexan parasites

Ghérardi A, Sarciron ME.

Pharmaceutical Department of Parasitology and Medical Mycology, Claude-Bernard University Lyon I, 8 Avenue Rockefeller, 69373 Lyon Cedex 08, France.

The need of intracellular parasites to retrieve nutrients and fulfill their energy requirements is achieved by manipulating the host's metabolism. With the spread of AIDS, research on purine metabolism has gained in importance with the aim to develop drugs against opportunistic infections. Many studies over the past ten years have yielded contradictory results, but this review tries to clarify these findings by exposing the latest data concerning purine transport and the specific activities of the major enzymes of the purine salvage pathway of Toxoplasma gondii, Plasmodium falciparum and Cryptosporidium parvum.

PMID: 17574921 [PubMed - as supplied by publisher]

Saturday, June 16, 2007

Epigenomic Modifications Predict Active Promoters and Gene Structure in Toxo

PLoS Pathog. 2007 Jun 8;3(6):e77 [Epub ahead of print]

Epigenomic Modifications Predict Active Promoters and Gene Structure in Toxoplasma gondii

Gissot M, Kelly KA, Ajioka JW, Greally JM, Kim K.

Mechanisms of gene regulation are poorly understood in Apicomplexa, a phylum that encompasses deadly human pathogens like Plasmodium and Toxoplasma. Initial studies suggest that epigenetic phenomena, including histone modifications and chromatin remodeling, have a profound effect upon gene expression and expression of virulence traits. Using the model organism Toxoplasma gondii, we characterized the epigenetic organization and transcription patterns of a contiguous 1% of the T. gondii genome using custom oligonucleotide microarrays. We show that methylation and acetylation of histones H3 and H4 are landmarks of active promoters in T. gondii that allow us to deduce the position and directionality of gene promoters with >95% accuracy. These histone methylation and acetylation "activation" marks are strongly associated with gene expression. We also demonstrate that the pattern of histone H3 arginine methylation distinguishes certain promoters, illustrating the complexity of the histone modification machinery in Toxoplasma. By integrating epigenetic data, gene prediction analysis, and gene expression data from the tachyzoite stage, we illustrate feasibility of creating an epigenomic map of T. gondii tachyzoite gene expression. Further, we illustrate the utility of the epigenomic map to empirically and biologically annotate the genome and show that this approach enables identification of previously unknown genes. Thus, our epigenomics approach provides novel insights into regulation of gene expression in the Apicomplexa. In addition, with its compact genome, genetic tractability, and discrete life cycle stages, T. gondii provides an important new model to study the evolutionarily conserved components of the histone code.

PMID: 17559302 [PubMed - as supplied by publisher]

Full-text Author Summary Screen PDF (584K)

Serpiginous choroiditis-like picture due to ocular toxoplasmosis

Ocul Immunol Inflamm. 2007 Mar-Apr;15(2):127-30.

Serpiginous choroiditis-like picture due to ocular toxoplasmosis

Mahendradas P, Kamath G, Mahalakshmi B, Shetty KB.

Department of Uveitis and Ocular Immunology, Narayana Nethralaya. Bangalore.

Purpose: To report an atypical presentation of ocular toxoplasmosis in the form of serpiginous choroiditis-like picture in an immunocompetent individual. Methods: Case report: A 32-year-old man with blurred vision in the left eye showed serpiginous choroiditis-like appearance. Fundus fluorescein angiography and relevant laboratory tests including anti-toxoplasma serology and polymerase chain reaction (PCR) in aqueous humor were performed. Results: The serology in blood and PCR in aqueous humor were positive for Toxoplasma gondii infection. The patient was treated with anti-toxoplasma antimicrobials and systemic steroids. At the end of six weeks of treatment the fundus lesions had healed well, with good visual recovery. Conclusions: Serpiginous choroiditis-like picture can be an atypical manifestation of ocular Toxoplasma gondii infection.

PMID: 17558839 [PubMed - in process]

Calcium ionophore-induced egress of Toxo shortly after host cell invasion

Vet Parasitol. 2007 Jun 6; [Epub ahead of print]

Calcium ionophore-induced egress of Toxoplasma gondii shortly after host cell invasion

Caldas LA, de Souza W, Attias M.

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, bloco G. Cidade Universitária cep, 21949-900 Rio de Janeiro, RJ, Brazil.

Calcium plays crucial roles in important events of Toxoplasma gondii life cycle, including motility, invasion and egress from the host cell. Calcium ionophore has been used to artificially trigger release of the parasites from infected cells. In this report we describe that calcium ionophore A21387 induced T. gondii egress from LLC-MK2 cells at times as early as 2h after entry. Addition of kinase inhibitors as staurosporine, wortmanine and genistein to the incubation medium significantly reduced ionophore-induced egress. The same occurred when the actin inhibitor cytochalasin D was used. Parasites egressed 2h post-infection from ionophore-treated cultures were unable of establishing infection in a new cell. S-VHS recording of egressing parasites showed that they assume an hourglass shape as they cross the plasma membrane, similar to the moving junction constriction observed during active invasion, and extrudes the conoid, similarly to what is also observed during invasion. Transmission and high resolution scanning electron microscopy revealed that the egressing tachyzoites are free from host cell derived membranes. These include plasma membrane and parasitophorous vacuole membranes as well as associated endoplasmic reticulum membranes. Taken together, these results indicate that although invasion and egress may share similar signaling pathways, as indicated by the effect of kinase and actin inhibitors, the tachyzoites move freely in the cytosol, a phenomenon very distinctive from invasion and that deserves attention.

PMID: 17560036 [PubMed - as supplied by publisher]

Cryoelectron tomography reveals periodic material at the inner side of subpellicular microtubules in apicomplexan parasites

J Exp Med. 2007 Jun 11;204(6):1281-7. Epub 2007 May 14.

Cryoelectron tomography reveals periodic material at the inner side of subpellicular microtubules in apicomplexan parasites

Cyrklaff M, Kudryashev M, Leis A, Leonard K, Baumeister W, Menard R, Meissner M, Frischknecht F.

Department of Molecular Structural Biology, Max Planck Institute for Biochemistry, 82152, Martinsried, Germany.

Microtubules are dynamic cytoskeletal structures important for cell division, polarity, and motility and are therefore major targets for anticancer and antiparasite drugs. In the invasive forms of apicomplexan parasites, which are highly polarized and often motile cells, exceptionally stable subpellicular microtubules determine the shape of the parasite, and serve as tracks for vesicle transport. We used cryoelectron tomography to image cytoplasmic structures in three dimensions within intact, rapidly frozen Plasmodium sporozoites. This approach revealed microtubule walls that are extended at the luminal side by an additional 3 nm compared to microtubules of mammalian cells. Fourier analysis revealed an 8-nm longitudinal periodicity of the luminal constituent, suggesting the presence of a molecule interacting with tubulin dimers. In silico generation and analysis of microtubule models confirmed this unexpected topology. Microtubules from extracted sporozoites and Toxoplasma gondii tachyzoites showed a similar density distribution, suggesting that the putative protein is conserved among Apicomplexa and serves to stabilize microtubules.

PMID: 17562819 [PubMed - in process]

SET8-Mediated Methylations of Histone H4 Lysine 20 Mark Silent Heterochromatic Domains in Apicomplexan Genomes

Mol Cell Biol. 2007 Jun 11; [Epub ahead of print]

SET8-Mediated Methylations of Histone H4 Lysine 20 Mark Silent Heterochromatic Domains in Apicomplexan Genomes

Sautel CF, Cannella D, Bastien O, Kieffer S, Aldebert D, Garin J, Tardieux I, Belrhali H, Hakimi MA.

UMR5163/CNRS-Joseph Fourier University, JEAN-ROGET INSTITUTE, GRENOBLE, F-38042 FRANCE; EMBL-Grenoble Outstation, GRENOBLE, F-38042 FRANCE, Institut Cochin - PARIS, F-75014 France, INSERM ERIT - M 0201, CEA, GRENOBLE, FRANCE.

Post-translational histone modifications modulate chromatin-templated processes in various biological systems. H4K20 methylation is considered to have an evolutionary ancient role in DNA repair/genome integrity, while its function in heterochromatin function/gene expression is thought to have arisen later during evolution. Here, we identify and characterize H4K20 methylases of the Set8 family in Plasmodium and Toxoplasma, two medically-important members of the Apicomplexan phylum of protozoa. Remarkably, parasite Set8-related proteins display H4K20 mono-, di- and trimethylase activity in striking contrast with the mono-methylase restricted human Set8. Structurally, few residues forming the substrate-specific channel dictate the enzyme methylation multiplicity. These enzymes are cell cycle regulated and focally enriched at pericentric and telomeric heterochromatin in both parasites. Collectively, our findings provide new insights into the evolution of Set8-mediated biochemical pathways suggesting that the heterochromatic function of the mark is not restricted to metazoans. Thus, these lower eukaryotes have developed a diverse panel of biological stages through their high capacity to differentiate and epigenetics only begins to emerge as a strong determinant of their biology.

PMID: 17562855 [PubMed - as supplied by publisher]

Thursday, June 07, 2007

Aspartic Proteases and a Novel, Dynamic and Cell-Cycle-Dependent Protease Localization in Toxo

Traffic. 2007 Jun 5; [Epub ahead of print]

A Family of Aspartic Proteases and a Novel, Dynamic and Cell-Cycle-Dependent Protease Localization in the Secretory Pathway of Toxoplasma gondii

Shea M, Jäkle U, Liu Q, Berry C, Joiner KA, Soldati-Favre D.

Department of Microbiology and Molecular Medicine, University of Geneva, 1211 Geneva, Switzerland.

Aspartic proteases are important virulence factors in pathogens like HIV, Candida albicans or Plasmodium falciparum. We report here the identification of seven putative aspartic proteases, TgASP1 to TgASP7, in the apicomplexan parasite Toxoplasma gondii. Bioinformatic and phylogenetic analysis of the TgASPs and other aspartic proteases from related Apicomplexa suggests the existence of five distinct groups of aspartic proteases with different evolutionary lineages. The members of each group share predicted biological features that validate the phylogeny. TgASP1 is expressed in tachyzoites, the rapidly dividing asexual stage of T.gondii. We present the proteolytic maturation and subcellular localization of this protease through the cell cycle. TgASP1 shows a novel punctate localization associated with the secretory system in non-dividing cells, and relocalizes dramatically and unambiguously to the nascent inner membrane complex of daughter cells at replication, before coalescing again at the end of division.

PMID: 17547703 [PubMed - as supplied by publisher]

Tuesday, June 05, 2007

Immobilization of the Type XIV Myosin Complex in Toxoplasma gondii

Mol Biol Cell. 2007 May 30; [Epub ahead of print]

Immobilization of the Type XIV Myosin Complex in Toxoplasma gondii

Johnson TM, Rajfur Z, Jacobson K, Beckers CJ.

Department of Cell and Developmental Biology, The University of North Carolina, Chapel Hill, NC 27599-7090.

Monitoring Editor: Yu-li Wang The substrate-dependent movement of apicomplexan parasites like Toxoplasma gondii and Plasmodium sp. is driven by the interaction of a type XIV myosin with F-actin. A complex containing the myosin-A heavy chain, a myosin light chain and the accessory protein GAP45 is attached to the membranes of the inner membrane complex (IMC) through its tight interaction with the integral membrane glycoprotein GAP50. In order for the interaction of this complex with F-actin to result in net parasite movement, it is necessary that one of them be immobilized with respect to the parasite and the other with respect to the substrate the parasite is moving on. We report here that the myosin motor complex of Toxoplasma is firmly immobilized in the plane of the IMC. This does not appear to be accomplished by direct interactions with cytoskeletal elements. Immobilization of the motor complex does appear to require cholesterol, however. Both the motor complex and the cholesterol are found in detergent-resistant membrane domains that encompass a large fraction of the inner membrane complex surface. The observation that the myosin XIV motor complex of Toxoplasma is immobilized within this cholesterol-rich membrane likely extends to closely related pathogens like Plasmodium and possibly other eukaryotes.

PMID: 17538016 [PubMed - as supplied by publisher]

Go there