BMC Genomics. 2010 Oct 25;11(1):603. [Epub ahead of print]
A novel multifunctional oligonucleotide microarray for Toxoplasma gondii
Bahl A, Davis PH, Behnke M, Dzierszinski F, Jagalur M, Chen F, Shanmugam D, White M, Kulp D, Roos DS.
Abstract
ABSTRACT:
BACKGROUND: Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a prohibitively expensive undertaking.
RESULTS: Taking advantage of available genome sequences and annotation for Toxoplasma gondii (the causative agent of opportunistic illness in immunocompromised individuals) and Plasmodium falciparum (a related parasite responsible for severe human malaria), we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for Toxoplasma, and single-nucleotide polymorphism (SNP)-based genotyping of both T. gondii and P. falciparum. Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis). The relatively small genomes of P. falciparum (23 Mb) and T. gondii (65 Mb), and the ability to isolate DNA from haploid stages, coupled with a careful analysis of signals from overlapping probes, permits a novel design enabling high confidence genotyping using only four probes per SNP. This strategy permits resolution of recombination points in the clonal progeny of sexual crosses, allowing detailed mapping of phenotypic traits. Recent sequencing of additional T. gondii isolates identifies >620K new SNPs, including >11K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating gene and transcript discovery, statistical analysis of expression profiles, etc. are also discussed.
CONCLUSIONS: In addition to providing an initial global view of the T. gondii transcriptome across major lineages and permitting detailed resolution of recombination points in a historical sexual cross, the multifunctional nature of this array also allowed opportunities to exploit probes for purposes beyond their intended use, enhancing analyses. This array is in widespread use by the T. gondii research community, and several aspects of the design strategy are likely to be useful for other pathogens.
PMID: 20974003 [PubMed - as supplied by publisher]
Thursday, October 28, 2010
Thursday, October 21, 2010
Generation of a Neutralizing Human Monoclonal Antibody Fab Fragment to SAG1 of Toxoplasma gondii Tachyzoites
Infect Immun. 2010 Oct 18. [Epub ahead of print]
Generation of a Neutralizing Human Monoclonal Antibody Fab Fragment to SAG1 of Toxoplasma gondii Tachyzoites
Fu YF, Feng M, Ohnishi K, Kimura T, Itoh J, Cheng XJ, Tachibana H.
Department of Medical Microbiology and Parasitology, Fudan University School of Medicine, Shanghai 200032, China; Department of Infectious Diseases, Tokyo Metropolitan Bokutoh General Hospital, Sumida-Ku, Tokyo 130-8575, Japan; Department of Infectious Diseases and Teaching and Research Support Center, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan.
Abstract
A combinatorial immunoglobulin gene library was constructed from lymphocytes in peripheral blood of a patient with toxoplasmosis and screened for production of human monoclonal antibody Fab fragments to recombinant SAG1 of Toxoplasma gondii. Two Fab clones, Tox203 and Tox1403, which consisted of a common heavy chain and different light chains, showed positive staining on the entire surface of tachyzoites in confocal microscopy. Sequence analysis of the heavy chain gene revealed that the closest germline V segments were VH3-23. The germline D segment was D1-7, and the closest germline J segment was JH4. In the light chain genes, the closest germline V segment was Vκ1-17 with the Jκ1 or Jκ4 segments. The dissociation constants of these Fab fragments with recombinant SAG1 were 3.09x10(-9) M for Tox203 and 2.01x10(-8) M for Tox1403, indicating that the affinity of Tox203 was 7 times higher than that of Tox1403. Preincubation of T. gondii tachyzoites with Tox203 significantly inhibited their attachment to cultured MDBK cells. Passive immunization of mice with Tox203 also significantly reduced mortality after challenge with T. gondii tachyzoites. This is the first report of bacterial expression of human monoclonal antibody Fab fragments to SAG1 of T. gondii. These results also demonstrate that human Fab fragments to SAG1 might be applicable for immunoprophylaxis of toxoplasmosis.
PMID: 20956568 [PubMed - as supplied by publisher]
Generation of a Neutralizing Human Monoclonal Antibody Fab Fragment to SAG1 of Toxoplasma gondii Tachyzoites
Fu YF, Feng M, Ohnishi K, Kimura T, Itoh J, Cheng XJ, Tachibana H.
Department of Medical Microbiology and Parasitology, Fudan University School of Medicine, Shanghai 200032, China; Department of Infectious Diseases, Tokyo Metropolitan Bokutoh General Hospital, Sumida-Ku, Tokyo 130-8575, Japan; Department of Infectious Diseases and Teaching and Research Support Center, Tokai University School of Medicine, Isehara, Kanagawa 259-1193, Japan.
Abstract
A combinatorial immunoglobulin gene library was constructed from lymphocytes in peripheral blood of a patient with toxoplasmosis and screened for production of human monoclonal antibody Fab fragments to recombinant SAG1 of Toxoplasma gondii. Two Fab clones, Tox203 and Tox1403, which consisted of a common heavy chain and different light chains, showed positive staining on the entire surface of tachyzoites in confocal microscopy. Sequence analysis of the heavy chain gene revealed that the closest germline V segments were VH3-23. The germline D segment was D1-7, and the closest germline J segment was JH4. In the light chain genes, the closest germline V segment was Vκ1-17 with the Jκ1 or Jκ4 segments. The dissociation constants of these Fab fragments with recombinant SAG1 were 3.09x10(-9) M for Tox203 and 2.01x10(-8) M for Tox1403, indicating that the affinity of Tox203 was 7 times higher than that of Tox1403. Preincubation of T. gondii tachyzoites with Tox203 significantly inhibited their attachment to cultured MDBK cells. Passive immunization of mice with Tox203 also significantly reduced mortality after challenge with T. gondii tachyzoites. This is the first report of bacterial expression of human monoclonal antibody Fab fragments to SAG1 of T. gondii. These results also demonstrate that human Fab fragments to SAG1 might be applicable for immunoprophylaxis of toxoplasmosis.
PMID: 20956568 [PubMed - as supplied by publisher]
Toxoplasma gondii primary infection in renal transplant recipients. Two case reports and literature review
Transpl Int. 2010 Oct 19. doi: 10.1111/j.1432-2277.2010.01173.x. [Epub ahead of print]
Toxoplasma gondii primary infection in renal transplant recipients. Two case reports and literature review
Martina MN, Cervera C, Esforzado N, Linares L, Torregrosa V, Sanclemente G, Hoyo I, Cofan F, Oppenheimer F, Miro JM, Campistol JM, Moreno A.
Renal Transplant Unit, Hospital Clinic of Barcelona, IDIBAPS, University of Barcelona, Barcelona, Spain Infectious Diseases Service, Hospital Clinic of Barcelona, IDIBAPS, University of Barcelona, Barcelona, Spain.
Abstract
Toxoplasmosis after solid organ transplantation is a complication associated with high morbidity and mortality. Universal prophylaxis with trimethoprim-sulfamethoxazole (TMP-SMX) is effective to prevent post-transplant toxoplasmosis. We report two cases of renal transplant recipients with negative antibodies against Toxoplasma gondii pretransplant who developed toxoplasmosis after TMP-SMX discontinuation. We have also performed a review of published cases of primary toxoplasmosis after renal transplantation. Of 20 cases reviewed, 11 were male and the mean age was 37.8 years (SD = 13.8). Donor serology for T. gondii was determined in 15 donors, two of them (13%) with negative immunoglobulin (Ig)G and four (27%) with positive IgG and IgM antibodies. Fever was present in 85% of primary toxoplasmosis and hematologic abnormalities were observed in 69% of the cases. Ten patients died (50%). All patients with fatal outcomes had clinical evidence of toxoplasmosis during the early post-transplant period (first 90 days), while no patient with late toxoplasmosis died (P = 0.003). Primary toxoplasmosis is associated with high mortality rates and TMP-SMX prophylaxis can delay the onset of symptoms resulting in an improvement of prognosis.
© 2010 The Authors. Transplant International © 2010 European Society for Organ Transplantation.
PMID: 20955469 [PubMed - as supplied by publisher]
Toxoplasma gondii primary infection in renal transplant recipients. Two case reports and literature review
Martina MN, Cervera C, Esforzado N, Linares L, Torregrosa V, Sanclemente G, Hoyo I, Cofan F, Oppenheimer F, Miro JM, Campistol JM, Moreno A.
Renal Transplant Unit, Hospital Clinic of Barcelona, IDIBAPS, University of Barcelona, Barcelona, Spain Infectious Diseases Service, Hospital Clinic of Barcelona, IDIBAPS, University of Barcelona, Barcelona, Spain.
Abstract
Toxoplasmosis after solid organ transplantation is a complication associated with high morbidity and mortality. Universal prophylaxis with trimethoprim-sulfamethoxazole (TMP-SMX) is effective to prevent post-transplant toxoplasmosis. We report two cases of renal transplant recipients with negative antibodies against Toxoplasma gondii pretransplant who developed toxoplasmosis after TMP-SMX discontinuation. We have also performed a review of published cases of primary toxoplasmosis after renal transplantation. Of 20 cases reviewed, 11 were male and the mean age was 37.8 years (SD = 13.8). Donor serology for T. gondii was determined in 15 donors, two of them (13%) with negative immunoglobulin (Ig)G and four (27%) with positive IgG and IgM antibodies. Fever was present in 85% of primary toxoplasmosis and hematologic abnormalities were observed in 69% of the cases. Ten patients died (50%). All patients with fatal outcomes had clinical evidence of toxoplasmosis during the early post-transplant period (first 90 days), while no patient with late toxoplasmosis died (P = 0.003). Primary toxoplasmosis is associated with high mortality rates and TMP-SMX prophylaxis can delay the onset of symptoms resulting in an improvement of prognosis.
© 2010 The Authors. Transplant International © 2010 European Society for Organ Transplantation.
PMID: 20955469 [PubMed - as supplied by publisher]
Intracellular Protozoan Parasites of Humans: The Role of Molecular Chaperones in Development and Pathogenesis
Protein Pept Lett. 2010 Oct 18. [Epub ahead of print]
Intracellular Protozoan Parasites of Humans: The Role of Molecular Chaperones in Development and Pathogenesis
Shonhai A, Maier AG, Przyborski JM, Blatch GL.
Department of Biochemistry & Microbiology, University of Zululand, Kwadlangezwa, South Africa. G.Blatch@ru.ac.za.
Abstract
Certain kinetoplastid (Leishmania spp. and Tryapnosoma cruzi) and apicomplexan parasites (Plasmodium falciparum and Toxoplasma gondii) are capable of invading human cells as part of their pathology. These parasites appear to have evolved a relatively expanded or diverse complement of genes encoding molecular chaperones. The gene families encoding heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) chaperones show significant expansion and diversity (especially for Leishmania spp. and T. cruzi), and in particular the Hsp40 family appears to be an extreme example of phylogenetic radiation. In general, Hsp40 proteins act as co-chaperones of Hsp70 chaperones, forming protein folding pathways that integrate with Hsp90 to ensure proteostasis in the cell. It is tempting to speculate that the diverse environmental insults that these parasites endure have resulted in the evolutionary selection of a diverse and expanded chaperone network. Hsp90 is involved in development and growth of all of these intracellular parasites, and so far represents the strongest candidate as a target for chemotherapeutic interventions. While there have been some excellent studies on the molecular and cell biology of Hsp70 proteins, relatively little is known about the biological function of Hsp70-Hsp40 interactions in these intracellular parasites. This review focuses on intracellular protozoan parasites of humans, and provides a critique of the role of heat shock proteins in development and pathogenesis, especially the molecular chaperones Hsp90, Hsp70 and Hsp40.
PMID: 20955165 [PubMed - as supplied by publisher]
Intracellular Protozoan Parasites of Humans: The Role of Molecular Chaperones in Development and Pathogenesis
Shonhai A, Maier AG, Przyborski JM, Blatch GL.
Department of Biochemistry & Microbiology, University of Zululand, Kwadlangezwa, South Africa. G.Blatch@ru.ac.za.
Abstract
Certain kinetoplastid (Leishmania spp. and Tryapnosoma cruzi) and apicomplexan parasites (Plasmodium falciparum and Toxoplasma gondii) are capable of invading human cells as part of their pathology. These parasites appear to have evolved a relatively expanded or diverse complement of genes encoding molecular chaperones. The gene families encoding heat shock protein 90 (Hsp90) and heat shock protein 70 (Hsp70) chaperones show significant expansion and diversity (especially for Leishmania spp. and T. cruzi), and in particular the Hsp40 family appears to be an extreme example of phylogenetic radiation. In general, Hsp40 proteins act as co-chaperones of Hsp70 chaperones, forming protein folding pathways that integrate with Hsp90 to ensure proteostasis in the cell. It is tempting to speculate that the diverse environmental insults that these parasites endure have resulted in the evolutionary selection of a diverse and expanded chaperone network. Hsp90 is involved in development and growth of all of these intracellular parasites, and so far represents the strongest candidate as a target for chemotherapeutic interventions. While there have been some excellent studies on the molecular and cell biology of Hsp70 proteins, relatively little is known about the biological function of Hsp70-Hsp40 interactions in these intracellular parasites. This review focuses on intracellular protozoan parasites of humans, and provides a critique of the role of heat shock proteins in development and pathogenesis, especially the molecular chaperones Hsp90, Hsp70 and Hsp40.
PMID: 20955165 [PubMed - as supplied by publisher]
Sunday, October 17, 2010
Concerted Action of Two Formins in Gliding Motility and Host Cell Invasion by Toxoplasma gondii.
PLoS Pathog. 2010 Oct 7;6(10). pii: e1001132.
Concerted Action of Two Formins in Gliding Motility and Host Cell Invasion by Toxoplasma gondii.
Daher W, Plattner F, Carlier MF, Soldati-Favre D.
Department of Microbiology and Molecular Medicine, CMU, University of Geneva, Geneva, Switzerland.
Abstract
The invasive forms of apicomplexan parasites share a conserved form of gliding motility that powers parasite migration across biological barriers, host cell invasion and egress from infected cells. Previous studies have established that the duration and direction of gliding motility are determined by actin polymerization; however, regulators of actin dynamics in apicomplexans remain poorly characterized. In the absence of a complete ARP2/3 complex, the formin homology 2 domain containing proteins and the accessory protein profilin are presumed to orchestrate actin polymerization during host cell invasion. Here, we have undertaken the biochemical and functional characterization of two Toxoplasma gondii formins and established that they act in concert as actin nucleators during invasion. The importance of TgFRM1 for parasite motility has been assessed by conditional gene disruption. The contribution of each formin individually and jointly was revealed by an approach based upon the expression of dominant mutants with modified FH2 domains impaired in actin binding but still able to dimerize with their respective endogenous formin. These mutated FH2 domains were fused to the ligand-controlled destabilization domain (DD-FKBP) to achieve conditional expression. This strategy proved unique in identifying the non-redundant and critical roles of both formins in invasion. These findings provide new insights into how controlled actin polymerization drives the directional movement required for productive penetration of parasites into host cells.
PMID: 20949068 [PubMed - in process]
Concerted Action of Two Formins in Gliding Motility and Host Cell Invasion by Toxoplasma gondii.
Daher W, Plattner F, Carlier MF, Soldati-Favre D.
Department of Microbiology and Molecular Medicine, CMU, University of Geneva, Geneva, Switzerland.
Abstract
The invasive forms of apicomplexan parasites share a conserved form of gliding motility that powers parasite migration across biological barriers, host cell invasion and egress from infected cells. Previous studies have established that the duration and direction of gliding motility are determined by actin polymerization; however, regulators of actin dynamics in apicomplexans remain poorly characterized. In the absence of a complete ARP2/3 complex, the formin homology 2 domain containing proteins and the accessory protein profilin are presumed to orchestrate actin polymerization during host cell invasion. Here, we have undertaken the biochemical and functional characterization of two Toxoplasma gondii formins and established that they act in concert as actin nucleators during invasion. The importance of TgFRM1 for parasite motility has been assessed by conditional gene disruption. The contribution of each formin individually and jointly was revealed by an approach based upon the expression of dominant mutants with modified FH2 domains impaired in actin binding but still able to dimerize with their respective endogenous formin. These mutated FH2 domains were fused to the ligand-controlled destabilization domain (DD-FKBP) to achieve conditional expression. This strategy proved unique in identifying the non-redundant and critical roles of both formins in invasion. These findings provide new insights into how controlled actin polymerization drives the directional movement required for productive penetration of parasites into host cells.
PMID: 20949068 [PubMed - in process]
Wednesday, October 13, 2010
Chronic infection with Toxoplasma gondii causes myenteric neuroplasticity of the jejunum in rats
Auton Neurosci. 2010 Oct 5. [Epub ahead of print]
Chronic infection with Toxoplasma gondii causes myenteric neuroplasticity of the jejunum in rats
Hermes-Uliana C, Pereira-Severi LS, Luerdes RB, Franco CL, da Silva AV, Araújo EJ, Sant'ana DD.
Programa de Pós-Graduação em Ciência Animal, Universidade Paranaense, (UNIPAR), Paraná, Brazil.
Abstract
Toxoplasma gondii is an aetiological agent of toxoplasmosis, which commonly causes diarrhoea in a number of species. This observation and the parasite's affinity for the nervous tissue support the theory that T. gondii infection may affect the myenteric neurons. The aim of this study was to evaluate the changes caused by T. gondii (genotype III) in the myenteric neurons of the jejunum in rats. Fifteen rats were distributed into three groups: control (CG), inoculated for 30days (G30) and inoculated for 90days (G90). Rats from the G30 and G90 groups received an oral inoculum with 500 oocysts from a genotype III (M7741) T. gondii strain. At 180days of age, all animals were anaesthetised and euthanised. Whole mounts were stained by using Giemsa (total population) and NADPH-diaphorase (nitrergic subpopulation) histochemistry. Maintenance of the width, length, area and neuronal density was observed; there was neuronal atrophy in the G30 group and a tendency to hypertrophy in the G90 group. Rats inoculated orally with sporulated oocysts did not show clinical illness or macroscopic or microscopic lesions, as do the majority of animal species. Therefore, infection was confirmed by a serum agglutination test; 30days of infection caused increased weight gain and atrophy of myenteric neurons. At 90days post-infection, weight gain became normal, and myenteric neurons hypertrophied.
PMID: 20932812 [PubMed - as supplied by publisher]
Chronic infection with Toxoplasma gondii causes myenteric neuroplasticity of the jejunum in rats
Hermes-Uliana C, Pereira-Severi LS, Luerdes RB, Franco CL, da Silva AV, Araújo EJ, Sant'ana DD.
Programa de Pós-Graduação em Ciência Animal, Universidade Paranaense, (UNIPAR), Paraná, Brazil.
Abstract
Toxoplasma gondii is an aetiological agent of toxoplasmosis, which commonly causes diarrhoea in a number of species. This observation and the parasite's affinity for the nervous tissue support the theory that T. gondii infection may affect the myenteric neurons. The aim of this study was to evaluate the changes caused by T. gondii (genotype III) in the myenteric neurons of the jejunum in rats. Fifteen rats were distributed into three groups: control (CG), inoculated for 30days (G30) and inoculated for 90days (G90). Rats from the G30 and G90 groups received an oral inoculum with 500 oocysts from a genotype III (M7741) T. gondii strain. At 180days of age, all animals were anaesthetised and euthanised. Whole mounts were stained by using Giemsa (total population) and NADPH-diaphorase (nitrergic subpopulation) histochemistry. Maintenance of the width, length, area and neuronal density was observed; there was neuronal atrophy in the G30 group and a tendency to hypertrophy in the G90 group. Rats inoculated orally with sporulated oocysts did not show clinical illness or macroscopic or microscopic lesions, as do the majority of animal species. Therefore, infection was confirmed by a serum agglutination test; 30days of infection caused increased weight gain and atrophy of myenteric neurons. At 90days post-infection, weight gain became normal, and myenteric neurons hypertrophied.
PMID: 20932812 [PubMed - as supplied by publisher]
Comprehensive proteomic analysis of membrane proteins in toxoplasma gondii
Mol Cell Proteomics. 2010 Oct 10. [Epub ahead of print]
Comprehensive proteomic analysis of membrane proteins in toxoplasma gondii
Che FY, Madrid-Aliste C, Burd B, Zhang H, Nieves E, Kim K, Fiser A, Angeletti RH, Weiss LM.
Albert Einstein College of Medicine, United States.
Abstract
Toxoplasma gondii (T.gondii) is an obligate intracellular protozoan parasite that is important human and animal pathogen. Experimental information on T.gondii membrane proteins is limited, and the majority of gen predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T.gondii is important not only for understanding this parasite's invasion mechanism(s), but also for the discovery of potentail drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T.gondii, employing three proteomics strategies: 1D gel LC-MSMS annlysis (one dimensional gel electrophoresis liquid chromatography tandem mass spectrometry), biotin labeling in conjunction with 1D gel LC-MSMS analysis, and a novel strategy that combines Three Layer "Sandwich" gel Electrophoresis (TLSGE) with multidimensional protein identification technology (MudPIT). A total of 2, 241 T.gondii proteins with at least one predicted transmembrane segment were identified and grouped into 841 sequentially non-redundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T.gondii genmoe. A large portion (42%) of the identified T. gondii membrane proteins are hypothetical proteins. Furthermore, many of the membrane proteins validated by mass spectrometry are unique to T.gondii or to the Apicomplexa, providing a set of gene predictions ripe for experimental investigation, and potentially siutable targets for the development of therapeutic strategies.
PMID: 20935347 [PubMed - as supplied by publisher]
Comprehensive proteomic analysis of membrane proteins in toxoplasma gondii
Che FY, Madrid-Aliste C, Burd B, Zhang H, Nieves E, Kim K, Fiser A, Angeletti RH, Weiss LM.
Albert Einstein College of Medicine, United States.
Abstract
Toxoplasma gondii (T.gondii) is an obligate intracellular protozoan parasite that is important human and animal pathogen. Experimental information on T.gondii membrane proteins is limited, and the majority of gen predictions with predicted transmembrane motifs are of unknown function. A systematic analysis of the membrane proteome of T.gondii is important not only for understanding this parasite's invasion mechanism(s), but also for the discovery of potentail drug targets and new preventative and therapeutic strategies. Here we report a comprehensive analysis of the membrane proteome of T.gondii, employing three proteomics strategies: 1D gel LC-MSMS annlysis (one dimensional gel electrophoresis liquid chromatography tandem mass spectrometry), biotin labeling in conjunction with 1D gel LC-MSMS analysis, and a novel strategy that combines Three Layer "Sandwich" gel Electrophoresis (TLSGE) with multidimensional protein identification technology (MudPIT). A total of 2, 241 T.gondii proteins with at least one predicted transmembrane segment were identified and grouped into 841 sequentially non-redundant protein clusters, which account for 21.8% of the predicted transmembrane protein clusters in the T.gondii genmoe. A large portion (42%) of the identified T. gondii membrane proteins are hypothetical proteins. Furthermore, many of the membrane proteins validated by mass spectrometry are unique to T.gondii or to the Apicomplexa, providing a set of gene predictions ripe for experimental investigation, and potentially siutable targets for the development of therapeutic strategies.
PMID: 20935347 [PubMed - as supplied by publisher]
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