J Org Chem. 2013 Mar 27. [Epub ahead of print]
Flexible synthesis and evaluation of diverse anti-Apicomplexa cyclic peptides
Traoré M, Mietton F, Maubon D, Peuchmaur M, Francisco Hilario F, Pereira de Freitas R, Bougdour A, Curt A, Maynadier M, Vial H, Pelloux H, Hakimi MA, Wong YS.
A modular approach to synthesize anti-Apicomplexa parasite inhibitors was developed and takes advantage of a pluripotent cyclic tetrapeptide scaffold capable of adjusting appendage and skeletal diversities in only few steps (1-3 steps). The diversification processes make use of selective radical coupling reactions and involve a new example of reductive carbon-nitrogen cleavage reaction with SmI2. The resulting bioactive cyclic peptides have revealed new insights into structural factors that govern selectivity between Apicomplexa parasites such as Toxoplasma and Plasmodium and human cells.
PMID: 23535138 [PubMed - as supplied by publisher]
Friday, March 29, 2013
Rapid cytoskeleton remodeling in dendritic cells following invasion by Toxoplasma gondii coincides with the onset of a hypermigratory phenotype
Cell Microbiol. 2013 Mar 28. doi: 10.1111/cmi.12145. [Epub ahead of print]
Rapid cytoskeleton remodeling in dendritic cells following invasion by Toxoplasma gondii coincides with the onset of a hypermigratory phenotype
Weidner JM, Kanatani S, Hernández-Castañeda MA, Fuks JM, Rethi B, Wallin RP, Barragan A.
Center for Infectious Medicine, Department of Medicine, Tumor- and Cell Biology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden; Swedish Institute for Communicable Disease Control, Stockholm, Sweden.
Host cell manipulation is an important feature of the obligate intracellular parasite Toxoplasma gondii. Recent reports have shown that the tachyzoite stages subvert dendritic cells (DC) as a conduit for dissemination (Trojan horse) during acute infection. To examine the cellular basis of these processes, we performed a detailed analysis of the early events following tachyzoite invasion of human monocyte-derived DC. We demonstrate that within minutes after tachyzoite penetration, profound morphological changes take place in DC that coincide with a migratory activation. Active parasite invasion of DC led to cytoskeletal actin redistribution with loss of adhesive podosome structures and redistribution of integrins (CD18 and CD11c), that concurred with the onset of DC hypermotility in vitro. Inhibition of parasite rhoptry secretion and invasion, but not inhibition of parasite or host cell protein synthesis, abrogated the onset of morphological changes and hypermotility in DC dose-dependently. Also, infected DC, but not by-stander DC, exhibited up-regulation of C-C chemokine receptor 7 (CCR7). Yet, the onset of parasite-induced DC hypermotility preceded chemotactic migratory responses in vitro. Collectively, present data reveal that invasion of DC by T. gondii initiates a series of regulated events, including rapid cytoskeleton rearrangements, hypermotility and chemotaxis, that promote the migratory activation of DC.
PMID: 23534541 [PubMed - as supplied by publisher]
Rapid cytoskeleton remodeling in dendritic cells following invasion by Toxoplasma gondii coincides with the onset of a hypermigratory phenotype
Weidner JM, Kanatani S, Hernández-Castañeda MA, Fuks JM, Rethi B, Wallin RP, Barragan A.
Center for Infectious Medicine, Department of Medicine, Tumor- and Cell Biology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden; Swedish Institute for Communicable Disease Control, Stockholm, Sweden.
Host cell manipulation is an important feature of the obligate intracellular parasite Toxoplasma gondii. Recent reports have shown that the tachyzoite stages subvert dendritic cells (DC) as a conduit for dissemination (Trojan horse) during acute infection. To examine the cellular basis of these processes, we performed a detailed analysis of the early events following tachyzoite invasion of human monocyte-derived DC. We demonstrate that within minutes after tachyzoite penetration, profound morphological changes take place in DC that coincide with a migratory activation. Active parasite invasion of DC led to cytoskeletal actin redistribution with loss of adhesive podosome structures and redistribution of integrins (CD18 and CD11c), that concurred with the onset of DC hypermotility in vitro. Inhibition of parasite rhoptry secretion and invasion, but not inhibition of parasite or host cell protein synthesis, abrogated the onset of morphological changes and hypermotility in DC dose-dependently. Also, infected DC, but not by-stander DC, exhibited up-regulation of C-C chemokine receptor 7 (CCR7). Yet, the onset of parasite-induced DC hypermotility preceded chemotactic migratory responses in vitro. Collectively, present data reveal that invasion of DC by T. gondii initiates a series of regulated events, including rapid cytoskeleton rearrangements, hypermotility and chemotaxis, that promote the migratory activation of DC.
PMID: 23534541 [PubMed - as supplied by publisher]
Thursday, March 28, 2013
Exploiting the unique ATP-binding pocket of Toxoplasma calcium-dependent protein kinase 1 to identify its substrates
ACS Chem Biol. 2013 Mar 26. [Epub ahead of print]
Exploiting the unique ATP-binding pocket of Toxoplasma calcium-dependent protein kinase 1 to identify its substrates
Lourido S, Jeschke GR, Turk BE, Sibley LD
Apicomplexan parasites rely on calcium as a second messenger to regulate a variety of essential cellular processes. Calcium-dependent protein kinases (CDPK), which transduce these signals, are conserved among apicomplexans but absent from mammalian hosts, making them attractive targets for therapeutic intervention. Despite their importance, the signaling pathways CDPK regulate remain poorly characterized, and their protein substrates are completely unknown. In Toxoplasma gondii, CDPK1 is required for calcium-regulated secretion from micronemes, thereby controlling motility, invasion, and egress from host cells. CDPK1 is unique among parasite and mammalian kinases in containing glycine at the key "gatekeeper" residue, which results in an expanded ATP-binding pocket. In the present study, we use a synthetic ATPγS analogue that displays steric complementarity to the ATP-binding pocket and hence allows identification of protein substrates based on selective thiophosphorylation. The specificity of this approach was validated by the concordance between the identified phosphorylation sites and the in vitro substrate preference of CDPK1. We further demonstrate that the phosphorylation of predicted substrates is dependent on CDPK1 both in vivo and in vitro. This combined strategy for identifying the targets of specific protein kinases provides a platform for defining the roles of CDPKs in apicomplexans.
PMID: 23530747 [PubMed - as supplied by publisher]
Exploiting the unique ATP-binding pocket of Toxoplasma calcium-dependent protein kinase 1 to identify its substrates
Lourido S, Jeschke GR, Turk BE, Sibley LD
Apicomplexan parasites rely on calcium as a second messenger to regulate a variety of essential cellular processes. Calcium-dependent protein kinases (CDPK), which transduce these signals, are conserved among apicomplexans but absent from mammalian hosts, making them attractive targets for therapeutic intervention. Despite their importance, the signaling pathways CDPK regulate remain poorly characterized, and their protein substrates are completely unknown. In Toxoplasma gondii, CDPK1 is required for calcium-regulated secretion from micronemes, thereby controlling motility, invasion, and egress from host cells. CDPK1 is unique among parasite and mammalian kinases in containing glycine at the key "gatekeeper" residue, which results in an expanded ATP-binding pocket. In the present study, we use a synthetic ATPγS analogue that displays steric complementarity to the ATP-binding pocket and hence allows identification of protein substrates based on selective thiophosphorylation. The specificity of this approach was validated by the concordance between the identified phosphorylation sites and the in vitro substrate preference of CDPK1. We further demonstrate that the phosphorylation of predicted substrates is dependent on CDPK1 both in vivo and in vitro. This combined strategy for identifying the targets of specific protein kinases provides a platform for defining the roles of CDPKs in apicomplexans.
PMID: 23530747 [PubMed - as supplied by publisher]
Toxoplasmosis and Polygenic Disease Susceptibility Genes
J Pathog. 2013;2013:965046. doi: 10.1155/2013/965046. Epub 2013 Mar 4.
Toxoplasmosis and Polygenic Disease Susceptibility Genes: Extensive Toxoplasma gondii Host/Pathogen Interactome Enrichment in Nine Psychiatric or Neurological Disorders
Carter CJ.
Polygenic Pathways, Flat 2, 40 Baldslow Road, Hastings, East Sussex TN34 2EY, UK.
Toxoplasma gondii is not only implicated in schizophrenia and related disorders, but also in Alzheimer's or Parkinson's disease, cancer, cardiac myopathies, and autoimmune disorders. During its life cycle, the pathogen interacts with ~3000 host genes or proteins. Susceptibility genes for multiple sclerosis, Alzheimer's disease, schizophrenia, bipolar disorder, depression, childhood obesity, Parkinson's disease, attention deficit hyperactivity disorder (P from 8.01E - 05 (ADHD) to 1.22E - 71) (multiple sclerosis), and autism (P = 0.013), but not anorexia or chronic fatigue are highly enriched in the human arm of this interactome and 18 (ADHD) to 33% (MS) of the susceptibility genes relate to it. The signalling pathways involved in the susceptibility gene/interactome overlaps are relatively specific and relevant to each disease suggesting a means whereby susceptibility genes could orient the attentions of a single pathogen towards disruption of the specific pathways that together contribute (positively or negatively) to the endophenotypes of different diseases. Conditional protein knockdown, orchestrated by T. gondii proteins or antibodies binding to those of the host (pathogen derived autoimmunity) and metabolite exchange, may contribute to this disruption. Susceptibility genes may thus be related to the causes and influencers of disease, rather than (and as well as) to the disease itself.
PMID: 23533776 [PubMed - in process]
Toxoplasmosis and Polygenic Disease Susceptibility Genes: Extensive Toxoplasma gondii Host/Pathogen Interactome Enrichment in Nine Psychiatric or Neurological Disorders
Carter CJ.
Polygenic Pathways, Flat 2, 40 Baldslow Road, Hastings, East Sussex TN34 2EY, UK.
Toxoplasma gondii is not only implicated in schizophrenia and related disorders, but also in Alzheimer's or Parkinson's disease, cancer, cardiac myopathies, and autoimmune disorders. During its life cycle, the pathogen interacts with ~3000 host genes or proteins. Susceptibility genes for multiple sclerosis, Alzheimer's disease, schizophrenia, bipolar disorder, depression, childhood obesity, Parkinson's disease, attention deficit hyperactivity disorder (P from 8.01E - 05 (ADHD) to 1.22E - 71) (multiple sclerosis), and autism (P = 0.013), but not anorexia or chronic fatigue are highly enriched in the human arm of this interactome and 18 (ADHD) to 33% (MS) of the susceptibility genes relate to it. The signalling pathways involved in the susceptibility gene/interactome overlaps are relatively specific and relevant to each disease suggesting a means whereby susceptibility genes could orient the attentions of a single pathogen towards disruption of the specific pathways that together contribute (positively or negatively) to the endophenotypes of different diseases. Conditional protein knockdown, orchestrated by T. gondii proteins or antibodies binding to those of the host (pathogen derived autoimmunity) and metabolite exchange, may contribute to this disruption. Susceptibility genes may thus be related to the causes and influencers of disease, rather than (and as well as) to the disease itself.
PMID: 23533776 [PubMed - in process]
Tuesday, March 26, 2013
Optogenetic modulation of an adenylate cyclase in Toxoplasma gondii demonstrates a requirement of the parasite cAMP for host-cell invasion and stage differentiation
J Biol Chem. 2013 Mar 22. [Epub ahead of print]
Optogenetic modulation of an adenylate cyclase in Toxoplasma gondii demonstrates a requirement of the parasite cAMP for host-cell invasion and stage differentiation
Hartmann A, Arroyo-Olarte RD, Imkeller K, Hegemann P, Lucius R, Gupta N
Humboldt University, Berlin, Germany.
Successful infection and transmission of the obligate intracellular parasite Toxoplasma gondii depends on its ability to switch between fast-replicating tachyzoite (acute) and quiescent bradyzoite (chronic) stages. An induction of cAMP in the parasitized host cells has been proposed to influence parasite differentiation. It is unknown whether the parasite or host cAMP is required to drive this phenomenon. An unequivocal research on cAMP-mediated signaling in such intertwined systems also requires a method for an efficient and spatial control of the cAMP pool in the pathogen or in the enclosing host cell. We have resolved these critical concerns by expressing a photo-activated adenylate cyclase that allows a light-sensitive control of the cytosolic cAMP in T. gondii, or in its human host cells. Using this method, we reveal multiple roles of the parasite-derived cAMP in host-cell invasion, stage-specific expression and asexual differentiation. An optogenetic method provides many desired advantages such as, it (i) allows rapid, transient and efficient induction of cAMP in the extracellular/intracellular and acute/chronic stages; (ii) circumvents difficulties often faced in cultures i.e., poor diffusion, premature degradation, steady activation and/or pleiotropic effects of cAMP agonists and antagonists; (iii) involves genetically-encoded enzyme expression, hence, inheritable to the progeny; and (iv) allows conditional and spatiotemporal control of cAMP. Importantly, a successful optogenetic application in Toxoplasma also illustrates its wider utility to many other genetically amenable two-organism systems such as, symbiotic and pathogen-host interactions.
PMID: 23525100 [PubMed - as supplied by publisher]
Optogenetic modulation of an adenylate cyclase in Toxoplasma gondii demonstrates a requirement of the parasite cAMP for host-cell invasion and stage differentiation
Hartmann A, Arroyo-Olarte RD, Imkeller K, Hegemann P, Lucius R, Gupta N
Humboldt University, Berlin, Germany.
Successful infection and transmission of the obligate intracellular parasite Toxoplasma gondii depends on its ability to switch between fast-replicating tachyzoite (acute) and quiescent bradyzoite (chronic) stages. An induction of cAMP in the parasitized host cells has been proposed to influence parasite differentiation. It is unknown whether the parasite or host cAMP is required to drive this phenomenon. An unequivocal research on cAMP-mediated signaling in such intertwined systems also requires a method for an efficient and spatial control of the cAMP pool in the pathogen or in the enclosing host cell. We have resolved these critical concerns by expressing a photo-activated adenylate cyclase that allows a light-sensitive control of the cytosolic cAMP in T. gondii, or in its human host cells. Using this method, we reveal multiple roles of the parasite-derived cAMP in host-cell invasion, stage-specific expression and asexual differentiation. An optogenetic method provides many desired advantages such as, it (i) allows rapid, transient and efficient induction of cAMP in the extracellular/intracellular and acute/chronic stages; (ii) circumvents difficulties often faced in cultures i.e., poor diffusion, premature degradation, steady activation and/or pleiotropic effects of cAMP agonists and antagonists; (iii) involves genetically-encoded enzyme expression, hence, inheritable to the progeny; and (iv) allows conditional and spatiotemporal control of cAMP. Importantly, a successful optogenetic application in Toxoplasma also illustrates its wider utility to many other genetically amenable two-organism systems such as, symbiotic and pathogen-host interactions.
PMID: 23525100 [PubMed - as supplied by publisher]
The crystal structure of Toxoplasma gondii nucleoside triphosphate diphosphohydrolase 1
Proteins. 2013 Mar 22. doi: 10.1002/prot.24288. [Epub ahead of print]
The crystal structure of Toxoplasma gondii nucleoside triphosphate diphosphohydrolase 1 represents a conformational intermediate in the reductive activation mechanism of the tetrameric enzyme
Krug U, Totzauer R, Sträter N.
From the Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, University of Leipzig, 04103, Leipzig, Germany.
Toxoplasma gondii nucleoside triphosphate diphosphohydrolase (NTPDase) 1 was crystallized in an intermediate tetrameric conformation. The crystal structure is similar to that of T. gondii NTPDase3 and represents an inactive conformation as the activating disulfide bridge is not reduced and the active site cleft between the two domains of each monomer is open. However, the arrangement of the monomers within the tetramer differs from that of the inactive form of NTPDase3 and may represent an intermediate conformation on the path of the closure motion of the tetramer induced upon activation.
PMID: 23526564 [PubMed - as supplied by publisher]
The crystal structure of Toxoplasma gondii nucleoside triphosphate diphosphohydrolase 1 represents a conformational intermediate in the reductive activation mechanism of the tetrameric enzyme
Krug U, Totzauer R, Sträter N.
From the Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, University of Leipzig, 04103, Leipzig, Germany.
Toxoplasma gondii nucleoside triphosphate diphosphohydrolase (NTPDase) 1 was crystallized in an intermediate tetrameric conformation. The crystal structure is similar to that of T. gondii NTPDase3 and represents an inactive conformation as the activating disulfide bridge is not reduced and the active site cleft between the two domains of each monomer is open. However, the arrangement of the monomers within the tetramer differs from that of the inactive form of NTPDase3 and may represent an intermediate conformation on the path of the closure motion of the tetramer induced upon activation.
PMID: 23526564 [PubMed - as supplied by publisher]
Toxoplasma gondii Triggers Phosphorylation and Nuclear Translocation of Dendritic Cell STAT1
PLoS One. 2013;8(3):e60215. doi: 10.1371/journal.pone.0060215. Epub 2013 Mar 20.
Toxoplasma gondii Triggers Phosphorylation and Nuclear Translocation of Dendritic Cell STAT1 while Simultaneously Blocking IFNγ-Induced STAT1 Transcriptional Activity
Schneider AG, Abi Abdallah DS, Butcher BA, Denkers EY.
Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.
The protozoan Toxoplasma gondii actively modulates cytokine-induced JAK/STAT signaling pathways to facilitate survival within the host, including blocking IFNγ-mediated STAT1-dependent proinflammatory gene expression. We sought to further characterize inhibition of STAT1 signaling in infected murine dendritic cells (DC) because this cell type has not previously been examined, yet is known to serve as an early target of in vivo infection. Unexpectedly, we discovered that T. gondii infection alone induced sustained STAT1 phosphorylation and nuclear translocation in DC in a parasite strain-independent manner. Maintenance of STAT1 phosphorylation required active invasion but intracellular parasite replication was dispensable. The parasite rhoptry protein ROP16, recently shown to mediate STAT3 and STAT6 phosphorylation, was not required for STAT1 phosphorylation. In combination with IFNγ, T. gondii induced synergistic STAT1 phosphorylation and binding of aberrant STAT1-containing complexes to IFNγ consensus sequence oligonucleotides. Despite these findings, parasite infection blocked STAT1 binding to the native promoters of the IFNγ-inducible genes Irf-1 and Lrg47, along with subsequent gene expression. These results reinforce the importance of parasite-mediated blockade of IFNγ responses in dendritic cells, while simultaneously showing that T. gondii alone induces STAT1 phosphorylation.
PMID: 23527309 [PubMed - in process]
Toxoplasma gondii Triggers Phosphorylation and Nuclear Translocation of Dendritic Cell STAT1 while Simultaneously Blocking IFNγ-Induced STAT1 Transcriptional Activity
Schneider AG, Abi Abdallah DS, Butcher BA, Denkers EY.
Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America.
The protozoan Toxoplasma gondii actively modulates cytokine-induced JAK/STAT signaling pathways to facilitate survival within the host, including blocking IFNγ-mediated STAT1-dependent proinflammatory gene expression. We sought to further characterize inhibition of STAT1 signaling in infected murine dendritic cells (DC) because this cell type has not previously been examined, yet is known to serve as an early target of in vivo infection. Unexpectedly, we discovered that T. gondii infection alone induced sustained STAT1 phosphorylation and nuclear translocation in DC in a parasite strain-independent manner. Maintenance of STAT1 phosphorylation required active invasion but intracellular parasite replication was dispensable. The parasite rhoptry protein ROP16, recently shown to mediate STAT3 and STAT6 phosphorylation, was not required for STAT1 phosphorylation. In combination with IFNγ, T. gondii induced synergistic STAT1 phosphorylation and binding of aberrant STAT1-containing complexes to IFNγ consensus sequence oligonucleotides. Despite these findings, parasite infection blocked STAT1 binding to the native promoters of the IFNγ-inducible genes Irf-1 and Lrg47, along with subsequent gene expression. These results reinforce the importance of parasite-mediated blockade of IFNγ responses in dendritic cells, while simultaneously showing that T. gondii alone induces STAT1 phosphorylation.
PMID: 23527309 [PubMed - in process]
Thursday, March 21, 2013
Risk factors for ocular toxoplasmosis in Brazil
Epidemiol Infect. 2013 Mar 18:1-7. [Epub ahead of print]
Risk factors for ocular toxoplasmosis in Brazil
Ferreira AI, De Mattos CC, Frederico FB, Meira CS, Almeida GC, Nakashima F, Bernardo CR, Pereira-Chioccola VL, De Mattos LC.
Immunogenetics Laboratory, Molecular Biology Department, Faculdade de Medicina de São José do Rio Preto (FAMERP), São José do Rio Preto, SP, Brazil
SUMMARY The aim of this study was to investigate risk factors for ocular toxoplasmosis (OT) in patients who received medical attention at a public health service. Three hundred and forty-nine consecutive patients, treated in the Outpatient Eye Clinic of Hospital de Base, São José do Rio Preto, São Paulo state, Brazil, were enrolled in this study. After an eye examination, enzyme-linked immunosorbent assay (ELISA) was used to determine anti-Toxoplasma gondii antibodies. The results showed that 25·5% of the patients were seronegative and 74·5% were seropositive for IgG anti-T. gondii antibodies; of these 27·3% had OT and 72·7% had other ocular diseases (OOD). The presence of cats or dogs [odds ratio (OR) 2·22, 95% confidence interval (CI) 1·24-3·98, P = 0·009] and consumption of raw or undercooked meat (OR 1·77, 95% CI 1·05-2·98, P = 0·03) were associated with infection but not with the development of OT. Age (OT 48·2 ± 21·2 years vs. OOD: 69·5 ± 14·7 years, P < 0·0001) and the low level of schooling/literacy (OT vs. OOD: OR 0·414, 95% CI 0·2231-0·7692, P = 0·007) were associated with OT. The presence of dogs and cats as well as eating raw/undercooked meat increases the risk of infection, but is not associated with the development of OT.
PMID: 23507508 [PubMed - as supplied by publisher]
Risk factors for ocular toxoplasmosis in Brazil
Ferreira AI, De Mattos CC, Frederico FB, Meira CS, Almeida GC, Nakashima F, Bernardo CR, Pereira-Chioccola VL, De Mattos LC.
Immunogenetics Laboratory, Molecular Biology Department, Faculdade de Medicina de São José do Rio Preto (FAMERP), São José do Rio Preto, SP, Brazil
SUMMARY The aim of this study was to investigate risk factors for ocular toxoplasmosis (OT) in patients who received medical attention at a public health service. Three hundred and forty-nine consecutive patients, treated in the Outpatient Eye Clinic of Hospital de Base, São José do Rio Preto, São Paulo state, Brazil, were enrolled in this study. After an eye examination, enzyme-linked immunosorbent assay (ELISA) was used to determine anti-Toxoplasma gondii antibodies. The results showed that 25·5% of the patients were seronegative and 74·5% were seropositive for IgG anti-T. gondii antibodies; of these 27·3% had OT and 72·7% had other ocular diseases (OOD). The presence of cats or dogs [odds ratio (OR) 2·22, 95% confidence interval (CI) 1·24-3·98, P = 0·009] and consumption of raw or undercooked meat (OR 1·77, 95% CI 1·05-2·98, P = 0·03) were associated with infection but not with the development of OT. Age (OT 48·2 ± 21·2 years vs. OOD: 69·5 ± 14·7 years, P < 0·0001) and the low level of schooling/literacy (OT vs. OOD: OR 0·414, 95% CI 0·2231-0·7692, P = 0·007) were associated with OT. The presence of dogs and cats as well as eating raw/undercooked meat increases the risk of infection, but is not associated with the development of OT.
PMID: 23507508 [PubMed - as supplied by publisher]
CD40 induces anti-Toxoplasma gondii activity in non-hematopoietic cells dependent on autophagy proteins
Infect Immun. 2013 Mar 18. [Epub ahead of print]
CD40 induces anti-Toxoplasma gondii activity in non-hematopoietic cells dependent on autophagy proteins
Van Grol J, Muniz-Feliciano L, Portillo JA, Bonilha VL, Subauste CS
Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106
Toxoplasma gondii infects both hematopoietic and non-hematopoietic cells and can cause cerebral and ocular toxoplasmosis, either as a result of congenital or post-natally acquired infections. Host protection likely acts at both cellular levels to control the parasite. CD40 is key for protection against cerebral and ocular toxoplasmosis. We determined if CD40 induces anti-T. gondii activity at the level of non-hematopoietic cells. Engagement of CD40 on various endothelial cells including human microvascular brain endothelial cells, human umbilical vein endothelial cells, a mouse endothelial cell line, as well as human and mouse retinal pigment epithelial cells resulted in killing of T. gondii. CD40 stimulation increased expression of the autophagy proteins Beclin 1 and LC3 II, enhanced autophagy flux and led to recruitment of LC3 around the parasite. The late endosomal/lysosomal marker LAMP-1 accumulated around the parasite in CD40 stimulated cells. This was accompanied by killing of T. gondii dependent on lysosomal enzymes. Accumulation of LAMP-1 and killing of T. gondii were dependent on the autophagy proteins Beclin 1 and Atg7. Together, these studies revealed that CD40 induces toxoplasmacidal activity in various non-hematopoietic cells dependent on proteins of the autophagy machinery.
PMID: 23509150 [PubMed - as supplied by publisher]
CD40 induces anti-Toxoplasma gondii activity in non-hematopoietic cells dependent on autophagy proteins
Van Grol J, Muniz-Feliciano L, Portillo JA, Bonilha VL, Subauste CS
Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106
Toxoplasma gondii infects both hematopoietic and non-hematopoietic cells and can cause cerebral and ocular toxoplasmosis, either as a result of congenital or post-natally acquired infections. Host protection likely acts at both cellular levels to control the parasite. CD40 is key for protection against cerebral and ocular toxoplasmosis. We determined if CD40 induces anti-T. gondii activity at the level of non-hematopoietic cells. Engagement of CD40 on various endothelial cells including human microvascular brain endothelial cells, human umbilical vein endothelial cells, a mouse endothelial cell line, as well as human and mouse retinal pigment epithelial cells resulted in killing of T. gondii. CD40 stimulation increased expression of the autophagy proteins Beclin 1 and LC3 II, enhanced autophagy flux and led to recruitment of LC3 around the parasite. The late endosomal/lysosomal marker LAMP-1 accumulated around the parasite in CD40 stimulated cells. This was accompanied by killing of T. gondii dependent on lysosomal enzymes. Accumulation of LAMP-1 and killing of T. gondii were dependent on the autophagy proteins Beclin 1 and Atg7. Together, these studies revealed that CD40 induces toxoplasmacidal activity in various non-hematopoietic cells dependent on proteins of the autophagy machinery.
PMID: 23509150 [PubMed - as supplied by publisher]
Wednesday, March 20, 2013
Global proteomic analysis of the oocyst/sporozoite of Toxoplasma gondii reveals commitment to a host-independent lifestyle
BMC Genomics. 2013 Mar 15;14(1):183. [Epub ahead of print]
Global proteomic analysis of the oocyst/sporozoite of Toxoplasma gondii reveals commitment to a host-independent lifestyle
Possenti A, Fratini F, Fantozzi L, Pozio E, Dubey JP, Ponzi M, Pizzi E, Spano F.
BACKGROUND:
Toxoplasmosis is caused by the apicomplexan parasite Toxoplasma gondii and can be acquired either congenitally or via the oral route. In the latter case, transmission is mediated by two distinct invasive stages, i.e., bradyzoites residing in tissue cysts or sporozoites contained in environmentally resistant oocysts shed by felids in their feces. The oocyst plays a central epidemiological role, yet this stage has been scarcely investigated at the molecular level and the knowledge of its expressed proteome is very limited.
RESULTS:
Using one-dimensional gel electrophoresis coupled to liquid chromatography-linked tandem mass spectrometry, we analysed total or fractionated protein extracts of partially sporulated T. gondii oocysts, producing a dataset of 1304 non reduntant proteins (~18% of the total predicted proteome), ~59% of which were classified according to the MIPS functional catalogue database. Notably, the comparison of the oocyst dataset with the extensively covered proteome of T. gondii tachyzoite, the invasive stage responsible for the clinical signs of toxoplasmosis, identified 154 putative oocyst/sporozoite-specific proteins, some of which were validated by Western blot. The analysis of this protein subset showed that, compared to tachyzoites, oocysts have a greater capability of de novo amino acid biosynthesis and are well equipped to fuel the Krebs cycle with the acetyl-CoA generated through fatty acid beta-oxidation and the degradation of branched amino acids.
CONCLUSIONS:
The study reported herein significantly expanded our knowledge of the proteome expressed by the oocyst/sporozoite of T. gondii, shedding light on a stage-specifc subset of proteins whose functional profile is consistent with the adaptation of T. gondii oocysts to the nutrient-poor and stressing extracellular environment.
PMID: 23496850 [PubMed - as supplied by publisher]
Global proteomic analysis of the oocyst/sporozoite of Toxoplasma gondii reveals commitment to a host-independent lifestyle
Possenti A, Fratini F, Fantozzi L, Pozio E, Dubey JP, Ponzi M, Pizzi E, Spano F.
BACKGROUND:
Toxoplasmosis is caused by the apicomplexan parasite Toxoplasma gondii and can be acquired either congenitally or via the oral route. In the latter case, transmission is mediated by two distinct invasive stages, i.e., bradyzoites residing in tissue cysts or sporozoites contained in environmentally resistant oocysts shed by felids in their feces. The oocyst plays a central epidemiological role, yet this stage has been scarcely investigated at the molecular level and the knowledge of its expressed proteome is very limited.
RESULTS:
Using one-dimensional gel electrophoresis coupled to liquid chromatography-linked tandem mass spectrometry, we analysed total or fractionated protein extracts of partially sporulated T. gondii oocysts, producing a dataset of 1304 non reduntant proteins (~18% of the total predicted proteome), ~59% of which were classified according to the MIPS functional catalogue database. Notably, the comparison of the oocyst dataset with the extensively covered proteome of T. gondii tachyzoite, the invasive stage responsible for the clinical signs of toxoplasmosis, identified 154 putative oocyst/sporozoite-specific proteins, some of which were validated by Western blot. The analysis of this protein subset showed that, compared to tachyzoites, oocysts have a greater capability of de novo amino acid biosynthesis and are well equipped to fuel the Krebs cycle with the acetyl-CoA generated through fatty acid beta-oxidation and the degradation of branched amino acids.
CONCLUSIONS:
The study reported herein significantly expanded our knowledge of the proteome expressed by the oocyst/sporozoite of T. gondii, shedding light on a stage-specifc subset of proteins whose functional profile is consistent with the adaptation of T. gondii oocysts to the nutrient-poor and stressing extracellular environment.
PMID: 23496850 [PubMed - as supplied by publisher]
The Toxoplasma Protein ARO Mediates the Apical Positioning of Rhoptry Organelles, a Prerequisite for Host Cell Invasion
Cell Host Microbe. 2013 Mar 13;13(3):289-301. doi: 10.1016/j.chom.2013.02.001.
The Toxoplasma Protein ARO Mediates the Apical Positioning of Rhoptry Organelles, a Prerequisite for Host Cell Invasion
Mueller C, Klages N, Jacot D, Santos JM, Cabrera A, Gilberger TW, Dubremetz JF, Soldati-Favre D.
Department of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 Rue Michel-Servet, 1211 Geneva 4, Switzerland.
Members of the phylum Apicomplexa actively enter host cells by a process involving the discharge of the apically localized microneme and rhoptry organelles. To unravel the processes involved in rhoptry organelle biogenesis, we focused on the Toxoplasma gondii armadillo repeats only protein (TgARO), a conserved acylated protein homogenously anchored to the rhoptry membrane. Conditional disruption of TgARO results in the random cytosolic dispersion of rhoptries and a severe defect in T. gondii invasion, with no effects on intracellular growth or host cell egress. Importantly, rhoptry displacement upon ARO depletion can be functionally complemented with wild-type TgARO but not an acylation mutant. TgARO interacts with myosin F, and inhibition of actin polymerization or myosin function also results in rhoptry dispersal, indicating that the apical positioning of rhoptries is an actomyosin-based process. Thus, TgARO mediates the apical localization of rhoptries, which is specifically required for host cell invasion.
PMID: 23498954 [PubMed - in process]
The Toxoplasma Protein ARO Mediates the Apical Positioning of Rhoptry Organelles, a Prerequisite for Host Cell Invasion
Mueller C, Klages N, Jacot D, Santos JM, Cabrera A, Gilberger TW, Dubremetz JF, Soldati-Favre D.
Department of Microbiology and Molecular Medicine, CMU, University of Geneva, 1 Rue Michel-Servet, 1211 Geneva 4, Switzerland.
Members of the phylum Apicomplexa actively enter host cells by a process involving the discharge of the apically localized microneme and rhoptry organelles. To unravel the processes involved in rhoptry organelle biogenesis, we focused on the Toxoplasma gondii armadillo repeats only protein (TgARO), a conserved acylated protein homogenously anchored to the rhoptry membrane. Conditional disruption of TgARO results in the random cytosolic dispersion of rhoptries and a severe defect in T. gondii invasion, with no effects on intracellular growth or host cell egress. Importantly, rhoptry displacement upon ARO depletion can be functionally complemented with wild-type TgARO but not an acylation mutant. TgARO interacts with myosin F, and inhibition of actin polymerization or myosin function also results in rhoptry dispersal, indicating that the apical positioning of rhoptries is an actomyosin-based process. Thus, TgARO mediates the apical localization of rhoptries, which is specifically required for host cell invasion.
PMID: 23498954 [PubMed - in process]
An Overexpression Screen of Toxoplasma gondii Rab-GTPases Reveals Distinct Transport Routes to the Micronemes
PLoS Pathog. 2013 Mar;9(3):e1003213. doi: 10.1371/journal.ppat.1003213. Epub 2013 Mar 7.
An Overexpression Screen of Toxoplasma gondii Rab-GTPases Reveals Distinct Transport Routes to the Micronemes
Kremer K, Kamin D, Rittweger E, Wilkes J, Flammer H, Mahler S, Heng J, Tonkin CJ, Langsley G, Hell SW, Carruthers VB, Ferguson DJ, Meissner M
Institute of Infection, Immunity and Inflammation, Wellcome Centre for Molecular Parasitology, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, United Kingdom.
The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA) is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules) that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED) we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles.
PMID: 23505371 [PubMed - in process]
An Overexpression Screen of Toxoplasma gondii Rab-GTPases Reveals Distinct Transport Routes to the Micronemes
Kremer K, Kamin D, Rittweger E, Wilkes J, Flammer H, Mahler S, Heng J, Tonkin CJ, Langsley G, Hell SW, Carruthers VB, Ferguson DJ, Meissner M
Institute of Infection, Immunity and Inflammation, Wellcome Centre for Molecular Parasitology, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, United Kingdom.
The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA) is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules) that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED) we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles.
PMID: 23505371 [PubMed - in process]
Sunday, March 17, 2013
Childhood parasitic infections endemic to the United States
Pediatr Clin North Am. 2013 Apr;60(2):471-85. doi: 10.1016/j.pcl.2012.12.011. Epub 2013 Jan 18.
Childhood parasitic infections endemic to the United States
Barry MA, Weatherhead JE, Hotez PJ, Woc-Colburn L.
Interdepartmental Program in Translational Biology and Molecular Medicine, National School of Tropical Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; BCM Medical Scientist Training Program, National School of Tropical Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Endemic parasitic infections in the United States are more frequent than is commonly perceived. Intestinal parasitic infection with Cryptosporidium, Dientamoeba, and Giardia occurs most often in children in northern states during the summer months. Zoonotic Toxocara and Toxoplasma parasitic infections are more frequent in southern states, in African Americans, and in populations with lower socioeconomic status. Approximately 300, 000 people in the United States have Trypanosoma cruzi infection. Local, vector-borne transmission of T cruzi and Leishmania infections has been documented in southern states. Parasitic diseases endemic to the United States are not uncommon but are understudied.
PMID: 23481112 [PubMed - in process]
Childhood parasitic infections endemic to the United States
Barry MA, Weatherhead JE, Hotez PJ, Woc-Colburn L.
Interdepartmental Program in Translational Biology and Molecular Medicine, National School of Tropical Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA; BCM Medical Scientist Training Program, National School of Tropical Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Endemic parasitic infections in the United States are more frequent than is commonly perceived. Intestinal parasitic infection with Cryptosporidium, Dientamoeba, and Giardia occurs most often in children in northern states during the summer months. Zoonotic Toxocara and Toxoplasma parasitic infections are more frequent in southern states, in African Americans, and in populations with lower socioeconomic status. Approximately 300, 000 people in the United States have Trypanosoma cruzi infection. Local, vector-borne transmission of T cruzi and Leishmania infections has been documented in southern states. Parasitic diseases endemic to the United States are not uncommon but are understudied.
PMID: 23481112 [PubMed - in process]
N-terminal palmitoylation is required for Toxoplasma gondii HSP20 inner membrane complex localization
Biochim Biophys Acta. 2013 Feb 25. pii: S0167-4889(13)00080-3. doi: 10.1016/j.bbamcr.2013.02.022. [Epub ahead of print]
N-terminal palmitoylation is required for Toxoplasma gondii HSP20 inner membrane complex localization
Napoli MG, Miguel ND, Lebrun M, Moreno SN, Angel SO, Corvi MM.
Laboratorio de Parasitología Molecular, Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomus (IIB-INTECH), Universidad Nacional de San Martín (UNSAM) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Intendente Marino Km 8,2 (B7130) Chascomús, Provincia de Buenos Aires, Argentina.
Toxoplasma gondii is an obligate intracellular parasite and the causative agent of toxoplasmosis. Protein palmitoylation is known to play roles in signal transduction and in enhancing the hydrophobicity of proteins thus contributing to their membrane association. Global inhibition of protein palmitoylation has been shown to affect T. gondii physiology and invasion of the host cell. However, the proteins affected by this modification have been understudied. This paper shows that the small heat shock protein 20 from T. gondii (TgHSP20) is synthesized as a mature protein in the cytosol and is palmitoylated in three cysteine residues. However, its localization at the inner membrane complex (IMC) is dependent only on N-terminal palmitoylation. Absence or incomplete N-terminal palmitoylation causes TgHSP20 to partially accumulate in a membranous structure. Interestingly, TgHSP20 palmitoylation is not responsible for its interaction with the daughter cells IMCs. Together, our data describe the importance of palmitoylation in protein targeting to the IMC in T. gondii.
PMID: 23485398 [PubMed - as supplied by publisher]
N-terminal palmitoylation is required for Toxoplasma gondii HSP20 inner membrane complex localization
Napoli MG, Miguel ND, Lebrun M, Moreno SN, Angel SO, Corvi MM.
Laboratorio de Parasitología Molecular, Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico de Chascomus (IIB-INTECH), Universidad Nacional de San Martín (UNSAM) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Intendente Marino Km 8,2 (B7130) Chascomús, Provincia de Buenos Aires, Argentina.
Toxoplasma gondii is an obligate intracellular parasite and the causative agent of toxoplasmosis. Protein palmitoylation is known to play roles in signal transduction and in enhancing the hydrophobicity of proteins thus contributing to their membrane association. Global inhibition of protein palmitoylation has been shown to affect T. gondii physiology and invasion of the host cell. However, the proteins affected by this modification have been understudied. This paper shows that the small heat shock protein 20 from T. gondii (TgHSP20) is synthesized as a mature protein in the cytosol and is palmitoylated in three cysteine residues. However, its localization at the inner membrane complex (IMC) is dependent only on N-terminal palmitoylation. Absence or incomplete N-terminal palmitoylation causes TgHSP20 to partially accumulate in a membranous structure. Interestingly, TgHSP20 palmitoylation is not responsible for its interaction with the daughter cells IMCs. Together, our data describe the importance of palmitoylation in protein targeting to the IMC in T. gondii.
PMID: 23485398 [PubMed - as supplied by publisher]
Monday, March 11, 2013
Optimizing small molecule inhibitors of calcium-dependent protein kinase 1 to prevent infection by Toxoplasma gondii
J Med Chem. 2013 Mar 7. [Epub ahead of print]
Optimizing small molecule inhibitors of calcium-dependent protein kinase 1 to prevent infection by Toxoplasma gondii
Lourido S, Zhang C, Lopez M, Tang K, Barks J, Wang Q, Wildman SA, Shokat K, Sibley LD.
Toxoplasma gondii is sensitive to bulky pyrazolo [3,4-d] pyrimidine (PP) inhibitors due to the presence of a Gly gatekeeper in the essential calcium dependent protein kinase 1 (CDPK1). Here we synthesized a number of new derivatives of 3-methyl-benzyl-PP (3-MB-PP, or 1). The potency of PP analogs in inhibiting CDPK1 enzyme activity in vitro (low nM IC50 values) and blocking parasite growth in host cell monolayers in vitro (low μM EC50 values) were highly correlated and occurred in a CDPK1-specific manner. Chemical modification of the PP scaffold to increase half-life in the presence of microsomes in vitro led to identification of compounds with enhanced stability while retaining activity. Several of these more potent compounds were able to prevent lethal infection with T. gondii in the mouse model. Collectively the strategies outlined here provide a route for development of more effective compounds for treatment of toxoplasmosis, and perhaps related parasitic diseases.
PMID: 23470217 [PubMed - as supplied by publisher]
Optimizing small molecule inhibitors of calcium-dependent protein kinase 1 to prevent infection by Toxoplasma gondii
Lourido S, Zhang C, Lopez M, Tang K, Barks J, Wang Q, Wildman SA, Shokat K, Sibley LD.
Toxoplasma gondii is sensitive to bulky pyrazolo [3,4-d] pyrimidine (PP) inhibitors due to the presence of a Gly gatekeeper in the essential calcium dependent protein kinase 1 (CDPK1). Here we synthesized a number of new derivatives of 3-methyl-benzyl-PP (3-MB-PP, or 1). The potency of PP analogs in inhibiting CDPK1 enzyme activity in vitro (low nM IC50 values) and blocking parasite growth in host cell monolayers in vitro (low μM EC50 values) were highly correlated and occurred in a CDPK1-specific manner. Chemical modification of the PP scaffold to increase half-life in the presence of microsomes in vitro led to identification of compounds with enhanced stability while retaining activity. Several of these more potent compounds were able to prevent lethal infection with T. gondii in the mouse model. Collectively the strategies outlined here provide a route for development of more effective compounds for treatment of toxoplasmosis, and perhaps related parasitic diseases.
PMID: 23470217 [PubMed - as supplied by publisher]
Friday, March 08, 2013
Apoptosis-like cell death pathways in the unicellular parasite Toxoplasma gondii following treatment with apoptosis inducers and chemotherapeutic agents
Apoptosis. 2013 Mar 7. [Epub ahead of print]
Apoptosis-like cell death pathways in the unicellular parasite Toxoplasma gondii following treatment with apoptosis inducers and chemotherapeutic agents: a proof-of-concept study
Ni Nyoman AD, Lüder CG
Institute for Medical Microbiology, University Medical Center, Georg-August-University, Kreuzbergring 57, 37075, Göttingen, Germany.
Ancient pathways of an apoptosis-like cell death have been identified in unicellular eukaryotes including protozoan parasites. Here, we examined programmed cell death in the apicomplexan Toxoplasma gondii which is a common intracellular pathogen of humans and warm-blooded animals. Treatment of extracellular T. gondii with various pro-apoptotic stimuli significantly induced DNA strand breaks as revealed by TUNEL and flow cytometry. Using staurosporine or miltefosine as pro-apoptotic stimuli, parasites also presented a reduced cell size, i.e. pyknosis and externalized phosphatidylserine while the plasma membrane remained intact. Importantly, staurosporine also induced DNA strand breaks in intracellular T. gondii. Data mining of the Toxoplasma genome resource identified 17 putative cell death-associated genes encoding proteases, a nuclease and several apoptosis regulators. Staurosporine-treated parasites but not controls strongly up-regulated several of these genes in a time-dependent fashion with a putative PDCD2 protein being more than 100-fold up-regulated. However, the mitochondrial membrane potential (ΔΨ m) remained intact and caspase-like activity increased only slightly during staurosporine-triggered cell death. As compared to staurosporine, the transcriptional response of parasites to miltefosine was more restricted but PDCD2 was again strongly induced. Furthermore, T. gondii lost their ΔΨ m and rapidly presented strong caspase-like activity during miltefosine treatment. Consequently, protease inhibitors abrogated miltefosine-induced but not staurosporine-induced Toxoplasma cell death. Finally, toxoplasmacidal drugs triggered DNA strand breaks in extracellular T. gondii. Interestingly, clindamycin also induced markers of an apoptosis-like cell death in intracellular parasites. Together, the data indicate that T. gondii possesses ancient apoptosis-like cell death machinery which can be triggered by chemotherapeutic agents.
PMID: 23468121 [PubMed - as supplied by publisher]
Apoptosis-like cell death pathways in the unicellular parasite Toxoplasma gondii following treatment with apoptosis inducers and chemotherapeutic agents: a proof-of-concept study
Ni Nyoman AD, Lüder CG
Institute for Medical Microbiology, University Medical Center, Georg-August-University, Kreuzbergring 57, 37075, Göttingen, Germany.
Ancient pathways of an apoptosis-like cell death have been identified in unicellular eukaryotes including protozoan parasites. Here, we examined programmed cell death in the apicomplexan Toxoplasma gondii which is a common intracellular pathogen of humans and warm-blooded animals. Treatment of extracellular T. gondii with various pro-apoptotic stimuli significantly induced DNA strand breaks as revealed by TUNEL and flow cytometry. Using staurosporine or miltefosine as pro-apoptotic stimuli, parasites also presented a reduced cell size, i.e. pyknosis and externalized phosphatidylserine while the plasma membrane remained intact. Importantly, staurosporine also induced DNA strand breaks in intracellular T. gondii. Data mining of the Toxoplasma genome resource identified 17 putative cell death-associated genes encoding proteases, a nuclease and several apoptosis regulators. Staurosporine-treated parasites but not controls strongly up-regulated several of these genes in a time-dependent fashion with a putative PDCD2 protein being more than 100-fold up-regulated. However, the mitochondrial membrane potential (ΔΨ m) remained intact and caspase-like activity increased only slightly during staurosporine-triggered cell death. As compared to staurosporine, the transcriptional response of parasites to miltefosine was more restricted but PDCD2 was again strongly induced. Furthermore, T. gondii lost their ΔΨ m and rapidly presented strong caspase-like activity during miltefosine treatment. Consequently, protease inhibitors abrogated miltefosine-induced but not staurosporine-induced Toxoplasma cell death. Finally, toxoplasmacidal drugs triggered DNA strand breaks in extracellular T. gondii. Interestingly, clindamycin also induced markers of an apoptosis-like cell death in intracellular parasites. Together, the data indicate that T. gondii possesses ancient apoptosis-like cell death machinery which can be triggered by chemotherapeutic agents.
PMID: 23468121 [PubMed - as supplied by publisher]
Thursday, March 07, 2013
Replication And Distribution Of Toxoplasma Gondii In The Small Intestine After Oral Infection With Tissue Cysts
Infect Immun. 2013 Mar 4. [Epub ahead of print]
Replication And Distribution Of Toxoplasma Gondii In The Small Intestine After Oral Infection With Tissue Cysts
Gregg B, Taylor BC, John B, Tait-Wojno ED, Girgis NM, Miller N, Wagage S, Roos DS, Hunter CA.
Departments of Biology Penn Genome Frontiers Institute.
Natural infection by Toxoplasma gondii occurs via oral ingestion of tissue cysts that rupture in the small intestine releasing zoites that infect locally before disseminating throughout the host. The studies presented here use fluorescent parasites combined with flow cytometry and multi-photon microscopy techniques to understand the events associated with parasite replication in the mucosa. At three days post-infection with tissue cysts, parasites were localized in small foci and flow cytometry revealed parasites present in macrophages, neutrophils, and monocytes in the lamina propria. By day six post-infection there were large foci of replicating parasites; however, foci unexpectedly varied in the number of villi involved and were associated with the presence of viable tachyzoites within the intestinal lumen. Consistent with the flow cytometry data, neutrophils and monocytes in the lamina propria were preferentially associated with parasite plaques. In contrast, dendritic cells comprised a small fraction of the infected immune cell population and were localized at the periphery of parasite plaques. Together, these findings reveal the formation of localized sites of parasite replication and inflammation early during infection and suggest that sustained replication of T. gondii in the gut may be a function of pathogen luminal spread.
PMID: 23460516 [PubMed - as supplied by publisher]
Replication And Distribution Of Toxoplasma Gondii In The Small Intestine After Oral Infection With Tissue Cysts
Gregg B, Taylor BC, John B, Tait-Wojno ED, Girgis NM, Miller N, Wagage S, Roos DS, Hunter CA.
Departments of Biology Penn Genome Frontiers Institute.
Natural infection by Toxoplasma gondii occurs via oral ingestion of tissue cysts that rupture in the small intestine releasing zoites that infect locally before disseminating throughout the host. The studies presented here use fluorescent parasites combined with flow cytometry and multi-photon microscopy techniques to understand the events associated with parasite replication in the mucosa. At three days post-infection with tissue cysts, parasites were localized in small foci and flow cytometry revealed parasites present in macrophages, neutrophils, and monocytes in the lamina propria. By day six post-infection there were large foci of replicating parasites; however, foci unexpectedly varied in the number of villi involved and were associated with the presence of viable tachyzoites within the intestinal lumen. Consistent with the flow cytometry data, neutrophils and monocytes in the lamina propria were preferentially associated with parasite plaques. In contrast, dendritic cells comprised a small fraction of the infected immune cell population and were localized at the periphery of parasite plaques. Together, these findings reveal the formation of localized sites of parasite replication and inflammation early during infection and suggest that sustained replication of T. gondii in the gut may be a function of pathogen luminal spread.
PMID: 23460516 [PubMed - as supplied by publisher]
Tuesday, March 05, 2013
Design, synthesis, and biological activity of diaryl ether inhibitors of Toxoplasma gondii enoyl reductase
Bioorg Med Chem Lett. 2013 Feb 13. pii: S0960-894X(13)00193-5. doi: 10.1016/j.bmcl.2013.02.019. [Epub ahead of print]
Design, synthesis, and biological activity of diaryl ether inhibitors of Toxoplasma gondii enoyl reductase
Cheng G, Muench SP, Zhou Y, Afanador GA, Mui EJ, Fomovska A, Lai BS, Prigge ST, Woods S, Roberts CW, Hickman MR, Lee PJ, Leed SE, Auschwitz JM, Rice DW, McLeod R.
Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, United States.
Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan's poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the B-ring modifications have additional interactions with the strongly conserved Asn130.
PMID: 23453069 [PubMed - as supplied by publisher]
Design, synthesis, and biological activity of diaryl ether inhibitors of Toxoplasma gondii enoyl reductase
Cheng G, Muench SP, Zhou Y, Afanador GA, Mui EJ, Fomovska A, Lai BS, Prigge ST, Woods S, Roberts CW, Hickman MR, Lee PJ, Leed SE, Auschwitz JM, Rice DW, McLeod R.
Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612, United States.
Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan's poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the B-ring modifications have additional interactions with the strongly conserved Asn130.
PMID: 23453069 [PubMed - as supplied by publisher]
Toxoplasma gondii Alba proteins are involved in translational control of gene expression
J Mol Biol. 2013 Feb 20. pii: S0022-2836(13)00103-4. doi: 10.1016/j.jmb.2013.01.039. [Epub ahead of print]
Toxoplasma gondii Alba proteins are involved in translational control of gene expression
Gissot M, Walker R, Delhaye S, Alayi TD, Huot L, Hot D, Callebaut I, Schaeffer-Reiss C, Van Dorsselaer A, Tomavo S.
Center for Infection and Immunity of Lille, CNRS UMR 8204, INSERM 1019, Université Lille Nord de France, Institut Pasteur de Lille, Lille, France.
Molecular mechanisms controlling gene expression in apicomplexan parasites remain poorly understood. Here, we report the characterisation of two Toxoplasma gondii homologues of the ancient archeal Alba proteins named TgAlba1 and TgAlba2. The targeted disruption of TgAlba1 and 2 genes in both virulent type I and avirulent type II strains of T. gondii reveals that TgAlba proteins may have an important role in regulating stress response. We found that although the steady state level of Tgalba2 transcript is increased in the ΔTgalba1 null mutant parasites, the cognate TgAlba2 protein is undetectable, suggesting that TgAlba1 is required for translation of TgAlba2. Using a Tandem Affinity Purification Tag strategy combined with proteomic analyses, we provide evidence that many factors known to be involved in the translation machinery are co-purified with TgAlba1 and TgAlba2. We further performed RNA pull down and microarray analyses to show that TgAlba1 and 2 bind to more than 30 RNAs including their own transcripts. Moreover, we demonstrate that the tight translational regulation of the TgAlba2 endogenous transcript relies on the presence of both its 3'UTR and that of the TgAlba1 protein. Thus, our findings on TgAlba1 and 2 are consistent with a role in gene-specific translation.
PMID: 23454356 [PubMed - as supplied by publisher]
Toxoplasma gondii Alba proteins are involved in translational control of gene expression
Gissot M, Walker R, Delhaye S, Alayi TD, Huot L, Hot D, Callebaut I, Schaeffer-Reiss C, Van Dorsselaer A, Tomavo S.
Center for Infection and Immunity of Lille, CNRS UMR 8204, INSERM 1019, Université Lille Nord de France, Institut Pasteur de Lille, Lille, France.
Molecular mechanisms controlling gene expression in apicomplexan parasites remain poorly understood. Here, we report the characterisation of two Toxoplasma gondii homologues of the ancient archeal Alba proteins named TgAlba1 and TgAlba2. The targeted disruption of TgAlba1 and 2 genes in both virulent type I and avirulent type II strains of T. gondii reveals that TgAlba proteins may have an important role in regulating stress response. We found that although the steady state level of Tgalba2 transcript is increased in the ΔTgalba1 null mutant parasites, the cognate TgAlba2 protein is undetectable, suggesting that TgAlba1 is required for translation of TgAlba2. Using a Tandem Affinity Purification Tag strategy combined with proteomic analyses, we provide evidence that many factors known to be involved in the translation machinery are co-purified with TgAlba1 and TgAlba2. We further performed RNA pull down and microarray analyses to show that TgAlba1 and 2 bind to more than 30 RNAs including their own transcripts. Moreover, we demonstrate that the tight translational regulation of the TgAlba2 endogenous transcript relies on the presence of both its 3'UTR and that of the TgAlba1 protein. Thus, our findings on TgAlba1 and 2 are consistent with a role in gene-specific translation.
PMID: 23454356 [PubMed - as supplied by publisher]
Monday, March 04, 2013
Homology modeling of dihydrofolate reductase from T. gondii bonded to antagonists: molecular docking and molecular dynamics simulations
Mol Biosyst. 2013 Feb 28. [Epub ahead of print]
Homology modeling of dihydrofolate reductase from T. gondii bonded to antagonists: molecular docking and molecular dynamics simulations
Pacheco Homem D, Flores R, Tosqui P, de Castro Rozada T, Abicht Basso E, Gasparotto Junior A, Augusto Vicente Seixas F.
State University of Maringá, Av. Colombo 5790, Maringá, PR, Brazil. favseixas@uem.br.
The aim of this work was to solve the structure of the enzyme dihydrofolate reductase from Toxoplasma gondii (TgondiiDHFR) as a target for drug discovery on account of recent reports of parasite's growing resistance to pyrimethamine (CP6), which is the reference pharmaceutical used to treat toxoplasmosis and malaria. The tertiary structure of the protein bonded to NADP+ and CP6 was solved by homology modeling. The best output model was subjected to conjugate gradient minimization and the comparison with templates shows important replacements at the inhibitor's binding site allowing selective drug design. CP6 redocking in TgondiiDHFR shows a ΔGbinding of -8.66 kcal mol-1, higher than those found for templates Plasmodium vivax (-9.01) and P. falciparum (-8.99). Virtual screening of ligands similar to CP6 was performed using the ZINC database and docking procedures were carried out. The result indicates the substances ZINC14966516, ZINC13685962, ZINC13685929 and ZINC13686062 with a ΔGbinding of -10.57, -10.09, -9.87, and -9.76 kcal mol-1, respectively, as the best choices. NPT molecular dynamics with the complexes indicates that they remained stable along the 10 ns simulation and they dock to TgondiiDHFR by salt bridges to the Asp 30 and to nine other residues in the contact region, which makes it more difficult for single mutations to acquire resistance. The contact frequency of protein residues with ligands suggests plausible explanations for site-directed mutagenesis studies regarding CP6 resistance described previously in the literature. All results indicate that the new ligands could be tested as pyrimethamine substitutes in the treatment of toxoplasmosis, in addition to other protozoonosis diseases.
PMID: 23450239 [PubMed - as supplied by publisher]
Homology modeling of dihydrofolate reductase from T. gondii bonded to antagonists: molecular docking and molecular dynamics simulations
Pacheco Homem D, Flores R, Tosqui P, de Castro Rozada T, Abicht Basso E, Gasparotto Junior A, Augusto Vicente Seixas F.
State University of Maringá, Av. Colombo 5790, Maringá, PR, Brazil. favseixas@uem.br.
The aim of this work was to solve the structure of the enzyme dihydrofolate reductase from Toxoplasma gondii (TgondiiDHFR) as a target for drug discovery on account of recent reports of parasite's growing resistance to pyrimethamine (CP6), which is the reference pharmaceutical used to treat toxoplasmosis and malaria. The tertiary structure of the protein bonded to NADP+ and CP6 was solved by homology modeling. The best output model was subjected to conjugate gradient minimization and the comparison with templates shows important replacements at the inhibitor's binding site allowing selective drug design. CP6 redocking in TgondiiDHFR shows a ΔGbinding of -8.66 kcal mol-1, higher than those found for templates Plasmodium vivax (-9.01) and P. falciparum (-8.99). Virtual screening of ligands similar to CP6 was performed using the ZINC database and docking procedures were carried out. The result indicates the substances ZINC14966516, ZINC13685962, ZINC13685929 and ZINC13686062 with a ΔGbinding of -10.57, -10.09, -9.87, and -9.76 kcal mol-1, respectively, as the best choices. NPT molecular dynamics with the complexes indicates that they remained stable along the 10 ns simulation and they dock to TgondiiDHFR by salt bridges to the Asp 30 and to nine other residues in the contact region, which makes it more difficult for single mutations to acquire resistance. The contact frequency of protein residues with ligands suggests plausible explanations for site-directed mutagenesis studies regarding CP6 resistance described previously in the literature. All results indicate that the new ligands could be tested as pyrimethamine substitutes in the treatment of toxoplasmosis, in addition to other protozoonosis diseases.
PMID: 23450239 [PubMed - as supplied by publisher]
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