Rev Soc Bras Med Trop. 2012 Aug;45(4):485-90.
In vitro action of antiparasitic drugs, especially artesunate, against Toxoplasma gondii
Gomes TC, Andrade Júnior HF, Lescano SA, Amato-Neto V.
Laboratório de Parasitologia Médica, Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, São Paulo, SP.
INTRODUCTION:
Toxoplasmosis is usually a benign infection, except in the event of ocular, central nervous system (CNS), or congenital disease and particularly when the patient is immunocompromised. Treatment consists of drugs that frequently cause adverse effects; thus, newer, more effective drugs are needed. In this study, the possible activity of artesunate, a drug successfully being used for the treatment of malaria, on Toxoplasma gondii growth in cell culture is evaluated and compared with the action of drugs that are already being used against this parasite.
METHODS:
LLC-MK2 cells were cultivated in RPMI medium, kept in disposable plastic bottles, and incubated at 36ºC with 5% CO2. Tachyzoites of the RH strain were used. The following drugs were tested: artesunate, cotrimoxazole, pentamidine, pyrimethamine, quinine, and trimethoprim. The effects of these drugs on tachyzoites and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software.
RESULTS:
Artesunate showed a mean tachyzoite inhibitory concentration (IC50) of 0.075µM and an LLC MK2 toxicity of 2.003µM. Pyrimethamine was effective at an IC50 of 0.482µM and a toxicity of 11.178µM. Trimethoprim alone was effective against the in vitro parasite. Cotrimoxazole also was effective against the parasite but at higher concentrations than those observed for artesunate and pyrimethamine. Pentamidine and quinine had no inhibitory effect over tachyzoites.
CONCLUSIONS:
Artesunate is proven in vitro to be a useful alternative for the treatment of toxoplasmosis, implying a subsequent in vivo effect and suggesting the mechanism of this drug against the parasite.
PMID: 22930046 [PubMed - in process]
Friday, August 31, 2012
Length of the Isoprenoid Product of the Bifunctional Toxoplasma gondii Farnesyl-diphosphate Synthase
Biochemistry. 2012 Aug 29. [Epub ahead of print]
The N-terminus and the Chain-length Determination (CLD) Domain Play a Role in the Length of the Isoprenoid Product of the Bifunctional Toxoplasma gondii Farnesyl-diphosphate Synthase
Li ZH, Cintrón R, Koon NA, Moreno SN.
Toxoplasma gondii possesses a bifunctional farnesyl diphosphate (FPP)/geranylgeranyl diphosphate (GGPP) synthase (TgFPPS) that synthesizes C15 and C20 isoprenoid diphosphates from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This enzyme has a unique arrangement of the 4th and 5th amino acid upstream to the First Aspartic Rich Domain (FARM) where the 4th amino acid is aromatic and the 5th is a cysteine. We mutated these amino acids converting the enzyme to an absolute FPPS by changing the cysteine to a tyrosine. The enzyme could be converted to an absolute GGPPS by changing both the 4th and 5th amino acids to alanines. We also constructed four mutated TgFPPSs whose regions around the first aspartate-rich motif were replaced with the corresponding regions of FPP synthases from Arabidopsis thaliana or Saccharomyces cerevisiae or with the corresponding regions of GGPP synthases from Homo sapiens or S. cerevisiae. We determined that the presence of a cysteine at the 4th position is essential for the TgFPPS bifunctionality. We also found that the length of the N-terminal domain has a role in determining the specificity and the length of the isoprenoid product. Phylogenetic analysis supports the grouping of this enzyme with other Type I FPPSs but the biochemical data indicates that TgFPPS has unique characteristics that differentiate it from mammalian FPPSs and GGPPSs and is therefore an important drug target.
PMID: 22931372
The N-terminus and the Chain-length Determination (CLD) Domain Play a Role in the Length of the Isoprenoid Product of the Bifunctional Toxoplasma gondii Farnesyl-diphosphate Synthase
Li ZH, Cintrón R, Koon NA, Moreno SN.
Toxoplasma gondii possesses a bifunctional farnesyl diphosphate (FPP)/geranylgeranyl diphosphate (GGPP) synthase (TgFPPS) that synthesizes C15 and C20 isoprenoid diphosphates from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This enzyme has a unique arrangement of the 4th and 5th amino acid upstream to the First Aspartic Rich Domain (FARM) where the 4th amino acid is aromatic and the 5th is a cysteine. We mutated these amino acids converting the enzyme to an absolute FPPS by changing the cysteine to a tyrosine. The enzyme could be converted to an absolute GGPPS by changing both the 4th and 5th amino acids to alanines. We also constructed four mutated TgFPPSs whose regions around the first aspartate-rich motif were replaced with the corresponding regions of FPP synthases from Arabidopsis thaliana or Saccharomyces cerevisiae or with the corresponding regions of GGPP synthases from Homo sapiens or S. cerevisiae. We determined that the presence of a cysteine at the 4th position is essential for the TgFPPS bifunctionality. We also found that the length of the N-terminal domain has a role in determining the specificity and the length of the isoprenoid product. Phylogenetic analysis supports the grouping of this enzyme with other Type I FPPSs but the biochemical data indicates that TgFPPS has unique characteristics that differentiate it from mammalian FPPSs and GGPPSs and is therefore an important drug target.
PMID: 22931372
Tuesday, August 28, 2012
Guanylate-binding proteins: niche recruiters for antimicrobial effectors
Immunity. 2012 Aug 24;37(2):191-3.
Guanylate-binding proteins: niche recruiters for antimicrobial effectors
Dupont CD, Hunter CA.
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 380 South University Avenue, Philadelphia, PA 19104, USA.
There are fundamental questions regarding how IFN-γ activates host cells to eliminate intracellular pathogens. In this issue of Immunity, Yamamoto et al. (2012) demonstrate a critical role for the p65 guanylate-binding proteins (GBPs) in this process during infection with Toxoplasma gondii.
PMID: 22921115 [PubMed - in process]
Guanylate-binding proteins: niche recruiters for antimicrobial effectors
Dupont CD, Hunter CA.
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 380 South University Avenue, Philadelphia, PA 19104, USA.
There are fundamental questions regarding how IFN-γ activates host cells to eliminate intracellular pathogens. In this issue of Immunity, Yamamoto et al. (2012) demonstrate a critical role for the p65 guanylate-binding proteins (GBPs) in this process during infection with Toxoplasma gondii.
PMID: 22921115 [PubMed - in process]
Thursday, August 23, 2012
Toxoplasma Co-opts Host Cells It Does Not Invade
PLoS Pathog. 2012 Jul;8(7):e1002825. Epub 2012 Jul 26.
Toxoplasma Co-opts Host Cells It Does Not Invade
Koshy AA, Dietrich HK, Christian DA, Melehani JH, Shastri AJ, Hunter CA, Boothroyd JC.
Department of Medicine (Infectious Disease), Stanford University School of Medicine, Stanford, California, United States of America.
Like many intracellular microbes, the protozoan parasite Toxoplasma gondii injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that in vitro the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting Toxoplasma-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where Toxoplasma encysts and persists. This phenomenon has important implications for how Toxoplasma globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large.
PMID: 22910631 [PubMed - in process]
Toxoplasma Co-opts Host Cells It Does Not Invade
Koshy AA, Dietrich HK, Christian DA, Melehani JH, Shastri AJ, Hunter CA, Boothroyd JC.
Department of Medicine (Infectious Disease), Stanford University School of Medicine, Stanford, California, United States of America.
Like many intracellular microbes, the protozoan parasite Toxoplasma gondii injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that in vitro the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting Toxoplasma-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where Toxoplasma encysts and persists. This phenomenon has important implications for how Toxoplasma globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large.
PMID: 22910631 [PubMed - in process]
Foxp4 is dispensable for T cell development, but required for robust recall responses
PLoS One. 2012;7(8):e42273. Epub 2012 Aug 13.
Foxp4 is dispensable for T cell development, but required for robust recall responses
Wiehagen KR, Corbo-Rodgers E, Li S, Staub ES, Hunter CA, Morrisey EE, Maltzman JS.
Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
Transcription factors regulate T cell fates at every stage of development and differentiation. Members of the Foxp family of forkhead transcription factors are essential for normal T lineage development; Foxp3 is required for T regulatory cell generation and function, and Foxp1 is necessary for generation and maintenance of naïve T cells. Foxp4, an additional member of the Foxp family, is highly homologous to Foxp1 and has been shown to dimerize with other Foxp proteins. We report the initial characterization of Foxp4 in T lymphocytes. Foxp4 is expressed in both thymocytes and peripheral CD4(+) and CD8(+) T cells. We used a CD4Cre mediated approach to evaluate the cell autonomous role for Foxp4 in murine T lymphocytes. T cell development, peripheral cellularity and cell surface phenotype are normal in the absence of Foxp4. Furthermore, Foxp3(+) T regulatory cells develop normally in Foxp4 deficient animals and naïve Foxp4 deficient CD4 T cells can differentiate to inducible T regulatory cells in vitro. In wild-type T cells, expression of Foxp4 increases following activation, but deletion of Foxp4 does not affect T cell proliferative responses or in vitro effector T cell differentiation. In vivo, despite effective control of Toxoplasma gondii and acute lymphocytic choriomeningitis virus infections, effector cytokine production during antigen specific recall responses are reduced in the absence of Foxp4. We conclude that Foxp4 is dispensable for T cell development, but necessary for normal T cell cytokine recall responses to antigen following pathogenic infection.
PMID: 22912696 [PubMed - in process]
Foxp4 is dispensable for T cell development, but required for robust recall responses
Wiehagen KR, Corbo-Rodgers E, Li S, Staub ES, Hunter CA, Morrisey EE, Maltzman JS.
Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
Transcription factors regulate T cell fates at every stage of development and differentiation. Members of the Foxp family of forkhead transcription factors are essential for normal T lineage development; Foxp3 is required for T regulatory cell generation and function, and Foxp1 is necessary for generation and maintenance of naïve T cells. Foxp4, an additional member of the Foxp family, is highly homologous to Foxp1 and has been shown to dimerize with other Foxp proteins. We report the initial characterization of Foxp4 in T lymphocytes. Foxp4 is expressed in both thymocytes and peripheral CD4(+) and CD8(+) T cells. We used a CD4Cre mediated approach to evaluate the cell autonomous role for Foxp4 in murine T lymphocytes. T cell development, peripheral cellularity and cell surface phenotype are normal in the absence of Foxp4. Furthermore, Foxp3(+) T regulatory cells develop normally in Foxp4 deficient animals and naïve Foxp4 deficient CD4 T cells can differentiate to inducible T regulatory cells in vitro. In wild-type T cells, expression of Foxp4 increases following activation, but deletion of Foxp4 does not affect T cell proliferative responses or in vitro effector T cell differentiation. In vivo, despite effective control of Toxoplasma gondii and acute lymphocytic choriomeningitis virus infections, effector cytokine production during antigen specific recall responses are reduced in the absence of Foxp4. We conclude that Foxp4 is dispensable for T cell development, but necessary for normal T cell cytokine recall responses to antigen following pathogenic infection.
PMID: 22912696 [PubMed - in process]
Wednesday, August 22, 2012
The arginine-rich N-terminal domain of ROP18 is necessary for vacuole targeting and virulence of Toxoplasma gondii
Cell Microbiol. 2012 Aug 20. doi: 10.1111/cmi.12022. [Epub ahead of print]
The arginine-rich N-terminal domain of ROP18 is necessary for vacuole targeting and virulence of Toxoplasma gondii
Fentress SJ, Steinfeldt T, Howard JC, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis MO, USA
Toxoplasma gondii uses specialized secretory organelles called rhoptries to deliver virulence determinants into the host cell during parasite invasion. One such determinant called rhoptry protein 18 (ROP18) is a polymorphic serine/threonine kinase that phosphorylates host targets to modulate acute virulence. Following secretion into the host cell, ROP18 traffics to the parasitophorous vacuole membrane (PVM) where it is tethered to the cytosolic face of this host-pathogen interface. However, the functional consequences of PVM association are not known. In this report, we show that ROP18 mutants altered in an arginine-rich domain upstream of the kinase domain fail to associate to the PVM following secretion from rhoptries. During infection, host cells up-regulate immunity-related GTPases that localize to and destroy the PVM surrounding the parasites. ROP18 disarms this host innate immune pathway by phosphorylating IRGs in a critical GTPase domain and preventing loading on the PVM. Vacuole-targeting mutants of ROP18 failed to phosphorylate Irga6 and were unable to divert IRGs from the PVM, despite retaining intrinsic kinase activity. As a consequence, these mutants were avirulent in a mouse model of acute toxoplasmosis. Thus, the association of ROP18 with the PVM, mediated by its N-terminal arginine-rich domain, is critical to its function as a virulence determinant.
PMID: 22906355 [PubMed - as supplied by publisher]
The arginine-rich N-terminal domain of ROP18 is necessary for vacuole targeting and virulence of Toxoplasma gondii
Fentress SJ, Steinfeldt T, Howard JC, Sibley LD.
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis MO, USA
Toxoplasma gondii uses specialized secretory organelles called rhoptries to deliver virulence determinants into the host cell during parasite invasion. One such determinant called rhoptry protein 18 (ROP18) is a polymorphic serine/threonine kinase that phosphorylates host targets to modulate acute virulence. Following secretion into the host cell, ROP18 traffics to the parasitophorous vacuole membrane (PVM) where it is tethered to the cytosolic face of this host-pathogen interface. However, the functional consequences of PVM association are not known. In this report, we show that ROP18 mutants altered in an arginine-rich domain upstream of the kinase domain fail to associate to the PVM following secretion from rhoptries. During infection, host cells up-regulate immunity-related GTPases that localize to and destroy the PVM surrounding the parasites. ROP18 disarms this host innate immune pathway by phosphorylating IRGs in a critical GTPase domain and preventing loading on the PVM. Vacuole-targeting mutants of ROP18 failed to phosphorylate Irga6 and were unable to divert IRGs from the PVM, despite retaining intrinsic kinase activity. As a consequence, these mutants were avirulent in a mouse model of acute toxoplasmosis. Thus, the association of ROP18 with the PVM, mediated by its N-terminal arginine-rich domain, is critical to its function as a virulence determinant.
PMID: 22906355 [PubMed - as supplied by publisher]
Sink or swim: lipid rafts in parasite pathogenesis
Trends Parasitol. 2012 Aug 17. [Epub ahead of print]
Sink or swim: lipid rafts in parasite pathogenesis
Goldston AM, Powell RR, Temesvari LA.
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA.
Lipid rafts, sterol- and sphingolipid-rich membrane microdomains, have been extensively studied in mammalian cells. Recently, lipid rafts have been shown to control virulence in a variety of parasites including Entamoeba histolytica, Giardia intestinalis, Leishmania spp., Plasmodium spp., Toxoplasma gondii, and Trypanosoma spp. Parasite rafts regulate adhesion to host and invasion, and parasite adhesion molecules often localize to rafts. Parasite rafts also control vesicle trafficking, motility, and cell signaling. Parasites disrupt host cell rafts; the dysregulation of host membrane function facilitates the establishment of infection and evasion of the host immune system. Discerning the mechanism by which lipid rafts regulate parasite pathogenesis is essential to our understanding of virulence. Such insight may guide the development of new drugs for disease management.
PMID: 22906512 [PubMed - as supplied by publisher]
Sink or swim: lipid rafts in parasite pathogenesis
Goldston AM, Powell RR, Temesvari LA.
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA.
Lipid rafts, sterol- and sphingolipid-rich membrane microdomains, have been extensively studied in mammalian cells. Recently, lipid rafts have been shown to control virulence in a variety of parasites including Entamoeba histolytica, Giardia intestinalis, Leishmania spp., Plasmodium spp., Toxoplasma gondii, and Trypanosoma spp. Parasite rafts regulate adhesion to host and invasion, and parasite adhesion molecules often localize to rafts. Parasite rafts also control vesicle trafficking, motility, and cell signaling. Parasites disrupt host cell rafts; the dysregulation of host membrane function facilitates the establishment of infection and evasion of the host immune system. Discerning the mechanism by which lipid rafts regulate parasite pathogenesis is essential to our understanding of virulence. Such insight may guide the development of new drugs for disease management.
PMID: 22906512 [PubMed - as supplied by publisher]
Vaccines against Toxoplasma gondii: Status, challenges and future direction
Hum Vaccin Immunother. 2012 Sep 1;8(9). [Epub ahead of print]
Vaccines against Toxoplasma gondii: Status, challenges and future direction
Liu Q, Singla L, Zhou H.
Department of Parasitology, Shandong University School of Medicine; Jinan, Shandong, China.
Toxoplasma gondii is a ubiquitous protozoan parasite that can infect a wide range of animals including humans. This single known species in the genus Toxoplasma is considered as one of the most successful eukaryotic pathogens in terms of the number of host species and percentage of animals infected worldwide. Effective vaccines may contribute toward preventing and controlling the spread of toxoplasmosis. The present communication addresses the current status of development of vaccines against T. gondii. Further discussion is made on the difficulties along with challenges, such as vaccine construct, mode of vaccine administration and standardization of immunization evaluation. Finally suggestions are made on possible directions for future research for development of vaccines against T. gondii.
PMID: 22906945 [PubMed - as supplied by publisher]
Vaccines against Toxoplasma gondii: Status, challenges and future direction
Liu Q, Singla L, Zhou H.
Department of Parasitology, Shandong University School of Medicine; Jinan, Shandong, China.
Toxoplasma gondii is a ubiquitous protozoan parasite that can infect a wide range of animals including humans. This single known species in the genus Toxoplasma is considered as one of the most successful eukaryotic pathogens in terms of the number of host species and percentage of animals infected worldwide. Effective vaccines may contribute toward preventing and controlling the spread of toxoplasmosis. The present communication addresses the current status of development of vaccines against T. gondii. Further discussion is made on the difficulties along with challenges, such as vaccine construct, mode of vaccine administration and standardization of immunization evaluation. Finally suggestions are made on possible directions for future research for development of vaccines against T. gondii.
PMID: 22906945 [PubMed - as supplied by publisher]
A focused small molecule screen identifies 14 compounds with distinct effects on Toxoplasma gondii
Antimicrob Agents Chemother. 2012 Aug 20. [Epub ahead of print]
A focused small molecule screen identifies 14 compounds with distinct effects on Toxoplasma gondii
Kamau ET, Srinivasan AR, Brown MJ, Fair MG, Caraher EC, Boyle JP.
University of Pittsburgh, Department of Biological Sciences, Pittsburgh, PA. 15260.
Toxoplasma gondii is a globally ubiquitous pathogen that can cause severe disease in immunocompromised humans and the developing fetus. Given the proven role of Toxoplasma secreted kinases in the interaction of Toxoplasma with its host cell, identification of novel kinase inhibitors could precipitate the development of new anti-Toxoplasma drugs and define new pathways important for parasite survival. We selected a small (527), but diverse, set of putative kinase inhibitors and screened them for effects on the growth of Toxoplasma in vitro. We identified and validated 14 noncytotoxic compounds, all of which have EC50s in the nanomolar to micromolar range. We further characterized 8 of these compounds, 4 inhibitors and 4 enhancers, by determining their effects on parasite motility, invasion, and the likely cellular target (parasite or host cell). Only two compounds had an effect on parasite motility and invasion. All the inhibitors appear to target the parasite and interestingly, two of the enhancers appear to rather target the host cell, suggesting modulation of host cell pathways beneficial for parasite growth. For the four inhibitors, we also tested their efficacy in a mouse model where one compound proved potent. Overall these 14 compounds represent a new and diverse set of small molecules that are likely targeting distinct parasite and host cell pathways. Future work will aim to characterize their molecular targets in both the host and parasite.
PMID: 22908155 [PubMed - as supplied by publisher]
A focused small molecule screen identifies 14 compounds with distinct effects on Toxoplasma gondii
Kamau ET, Srinivasan AR, Brown MJ, Fair MG, Caraher EC, Boyle JP.
University of Pittsburgh, Department of Biological Sciences, Pittsburgh, PA. 15260.
Toxoplasma gondii is a globally ubiquitous pathogen that can cause severe disease in immunocompromised humans and the developing fetus. Given the proven role of Toxoplasma secreted kinases in the interaction of Toxoplasma with its host cell, identification of novel kinase inhibitors could precipitate the development of new anti-Toxoplasma drugs and define new pathways important for parasite survival. We selected a small (527), but diverse, set of putative kinase inhibitors and screened them for effects on the growth of Toxoplasma in vitro. We identified and validated 14 noncytotoxic compounds, all of which have EC50s in the nanomolar to micromolar range. We further characterized 8 of these compounds, 4 inhibitors and 4 enhancers, by determining their effects on parasite motility, invasion, and the likely cellular target (parasite or host cell). Only two compounds had an effect on parasite motility and invasion. All the inhibitors appear to target the parasite and interestingly, two of the enhancers appear to rather target the host cell, suggesting modulation of host cell pathways beneficial for parasite growth. For the four inhibitors, we also tested their efficacy in a mouse model where one compound proved potent. Overall these 14 compounds represent a new and diverse set of small molecules that are likely targeting distinct parasite and host cell pathways. Future work will aim to characterize their molecular targets in both the host and parasite.
PMID: 22908155 [PubMed - as supplied by publisher]
Tuesday, August 21, 2012
Gene transfection of Toxoplasma gondii using PEI/DNA polyplexes
J Microbiol Methods. 2012 Aug 10. [Epub ahead of print]
Gene transfection of Toxoplasma gondii using PEI/DNA polyplexes
Salehi N, Peng CA
The purpose of this study was to explore the potential of using cationic polyethylenimine (PEI) to deliver green fluorescent protein (GFP) to protozoan parasite Toxoplasma gondii. PEI/DNA polyplexes were formed using branched PEI and pEGFP-N1 plasmid with various N/P ratios that ranged from 5 to 50. With the increment of N/P ratio, the average size of formed PEI/DNA polyplexes determined by dynamic light scattering analysis decreased from 306 to 203nm, while the surface charge of polyplexes obtained by zeta potential measurements increased from 20.2 to 36.7mV. Gene transfection efficiency modulated by N/P ratio was determined, indicating PEI/DNA polyplexes were capable of transfecting parasites. The maximal GFP expression was observed 8h post-transfection using N/P ratio of 30. To demonstrate the infectivity and potential use of GFP-expressing T. gondii, transfected parasites were inoculated to the monolayer of human foreskin fibroblast (HFF) cells. GFP-expressing tachyzoites were observed in intracellular milieu of the infected HFF cells one day after the infection. After 12-day culture, the bradyzoites expressing GFP within cysts were clearly visualized extracellularly. Our results revealed that PEI can be harnessed as an effective and inexpensive reagent to construct GFP-expressing T. gondii which has potential uses such as the study of interconversion stages and antimicrobial drug screening.
PMID: 22902310 [PubMed - as supplied by publisher]
Gene transfection of Toxoplasma gondii using PEI/DNA polyplexes
Salehi N, Peng CA
The purpose of this study was to explore the potential of using cationic polyethylenimine (PEI) to deliver green fluorescent protein (GFP) to protozoan parasite Toxoplasma gondii. PEI/DNA polyplexes were formed using branched PEI and pEGFP-N1 plasmid with various N/P ratios that ranged from 5 to 50. With the increment of N/P ratio, the average size of formed PEI/DNA polyplexes determined by dynamic light scattering analysis decreased from 306 to 203nm, while the surface charge of polyplexes obtained by zeta potential measurements increased from 20.2 to 36.7mV. Gene transfection efficiency modulated by N/P ratio was determined, indicating PEI/DNA polyplexes were capable of transfecting parasites. The maximal GFP expression was observed 8h post-transfection using N/P ratio of 30. To demonstrate the infectivity and potential use of GFP-expressing T. gondii, transfected parasites were inoculated to the monolayer of human foreskin fibroblast (HFF) cells. GFP-expressing tachyzoites were observed in intracellular milieu of the infected HFF cells one day after the infection. After 12-day culture, the bradyzoites expressing GFP within cysts were clearly visualized extracellularly. Our results revealed that PEI can be harnessed as an effective and inexpensive reagent to construct GFP-expressing T. gondii which has potential uses such as the study of interconversion stages and antimicrobial drug screening.
PMID: 22902310 [PubMed - as supplied by publisher]
Friday, August 17, 2012
Microneme protein 5 regulates the activity of Toxoplasma subtilisin 1 by mimicking a subtilisin prodomain
J Biol Chem. 2012 Aug 15. [Epub ahead of print]
Microneme protein 5 regulates the activity of Toxoplasma subtilisin 1 by mimicking a subtilisin prodomain
Saouros S, Dou Z, Henry M, Marchant J, Carruthers VB, Matthews S.
Imperial College London, United Kingdom
Toxoplasma gondii is the model parasite of the phylum Apicomplexa, which contains obligate intracellular parasites of medical and veterinary importance. Apicomplexans invade host cells by a multi-step process involving the secretion of adhesive microneme protein (MIC) complexes. The subtilisin protease TgSUB1 trims several MICs on the parasite surface to activate gliding motility and host invasion. Although a previous study showed that expression of the secretory protein TgMIC5 suppresses TgSUB1 activity, the mechanism was unknown. Herein, we solve the three dimensional structure of TgMIC5 by Nuclear Magnetic Resonance (NMR), revealing that it mimics a subtilisin prodomain including a flexible C-terminal peptide that may insert into the subtilisin active site. We show that TgMIC5 is an almost fifty-fold more potent inhibitor of TgSUB1 activity than the small molecule inhibitor N-[N-(N-Acetyl-L-leucyl)-L-leucyl]-L-norleucine (ALLN). Moreover, we demonstrate that TgMIC5 is retained on the parasite plasma membrane via its physical interaction with the membrane anchored TgSUB1.
PMID: 22896704 [PubMed - as supplied by publisher]
Microneme protein 5 regulates the activity of Toxoplasma subtilisin 1 by mimicking a subtilisin prodomain
Saouros S, Dou Z, Henry M, Marchant J, Carruthers VB, Matthews S.
Imperial College London, United Kingdom
Toxoplasma gondii is the model parasite of the phylum Apicomplexa, which contains obligate intracellular parasites of medical and veterinary importance. Apicomplexans invade host cells by a multi-step process involving the secretion of adhesive microneme protein (MIC) complexes. The subtilisin protease TgSUB1 trims several MICs on the parasite surface to activate gliding motility and host invasion. Although a previous study showed that expression of the secretory protein TgMIC5 suppresses TgSUB1 activity, the mechanism was unknown. Herein, we solve the three dimensional structure of TgMIC5 by Nuclear Magnetic Resonance (NMR), revealing that it mimics a subtilisin prodomain including a flexible C-terminal peptide that may insert into the subtilisin active site. We show that TgMIC5 is an almost fifty-fold more potent inhibitor of TgSUB1 activity than the small molecule inhibitor N-[N-(N-Acetyl-L-leucyl)-L-leucyl]-L-norleucine (ALLN). Moreover, we demonstrate that TgMIC5 is retained on the parasite plasma membrane via its physical interaction with the membrane anchored TgSUB1.
PMID: 22896704 [PubMed - as supplied by publisher]
Trefoil Factor 2 Negatively Regulates Type 1 Immunity against Toxoplasma gondii
J Immunol. 2012 Aug 15. [Epub ahead of print]
Trefoil Factor 2 Negatively Regulates Type 1 Immunity against Toxoplasma gondii
McBerry C, Egan CE, Rani R, Yang Y, Wu D, Boespflug N, Boon L, Butcher B, Mirpuri J, Hogan SP, Denkers EY, Aliberti J, Herbert DR.
Division of Molecular Immunology, Cincinnati Children's Research Foundation, Cincinnati, OH 45229
IL-12-mediated type 1 inflammation confers host protection against the parasitic protozoan Toxoplasma gondii. However, production of IFN-γ, another type 1 inflammatory cytokine, also drives lethality from excessive injury to the intestinal epithelium. As mechanisms that restore epithelial barrier function following infection remain poorly understood, this study investigated the role of trefoil factor 2 (TFF2), a well-established regulator of mucosal tissue repair. Paradoxically, TFF2 antagonized IL-12 release from dendritic cells (DCs) and macrophages, which protected TFF2-deficient (TFF2(-/-)) mice from T. gondii pathogenesis. Dysregulated intestinal homeostasis in naive TFF2(-/-) mice correlated with increased IL-12/23p40 levels and enhanced T cell recruitment at baseline. Infected TFF2(-/-) mice displayed low rates of parasite replication and reduced gut immunopathology, whereas wild-type (WT) mice experienced disseminated infection and lethal ileitis. p38 MAPK activation and IL-12p70 production was more robust from TFF2(-/-)CD8(+) DC compared with WT CD8(+) DC and treatment of WT DC with rTFF2 suppressed TLR-induced IL-12/23p40 production. Neutralization of IFN-γ and IL-12 in TFF2(-/-) animals abrogated resistance shown by enhanced parasite replication and infection-induced morbidity. Hence, TFF2 regulated intestinal barrier function and type 1 cytokine release from myeloid phagocytes, which dictated the outcome of oral T. gondii infection in mice.
PMID: 22896633 [PubMed - as supplied by publisher]
Trefoil Factor 2 Negatively Regulates Type 1 Immunity against Toxoplasma gondii
McBerry C, Egan CE, Rani R, Yang Y, Wu D, Boespflug N, Boon L, Butcher B, Mirpuri J, Hogan SP, Denkers EY, Aliberti J, Herbert DR.
Division of Molecular Immunology, Cincinnati Children's Research Foundation, Cincinnati, OH 45229
IL-12-mediated type 1 inflammation confers host protection against the parasitic protozoan Toxoplasma gondii. However, production of IFN-γ, another type 1 inflammatory cytokine, also drives lethality from excessive injury to the intestinal epithelium. As mechanisms that restore epithelial barrier function following infection remain poorly understood, this study investigated the role of trefoil factor 2 (TFF2), a well-established regulator of mucosal tissue repair. Paradoxically, TFF2 antagonized IL-12 release from dendritic cells (DCs) and macrophages, which protected TFF2-deficient (TFF2(-/-)) mice from T. gondii pathogenesis. Dysregulated intestinal homeostasis in naive TFF2(-/-) mice correlated with increased IL-12/23p40 levels and enhanced T cell recruitment at baseline. Infected TFF2(-/-) mice displayed low rates of parasite replication and reduced gut immunopathology, whereas wild-type (WT) mice experienced disseminated infection and lethal ileitis. p38 MAPK activation and IL-12p70 production was more robust from TFF2(-/-)CD8(+) DC compared with WT CD8(+) DC and treatment of WT DC with rTFF2 suppressed TLR-induced IL-12/23p40 production. Neutralization of IFN-γ and IL-12 in TFF2(-/-) animals abrogated resistance shown by enhanced parasite replication and infection-induced morbidity. Hence, TFF2 regulated intestinal barrier function and type 1 cytokine release from myeloid phagocytes, which dictated the outcome of oral T. gondii infection in mice.
PMID: 22896633 [PubMed - as supplied by publisher]
Thursday, August 16, 2012
Recombinant ROP2, ROP4, GRA4 and SAG1 antigen-cocktails as possible tools for immunoprophylaxis of toxoplasmosis: What's next?
Bioengineered. 2012 Nov 1;3(6). [Epub ahead of print]
Recombinant ROP2, ROP4, GRA4 and SAG1 antigen-cocktails as possible tools for immunoprophylaxis of toxoplasmosis: What's next?
Dziadek B, Brzostek A.
Department of Immunoparasitology; University of Lodz; Lodz, Poland.
Toxoplasmosis is a globally distributed foodborne zoonosis caused by a protozoan parasite Toxoplasma gondii. Usually asymptomatic in immunocompetent humans, toxoplasmosis is a serious clinical and veterinary problem often leading to lethal damage in an infected host. In order to overcome the exceptionally strong clinical and socio-economic impact of Toxoplasma infection, the construction of an effective vaccine inducing full immunoprotection against the parasite is an urgent issue. In the last two decades many live attenuated, subunit and DNA-based vaccines against toxoplasmosis have been studied, however only partial protection conferred by vaccination against chronic as well as acute infection has been achieved. Among various immunization strategies, no viable subunit vaccines based on recombinant secretory (ROP2, ROP4, GRA4) and surface (SAG1) T. gondii proteins have been found as attractive tools for further studies. This is due to their high, but still partial, protective efficacy correlated with the induction of cellular and humoral immune responses.
PMID: 22892593 [PubMed - as supplied by publisher]
Recombinant ROP2, ROP4, GRA4 and SAG1 antigen-cocktails as possible tools for immunoprophylaxis of toxoplasmosis: What's next?
Dziadek B, Brzostek A.
Department of Immunoparasitology; University of Lodz; Lodz, Poland.
Toxoplasmosis is a globally distributed foodborne zoonosis caused by a protozoan parasite Toxoplasma gondii. Usually asymptomatic in immunocompetent humans, toxoplasmosis is a serious clinical and veterinary problem often leading to lethal damage in an infected host. In order to overcome the exceptionally strong clinical and socio-economic impact of Toxoplasma infection, the construction of an effective vaccine inducing full immunoprotection against the parasite is an urgent issue. In the last two decades many live attenuated, subunit and DNA-based vaccines against toxoplasmosis have been studied, however only partial protection conferred by vaccination against chronic as well as acute infection has been achieved. Among various immunization strategies, no viable subunit vaccines based on recombinant secretory (ROP2, ROP4, GRA4) and surface (SAG1) T. gondii proteins have been found as attractive tools for further studies. This is due to their high, but still partial, protective efficacy correlated with the induction of cellular and humoral immune responses.
PMID: 22892593 [PubMed - as supplied by publisher]
Molecular target validation, antimicrobial delivery, and potential treatment of Toxoplasma gondii infection
Proc Natl Acad Sci U S A. 2012 Aug 13. [Epub ahead of print]
Molecular target validation, antimicrobial delivery, and potential treatment of Toxoplasma gondii infectio
Lai BS, Witola WH, El Bissati K, Zhou Y, Mui E, Fomovska A, McLeod R.
Department of Surgery, University of Chicago, Chicago, IL 60637.
Toxoplasma gondii persistently infects over two billion people worldwide. It can cause substantial morbidity and mortality. Existing treatments have associated toxicities and hypersensitivity and do not eliminate encysted bradyzoites that recrudesce. New, improved medicines are needed. Transductive peptides carry small molecule cargos across multiple membranes to enter intracellular tachyzoites and encysted bradyzoites. They also carry cargos into retina when applied topically to eyes, and cross blood brain barrier when administered intravenously. Phosphorodiamidate morpholino oligomers (PMO) inhibit gene expression in a sequence-specific manner. Herein, effect of transductive peptide conjugated PMO (PPMO) on tachyzoite protein expression and replication in vitro and in vivo was studied. Initially, sequence-specific PPMO successfully reduced transfected T. gondii's fluorescence and luminescence. PPMO directed against T. gondii's dihydrofolate reductase (DHFR), an enzyme necessary for folate synthesis, limited tachyzoite replication. Rescue with exogenous folate demonstrated DHFR PPMO's specificity. PPMO directed against enoyl-ACP reductase (ENR), an enzyme of type II fatty acid synthesis that is structurally distinct in T. gondii from ENR in mammalian cells was investigated. PPMO directed against plant-like Apetela 2 (AP2) domain transcription factor XI-3 (AP2XI-3), not present in human cells, was characterized. ENR and AP2XI-3 PPMO each restricted intracellular parasite replication validating these molecular targets in tachyzoites. DHFR-specific PPMO administered to infected mice diminished parasite burden. Thus, these antisense oligomers are a versatile approach to validate T. gondii molecular targets, reduce essential T. gondii proteins in vitro and in vivo, and have potential for development as curative medicines.
PMID: 22891343 [PubMed - as supplied by publisher]
Molecular target validation, antimicrobial delivery, and potential treatment of Toxoplasma gondii infectio
Lai BS, Witola WH, El Bissati K, Zhou Y, Mui E, Fomovska A, McLeod R.
Department of Surgery, University of Chicago, Chicago, IL 60637.
Toxoplasma gondii persistently infects over two billion people worldwide. It can cause substantial morbidity and mortality. Existing treatments have associated toxicities and hypersensitivity and do not eliminate encysted bradyzoites that recrudesce. New, improved medicines are needed. Transductive peptides carry small molecule cargos across multiple membranes to enter intracellular tachyzoites and encysted bradyzoites. They also carry cargos into retina when applied topically to eyes, and cross blood brain barrier when administered intravenously. Phosphorodiamidate morpholino oligomers (PMO) inhibit gene expression in a sequence-specific manner. Herein, effect of transductive peptide conjugated PMO (PPMO) on tachyzoite protein expression and replication in vitro and in vivo was studied. Initially, sequence-specific PPMO successfully reduced transfected T. gondii's fluorescence and luminescence. PPMO directed against T. gondii's dihydrofolate reductase (DHFR), an enzyme necessary for folate synthesis, limited tachyzoite replication. Rescue with exogenous folate demonstrated DHFR PPMO's specificity. PPMO directed against enoyl-ACP reductase (ENR), an enzyme of type II fatty acid synthesis that is structurally distinct in T. gondii from ENR in mammalian cells was investigated. PPMO directed against plant-like Apetela 2 (AP2) domain transcription factor XI-3 (AP2XI-3), not present in human cells, was characterized. ENR and AP2XI-3 PPMO each restricted intracellular parasite replication validating these molecular targets in tachyzoites. DHFR-specific PPMO administered to infected mice diminished parasite burden. Thus, these antisense oligomers are a versatile approach to validate T. gondii molecular targets, reduce essential T. gondii proteins in vitro and in vivo, and have potential for development as curative medicines.
PMID: 22891343 [PubMed - as supplied by publisher]
Tuesday, August 14, 2012
A mathematical model for within-host Toxoplasma gondii invasion dynamics
Math Biosci Eng. 2012 Jul 1;9(3):647-62. doi: 10.3934/mbe.2012.9.647.
A mathematical model for within-host Toxoplasma gondii invasion dynamics
Sullivan A, Agusto F, Bewick S, Su C, Lenhart S, Zhao X.
Department of Mechanical, Aerospace, and Biomedical Engineering, University of Tennessee, Knoxville, TN 37996, United States. asulli17@utk.edu.
Toxoplasma gondii (T. gondii) is a protozoan parasite that infects a wide range of intermediate hosts, including all mammals and birds. Up to 20% of the human population in the US and 30% in the world are chronically infected. This paper presents a mathematical model to describe intra-host dynamics of T. gondii infection. The model considers the invasion process, egress kinetics, interconversion between fast-replicating tachyzoite stage and slowly replicating bradyzoite stage, as well as the host's immune response. Analytical and numerical studies of the model can help to understand the influences of various parameters to the transient and steady-state dynamics of the disease infection.
PMID: 22881030
A mathematical model for within-host Toxoplasma gondii invasion dynamics
Sullivan A, Agusto F, Bewick S, Su C, Lenhart S, Zhao X.
Department of Mechanical, Aerospace, and Biomedical Engineering, University of Tennessee, Knoxville, TN 37996, United States. asulli17@utk.edu.
Toxoplasma gondii (T. gondii) is a protozoan parasite that infects a wide range of intermediate hosts, including all mammals and birds. Up to 20% of the human population in the US and 30% in the world are chronically infected. This paper presents a mathematical model to describe intra-host dynamics of T. gondii infection. The model considers the invasion process, egress kinetics, interconversion between fast-replicating tachyzoite stage and slowly replicating bradyzoite stage, as well as the host's immune response. Analytical and numerical studies of the model can help to understand the influences of various parameters to the transient and steady-state dynamics of the disease infection.
PMID: 22881030
IL-7 and IL-15 does not synergize during CD8 T cell recall response against an obligate intracellular parasite
Microbes Infect. 2012 Aug 1. [Epub ahead of print]
IL-7 and IL-15 does not synergize during CD8 T cell recall response against an obligate intracellular parasite
Bharda R, Khan IA.
Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington, DC 20037, USA.
Long-term protection against Toxoplasma gondii is dependent on robust CD8(+) T cell immunity. In the absence of this response, the host is unable to maintain chronicity, which results in recrudescence of infection and possible death. Factors needed for the persistence of protective CD8(+) T cells against the parasite need to be evaluated. Previous studies from our laboratory have reported that synergism between γ chain cytokines like IL-7 and IL-15 is critical for the generation of CD8(+) T cell response needed for protection during acute infection. In this study we report that the situation is different during the recall response where CD8(+) T cell response is almost entirely dependent on IL-15, with IL-7 at best playing a minor role. In the absence of IL-15, CD8(+) T cells fail to respond optimally to parasitic re-challenge and hosts are unable to control their replication, which leads to their death. Thus T. gondii infection may represent a unique situation where CD8(+) T cell response during secondary challenge is primarily dependent on IL-15 with other γ chain cytokines having nominal effect. These findings provide important information regarding factors involved in the generation of protective immunity against T. gondii with strong implications in developing immunotherapeutic agents against the pathogen.
PMID: 22885140
IL-7 and IL-15 does not synergize during CD8 T cell recall response against an obligate intracellular parasite
Bharda R, Khan IA.
Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington, DC 20037, USA.
Long-term protection against Toxoplasma gondii is dependent on robust CD8(+) T cell immunity. In the absence of this response, the host is unable to maintain chronicity, which results in recrudescence of infection and possible death. Factors needed for the persistence of protective CD8(+) T cells against the parasite need to be evaluated. Previous studies from our laboratory have reported that synergism between γ chain cytokines like IL-7 and IL-15 is critical for the generation of CD8(+) T cell response needed for protection during acute infection. In this study we report that the situation is different during the recall response where CD8(+) T cell response is almost entirely dependent on IL-15, with IL-7 at best playing a minor role. In the absence of IL-15, CD8(+) T cells fail to respond optimally to parasitic re-challenge and hosts are unable to control their replication, which leads to their death. Thus T. gondii infection may represent a unique situation where CD8(+) T cell response during secondary challenge is primarily dependent on IL-15 with other γ chain cytokines having nominal effect. These findings provide important information regarding factors involved in the generation of protective immunity against T. gondii with strong implications in developing immunotherapeutic agents against the pathogen.
PMID: 22885140
Saturday, August 11, 2012
Cytoskeleton Assembly in Toxoplasma gondii Cell Division
Int Rev Cell Mol Biol. 2012;298:1-31.
Cytoskeleton Assembly in Toxoplasma gondii Cell Division
Anderson-White B, Beck JR, Chen CT, Meissner M, Bradley PJ, Gubbels MJ.
Department of Biology, Boston College, Chestnut Hill, Massachusetts, USA.
Cell division across members of the protozoan parasite phylum Apicomplexa displays a surprising diversity between different species as well as between different life stages of the same parasite. In most cases, infection of a host cell by a single parasite results in the formation of a polyploid cell from which individual daughters bud in a process dependent on a final round of mitosis. Unlike other apicomplexans, Toxoplasma gondii divides by a binary process consisting of internal budding that results in only two daughter cells per round of division. Since T. gondii is experimentally accessible and displays the simplest division mode, it has manifested itself as a model for apicomplexan daughter formation. Here, we review newly emerging insights in the prominent role that assembly of the cortical cytoskeletal scaffold plays in the process of daughter parasite formation.
PMID: 22878103
Cytoskeleton Assembly in Toxoplasma gondii Cell Division
Anderson-White B, Beck JR, Chen CT, Meissner M, Bradley PJ, Gubbels MJ.
Department of Biology, Boston College, Chestnut Hill, Massachusetts, USA.
Cell division across members of the protozoan parasite phylum Apicomplexa displays a surprising diversity between different species as well as between different life stages of the same parasite. In most cases, infection of a host cell by a single parasite results in the formation of a polyploid cell from which individual daughters bud in a process dependent on a final round of mitosis. Unlike other apicomplexans, Toxoplasma gondii divides by a binary process consisting of internal budding that results in only two daughter cells per round of division. Since T. gondii is experimentally accessible and displays the simplest division mode, it has manifested itself as a model for apicomplexan daughter formation. Here, we review newly emerging insights in the prominent role that assembly of the cortical cytoskeletal scaffold plays in the process of daughter parasite formation.
PMID: 22878103
Saturday, August 04, 2012
Characterization of Rad51 from Apicomplexan Parasite Toxoplasma gondii: An Implication for Inefficient Gene Targeting
PLoS One. 2012;7(7):e41925. Epub 2012 Jul 30.
Characterization of Rad51 from Apicomplexan Parasite Toxoplasma gondii: An Implication for Inefficient Gene Targeting
Achanta SS, Varunan SM, Bhattacharyya S, Bhattacharyya MK.
Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Andhra Pradesh, India
Repairing double strand breaks (DSBs) is absolutely essential for the survival of obligate intracellular parasite Toxoplasma gondii. Thus, DSB repair mechanisms could be excellent targets for chemotherapeutic interventions. Recent genetic and bioinformatics analyses confirm the presence of both homologous recombination (HR) as well as non homologous end joining (NHEJ) proteins in this lower eukaryote. In order to get mechanistic insights into the HR mediated DSB repair pathway in this parasite, we have characterized the key protein involved in homologous recombination, namely TgRad51, at the biochemical and genetic levels. We have purified recombinant TgRad51 protein to 99% homogeneity and have characterized it biochemically. The ATP hydrolysis activity of TgRad51 shows a higher K(M) and much lower k(cat) compared to bacterial RecA or Rad51 from other related protozoan parasites. Taking yeast as a surrogate model system we have shown that TgRad51 is less efficient in gene conversion mechanism. Further, we have found that TgRad51 mediated gene integration is more prone towards random genetic loci rather than targeted locus. We hypothesize that compromised ATPase activity of TgRad51 is responsible for inefficient gene targeting and poor gene conversion efficiency in this protozoan parasite. With increase in homologous flanking regions almost three fold increments in targeted gene integration is observed, which is similar to the trend found with ScRad51. Our findings not only help us in understanding the reason behind inefficient gene targeting in T. gondii but also could be exploited to facilitate high throughput knockout as well as epitope tagging of Toxoplasma genes.
PMID: 22860032
Characterization of Rad51 from Apicomplexan Parasite Toxoplasma gondii: An Implication for Inefficient Gene Targeting
Achanta SS, Varunan SM, Bhattacharyya S, Bhattacharyya MK.
Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Andhra Pradesh, India
Repairing double strand breaks (DSBs) is absolutely essential for the survival of obligate intracellular parasite Toxoplasma gondii. Thus, DSB repair mechanisms could be excellent targets for chemotherapeutic interventions. Recent genetic and bioinformatics analyses confirm the presence of both homologous recombination (HR) as well as non homologous end joining (NHEJ) proteins in this lower eukaryote. In order to get mechanistic insights into the HR mediated DSB repair pathway in this parasite, we have characterized the key protein involved in homologous recombination, namely TgRad51, at the biochemical and genetic levels. We have purified recombinant TgRad51 protein to 99% homogeneity and have characterized it biochemically. The ATP hydrolysis activity of TgRad51 shows a higher K(M) and much lower k(cat) compared to bacterial RecA or Rad51 from other related protozoan parasites. Taking yeast as a surrogate model system we have shown that TgRad51 is less efficient in gene conversion mechanism. Further, we have found that TgRad51 mediated gene integration is more prone towards random genetic loci rather than targeted locus. We hypothesize that compromised ATPase activity of TgRad51 is responsible for inefficient gene targeting and poor gene conversion efficiency in this protozoan parasite. With increase in homologous flanking regions almost three fold increments in targeted gene integration is observed, which is similar to the trend found with ScRad51. Our findings not only help us in understanding the reason behind inefficient gene targeting in T. gondii but also could be exploited to facilitate high throughput knockout as well as epitope tagging of Toxoplasma genes.
PMID: 22860032
Thursday, August 02, 2012
Admixture and recombination among Toxoplasma gondii lineages explain global genome diversity
Proc Natl Acad Sci U S A. 2012 Jul 30. [Epub ahead of print]
Admixture and recombination among Toxoplasma gondii lineages explain global genome diversity
Minot S, Melo MB, Li F, Lu D, Niedelman W, Levine SS, Saeij JP.
Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02139
Toxoplasma gondii is a highly successful protozoan parasite that infects all warm-blooded animals and causes severe disease in immunocompromised and immune-naïve humans. It has an unusual global population structure: In North America and Europe, isolated strains fall predominantly into four largely clonal lineages, but in South America there is great genetic diversity and the North American clonal lineages are rarely found. Genetic variation between Toxoplasma strains determines differences in virulence, modulation of host-signaling pathways, growth, dissemination, and disease severity in mice and likely in humans. Most studies on Toxoplasma genetic variation have focused on either a few loci in many strains or low-resolution genome analysis of three clonal lineages. We use whole-genome sequencing to identify a large number of SNPs between 10 Toxoplasma strains from Europe and North and South America. These were used to identify haplotype blocks (genomic regions) shared between strains and construct a Toxoplasma haplotype map. Additional SNP analysis of RNA-sequencing data of 26 Toxoplasma strains, representing global diversity, allowed us to construct a comprehensive genealogy for Toxoplasma gondii that incorporates sexual recombination. These data show that most current isolates are recent recombinants and cannot be easily grouped into a limited number of haplogroups. A complex picture emerges in which some genomic regions have not been recently exchanged between any strains, and others recently spread from one strain to many others.
PMID: 22847430
Admixture and recombination among Toxoplasma gondii lineages explain global genome diversity
Minot S, Melo MB, Li F, Lu D, Niedelman W, Levine SS, Saeij JP.
Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02139
Toxoplasma gondii is a highly successful protozoan parasite that infects all warm-blooded animals and causes severe disease in immunocompromised and immune-naïve humans. It has an unusual global population structure: In North America and Europe, isolated strains fall predominantly into four largely clonal lineages, but in South America there is great genetic diversity and the North American clonal lineages are rarely found. Genetic variation between Toxoplasma strains determines differences in virulence, modulation of host-signaling pathways, growth, dissemination, and disease severity in mice and likely in humans. Most studies on Toxoplasma genetic variation have focused on either a few loci in many strains or low-resolution genome analysis of three clonal lineages. We use whole-genome sequencing to identify a large number of SNPs between 10 Toxoplasma strains from Europe and North and South America. These were used to identify haplotype blocks (genomic regions) shared between strains and construct a Toxoplasma haplotype map. Additional SNP analysis of RNA-sequencing data of 26 Toxoplasma strains, representing global diversity, allowed us to construct a comprehensive genealogy for Toxoplasma gondii that incorporates sexual recombination. These data show that most current isolates are recent recombinants and cannot be easily grouped into a limited number of haplogroups. A complex picture emerges in which some genomic regions have not been recently exchanged between any strains, and others recently spread from one strain to many others.
PMID: 22847430
Analysis of Monensin Sensitivity in Toxoplasma gondii Reveals Autophagy as a Mechanism for Drug Induced Death
PLoS One. 2012;7(7):e42107. Epub 2012 Jul 25.
Analysis of Monensin Sensitivity in Toxoplasma gondii Reveals Autophagy as a Mechanism for Drug Induced Death
Lavine MD, Arrizabalaga G.
Department of Biological Sciences, University of Idaho, Moscow, Idaho, United States of America.
Understanding the mechanisms by which anti-parasitic drugs alter the physiology and ultimately kill is an important area of investigation. Development of novel parasitic drugs, as well as the continued utilization of existing drugs in the face of resistant parasite populations, requires such knowledge. Here we show that the anti-coccidial drug monensin kills Toxoplasma gondii by inducing autophagy in the parasites, a novel mechanism of cell death in response to an antimicrobial drug. Monensin treatment results autophagy, as shown by translocation of ATG8 to autophagosomes, as well as causing marked morphological changes in the parasites' mitochondria. Use of the autophagy inhibitor 3-methyladenine blocks autophagy and mitochondrial alterations, and enhances parasite survival, in monensin-exposed parasites, although it does not block other monensin-induced effects on the parasites, such as late S-phase cell cycle arrest. Monensin does not induce autophagy in a parasite strain deficient in the mitochondrial DNA repair enzyme TgMSH-1 an enzyme that mediates monensin-induced late S-phase arrest. TgMSH-1 therefore either mediates cell cycle arrest and autophagy independently, or autophagy occurs downstream of cell cycle arrest in a manner analogous to apoptosis of cells arrested in G(2) of the cell cycle. Overall, our results point to autophagy as a potentially important mode of cell death of protozoan parasites in response to antimicrobial drugs and indicate that disruption of the autophagy pathway could result in drug resistance.
PMID: 22848721 [PubMed - in process]
Analysis of Monensin Sensitivity in Toxoplasma gondii Reveals Autophagy as a Mechanism for Drug Induced Death
Lavine MD, Arrizabalaga G.
Department of Biological Sciences, University of Idaho, Moscow, Idaho, United States of America.
Understanding the mechanisms by which anti-parasitic drugs alter the physiology and ultimately kill is an important area of investigation. Development of novel parasitic drugs, as well as the continued utilization of existing drugs in the face of resistant parasite populations, requires such knowledge. Here we show that the anti-coccidial drug monensin kills Toxoplasma gondii by inducing autophagy in the parasites, a novel mechanism of cell death in response to an antimicrobial drug. Monensin treatment results autophagy, as shown by translocation of ATG8 to autophagosomes, as well as causing marked morphological changes in the parasites' mitochondria. Use of the autophagy inhibitor 3-methyladenine blocks autophagy and mitochondrial alterations, and enhances parasite survival, in monensin-exposed parasites, although it does not block other monensin-induced effects on the parasites, such as late S-phase cell cycle arrest. Monensin does not induce autophagy in a parasite strain deficient in the mitochondrial DNA repair enzyme TgMSH-1 an enzyme that mediates monensin-induced late S-phase arrest. TgMSH-1 therefore either mediates cell cycle arrest and autophagy independently, or autophagy occurs downstream of cell cycle arrest in a manner analogous to apoptosis of cells arrested in G(2) of the cell cycle. Overall, our results point to autophagy as a potentially important mode of cell death of protozoan parasites in response to antimicrobial drugs and indicate that disruption of the autophagy pathway could result in drug resistance.
PMID: 22848721 [PubMed - in process]
Toxoplasma gondii antigens recognized by IgG antibodies differ between mice with and without active proliferation of tachyzoites in the brain during the chronic stage of infection
Infect Immun. 2012 Jul 30. [Epub ahead of print]
Toxoplasma gondii antigens recognized by IgG antibodies differ between mice with and without active proliferation of tachyzoites in the brain during the chronic stage of infection
Hester J, Mullins J, Sa Q, Payne L, Mercier C, Cesbron-Delauw MF, Suzuki Y.
Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536.
We examined whether tachyzoite proliferation in the brain of immunocompetent hosts during the chronic stage of infection with Toxoplasma gondii induces production of IgG antibodies that recognize parasite antigens different from those recognized by the antibodies of infected hosts that do not have the tachyzoite growth. For this purpose, two groups of CBA/J mice, which display continuous tachyzoite growth in their brains during the later stage of infection, were infected, and one group received treatment with sulfadiazine to prevent tachyzoite proliferation during the chronic stage of infection. T. gondii antigens recognized by the IgG antibodies from these two groups of mice were compared using immunoblotting following separation of tachyzoite antigens by two-dimensional gel electrophoresis. Several antigens, including the microneme protein MIC2, the cyst matrix protein MAG1, the dense granule proteins GRA4, and GRA7, were commonly recognized by IgG antibodies from both groups of mice. There were multiple antigens recognized mostly by IgG antibodies of only one group of mice, either with or without cerebral tachyzoite growth. The antigens recognized only by or mostly by the antibodies of mice with cerebral tachyzoite growth include MIC6, the rhoptry protein ROP1, GRA2, one heat shock protein HSP90, one (putative) heat shock protein HSP70, and the myosin heavy chain. These results indicate that IgG antibody levels increase only to selected T. gondii antigens in association with cerebral tachyzoite proliferation (reactivation of infection) in immunocompetent hosts with chronic infection.
PMID: 22851753 [PubMed - as supplied by publisher]
Toxoplasma gondii antigens recognized by IgG antibodies differ between mice with and without active proliferation of tachyzoites in the brain during the chronic stage of infection
Hester J, Mullins J, Sa Q, Payne L, Mercier C, Cesbron-Delauw MF, Suzuki Y.
Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536.
We examined whether tachyzoite proliferation in the brain of immunocompetent hosts during the chronic stage of infection with Toxoplasma gondii induces production of IgG antibodies that recognize parasite antigens different from those recognized by the antibodies of infected hosts that do not have the tachyzoite growth. For this purpose, two groups of CBA/J mice, which display continuous tachyzoite growth in their brains during the later stage of infection, were infected, and one group received treatment with sulfadiazine to prevent tachyzoite proliferation during the chronic stage of infection. T. gondii antigens recognized by the IgG antibodies from these two groups of mice were compared using immunoblotting following separation of tachyzoite antigens by two-dimensional gel electrophoresis. Several antigens, including the microneme protein MIC2, the cyst matrix protein MAG1, the dense granule proteins GRA4, and GRA7, were commonly recognized by IgG antibodies from both groups of mice. There were multiple antigens recognized mostly by IgG antibodies of only one group of mice, either with or without cerebral tachyzoite growth. The antigens recognized only by or mostly by the antibodies of mice with cerebral tachyzoite growth include MIC6, the rhoptry protein ROP1, GRA2, one heat shock protein HSP90, one (putative) heat shock protein HSP70, and the myosin heavy chain. These results indicate that IgG antibody levels increase only to selected T. gondii antigens in association with cerebral tachyzoite proliferation (reactivation of infection) in immunocompetent hosts with chronic infection.
PMID: 22851753 [PubMed - as supplied by publisher]
Infection with Toxoplasma gondii alters lymphotoxin expression associated with changes in splenic architecture
Infect Immun. 2012 Jul 30. [Epub ahead of print]
Infection with Toxoplasma gondii alters lymphotoxin expression associated with changes in splenic architecture
Glatman Zaretsky A, Silver JS, Siwicki M, Durham A, Ware CF, Hunter CA.
Department of Pathobiology, University of Pennsylvania, Philadelphia, PA 19104.
B cell responses are required for resistance to Toxoplasma gondii, however, the events that lead to production of class-switched antibodies during T. gondii infection have not been defined. Indeed, mice challenged with this parasite exhibited an expansion of T follicular helper cells and germinal center B cells in the spleen. Unexpectedly, this was not associated with germinal center formation and was instead accompanied by profound changes in splenic organization. This phenomenon was transient and correlated with a decrease in expression of effector proteins that contribute to splenic organization, including lymphotoxin α and β. The importance of lymphotoxin was confirmed as the use of a lymphotoxin β receptor agonist results in a partial restoration of splenic structure. Splenectomized mice were used to test the splenic contribution to the antibody response during T. gondii infection. Analysis of splenectomized mice revealed delayed kinetics in the production of parasite-specific antibody, but the mice did eventually develop normal levels of parasite-specific antibody. Together, these studies provide a better understanding of how infection with T. gondii impacts the customized structures required for the optimal humoral responses to this parasite and the role of lymphotoxin in these events.
PMID: 22851754 [PubMed - as supplied by publisher]
Infection with Toxoplasma gondii alters lymphotoxin expression associated with changes in splenic architecture
Glatman Zaretsky A, Silver JS, Siwicki M, Durham A, Ware CF, Hunter CA.
Department of Pathobiology, University of Pennsylvania, Philadelphia, PA 19104.
B cell responses are required for resistance to Toxoplasma gondii, however, the events that lead to production of class-switched antibodies during T. gondii infection have not been defined. Indeed, mice challenged with this parasite exhibited an expansion of T follicular helper cells and germinal center B cells in the spleen. Unexpectedly, this was not associated with germinal center formation and was instead accompanied by profound changes in splenic organization. This phenomenon was transient and correlated with a decrease in expression of effector proteins that contribute to splenic organization, including lymphotoxin α and β. The importance of lymphotoxin was confirmed as the use of a lymphotoxin β receptor agonist results in a partial restoration of splenic structure. Splenectomized mice were used to test the splenic contribution to the antibody response during T. gondii infection. Analysis of splenectomized mice revealed delayed kinetics in the production of parasite-specific antibody, but the mice did eventually develop normal levels of parasite-specific antibody. Together, these studies provide a better understanding of how infection with T. gondii impacts the customized structures required for the optimal humoral responses to this parasite and the role of lymphotoxin in these events.
PMID: 22851754 [PubMed - as supplied by publisher]
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