Take you, me and someone sitting near you at lunch. Chances are at least one of us has Toxoplasma parasites in the brain, alive but safely inactivated by the immune system.
But if the immune system is compromised — say in cases of HIV or chemotherapy — the parasite can emerge from its latent state and resume replicating, creating a serious and sometimes fatal infection. It also poses a risk of congenital birth defects in pregnant women.
Now IU School of Medicine researchers Bill Sullivan, Ph.D., Ron Wek, Ph.D., Christian Konrad, Ph.D., and Sherry Queener, Ph.D., report that guanabenz, a drug used to treat hypertension, may work as a “sleeping pill” to keep the Toxoplasma parasite under control.
In a study published in the April 2013 edition of Antimicrobial Agents and Chemotherapy, the researchers tested the effects of guanabenz on parasites in cell culture and found that it...
Read the full article here.
Friday, June 28, 2013
Thursday, June 27, 2013
Mechanics of the Toxoplasma gondii oocyst wall
Proc Natl Acad Sci U S A. 2013 Jun 24. [Epub ahead of print]
Mechanics of the Toxoplasma gondii oocyst wall
Dumètre A, Dubey JP, Ferguson DJ, Bongrand P, Azas N, Puech PH.
Unité Mixte de Recherche (UMR) Ministère de la Défense 3 Infections Parasitaires, Transmission, Physiopathologie et Thérapeutique, Faculté de Pharmacie, Aix-Marseille Université, 13385 Marseille Cedex 05, France.
The ability of microorganisms to survive under extreme conditions is closely related to the physicochemical properties of their wall. In the ubiquitous protozoan parasite Toxoplasma gondii, the oocyst stage possesses a bilayered wall that protects the dormant but potentially infective parasites from harsh environmental conditions until their ingestion by the host. None of the common disinfectants are effective in killing the parasite because the oocyst wall acts as a primary barrier to physical and chemical attacks. Here, we address the structure and chemistry of the wall of the T. gondii oocyst by combining wall surface treatments, fluorescence imaging, EM, and measurements of its mechanical characteristics by using atomic force microscopy. Elasticity and indentation measurements indicated that the oocyst wall resembles common plastic materials, based on the Young moduli, E, evaluated by atomic force microscopy. Our study demonstrates that the inner layer is as robust as the bilayered wall itself. Besides wall mechanics, our results suggest important differences regarding the nonspecific adhesive properties of each layer. All together, these findings suggest a key biological role for the oocyst wall mechanics in maintaining the integrity of the T. gondii oocysts in the environment or after exposure to disinfectants, and therefore their potential infectivity to humans and animals.
KEYWORDS:
Toxoplasma infectivity, oocyst integrity, oocyst resistance, oocyst structure, transmission
PMID: 23798399 [PubMed - as supplied by publisher]
Mechanics of the Toxoplasma gondii oocyst wall
Dumètre A, Dubey JP, Ferguson DJ, Bongrand P, Azas N, Puech PH.
Unité Mixte de Recherche (UMR) Ministère de la Défense 3 Infections Parasitaires, Transmission, Physiopathologie et Thérapeutique, Faculté de Pharmacie, Aix-Marseille Université, 13385 Marseille Cedex 05, France.
The ability of microorganisms to survive under extreme conditions is closely related to the physicochemical properties of their wall. In the ubiquitous protozoan parasite Toxoplasma gondii, the oocyst stage possesses a bilayered wall that protects the dormant but potentially infective parasites from harsh environmental conditions until their ingestion by the host. None of the common disinfectants are effective in killing the parasite because the oocyst wall acts as a primary barrier to physical and chemical attacks. Here, we address the structure and chemistry of the wall of the T. gondii oocyst by combining wall surface treatments, fluorescence imaging, EM, and measurements of its mechanical characteristics by using atomic force microscopy. Elasticity and indentation measurements indicated that the oocyst wall resembles common plastic materials, based on the Young moduli, E, evaluated by atomic force microscopy. Our study demonstrates that the inner layer is as robust as the bilayered wall itself. Besides wall mechanics, our results suggest important differences regarding the nonspecific adhesive properties of each layer. All together, these findings suggest a key biological role for the oocyst wall mechanics in maintaining the integrity of the T. gondii oocysts in the environment or after exposure to disinfectants, and therefore their potential infectivity to humans and animals.
KEYWORDS:
Toxoplasma infectivity, oocyst integrity, oocyst resistance, oocyst structure, transmission
PMID: 23798399 [PubMed - as supplied by publisher]
Wednesday, June 26, 2013
Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during Plasmodium falciparum intraerythrocytic development
Mol Microbiol. 2013 Jun 24. doi: 10.1111/mmi.12303. [Epub ahead of print]
Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during Plasmodium falciparum intraerythrocytic development
Miao J, Lawrence M, Jeffers V, Zhao F, Parker D, Ge Y, Sullivan WJ Jr, Cui L.
Department of Entomology, Pennsylvania State University, 501 ASI Building, University Park, PA 16802.
Lysine acetylation has emerged as a major posttranslational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine-acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion, and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely-related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium-specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites.
PMID: 23796209 [PubMed - as supplied by publisher]
Extensive lysine acetylation occurs in evolutionarily conserved metabolic pathways and parasite-specific functions during Plasmodium falciparum intraerythrocytic development
Miao J, Lawrence M, Jeffers V, Zhao F, Parker D, Ge Y, Sullivan WJ Jr, Cui L.
Department of Entomology, Pennsylvania State University, 501 ASI Building, University Park, PA 16802.
Lysine acetylation has emerged as a major posttranslational modification involved in diverse cellular functions. Using a combination of immunoisolation and liquid chromatography coupled to accurate mass spectrometry, we determined the first acetylome of the human malaria parasite Plasmodium falciparum during its active proliferation in erythrocytes with 421 acetylation sites identified in 230 proteins. Lysine-acetylated proteins are distributed in the nucleus, cytoplasm, mitochondrion, and apicoplast. Whereas occurrence of lysine acetylation in a similarly wide range of cellular functions suggests conservation of lysine acetylation through evolution, the Plasmodium acetylome also revealed significant divergence from those of other eukaryotes and even the closely-related parasite Toxoplasma. This divergence is reflected in the acetylation of a large number of Plasmodium-specific proteins and different acetylation sites in evolutionarily conserved acetylated proteins. A prominent example is the abundant acetylation of proteins in the glycolysis pathway but relatively deficient acetylation of enzymes in the citrate cycle. Using specific transgenic lines and inhibitors, we determined that the acetyltransferase PfMYST and lysine deacetylases play important roles in regulating the dynamics of cytoplasmic protein acetylation. The Plasmodium acetylome provides an exciting start point for further exploration of functions of acetylation in the biology of malaria parasites.
PMID: 23796209 [PubMed - as supplied by publisher]
Friday, June 21, 2013
An Apicoplast Localized Ubiquitylation System Is Required for the Import of Nuclear-encoded Plastid Proteins
PLoS Pathog. 2013 Jun;9(6):e1003426. doi: 10.1371/journal.ppat.1003426. Epub 2013 Jun 13.
An Apicoplast Localized Ubiquitylation System Is Required for the Import of Nuclear-encoded Plastid Proteins
Agrawal S, Chung DW, Ponts N, van Dooren GG, Prudhomme J, Brooks CF, Rodrigues EM, Tan JC, Ferdig MT, Striepen B, Le Roch KG.
Department of Cellular Biology, University of Georgia, Athens, Georgia, United States of America.
Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD) has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.
PMID: 23785288 [PubMed - in process]
An Apicoplast Localized Ubiquitylation System Is Required for the Import of Nuclear-encoded Plastid Proteins
Agrawal S, Chung DW, Ponts N, van Dooren GG, Prudhomme J, Brooks CF, Rodrigues EM, Tan JC, Ferdig MT, Striepen B, Le Roch KG.
Department of Cellular Biology, University of Georgia, Athens, Georgia, United States of America.
Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD) has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.
PMID: 23785288 [PubMed - in process]
IRG and GBP Host Resistance Factors Target Aberrant, "Non-self" Vacuoles Characterized by the Missing of "Self" IRGM Proteins
PLoS Pathog. 2013 Jun;9(6):e1003414. doi: 10.1371/journal.ppat.1003414. Epub 2013 Jun 13.
IRG and GBP Host Resistance Factors Target Aberrant, "Non-self" Vacuoles Characterized by the Missing of "Self" IRGM Proteins
Haldar AK, Saka HA, Piro AS, Dunn JD, Henry SC, Taylor GA, Frickel EM, Valdivia RH, Coers J.
Departments of Molecular Genetics and Microbiology, and Immunology, Duke University Medical Center, Durham, North Carolina, United States of America.
Interferon-inducible GTPases of the Immunity Related GTPase (IRG) and Guanylate Binding Protein (GBP) families provide resistance to intracellular pathogenic microbes. IRGs and GBPs stably associate with pathogen-containing vacuoles (PVs) and elicit immune pathways directed at the targeted vacuoles. Targeting of Interferon-inducible GTPases to PVs requires the formation of higher-order protein oligomers, a process negatively regulated by a subclass of IRG proteins called IRGMs. We found that the paralogous IRGM proteins Irgm1 and Irgm3 fail to robustly associate with "non-self" PVs containing either the bacterial pathogen Chlamydia trachomatis or the protozoan pathogen Toxoplasma gondii. Instead, Irgm1 and Irgm3 reside on "self" organelles including lipid droplets (LDs). Whereas IRGM-positive LDs are guarded against the stable association with other IRGs and GBPs, we demonstrate that IRGM-stripped LDs become high affinity binding substrates for IRG and GBP proteins. These data reveal that intracellular immune recognition of organelle-like structures by IRG and GBP proteins is partly dictated by the missing of "self" IRGM proteins from these structures.
PMID: 23785284 [PubMed - in process]
IRG and GBP Host Resistance Factors Target Aberrant, "Non-self" Vacuoles Characterized by the Missing of "Self" IRGM Proteins
Haldar AK, Saka HA, Piro AS, Dunn JD, Henry SC, Taylor GA, Frickel EM, Valdivia RH, Coers J.
Departments of Molecular Genetics and Microbiology, and Immunology, Duke University Medical Center, Durham, North Carolina, United States of America.
Interferon-inducible GTPases of the Immunity Related GTPase (IRG) and Guanylate Binding Protein (GBP) families provide resistance to intracellular pathogenic microbes. IRGs and GBPs stably associate with pathogen-containing vacuoles (PVs) and elicit immune pathways directed at the targeted vacuoles. Targeting of Interferon-inducible GTPases to PVs requires the formation of higher-order protein oligomers, a process negatively regulated by a subclass of IRG proteins called IRGMs. We found that the paralogous IRGM proteins Irgm1 and Irgm3 fail to robustly associate with "non-self" PVs containing either the bacterial pathogen Chlamydia trachomatis or the protozoan pathogen Toxoplasma gondii. Instead, Irgm1 and Irgm3 reside on "self" organelles including lipid droplets (LDs). Whereas IRGM-positive LDs are guarded against the stable association with other IRGs and GBPs, we demonstrate that IRGM-stripped LDs become high affinity binding substrates for IRG and GBP proteins. These data reveal that intracellular immune recognition of organelle-like structures by IRG and GBP proteins is partly dictated by the missing of "self" IRGM proteins from these structures.
PMID: 23785284 [PubMed - in process]
Wednesday, June 19, 2013
Identification and characterization of Toxoplasma gondii aspartic protease 1 as a novel vaccine candidate against toxoplasmosis
Parasit Vectors. 2013 Jun 14;6(1):175. [Epub ahead of print]
Identification and characterization of Toxoplasma gondii aspartic protease 1 as a novel vaccine candidate against toxoplasmosis
Zhao G, Zhou A, Lu G, Meng M, Sun M, Bai Y, Han Y, Wang L, Zhou H, Cong H, Zhao Q, Zhu XQ, He S.
BACKGROUND:
Toxoplasma gondii is an obligate intracellular parasite that can pose a serious threat to human health by causing toxoplasmosis. There are no drugs that target the chronic cyst stage of this infection; therefore, development of an effective vaccine would be an important advance. Aspartic proteases play essential roles in the T. gondii lifecycle. The parasite has four aspartic protease encoding genes, which are called toxomepsin 1, 2, 3 and 5 (TgASP1, 2, 3 and 5, respectively).
METHODS:
Bioinformatics approaches have enabled us to identify several promising linear-B cell epitopes and potential Th-cell epitopes on TgASP1, thus supporting its potential as a DNA vaccine against toxoplasmosis. We expressed TgASP1 in Escherichia coli and used the purified protein to immunize BALB/c mice. The antibodies obtained were used to determine where TgASP1 was localized in the parasite. We also made a TgASP1 DNA vaccine construct and evaluated it for the level of protection conferred to mice against infection with the virulent RH strain of T. gondii.
RESULTS:
TgASP1 appears to be a membrane protein located primarily at the tip of the T. gondii tachyzoite. Investigation of its potential as a DNA vaccine showed that it elicited strong humoral and cellular immune responses in mice, and that these responses were mediated by Th-1 cells. Mice immunized with the vaccine had greater levels of protection against mortality following challenge with T. gondii RH tachyzoites than did those immunized with PBS or the empty vector control.
CONCLUSIONS:
TgASP1 is a novel candidate DNA vaccine that merits further investigation.
PMID: 23768047 [PubMed - as supplied by publisher]
Identification and characterization of Toxoplasma gondii aspartic protease 1 as a novel vaccine candidate against toxoplasmosis
Zhao G, Zhou A, Lu G, Meng M, Sun M, Bai Y, Han Y, Wang L, Zhou H, Cong H, Zhao Q, Zhu XQ, He S.
BACKGROUND:
Toxoplasma gondii is an obligate intracellular parasite that can pose a serious threat to human health by causing toxoplasmosis. There are no drugs that target the chronic cyst stage of this infection; therefore, development of an effective vaccine would be an important advance. Aspartic proteases play essential roles in the T. gondii lifecycle. The parasite has four aspartic protease encoding genes, which are called toxomepsin 1, 2, 3 and 5 (TgASP1, 2, 3 and 5, respectively).
METHODS:
Bioinformatics approaches have enabled us to identify several promising linear-B cell epitopes and potential Th-cell epitopes on TgASP1, thus supporting its potential as a DNA vaccine against toxoplasmosis. We expressed TgASP1 in Escherichia coli and used the purified protein to immunize BALB/c mice. The antibodies obtained were used to determine where TgASP1 was localized in the parasite. We also made a TgASP1 DNA vaccine construct and evaluated it for the level of protection conferred to mice against infection with the virulent RH strain of T. gondii.
RESULTS:
TgASP1 appears to be a membrane protein located primarily at the tip of the T. gondii tachyzoite. Investigation of its potential as a DNA vaccine showed that it elicited strong humoral and cellular immune responses in mice, and that these responses were mediated by Th-1 cells. Mice immunized with the vaccine had greater levels of protection against mortality following challenge with T. gondii RH tachyzoites than did those immunized with PBS or the empty vector control.
CONCLUSIONS:
TgASP1 is a novel candidate DNA vaccine that merits further investigation.
PMID: 23768047 [PubMed - as supplied by publisher]
Comparative analysis of anti-toxoplasmic activity of antipsychotic drugs and valproate
Eur Arch Psychiatry Clin Neurosci. 2013 Jun 15. [Epub ahead of print]
Comparative analysis of anti-toxoplasmic activity of antipsychotic drugs and valproate
Fond G, Macgregor A, Tamouza R, Hamdani N, Meary A, Leboyer M, Dubremetz JF.
Fondation FondaMental Fondation de Coopération Scientifique en Santé Mentale, Eq Psychiatrie Génétique, INSERM U955, DHU Pepsy, Pôle de Psychiatrie du Groupe des Hôpitaux Universitaires de Mondor, Université Paris Est-Créteil, 94000, Créteil, France, guillaume.fond@gmail.com.
Recent studies have shown a strong link between Toxoplasma gondii infection and psychiatric disorders, especially schizophrenia and bipolar disorders (odd ratio ≈2.7 for each disorder). Antipsychotic drugs and mood stabilizers may have anti-toxoplasmic activity that potentially may be associated with better effectiveness in these disorders, but previous results have been few in number and conflicting. We therefore sought to determine which daily prescribed antipsychotics and mood stabilizer have the best anti-toxoplasmic activity during the development phase of the parasite. In the present study, we examined the effects of commonly used antipsychotic drugs (amisulpride, cyamemazine, fluphenazine, haloperidol, levomepromazine, loxapine, olanzapine, risperidone and tiapride) and one mood-stabilizing agent (valproate) on toxoplasmic activity. We replicated that fluphenazine has a high anti-toxoplasmic activity, but it does not seem to be a phenothiazine-specific class effect: indeed, we found that another first-generation antipsychotic, zuclopenthixol, has a high anti-toxoplasmic activity. Valproate, tiapride and amisulpride have no anti-toxoplasmic activity on parasite growth, and the other antipsychotic drugs showed low or intermediate anti-toxoplasmic activity. As it is not possible to know the intracellular concentrations of antipsychotics in the brain, further clinical studies are warranted to determine whether these in vitro findings have potential implications in treatment of toxo-positive patients with schizophrenia. These findings may be potentially relevant for the choice of the first-line antipsychotic drug or mood stabilizer in previously infected patients.
PMID: 23771405 [PubMed - as supplied by publisher]
Modification of Triclosan Scaffold in Search of Improved Inhibitors for Enoyl-Acyl Carrier Protein (ACP) Reductase in Toxoplasma gondii
ChemMedChem. 2013 Jun 14. doi: 10.1002/cmdc.201300050. [Epub ahead of print]
Modification of Triclosan Scaffold in Search of Improved Inhibitors for Enoyl-Acyl Carrier Protein (ACP) Reductase in Toxoplasma gondii
Stec J, Fomovska A, Afanador GA, Muench SP, Zhou Y, Lai BS, El Bissati K, Hickman MR, Lee PJ, Leed SE, Auschwitz JM, Sommervile C, Woods S, Roberts CW, Rice D, Prigge ST, McLeod R, Kozikowski AP.
Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612 (USA); Chicago State University, College of Pharmacy, DH 203-3, 9501 South King Drive, Chicago, IL 60628 (USA).
Through our focused effort to discover new and effective agents against toxoplasmosis, a structure-based drug design approach was used to develop a series of potent inhibitors of the enoyl-acyl carrier protein (ACP) reductase (ENR) enzyme in Toxoplasma gondii (TgENR). Modifications to positions 5 and 4' of the well-known ENR inhibitor triclosan afforded a series of 29 new analogues. Among the resulting compounds, many showed high potency and improved physicochemical properties in comparison with the lead. The most potent compounds 16 a and 16 c have IC50 values of 250 nM against Toxoplasma gondii tachyzoites without apparent toxicity to the host cells. Their IC50 values against recombinant TgENR were found to be 43 and 26 nM, respectively. Additionally, 11 other analogues in this series had IC50 values ranging from 17 to 130 nM in the enzyme-based assay. With respect to their excellent in vitro activity as well as improved drug-like properties, the lead compounds 16 a and 16 c are deemed to be excellent starting points for the development of new medicines to effectively treat Toxoplasma gondii infections.
PMID: 23776166 [PubMed - as supplied by publisher]
Modification of Triclosan Scaffold in Search of Improved Inhibitors for Enoyl-Acyl Carrier Protein (ACP) Reductase in Toxoplasma gondii
Stec J, Fomovska A, Afanador GA, Muench SP, Zhou Y, Lai BS, El Bissati K, Hickman MR, Lee PJ, Leed SE, Auschwitz JM, Sommervile C, Woods S, Roberts CW, Rice D, Prigge ST, McLeod R, Kozikowski AP.
Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL 60612 (USA); Chicago State University, College of Pharmacy, DH 203-3, 9501 South King Drive, Chicago, IL 60628 (USA).
Through our focused effort to discover new and effective agents against toxoplasmosis, a structure-based drug design approach was used to develop a series of potent inhibitors of the enoyl-acyl carrier protein (ACP) reductase (ENR) enzyme in Toxoplasma gondii (TgENR). Modifications to positions 5 and 4' of the well-known ENR inhibitor triclosan afforded a series of 29 new analogues. Among the resulting compounds, many showed high potency and improved physicochemical properties in comparison with the lead. The most potent compounds 16 a and 16 c have IC50 values of 250 nM against Toxoplasma gondii tachyzoites without apparent toxicity to the host cells. Their IC50 values against recombinant TgENR were found to be 43 and 26 nM, respectively. Additionally, 11 other analogues in this series had IC50 values ranging from 17 to 130 nM in the enzyme-based assay. With respect to their excellent in vitro activity as well as improved drug-like properties, the lead compounds 16 a and 16 c are deemed to be excellent starting points for the development of new medicines to effectively treat Toxoplasma gondii infections.
PMID: 23776166 [PubMed - as supplied by publisher]
Friday, June 14, 2013
Drug repurposing: mining protozoan proteomes for targets of known bioactive compounds
J Am Med Inform Assoc. 2013 Jun 11. [Epub ahead of print]
Drug repurposing: mining protozoan proteomes for targets of known bioactive compounds
Sateriale A, Bessoff K, Sarkar IN, Huston CD.
Cell, Molecular, and Biomedical Sciences Graduate Program, University of Vermont College of Medicine, Burlington, Vermont, USA.
OBJECTIVE:
To identify potential opportunities for drug repurposing by developing an automated approach to pre-screen the predicted proteomes of any organism against databases of known drug targets using only freely available resources.
MATERIALS AND METHODS:
We employed a combination of Ruby scripts that leverage data from the DrugBank and ChEMBL databases, MySQL, and BLAST to predict potential drugs and their targets from 13 published genomes. Results from a previous cell-based screen to identify inhibitors of Cryptosporidium parvum growth were used to validate our in-silico prediction method.
RESULTS:
In-vitro validation of these results, using a cell-based C parvum growth assay, showed that the predicted inhibitors were significantly more likely than expected by chance to have confirmed activity, with 8.9-15.6% of predicted inhibitors confirmed depending on the drug target database used. This method was then used to predict inhibitors for the following 13 disease-causing protozoan parasites, including: C parvum, Entamoeba histolytica, Giardia intestinalis, Leishmania braziliensis, Leishmania donovani, Leishmania major, Naegleria gruberi (in proxy of Naegleria fowleri), Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brucei and Trypanosoma cruzi.
CONCLUSIONS:
Although proteome-wide screens for drug targets have disadvantages, in-silico methods can be developed that are fast, broad, inexpensive, and effective. In-vitro validation of our results for C parvum indicate that the method presented here can be used to construct a library for more directed small molecule screening, or pipelined into structural modeling and docking programs to facilitate target-based drug development.
KEYWORDS:
computational biology, drug repositioning, drug therapy, parasitology, proteomics
PMID: 23757409 [PubMed - as supplied by publisher]
Drug repurposing: mining protozoan proteomes for targets of known bioactive compounds
Sateriale A, Bessoff K, Sarkar IN, Huston CD.
Cell, Molecular, and Biomedical Sciences Graduate Program, University of Vermont College of Medicine, Burlington, Vermont, USA.
OBJECTIVE:
To identify potential opportunities for drug repurposing by developing an automated approach to pre-screen the predicted proteomes of any organism against databases of known drug targets using only freely available resources.
MATERIALS AND METHODS:
We employed a combination of Ruby scripts that leverage data from the DrugBank and ChEMBL databases, MySQL, and BLAST to predict potential drugs and their targets from 13 published genomes. Results from a previous cell-based screen to identify inhibitors of Cryptosporidium parvum growth were used to validate our in-silico prediction method.
RESULTS:
In-vitro validation of these results, using a cell-based C parvum growth assay, showed that the predicted inhibitors were significantly more likely than expected by chance to have confirmed activity, with 8.9-15.6% of predicted inhibitors confirmed depending on the drug target database used. This method was then used to predict inhibitors for the following 13 disease-causing protozoan parasites, including: C parvum, Entamoeba histolytica, Giardia intestinalis, Leishmania braziliensis, Leishmania donovani, Leishmania major, Naegleria gruberi (in proxy of Naegleria fowleri), Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brucei and Trypanosoma cruzi.
CONCLUSIONS:
Although proteome-wide screens for drug targets have disadvantages, in-silico methods can be developed that are fast, broad, inexpensive, and effective. In-vitro validation of our results for C parvum indicate that the method presented here can be used to construct a library for more directed small molecule screening, or pipelined into structural modeling and docking programs to facilitate target-based drug development.
KEYWORDS:
computational biology, drug repositioning, drug therapy, parasitology, proteomics
PMID: 23757409 [PubMed - as supplied by publisher]
Thursday, June 13, 2013
The shikimate pathway in apicomplexan parasites: Implications for drug development
Front Biosci. 2013 Jun 1;18:944-69.
The shikimate pathway in apicomplexan parasites: Implications for drug development
Derrer B, Macheroux P, Kappes B.
Institute for Medical Biotechnology, Friedrich-Alexander University Erlangen-Nuremberg.
The shikimate pathway provides basic building blocks for a variety of aromatic compounds including aromatic amino acids, ubiquinone, folate and compounds of the secondary metabolism. The seven enzymatic reactions of the pathway lead to the generation of chorismate from simple products of the carbohydrate metabolism, namely erythrose 4-phosphate and phosphoenolpyruvate. The shikimate pathway is present in plants, bacteria, fungi and chromalveolata to which the apicomplexan parasites belong. As it is absent from humans, the enzymes of the shikimate pathway are attractive targets for antimicrobial drug development. Inhibition of the pathway is effective in controlling growth of certain apicomplexan parasites including the malaria parasite Plasmodium falciparum. Yet, despite being an attractive drug target, our knowledge of the shikimate pathway in this parasite group is lacking. The current review summarizes the available information and discusses aspects of the genetic organization of the shikimate pathway in apicomplexan parasites. Compounds acting on shikimate pathway enzymes will be presented and discussed in light of their impact for antiapicomplexan/antiplasmodial drug development.
PMID: 23747859 [PubMed - in process]
The shikimate pathway in apicomplexan parasites: Implications for drug development
Derrer B, Macheroux P, Kappes B.
Institute for Medical Biotechnology, Friedrich-Alexander University Erlangen-Nuremberg.
The shikimate pathway provides basic building blocks for a variety of aromatic compounds including aromatic amino acids, ubiquinone, folate and compounds of the secondary metabolism. The seven enzymatic reactions of the pathway lead to the generation of chorismate from simple products of the carbohydrate metabolism, namely erythrose 4-phosphate and phosphoenolpyruvate. The shikimate pathway is present in plants, bacteria, fungi and chromalveolata to which the apicomplexan parasites belong. As it is absent from humans, the enzymes of the shikimate pathway are attractive targets for antimicrobial drug development. Inhibition of the pathway is effective in controlling growth of certain apicomplexan parasites including the malaria parasite Plasmodium falciparum. Yet, despite being an attractive drug target, our knowledge of the shikimate pathway in this parasite group is lacking. The current review summarizes the available information and discusses aspects of the genetic organization of the shikimate pathway in apicomplexan parasites. Compounds acting on shikimate pathway enzymes will be presented and discussed in light of their impact for antiapicomplexan/antiplasmodial drug development.
PMID: 23747859 [PubMed - in process]
Identification of dihydroorotate dehydrogenase as a relevant drug target for 1-hydroxyquinolones in Toxoplasma gondii
Mol Biochem Parasitol. 2013 Jun 5. pii: S0166-6851(13)00085-6. doi: 10.1016/j.molbiopara.2013.05.008. [Epub ahead of print]
Identification of dihydroorotate dehydrogenase as a relevant drug target for 1-hydroxyquinolones in Toxoplasma gondii
Hegewald J, Gross U, Bohne W.
Institute of Medical Microbiology, University Medical Center Göttingen, Kreuzbergring 57, Göttingen D-37075; Germany.
1-hydroxyquinolones as for example 1-hydroxy-2-dodecyl-4(1)quinolone (HDQ) are effective growth inhibitors for Toxoplasma gondii. These compounds were shown to interfere with the respiratory chain function by inhibition of type II NADH dehydrogenase activity. With the aid of partial drug resistant mutants we identified in this study the fourth enzyme of the de novo pyrimidine synthesis pathway, the T. gondii dihydroorotate dehydrogenase (TgDHODH), as an additional 1-hydroxyquinolone target. A single point mutation was found in the TgDHODH coding sequence of drug resistant clones that changes a conserved Asn into Ser in the vicinity of the dihydroorotate binding site. This mutation is sufficient to confer the partial drug resistance phenotype as shown by allele replacement. Enzyme kinetics revealed that 1-hydroxyquinolones inhibit wild type TgDHODH with IC50s in the nanomolar range, while the IC50s for the N302S mutant were significantly increased. Furthermore, inhibition kinetics revealed that 1-hydroxyquinolones act as competitive inhibitors for the electron acceptor QD, but as uncompetitive inhibitors for dihydroorotate. Moreover, heterologous expression of the ubiquinone independent DHODH from Saccharomyces cerevisiae in T. gondii also leads to partial 1-hydroxyquinolone resistance. Our data suggest that inhibition of TgDHODH activity significantly contributes to the growth inhibiting potential of 1-hydroxyquinolones in T. gondii.
PMID: 23747278 [PubMed - as supplied by publisher]
Identification of dihydroorotate dehydrogenase as a relevant drug target for 1-hydroxyquinolones in Toxoplasma gondii
Hegewald J, Gross U, Bohne W.
Institute of Medical Microbiology, University Medical Center Göttingen, Kreuzbergring 57, Göttingen D-37075; Germany.
1-hydroxyquinolones as for example 1-hydroxy-2-dodecyl-4(1)quinolone (HDQ) are effective growth inhibitors for Toxoplasma gondii. These compounds were shown to interfere with the respiratory chain function by inhibition of type II NADH dehydrogenase activity. With the aid of partial drug resistant mutants we identified in this study the fourth enzyme of the de novo pyrimidine synthesis pathway, the T. gondii dihydroorotate dehydrogenase (TgDHODH), as an additional 1-hydroxyquinolone target. A single point mutation was found in the TgDHODH coding sequence of drug resistant clones that changes a conserved Asn into Ser in the vicinity of the dihydroorotate binding site. This mutation is sufficient to confer the partial drug resistance phenotype as shown by allele replacement. Enzyme kinetics revealed that 1-hydroxyquinolones inhibit wild type TgDHODH with IC50s in the nanomolar range, while the IC50s for the N302S mutant were significantly increased. Furthermore, inhibition kinetics revealed that 1-hydroxyquinolones act as competitive inhibitors for the electron acceptor QD, but as uncompetitive inhibitors for dihydroorotate. Moreover, heterologous expression of the ubiquinone independent DHODH from Saccharomyces cerevisiae in T. gondii also leads to partial 1-hydroxyquinolone resistance. Our data suggest that inhibition of TgDHODH activity significantly contributes to the growth inhibiting potential of 1-hydroxyquinolones in T. gondii.
PMID: 23747278 [PubMed - as supplied by publisher]
Regulation of ATG8 membrane association by ATG4 in the parasitic protist Toxoplasma gondii
Autophagy. 2013 Jun 6;9(9). [Epub ahead of print]
Regulation of ATG8 membrane association by ATG4 in the parasitic protist Toxoplasma gondii
Kong-Hap MA, Mouammine A, Daher W, Berry L, Lebrun M, Dubremetz JF, Besteiro S.
UMR 5235 CNRS; Universités de Montpellier 1 et 2; Dynamique des Interactions Membranaires Normales et Pathologiques; Montpellier, France.
In the process of autophagy, the Atg8 protein is conjugated, through a ubiquitin-like system, to the lipid phosphatidylethanolamine (PE) to associate with the membrane of forming autophagosomes. There, it plays a crucial role in the genesis of these organelles and in autophagy in general. In most eukaryotes, the cysteine peptidase Atg4 processes the C terminus of cytosolic Atg8 to regulate its association with autophagosomal membranes and also delipidates Atg8 to release this protein from membranes. The parasitic protist Toxoplasma gondii contains a functional, yet apparently reduced, autophagic machinery. T. gondii Atg8 homolog, in addition to a cytosolic and occasionally autophagosomal localization, also localizes to the apicoplast, a nonphotosynthetic plastid bounded by four membranes. Our attempts to interfere with TgATG8 function showed that it appears to be essential for parasite multiplication inside its host cell. This protein also displays a peculiar C terminus that does not seem to necessitate processing prior to membrane association and yet an unusually large Toxoplasma homolog of ATG4 is predicted in the parasite genome. A TgATG4 conditional expression mutant that we have generated is severely affected in growth, and displays significant alterations at the organellar level, noticeably with a fragmentation of the mitochondrial network and a loss of the apicoplast. TgATG4-depleted parasites appear to be defective in the recycling of membrane-bound TgATG8. Overall, our data highlight a role for the TgATG8 conjugation pathway in maintaining the homeostasis of the parasite's organelles and suggest that Toxoplasma has evolved a specialized autophagic machinery with original regulation.
PMID: 23748741 [PubMed - as supplied by publisher]
Regulation of ATG8 membrane association by ATG4 in the parasitic protist Toxoplasma gondii
Kong-Hap MA, Mouammine A, Daher W, Berry L, Lebrun M, Dubremetz JF, Besteiro S.
UMR 5235 CNRS; Universités de Montpellier 1 et 2; Dynamique des Interactions Membranaires Normales et Pathologiques; Montpellier, France.
In the process of autophagy, the Atg8 protein is conjugated, through a ubiquitin-like system, to the lipid phosphatidylethanolamine (PE) to associate with the membrane of forming autophagosomes. There, it plays a crucial role in the genesis of these organelles and in autophagy in general. In most eukaryotes, the cysteine peptidase Atg4 processes the C terminus of cytosolic Atg8 to regulate its association with autophagosomal membranes and also delipidates Atg8 to release this protein from membranes. The parasitic protist Toxoplasma gondii contains a functional, yet apparently reduced, autophagic machinery. T. gondii Atg8 homolog, in addition to a cytosolic and occasionally autophagosomal localization, also localizes to the apicoplast, a nonphotosynthetic plastid bounded by four membranes. Our attempts to interfere with TgATG8 function showed that it appears to be essential for parasite multiplication inside its host cell. This protein also displays a peculiar C terminus that does not seem to necessitate processing prior to membrane association and yet an unusually large Toxoplasma homolog of ATG4 is predicted in the parasite genome. A TgATG4 conditional expression mutant that we have generated is severely affected in growth, and displays significant alterations at the organellar level, noticeably with a fragmentation of the mitochondrial network and a loss of the apicoplast. TgATG4-depleted parasites appear to be defective in the recycling of membrane-bound TgATG8. Overall, our data highlight a role for the TgATG8 conjugation pathway in maintaining the homeostasis of the parasite's organelles and suggest that Toxoplasma has evolved a specialized autophagic machinery with original regulation.
PMID: 23748741 [PubMed - as supplied by publisher]
Endothelial cell invasion by Toxoplasma gondii: differences between cell types and parasite strains
Parasitol Res. 2013 Jun 10. [Epub ahead of print]
Endothelial cell invasion by Toxoplasma gondii: differences between cell types and parasite strains
Cañedo-Solares I, Calzada-Ruiz M, Ortiz-Alegría LB, Ortiz-Muñiz AR, Correa D.
Laboratorio de Inmunología Experimental, Subdirección de Medicina Experimental, Instituto Nacional de Pediatría Secretaría de Salud, Mexico, Distrito Federal, Mexico.
Toxoplasma gondii disseminates and causes congenital infection by invasion of the endothelial cells. The aim of this study was to analyze the ability of two strains to invade two endothelial cell types. Tachyzoites of the RH and ME49 strains were expanded in Balb/c and C57BL6-RAG2-/- mice, respectively. Tachyzoites were harvested from 72 h Vero cell cultures and incubated for 30 min to 4 h at 10:1 parasite/cell ratio in 24-well plates, containing monolayers of either HMEC-1 line or human umbilical cells (HUVECs). The number of infected cells and parasitic vacuoles per infected cell were counted in Wright stained slides. A slow increase in the proportion of infected cells occurred but varied according to cell type-parasite strain combination: ME49 tachyzoites invaded up to 63 % HMEC-1 cells, while RH parasites infected up to 19 % HUVECs. ME49 and RH tachyzoites invaded 49 and 46 % HUVECs and HMEC-1 cells, respectively. Reinvasion and formation of new parasitophorous vacuoles of infected cells was more frequent than invasion of noninfected cells. The results support that the factors influencing invasion, and thus dissemination and vertical transmission, are parasite type, host cell type/subtype, and activation state. Interestingly, T. gondii virulence does not seem to relay on its invasion efficiency, but probably on replication speed.
PMID: 23749089 [PubMed - as supplied by publisher]
Endothelial cell invasion by Toxoplasma gondii: differences between cell types and parasite strains
Cañedo-Solares I, Calzada-Ruiz M, Ortiz-Alegría LB, Ortiz-Muñiz AR, Correa D.
Laboratorio de Inmunología Experimental, Subdirección de Medicina Experimental, Instituto Nacional de Pediatría Secretaría de Salud, Mexico, Distrito Federal, Mexico.
Toxoplasma gondii disseminates and causes congenital infection by invasion of the endothelial cells. The aim of this study was to analyze the ability of two strains to invade two endothelial cell types. Tachyzoites of the RH and ME49 strains were expanded in Balb/c and C57BL6-RAG2-/- mice, respectively. Tachyzoites were harvested from 72 h Vero cell cultures and incubated for 30 min to 4 h at 10:1 parasite/cell ratio in 24-well plates, containing monolayers of either HMEC-1 line or human umbilical cells (HUVECs). The number of infected cells and parasitic vacuoles per infected cell were counted in Wright stained slides. A slow increase in the proportion of infected cells occurred but varied according to cell type-parasite strain combination: ME49 tachyzoites invaded up to 63 % HMEC-1 cells, while RH parasites infected up to 19 % HUVECs. ME49 and RH tachyzoites invaded 49 and 46 % HUVECs and HMEC-1 cells, respectively. Reinvasion and formation of new parasitophorous vacuoles of infected cells was more frequent than invasion of noninfected cells. The results support that the factors influencing invasion, and thus dissemination and vertical transmission, are parasite type, host cell type/subtype, and activation state. Interestingly, T. gondii virulence does not seem to relay on its invasion efficiency, but probably on replication speed.
PMID: 23749089 [PubMed - as supplied by publisher]
TLR-independent neutrophil-derived IFN-γ is important for host resistance to intracellular pathogens
Proc Natl Acad Sci U S A. 2013 Jun 10. [Epub ahead of print]
TLR-independent neutrophil-derived IFN-γ is important for host resistance to intracellular pathogens
Sturge CR, Benson A, Raetz M, Wilhelm CL, Mirpuri J, Vitetta ES, Yarovinsky F.
Departments of Immunology and Microbiology, and Cancer Immunobiology Center, University of Texas Southwestern Medical School, Dallas, TX 75390.
IFN-γ is a major cytokine that is critical for host resistance to a broad range of intracellular pathogens. Production of IFN-γ by natural killer and T cells is initiated by the recognition of pathogens by Toll-like receptors (TLRs). In an experimental model of toxoplasmosis, we have identified the presence of a nonlymphoid source of IFN-γ that was particularly evident in the absence of TLR-mediated recognition of Toxoplasma gondii. Genetically altered mice lacking all lymphoid cells due to deficiencies in Recombination Activating Gene 2 and IL-2Rγc genes also produced IFN-γ in response to the protozoan parasite. Flow-cytometry and morphological examinations of non-NK/non-T IFN-γ+ cells identified neutrophils as the cell type capable of producing IFN-γ. Selective elimination of neutrophils in TLR11-/- mice infected with the parasite resulted in acute susceptibility similar to that observed in IFN-γ-deficient mice. Similarly, Salmonella typhimurium infection of TLR-deficient mice induces the appearance of IFN-γ+ neutrophils. Thus, neutrophils are a crucial source for IFN-γ that is required for TLR-independent host protection against intracellular pathogens.
PMID: 23754402 [PubMed - as supplied by publisher]
TLR-independent neutrophil-derived IFN-γ is important for host resistance to intracellular pathogens
Sturge CR, Benson A, Raetz M, Wilhelm CL, Mirpuri J, Vitetta ES, Yarovinsky F.
Departments of Immunology and Microbiology, and Cancer Immunobiology Center, University of Texas Southwestern Medical School, Dallas, TX 75390.
IFN-γ is a major cytokine that is critical for host resistance to a broad range of intracellular pathogens. Production of IFN-γ by natural killer and T cells is initiated by the recognition of pathogens by Toll-like receptors (TLRs). In an experimental model of toxoplasmosis, we have identified the presence of a nonlymphoid source of IFN-γ that was particularly evident in the absence of TLR-mediated recognition of Toxoplasma gondii. Genetically altered mice lacking all lymphoid cells due to deficiencies in Recombination Activating Gene 2 and IL-2Rγc genes also produced IFN-γ in response to the protozoan parasite. Flow-cytometry and morphological examinations of non-NK/non-T IFN-γ+ cells identified neutrophils as the cell type capable of producing IFN-γ. Selective elimination of neutrophils in TLR11-/- mice infected with the parasite resulted in acute susceptibility similar to that observed in IFN-γ-deficient mice. Similarly, Salmonella typhimurium infection of TLR-deficient mice induces the appearance of IFN-γ+ neutrophils. Thus, neutrophils are a crucial source for IFN-γ that is required for TLR-independent host protection against intracellular pathogens.
PMID: 23754402 [PubMed - as supplied by publisher]
Saturday, June 08, 2013
Structural and evolutionary adaptation of rhoptry kinases and pseudokinases, a family of coccidian virulence factors
BMC Evol Biol. 2013 Jun 6;13(1):117. [Epub ahead of print]
Structural and evolutionary adaptation of rhoptry kinases and pseudokinases, a family of coccidian virulence factors
Talevich E, Kannan N.
Background The widespread protozoan parasite Toxoplasma gondii interferes with host cell functions by exporting the contents of a unique apical organelle, the rhoptry. Among the mix of secreted proteins are an expanded, lineage-specific family of protein kinases termed rhoptry kinases (ROPKs), several of which have been shown to be key virulence factors, including the pseudokinase ROP5. The extent and details of the diversification of this protein family are poorly understood. Results In this study, we comprehensively catalogued the ROPK family in the genomes of Toxoplasma gondii, Neospora caninum and Eimeria tenella, as well as portions of the unfinished genome of Sarcocystis neurona, and classified the identified genes into 42 distinct subfamilies. We systematically compared the rhoptry kinase protein sequences and structures to each other and to the broader superfamily of eukaryotic protein kinases to study the patterns of diversification and neofunctionalization in the ROPK family and its subfamilies. We identified three ROPK sub-clades of particular interest: those bearing a structurally conserved N-terminal extension to the kinase domain (NTE), an E. tenella-specific expansion, and a basal cluster including ROP35 and BPK1 that we term ROPKL. Structural analysis in light of the solved structures ROP2, ROP5, ROP8 and in comparison to typical eukaryotic protein kinases revealed ROPK-specific conservation patterns in two key regions of the kinase domain, surrounding a ROPK-conserved insert in the kinase hinge region and a disulfide bridge in the kinase substrate-binding lobe. We also examined conservation patterns specific to the NTE-bearing clade. We discuss the possible functional consequences of each. Conclusions Our work sheds light on several important but previously unrecognized features shared among rhoptry kinases, as well as the essential differences between active and degenerate protein kinases. We identify the most distinctive ROPK-specific features conserved across both active kinases and pseudokinases, and discuss these in terms of sequence motifs, evolutionary context, structural impact and potential functional relevance. By characterizing the proteins that enable these parasites to invade the host cell and co-opt its signaling mechanisms, we provide guidance on potential therapeutic targets for the diseases caused by coccidian parasites.
PMID: 23742205 [PubMed - as supplied by publisher]
Structural and evolutionary adaptation of rhoptry kinases and pseudokinases, a family of coccidian virulence factors
Talevich E, Kannan N.
Background The widespread protozoan parasite Toxoplasma gondii interferes with host cell functions by exporting the contents of a unique apical organelle, the rhoptry. Among the mix of secreted proteins are an expanded, lineage-specific family of protein kinases termed rhoptry kinases (ROPKs), several of which have been shown to be key virulence factors, including the pseudokinase ROP5. The extent and details of the diversification of this protein family are poorly understood. Results In this study, we comprehensively catalogued the ROPK family in the genomes of Toxoplasma gondii, Neospora caninum and Eimeria tenella, as well as portions of the unfinished genome of Sarcocystis neurona, and classified the identified genes into 42 distinct subfamilies. We systematically compared the rhoptry kinase protein sequences and structures to each other and to the broader superfamily of eukaryotic protein kinases to study the patterns of diversification and neofunctionalization in the ROPK family and its subfamilies. We identified three ROPK sub-clades of particular interest: those bearing a structurally conserved N-terminal extension to the kinase domain (NTE), an E. tenella-specific expansion, and a basal cluster including ROP35 and BPK1 that we term ROPKL. Structural analysis in light of the solved structures ROP2, ROP5, ROP8 and in comparison to typical eukaryotic protein kinases revealed ROPK-specific conservation patterns in two key regions of the kinase domain, surrounding a ROPK-conserved insert in the kinase hinge region and a disulfide bridge in the kinase substrate-binding lobe. We also examined conservation patterns specific to the NTE-bearing clade. We discuss the possible functional consequences of each. Conclusions Our work sheds light on several important but previously unrecognized features shared among rhoptry kinases, as well as the essential differences between active and degenerate protein kinases. We identify the most distinctive ROPK-specific features conserved across both active kinases and pseudokinases, and discuss these in terms of sequence motifs, evolutionary context, structural impact and potential functional relevance. By characterizing the proteins that enable these parasites to invade the host cell and co-opt its signaling mechanisms, we provide guidance on potential therapeutic targets for the diseases caused by coccidian parasites.
PMID: 23742205 [PubMed - as supplied by publisher]
Friday, June 07, 2013
A Novel High Throughput Invasion Screen Identifies Host Actin Regulators Required for Efficient Cell Entry by Toxoplasma gondii
PLoS One. 2013 May 31;8(5):e64693. doi: 10.1371/journal.pone.0064693. Print 2013.
A Novel High Throughput Invasion Screen Identifies Host Actin Regulators Required for Efficient Cell Entry by Toxoplasma gondii
Gaji RY, Huynh MH, Carruthers VB.
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
Toxoplasma gondii critically relies on cell invasion as a survival strategy to evade immune clearance during infection. Although it was widely thought that Toxoplasma entry is parasite directed and that the host cell is largely a passive victim, recent studies have suggested that host components such as microfilaments and microtubules indeed contribute to entry. Hence to identify additional host factors, we performed a high-throughput siRNA screen of a human siRNA library targeting druggable proteins using a novel inducible luciferase based invasion assay. The top 100 hits from the primary screen that showed the strongest decreases in invasion were subjected to confirmation by secondary screening, revealing 24 proteins that are potentially involved in Toxoplasma entry into host cells. Interestingly, 6 of the hits appear to affect parasite invasion by modifying host cell actin dynamics, resulting in increased deposition of F-actin at the periphery of the cell. These findings support the emerging notion that host actin dynamics are important for Toxoplasma invasion along with identifying several novel host factors that potentially participate in parasite entry.
PMID: 23741372 [PubMed - in process]
A Novel High Throughput Invasion Screen Identifies Host Actin Regulators Required for Efficient Cell Entry by Toxoplasma gondii
Gaji RY, Huynh MH, Carruthers VB.
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
Toxoplasma gondii critically relies on cell invasion as a survival strategy to evade immune clearance during infection. Although it was widely thought that Toxoplasma entry is parasite directed and that the host cell is largely a passive victim, recent studies have suggested that host components such as microfilaments and microtubules indeed contribute to entry. Hence to identify additional host factors, we performed a high-throughput siRNA screen of a human siRNA library targeting druggable proteins using a novel inducible luciferase based invasion assay. The top 100 hits from the primary screen that showed the strongest decreases in invasion were subjected to confirmation by secondary screening, revealing 24 proteins that are potentially involved in Toxoplasma entry into host cells. Interestingly, 6 of the hits appear to affect parasite invasion by modifying host cell actin dynamics, resulting in increased deposition of F-actin at the periphery of the cell. These findings support the emerging notion that host actin dynamics are important for Toxoplasma invasion along with identifying several novel host factors that potentially participate in parasite entry.
PMID: 23741372 [PubMed - in process]
Priming effects of tumor necrosis factor-α on production of reactive oxygen species during Toxoplasma gondii stimulation and receptor gene expression in differentiated HL-60 cells
J Infect Chemother. 2013 Jun 6. [Epub ahead of print]
Priming effects of tumor necrosis factor-α on production of reactive oxygen species during Toxoplasma gondii stimulation and receptor gene expression in differentiated HL-60 cells
Kikuchi-Ueda T, Ubagai T, Ono Y.
Department of Immunology and Microbiology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo, 173-8605, Japan, takane@med.teikyo-u.ac.jp.
Neutrophils are among the principal effector cells that protect against infectious agents, in part by producing reactive oxygen species (ROS) via the actions of tumor necrosis factor-α (TNF-α). In this study, we investigated whether HL-60 cells that had been differentiated into neutrophil-like cells by all-trans retinoic acid could be primed with TNF-α similar to human neutrophils. Our results showed that when differentiated HL-60 (dHL-60) cells were primed with TNF-α for 10 min, ROS production induced by zymosan A or phorbol myristate acetate (PMA) was enhanced in a TNF-α-dose-dependent manner. In addition, when dHL-60 cells were stimulated with live tachyzoites of Toxoplasma gondii after TNF-α priming, ROS production was also enhanced. Thus, dHL-60, similar to neutrophils, produced ROS after PMA, zymosan A, or T. gondii stimulation. Furthermore, we examined gene expression in dHL-60 cells after TNF-α treatment. The pro-inflammatory cytokine IL-6 was up-regulated more than 1.6-fold by 0.1 ng/mL TNF-α. Endogenous TNF-α was down-regulated by priming. IL-8 receptors genes were not affected by priming with 0.1 ng/mL or 1 ng/mL TNF-α. Complement receptor (CR) 1 and CR3 gene expression was not affected by TNF-α priming for 10 min. However, when the priming period was extended to 1 h, CR1 and CR3 genes were up-regulated 1.3 and 1.4-fold, respectively. Expression of the cell-surface CR3 (CD11b) was not significantly affected by TNF-α for 15 min but was slightly enhanced after priming for 2 h. These results suggest that dHL-60 cells may be used as a substitute for neutrophils when evaluating the effects of cytokines or immunomodulator agents.
PMID: 23740089 [PubMed - as supplied by publisher]
Priming effects of tumor necrosis factor-α on production of reactive oxygen species during Toxoplasma gondii stimulation and receptor gene expression in differentiated HL-60 cells
Kikuchi-Ueda T, Ubagai T, Ono Y.
Department of Immunology and Microbiology, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo, 173-8605, Japan, takane@med.teikyo-u.ac.jp.
Neutrophils are among the principal effector cells that protect against infectious agents, in part by producing reactive oxygen species (ROS) via the actions of tumor necrosis factor-α (TNF-α). In this study, we investigated whether HL-60 cells that had been differentiated into neutrophil-like cells by all-trans retinoic acid could be primed with TNF-α similar to human neutrophils. Our results showed that when differentiated HL-60 (dHL-60) cells were primed with TNF-α for 10 min, ROS production induced by zymosan A or phorbol myristate acetate (PMA) was enhanced in a TNF-α-dose-dependent manner. In addition, when dHL-60 cells were stimulated with live tachyzoites of Toxoplasma gondii after TNF-α priming, ROS production was also enhanced. Thus, dHL-60, similar to neutrophils, produced ROS after PMA, zymosan A, or T. gondii stimulation. Furthermore, we examined gene expression in dHL-60 cells after TNF-α treatment. The pro-inflammatory cytokine IL-6 was up-regulated more than 1.6-fold by 0.1 ng/mL TNF-α. Endogenous TNF-α was down-regulated by priming. IL-8 receptors genes were not affected by priming with 0.1 ng/mL or 1 ng/mL TNF-α. Complement receptor (CR) 1 and CR3 gene expression was not affected by TNF-α priming for 10 min. However, when the priming period was extended to 1 h, CR1 and CR3 genes were up-regulated 1.3 and 1.4-fold, respectively. Expression of the cell-surface CR3 (CD11b) was not significantly affected by TNF-α for 15 min but was slightly enhanced after priming for 2 h. These results suggest that dHL-60 cells may be used as a substitute for neutrophils when evaluating the effects of cytokines or immunomodulator agents.
PMID: 23740089 [PubMed - as supplied by publisher]
SAG2A protein from Toxoplasma gondii interacts with both innate and adaptive immune compartments of infected hosts
Parasit Vectors. 2013 Jun 5;6(1):163. [Epub ahead of print]
SAG2A protein from Toxoplasma gondii interacts with both innate and adaptive immune compartments of infected hosts
Macêdo AG Junior, Cunha JP Junior, Cardoso TH, Silva MV, Santiago FM, Silva JS, Pirovani CP, Silva DA, Mineo JR, Mineo TW.
BACKGROUND:
Toxoplasma gondii is an intracellular parasite that causes relevant clinical disease in humans and animals. Several studies have been performed in order to understand the interactions between proteins of the parasite and host cells. SAG2A is a 22 kDa protein that is mainly found in the surface of tachyzoites. In the present work, our aim was to correlate the predicted three-dimensional structure of this protein with the immune system of infected hosts.
METHODS:
To accomplish our goals, we performed in silico analysis of the amino acid sequence of SAG2A, correlating the predictions with in vitro stimulation of antigen presenting cells and serological assays.
RESULTS:
Structure modeling predicts that SAG2A protein possesses an unfolded C-terminal end, which varies its conformation within distinct strain types of T. gondii. This structure within the protein shelters a known B-cell immunodominant epitope, which presents low identity with its closest phyllogenetically related protein, an orthologue predicted in Neospora caninum. In agreement with the in silico observations, sera of known T. gondii infected mice and goats recognized recombinant SAG2A, whereas no serological cross-reactivity was observed with samples from N. caninum animals. Additionally, the C-terminal end of the protein was able to down-modulate pro-inflammatory responses of activated macrophages and dendritic cells.
CONCLUSIONS:
Altogether, we demonstrate herein that recombinant SAG2A protein from T. gondii is immunologically relevant in the host-parasite interface and may be targeted in therapeutic and diagnostic procedures designed against the infection.
PMID: 23735002 [PubMed - as supplied by publisher]
SAG2A protein from Toxoplasma gondii interacts with both innate and adaptive immune compartments of infected hosts
Macêdo AG Junior, Cunha JP Junior, Cardoso TH, Silva MV, Santiago FM, Silva JS, Pirovani CP, Silva DA, Mineo JR, Mineo TW.
BACKGROUND:
Toxoplasma gondii is an intracellular parasite that causes relevant clinical disease in humans and animals. Several studies have been performed in order to understand the interactions between proteins of the parasite and host cells. SAG2A is a 22 kDa protein that is mainly found in the surface of tachyzoites. In the present work, our aim was to correlate the predicted three-dimensional structure of this protein with the immune system of infected hosts.
METHODS:
To accomplish our goals, we performed in silico analysis of the amino acid sequence of SAG2A, correlating the predictions with in vitro stimulation of antigen presenting cells and serological assays.
RESULTS:
Structure modeling predicts that SAG2A protein possesses an unfolded C-terminal end, which varies its conformation within distinct strain types of T. gondii. This structure within the protein shelters a known B-cell immunodominant epitope, which presents low identity with its closest phyllogenetically related protein, an orthologue predicted in Neospora caninum. In agreement with the in silico observations, sera of known T. gondii infected mice and goats recognized recombinant SAG2A, whereas no serological cross-reactivity was observed with samples from N. caninum animals. Additionally, the C-terminal end of the protein was able to down-modulate pro-inflammatory responses of activated macrophages and dendritic cells.
CONCLUSIONS:
Altogether, we demonstrate herein that recombinant SAG2A protein from T. gondii is immunologically relevant in the host-parasite interface and may be targeted in therapeutic and diagnostic procedures designed against the infection.
PMID: 23735002 [PubMed - as supplied by publisher]
Genome-wide comparative analysis revealed significant transcriptome changes in mice after Toxoplasma gondii infection
Parasit Vectors. 2013 Jun 4;6(1):161. [Epub ahead of print]
Genome-wide comparative analysis revealed significant transcriptome changes in mice after Toxoplasma gondii infection
Jia B, Lu H, Liu Q, Yin J, Jiang N, Chen Q.
BACKGROUND:
Toxoplasma gondii is an intracellular parasite that can modulate host responses and presumably host behavior. Host responses as well as pathogenesis vary depending on the parasite strains that are responsible for infection. In immune competent individuals, T. gondii preferentially infects tissues of the central nervous systems (CNS), which might be an additional factor in certain psychiatric disorders. While in immune-compromised individuals and pregnant women, the parasite can cause life-threatening infections. With the availability of the genome-wide investigation platform, the global responses in gene expression of the host after T. gondii infection can be systematically investigated.
METHODS:
Total RNA of brain tissues and peripheral lymphocytes of BALB/C mice infected with RH and ME 49 strain T. gondii as well as that of healthy mice were purified and converted to cRNA with incorporated Cy5-CTP (experimental samples), or Cy3-CTP (control samples). The labeled cRNA probes were hybridized to the Whole Mouse Genome Microarray. The impact of parasite infection on gene expression in both brain tissues and peripheral lymphocytes were analyzed. Differentially expressed genes were revalidated with real-time quantitative reverse transcriptase-polymerase chain reaction (Q-PCR).
RESULTS:
Data indicated that the genes associated with immunity were up-regulated after infection by the two parasite strains, but significant up-regulation was observed in both brain tissues and peripheral lymphocytes of mice infected with ME49 strain compared to that infected by RH strain. The pathways related to pathogenesis of the nervous system were more significantly up-regulated in mice infected with RH strain.
CONCLUSIONS:
Genetically distinct T. gondii strains showed clear differences in modulation of host pathophysiological and immunological responses in both brain tissue and peripheral lymphocytes. It was likely that some of the host responses to T. gondii infection were universal, but the immune response and CNS reaction were in a strain-specific manner.
PMID: 23734932 [PubMed - as supplied by publisher]
Genome-wide comparative analysis revealed significant transcriptome changes in mice after Toxoplasma gondii infection
Jia B, Lu H, Liu Q, Yin J, Jiang N, Chen Q.
BACKGROUND:
Toxoplasma gondii is an intracellular parasite that can modulate host responses and presumably host behavior. Host responses as well as pathogenesis vary depending on the parasite strains that are responsible for infection. In immune competent individuals, T. gondii preferentially infects tissues of the central nervous systems (CNS), which might be an additional factor in certain psychiatric disorders. While in immune-compromised individuals and pregnant women, the parasite can cause life-threatening infections. With the availability of the genome-wide investigation platform, the global responses in gene expression of the host after T. gondii infection can be systematically investigated.
METHODS:
Total RNA of brain tissues and peripheral lymphocytes of BALB/C mice infected with RH and ME 49 strain T. gondii as well as that of healthy mice were purified and converted to cRNA with incorporated Cy5-CTP (experimental samples), or Cy3-CTP (control samples). The labeled cRNA probes were hybridized to the Whole Mouse Genome Microarray. The impact of parasite infection on gene expression in both brain tissues and peripheral lymphocytes were analyzed. Differentially expressed genes were revalidated with real-time quantitative reverse transcriptase-polymerase chain reaction (Q-PCR).
RESULTS:
Data indicated that the genes associated with immunity were up-regulated after infection by the two parasite strains, but significant up-regulation was observed in both brain tissues and peripheral lymphocytes of mice infected with ME49 strain compared to that infected by RH strain. The pathways related to pathogenesis of the nervous system were more significantly up-regulated in mice infected with RH strain.
CONCLUSIONS:
Genetically distinct T. gondii strains showed clear differences in modulation of host pathophysiological and immunological responses in both brain tissue and peripheral lymphocytes. It was likely that some of the host responses to T. gondii infection were universal, but the immune response and CNS reaction were in a strain-specific manner.
PMID: 23734932 [PubMed - as supplied by publisher]
Wednesday, June 05, 2013
Invasion factors of apicomplexan parasites: essential or redundant?
Curr Opin Microbiol. 2013 May 30. pii: S1369-5274(13)00069-6. doi: 10.1016/j.mib.2013.05.002. [Epub ahead of print]
Invasion factors of apicomplexan parasites: essential or redundant?
Meissner M, Ferguson DJ, Frischknecht F.
Welcome Trust and University of Glasgow, Glasgow Biomedical Research Centre, Office b-613, Glasgow G12 8QQ, United Kingdom.Electronic address: markus.meissner@glasgow.ac.uk
Apicomplexa are obligate intracellular parasites that cause several human and veterinary diseases worldwide. In contrast to most intracellular pathogens these protozoans are believed to invade a rather passive host cell in a process, that is, tightly linked to the ability of the parasites to move by gliding motility. Indeed specific inhibitors against components of the gliding machinery and the analysis of knockdown mutants demonstrate a linkage of gliding motility and invasion. Intriguingly, new data show that it is possible to block gliding motility, while host cell invasion still occurs. This suggests that either the current models established for host cell invasion need to be critically revised or that alternative, motor independent mechanisms are in place including a more active role of the host cell that can complement a missing actin-myosin-system. Here we discuss some of the discrepancies that need to be addressed for a better understanding of invasion.
PMID: 23727286 [PubMed - as supplied by publisher]
Invasion factors of apicomplexan parasites: essential or redundant?
Meissner M, Ferguson DJ, Frischknecht F.
Welcome Trust and University of Glasgow, Glasgow Biomedical Research Centre, Office b-613, Glasgow G12 8QQ, United Kingdom.Electronic address: markus.meissner@glasgow.ac.uk
Apicomplexa are obligate intracellular parasites that cause several human and veterinary diseases worldwide. In contrast to most intracellular pathogens these protozoans are believed to invade a rather passive host cell in a process, that is, tightly linked to the ability of the parasites to move by gliding motility. Indeed specific inhibitors against components of the gliding machinery and the analysis of knockdown mutants demonstrate a linkage of gliding motility and invasion. Intriguingly, new data show that it is possible to block gliding motility, while host cell invasion still occurs. This suggests that either the current models established for host cell invasion need to be critically revised or that alternative, motor independent mechanisms are in place including a more active role of the host cell that can complement a missing actin-myosin-system. Here we discuss some of the discrepancies that need to be addressed for a better understanding of invasion.
PMID: 23727286 [PubMed - as supplied by publisher]
Effects of pH, Sodium Chloride, and Curing Salt on the Infectivity of Toxoplasma gondii Tissue Cysts
J Food Prot. 2013 Jun;76(6):1056-61. doi: 10.4315/0362-028X.JFP-12-519
Effects of pH, Sodium Chloride, and Curing Salt on the Infectivity of Toxoplasma gondii Tissue Cysts
Pott S, Koethe M, Bangoura B, Zöller B, Daugschies A, Straubinger RK, Fehlhaber K, Ludewig M.
Institute of Food Hygiene, University of Leipzig, An den Tierkliniken 1, 04103 Leipzig, Germany.
Toxoplasma gondii is one of the most common zoonotic parasites in the world. The parasite causes no or mild symptoms in immunocompetent humans. However, a high potential hazard exists for seronegative pregnant women and immunocompromised patients. The consumption of meat containing tissue cysts or oocyst-contaminated vegetables and fruits or the handling of cat feces poses a high risk of infection with T. gondii. It is known that raw minced meat, raw fresh sausages, and locally produced raw meat products are possible causes of T. gondii infection. The infectivity of T. gondii tissue cysts in meat products depends, among other factors, on the pH and the salt concentration. Therefore, the impact of these two factors on the tissue cysts was examined. For this purpose, dissected musculature and brain from experimentally infected mice (donor mice) were placed in a cell culture medium (RPMI 1640). The medium was adjusted to different pH values (pH 5, 6, and 7) with lactic acid and to different salt concentrations (2.0, 2.5, and 3.0%) with sodium chloride (NaCl) or nitrite-enriched curing salt (NCS) for the various tests. After storage at 4°C for different time periods, the materials were fed to bioassay mice. Later, the brains were examined for presence of T. gondii to assess the infectivity. The data show that T. gondii tissue cysts have a high pH tolerance. Cysts were infectious in the muscle for up to 26 days (pH 5). In contrast to their tolerance to pH, cysts were very sensitive to salt. Muscle cysts survived at an NaCl concentration of up to 2.0% only, and for no longer than 8 days. At NaCl concentrations of 2.5 and 3.0%, the cysts lost their infectivity after 1 day. When NCS instead of NaCl was used under the same conditions, T. gondii muscle cysts retained infectivity for only 4 days at 2.0%. Consequently, NCS (NaCl plus 0.5% nitrite) has a stronger effect on T. gondii cysts than does common table salt. Sausages produced with low NaCl concentration and short contact times pose a potential risk for susceptible individuals.
PMID: 23726205 [PubMed - in process]
Recent insights into apicomplexan parasite egress provide new views to a kill
Curr Opin Microbiol. 2013 May 28. pii: S1369-5274(13)00065-9. doi: 10.1016/j.mib.2013.04.008. [Epub ahead of print]
Recent insights into apicomplexan parasite egress provide new views to a kill
Blackman MJ, Carruthers VB.
Division of Parasitology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
A hallmark of apicomplexan pathogens such as Plasmodium, Toxoplasma and Cryptosporidium is that they invade, replicate within, and then egress from their host cells. Egress usually results in lysis of the host cell, with deleterious consequences for the host. In the case of malaria, for example, much of the disease pathology is associated with cyclical waves of host erythrocyte destruction. This review highlights recent advances in mapping the signaling pathways that lead to egress and the parasite molecules involved in responding to and transmitting those signals. The review also discusses new findings for effector molecules that mediate disruption of the bounding membranes that enclose the intracellular parasite and the manner in which membrane rupture occurs to finally release invasive forms of the parasite.
PMID: 23725669 [PubMed - as supplied by publisher]
Recent insights into apicomplexan parasite egress provide new views to a kill
Blackman MJ, Carruthers VB.
Division of Parasitology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom.
A hallmark of apicomplexan pathogens such as Plasmodium, Toxoplasma and Cryptosporidium is that they invade, replicate within, and then egress from their host cells. Egress usually results in lysis of the host cell, with deleterious consequences for the host. In the case of malaria, for example, much of the disease pathology is associated with cyclical waves of host erythrocyte destruction. This review highlights recent advances in mapping the signaling pathways that lead to egress and the parasite molecules involved in responding to and transmitting those signals. The review also discusses new findings for effector molecules that mediate disruption of the bounding membranes that enclose the intracellular parasite and the manner in which membrane rupture occurs to finally release invasive forms of the parasite.
PMID: 23725669 [PubMed - as supplied by publisher]
The Toxoplasma gondii centrosome is the platform for internal daughter budding as revealed by a Nek1 kinase mutant
J Cell Sci. 2013 May 31. [Epub ahead of print]
The Toxoplasma gondii centrosome is the platform for internal daughter budding as revealed by a Nek1 kinase mutant
Chen CT, Gubbels MJ.
The pathology and severity of toxoplasmosis results from the rapid replication cycle of the apicomplexan parasite Toxoplasma gondii. The tachyzoites divide asexually through endodyogeny, wherein two daughter cells bud inside the mother cell. Before mitosis is completed, the daughter buds form around the duplicated centrosomes, and subsequently elongate to serve as the scaffold for organellogenesis and organelle partitioning. The molecular control mechanism of this process is poorly understood. Here, we characterized an ortholog of NIMA-related kinase (Nek) in T. gondii identified in a chemical mutagenesis screen. A temperature sensitive mutant, V-A15, possesses a Cys316Arg mutation in TgNek1 (a novel mutant allele in Neks), which is responsible for growth defects at the restrictive temperature. Phenotypic analysis of V-A15 indicated that TgNek1 is essential for centrosome splitting, proper formation of daughter cells, and faithful segregation of genetic material. In vitro kinase assays showed that the mutation abolishes the kinase activity of TgNek1. TgNek1 is recruited to the spindle pole prior to mitosis and localizes to the duplicated centrosomes on the face of the spindle poles in a cell-cycle-dependent manner. Mutational analysis of the activation loop suggests localization and activity are spatio-temporally regulated by differential phosphorylation. Collectively, our results identified a novel temperature sensitive allele for a Nek kinase, which in Toxoplasma highlights its essential function in centrosome splitting. Moreover, these results conclusively show for the first time that Toxoplasma bud assembly is facilitated by the centrosome since defective centrosome splitting results in single daughter cell budding.
PMID: 23729737 [PubMed - as supplied by publisher]
The Toxoplasma gondii centrosome is the platform for internal daughter budding as revealed by a Nek1 kinase mutant
Chen CT, Gubbels MJ.
The pathology and severity of toxoplasmosis results from the rapid replication cycle of the apicomplexan parasite Toxoplasma gondii. The tachyzoites divide asexually through endodyogeny, wherein two daughter cells bud inside the mother cell. Before mitosis is completed, the daughter buds form around the duplicated centrosomes, and subsequently elongate to serve as the scaffold for organellogenesis and organelle partitioning. The molecular control mechanism of this process is poorly understood. Here, we characterized an ortholog of NIMA-related kinase (Nek) in T. gondii identified in a chemical mutagenesis screen. A temperature sensitive mutant, V-A15, possesses a Cys316Arg mutation in TgNek1 (a novel mutant allele in Neks), which is responsible for growth defects at the restrictive temperature. Phenotypic analysis of V-A15 indicated that TgNek1 is essential for centrosome splitting, proper formation of daughter cells, and faithful segregation of genetic material. In vitro kinase assays showed that the mutation abolishes the kinase activity of TgNek1. TgNek1 is recruited to the spindle pole prior to mitosis and localizes to the duplicated centrosomes on the face of the spindle poles in a cell-cycle-dependent manner. Mutational analysis of the activation loop suggests localization and activity are spatio-temporally regulated by differential phosphorylation. Collectively, our results identified a novel temperature sensitive allele for a Nek kinase, which in Toxoplasma highlights its essential function in centrosome splitting. Moreover, these results conclusively show for the first time that Toxoplasma bud assembly is facilitated by the centrosome since defective centrosome splitting results in single daughter cell budding.
PMID: 23729737 [PubMed - as supplied by publisher]
The glideosome, a unique machinery that assists the Apicomplexa in gliding into host cells
Med Sci (Paris). 2013 May;29(5):515-22. doi: 10.1051/medsci/2013295015. Epub 2013 May 28.
The glideosome, a unique machinery that assists the Apicomplexa in gliding into host cells
[Article in French]
Frénal K, Soldati-Favre D.
Département de microbiologie et médecine moléculaire, faculté de médecine, université de Genève, centre médical universitaire, 1 rue Michel Servet, 1211 Genève, Suisse.
Protozoan parasites belonging to the phylum Apicomplexa are of considerable medical and veterinary significance. These obligate intracellular parasites use a unique form of locomotion to traverse biological barriers and actively invade in and egress from host cells. An actin-myosin-based complex named "glideosome" drives this unusual substrate-dependent motility, which is essential for the establishment of the infection. The mechanisms involved in motility, invasion and egress are conserved throughout the phylum. This article describes the current knowledge on the invasion process of two experimentally tractable apicomplexan parasites: Toxoplasma gondii and Plasmodium falciparum.
PMID: 23732101 [PubMed - in process]
The glideosome, a unique machinery that assists the Apicomplexa in gliding into host cells
[Article in French]
Frénal K, Soldati-Favre D.
Département de microbiologie et médecine moléculaire, faculté de médecine, université de Genève, centre médical universitaire, 1 rue Michel Servet, 1211 Genève, Suisse.
Protozoan parasites belonging to the phylum Apicomplexa are of considerable medical and veterinary significance. These obligate intracellular parasites use a unique form of locomotion to traverse biological barriers and actively invade in and egress from host cells. An actin-myosin-based complex named "glideosome" drives this unusual substrate-dependent motility, which is essential for the establishment of the infection. The mechanisms involved in motility, invasion and egress are conserved throughout the phylum. This article describes the current knowledge on the invasion process of two experimentally tractable apicomplexan parasites: Toxoplasma gondii and Plasmodium falciparum.
PMID: 23732101 [PubMed - in process]
Saturday, June 01, 2013
Enzymatically active Rho and Rac small-GTPases are involved in the establishment of the vacuolar membrane after Toxoplasma gondii invasion of host cells
BMC Microbiol. 2013 May 30;13(1):125. [Epub ahead of print]
Enzymatically active Rho and Rac small-GTPases are involved in the establishment of the vacuolar membrane after Toxoplasma gondii invasion of host cells
Na RH, Zhu GH, Luo JX, Meng XJ, Cui L, Peng HJ, Chen XG, Gomez-Cambronero J.
BACKGROUND:
GTPases are the family of hydrolases that bind and hydrolyze guanosine triphosphate. The large Immunity-related GTPases and the small GTPase ADP-ribosylation factor-6 in host cells are known to accumulate on the parasitophorous vacuole membrane (PVM) of Toxoplasma gondii and play critical roles in this parasite infection, but these GTPases cannot explain the full extent of infection.
RESULTS:
In this research, RhoA and Rac1 GTPases from the host cell were found to accumulate on the PVM regardless of the virulence of the T. gondii strains after T. gondii invasion, and this accumulation was dependent on their GTPase activity. The real-time micrography of T. gondii tachyzoites invading COS-7 cells overexpressing CFP-RhoA showed that this GTPase was recruited to the PVM at the very beginning of the invasion through the host cell membrane or from the cytosol. Host cell RhoA and Rac1 were also activated after T. gondii tachyzoites invasion, which was needed for host cell cytoskeleton reorganization to facilitate intracellular pathogens invasion. The decisive domains for the RhoA accumulation on the PVM included the GTP/Mg2+ binding site, the mDia effector interaction site, the G1 box, the G2 box and the G5 box, respectively, which were related to the binding of GTP for enzymatic activity and mDia for the regulation of microtubules. The recruited CFP-RhoA on the PVM could not be activated by epithelial growth factor (EGF) and no translocation was observed, unlike the unassociated RhoA in the host cell cytosol that migrated to the cell membrane towards the EGF activation spot. This result supported the hypothesis that the recruited RhoA or Rac1 on the PVM were in the GTP-bound active form. Wild-type RhoA or Rac1 overexpressed cells had almost the same infection rates by T. gondii as the mock-treated cells, while RhoA-N19 or Rac1-N17 transfected cells and RhoA, Rac1 or RhoA + Rac1 siRNA-treated cells showed significantly diminished infection rates compared to mock cells.
CONCLUSIONS:
The accumulation of the RhoA and Rac1 on the PVM and the requisite of their normal GTPase activity for efficient invasion implied their involvement and function in T. gondii invasion.
PMID: 23721065 [PubMed - as supplied by publisher]
Enzymatically active Rho and Rac small-GTPases are involved in the establishment of the vacuolar membrane after Toxoplasma gondii invasion of host cells
Na RH, Zhu GH, Luo JX, Meng XJ, Cui L, Peng HJ, Chen XG, Gomez-Cambronero J.
BACKGROUND:
GTPases are the family of hydrolases that bind and hydrolyze guanosine triphosphate. The large Immunity-related GTPases and the small GTPase ADP-ribosylation factor-6 in host cells are known to accumulate on the parasitophorous vacuole membrane (PVM) of Toxoplasma gondii and play critical roles in this parasite infection, but these GTPases cannot explain the full extent of infection.
RESULTS:
In this research, RhoA and Rac1 GTPases from the host cell were found to accumulate on the PVM regardless of the virulence of the T. gondii strains after T. gondii invasion, and this accumulation was dependent on their GTPase activity. The real-time micrography of T. gondii tachyzoites invading COS-7 cells overexpressing CFP-RhoA showed that this GTPase was recruited to the PVM at the very beginning of the invasion through the host cell membrane or from the cytosol. Host cell RhoA and Rac1 were also activated after T. gondii tachyzoites invasion, which was needed for host cell cytoskeleton reorganization to facilitate intracellular pathogens invasion. The decisive domains for the RhoA accumulation on the PVM included the GTP/Mg2+ binding site, the mDia effector interaction site, the G1 box, the G2 box and the G5 box, respectively, which were related to the binding of GTP for enzymatic activity and mDia for the regulation of microtubules. The recruited CFP-RhoA on the PVM could not be activated by epithelial growth factor (EGF) and no translocation was observed, unlike the unassociated RhoA in the host cell cytosol that migrated to the cell membrane towards the EGF activation spot. This result supported the hypothesis that the recruited RhoA or Rac1 on the PVM were in the GTP-bound active form. Wild-type RhoA or Rac1 overexpressed cells had almost the same infection rates by T. gondii as the mock-treated cells, while RhoA-N19 or Rac1-N17 transfected cells and RhoA, Rac1 or RhoA + Rac1 siRNA-treated cells showed significantly diminished infection rates compared to mock cells.
CONCLUSIONS:
The accumulation of the RhoA and Rac1 on the PVM and the requisite of their normal GTPase activity for efficient invasion implied their involvement and function in T. gondii invasion.
PMID: 23721065 [PubMed - as supplied by publisher]
Characterization of mouse brain microRNAs after infection with cyst-forming Toxoplasma gondii
Parasit Vectors. 2013 May 29;6(1):154. [Epub ahead of print]
Characterization of mouse brain microRNAs after infection with cyst-forming Toxoplasma gondii
Xu MJ, Zhou DH, Nisbet AJ, Huang SY, Fan YF, Zhu XQ.
BACKGROUND:
The obligate intracellular parasite Toxoplasma gondii can interfere with host cell signaling pathways, alter host defense systems and cell cycle control, and establish a chronic infection in the central nervous system. T. gondii infection may alter the expression profile of host microRNAs (miRNAs) which have key regulatory functions at the post-transcriptional level.
METHODS:
Using high-throughput sequencing and real-time quantitative PCR technology, we compared the miRNA expression profiles of uninfected mouse brains with brains from mice at 14 days and 21 days after infection with cyst-forming T. gondii (Type II).
RESULTS:
A total of 51.30 million raw reads were obtained from all samples and 495 (14d infected mouse sample), 511 (14d sham-infected control), 504 (21d infected mouse sample) and 514 (21d sham-infected control) miRNA candidates identified. Among these, 414 miRNAs were consistent across all the studied groups, 17 were specific to the 14d infected group and 32 were specific to the 21d infected group. In addition, 9 miRNAs were common to both the 14d- and 21d-infected groups. Enrichment analysis for the targets of these miRNAs showed a high percentage of "protein tag" functions. Immune related targets including chemokines, cytokines, growth factors and interleukins were also found.
CONCLUSIONS:
These results not only showed that the miRNA expression of the host can be changed by the invasion of cyst-forming T. gondii, but also indicated that the host attempts to respond using two tactics: marking proteins with "protein tags" and adaptation of immune related systems.
PMID: 23718711 [PubMed - as supplied by publisher]
Characterization of mouse brain microRNAs after infection with cyst-forming Toxoplasma gondii
Xu MJ, Zhou DH, Nisbet AJ, Huang SY, Fan YF, Zhu XQ.
BACKGROUND:
The obligate intracellular parasite Toxoplasma gondii can interfere with host cell signaling pathways, alter host defense systems and cell cycle control, and establish a chronic infection in the central nervous system. T. gondii infection may alter the expression profile of host microRNAs (miRNAs) which have key regulatory functions at the post-transcriptional level.
METHODS:
Using high-throughput sequencing and real-time quantitative PCR technology, we compared the miRNA expression profiles of uninfected mouse brains with brains from mice at 14 days and 21 days after infection with cyst-forming T. gondii (Type II).
RESULTS:
A total of 51.30 million raw reads were obtained from all samples and 495 (14d infected mouse sample), 511 (14d sham-infected control), 504 (21d infected mouse sample) and 514 (21d sham-infected control) miRNA candidates identified. Among these, 414 miRNAs were consistent across all the studied groups, 17 were specific to the 14d infected group and 32 were specific to the 21d infected group. In addition, 9 miRNAs were common to both the 14d- and 21d-infected groups. Enrichment analysis for the targets of these miRNAs showed a high percentage of "protein tag" functions. Immune related targets including chemokines, cytokines, growth factors and interleukins were also found.
CONCLUSIONS:
These results not only showed that the miRNA expression of the host can be changed by the invasion of cyst-forming T. gondii, but also indicated that the host attempts to respond using two tactics: marking proteins with "protein tags" and adaptation of immune related systems.
PMID: 23718711 [PubMed - as supplied by publisher]
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