J Immunol. 2013 Jan 28. [Epub ahead of print]
IL-2 Produced by CD8+ Immune T Cells Can Augment Their IFN-γ Production Independently from Their Proliferation in the Secondary Response to an Intracellular Pathogen
Sa Q, Woodward J, Suzuki Y.
Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY 40536.
Chronic infection with Toxoplasma gondii induces a potent resistance against reinfection, and IFN-γ production by CD8(+) T cells is crucial for the protective immunity. However, the molecular mechanisms that regulate the secondary response remain to be elucidated. In the current study, we examined the role of IL-2 in IFN-γ production by CD8(+) immune T cells in their secondary responses using T. gondii-specific CD8(+) T cell hybridomas and splenic CD8(+) immune T cells from chronically infected mice. The majority (92%) of CD8(+) T cell hybridomas produced large amounts of IFN-γ only when a low amount (0.5 ng/ml) of exogenous IL-2 was provided in combination with T. gondii Ags. Inhibition of cell proliferation by mitomycin C did not affect the enhancing effect of IL-2 on the IFN-γ production, and significant increases in transcription factor T-bet expression were associated with the IL-2-mediated IFN-γ amplification. Splenic CD8(+) immune T cells produced similar low levels of IL-2 in the secondary response to T. gondii, and a blocking of IL-2 signaling by anti-IL-2Rα Ab or inhibitors of JAK1 and JAK3 significantly reduced IFN-γ production of the T cells. This IL-2-mediated upregulation of IFN-γ production was observed in mitomycin C-treated CD8(+) immune T cells, thus independent from their cell division. Therefore, endogenous IL-2 produced by CD8(+) immune T cells can play an important autocrine-enhancing role on their IFN-γ production in the secondary responses to T. gondii, suggesting an importance of induction of CD8(+) immune T cells with an appropriate IL-2 production for vaccine development.
PMID: 23359502 [PubMed - as supplied by publisher]
Wednesday, January 30, 2013
A HT/PEXEL motif in Toxoplasma dense granule proteins is a signal for protein cleavage but not export into the host cell
Traffic. 2013 Jan 28. doi: 10.1111/tra.12049. [Epub ahead of print]
A HT/PEXEL motif in Toxoplasma dense granule proteins is a signal for protein cleavage but not export into the host cell
Hsiao CH, Luisa Hiller N, Haldar K, Knoll LJ
Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, WI.
Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium, secrete proteins for attachment, invasion and modulation of their host cells. The host targeting (HT), also known as the Plasmodium export element (PEXEL), directs Plasmodium proteins into erythrocytes to remodel the host cell and establish infection. Bioinformatic analysis of Toxoplasma revealed a HT/PEXEL-like motif at the N-terminal of several hypothetical unknown and dense granule proteins. Hemagglutinin (HA)-tagged versions of these uncharacterized proteins show co-localization with dense granule proteins found on the parasitophorous vacuole membrane (PVM). In contrast to Plasmodium, these Toxoplasma HT/PEXEL containing proteins are not exported into the host cell. Site directed mutagenesis of the Toxoplasma HT/PEXEL motif, RxLxD/E, shows that the arginine and leucine residues are permissible for protein cleavage. Mutations within the HT/PEXEL motif that prevent protein cleavage still allow for targeting to the PV but the proteins have a reduced association with the PVM. Addition of a Myc tag before and after the cleavage site shows that processed HT/PEXEL protein has increased PVM association. These findings suggest that while Toxoplasma and Plasmodium share similar HT/PEXEL motifs, Toxoplasma HT/PEXEL containing proteins interact with but do not cross the PVM.
PMID: 23356236 [PubMed - as supplied by publisher]
A HT/PEXEL motif in Toxoplasma dense granule proteins is a signal for protein cleavage but not export into the host cell
Hsiao CH, Luisa Hiller N, Haldar K, Knoll LJ
Department of Medical Microbiology & Immunology, University of Wisconsin-Madison, Madison, WI.
Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium, secrete proteins for attachment, invasion and modulation of their host cells. The host targeting (HT), also known as the Plasmodium export element (PEXEL), directs Plasmodium proteins into erythrocytes to remodel the host cell and establish infection. Bioinformatic analysis of Toxoplasma revealed a HT/PEXEL-like motif at the N-terminal of several hypothetical unknown and dense granule proteins. Hemagglutinin (HA)-tagged versions of these uncharacterized proteins show co-localization with dense granule proteins found on the parasitophorous vacuole membrane (PVM). In contrast to Plasmodium, these Toxoplasma HT/PEXEL containing proteins are not exported into the host cell. Site directed mutagenesis of the Toxoplasma HT/PEXEL motif, RxLxD/E, shows that the arginine and leucine residues are permissible for protein cleavage. Mutations within the HT/PEXEL motif that prevent protein cleavage still allow for targeting to the PV but the proteins have a reduced association with the PVM. Addition of a Myc tag before and after the cleavage site shows that processed HT/PEXEL protein has increased PVM association. These findings suggest that while Toxoplasma and Plasmodium share similar HT/PEXEL motifs, Toxoplasma HT/PEXEL containing proteins interact with but do not cross the PVM.
PMID: 23356236 [PubMed - as supplied by publisher]
Wednesday, January 23, 2013
A Host GPCR Signaling Network Required for the Cytolysis of Infected Cells Facilitates Release of Apicomplexan Parasites
Cell Host Microbe. 2013 Jan 16;13(1):15-28. doi: 10.1016/j.chom.2012.12.001. Epub 2013 Jan 16.
A Host GPCR Signaling Network Required for the Cytolysis of Infected Cells Facilitates Release of Apicomplexan Parasites
Millholland MG, Mishra S, Dupont CD, Love MS, Patel B, Shilling D, Kazanietz MG, Foskett JK, Hunter CA, Sinnis P, Greenbaum DC.
Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Following intracellular replication, the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii cause host cell cytolysis to facilitate parasite release and disease progression. Parasite exit from infected cells requires the interplay of parasite-derived proteins and host actin cytoskeletal changes; however, the host proteins underlying these changes remain obscure. We report the identification of a Gα(q)-coupled host-signaling cascade required for the egress of both P. falciparum and T. gondii. Gα(q)-coupled signaling results in protein kinase C (PKC)-mediated loss of the host cytoskeletal protein adducin and weakening of the cellular cytoskeleton. This cytoskeletal compromise induces catastrophic Ca(2+) influx mediated by the mechanosensitive cation channel TRPC6, which activates host calpain that proteolyzes the host cytoskeleton allowing parasite release. Reinforcing the feasibility of targeting host proteins as an antiparasitic strategy, mammalian PKC inhibitors demonstrated activity in murine models of malaria and toxoplasmosis. Importantly, an orally bioavailable PKC inhibitor prolonged survival in an experimental cerebral malaria model.
PMID: 23332153 [PubMed - in process]
A Host GPCR Signaling Network Required for the Cytolysis of Infected Cells Facilitates Release of Apicomplexan Parasites
Millholland MG, Mishra S, Dupont CD, Love MS, Patel B, Shilling D, Kazanietz MG, Foskett JK, Hunter CA, Sinnis P, Greenbaum DC.
Department of Pharmacology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Following intracellular replication, the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii cause host cell cytolysis to facilitate parasite release and disease progression. Parasite exit from infected cells requires the interplay of parasite-derived proteins and host actin cytoskeletal changes; however, the host proteins underlying these changes remain obscure. We report the identification of a Gα(q)-coupled host-signaling cascade required for the egress of both P. falciparum and T. gondii. Gα(q)-coupled signaling results in protein kinase C (PKC)-mediated loss of the host cytoskeletal protein adducin and weakening of the cellular cytoskeleton. This cytoskeletal compromise induces catastrophic Ca(2+) influx mediated by the mechanosensitive cation channel TRPC6, which activates host calpain that proteolyzes the host cytoskeleton allowing parasite release. Reinforcing the feasibility of targeting host proteins as an antiparasitic strategy, mammalian PKC inhibitors demonstrated activity in murine models of malaria and toxoplasmosis. Importantly, an orally bioavailable PKC inhibitor prolonged survival in an experimental cerebral malaria model.
PMID: 23332153 [PubMed - in process]
Tuesday, January 08, 2013
Combined Action of Nucleic Acid-Sensing Toll-like Receptors and TLR11/TLR12 Heterodimers Imparts Resistance to Toxoplasma gondii in Mice
Cell Host Microbe. 2013 Jan 1. pii: S1931-3128(12)00429-5. doi: 10.1016/j.chom.2012.12.003. [Epub ahead of print]
Combined Action of Nucleic Acid-Sensing Toll-like Receptors and TLR11/TLR12 Heterodimers Imparts Resistance to Toxoplasma gondii in Mice
Andrade WA, Souza MD, Ramos-Martinez E, Nagpal K, Dutra MS, Melo MB, Bartholomeu DC, Ghosh S, Golenbock DT, Gazzinelli RT.
University of Massachusetts Medical School, Worcester, MA 01605, USA; Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil.
"Triple-defective" (3d) mice carrying a mutation in UNC93B1, a chaperone for the endosomal nucleic acid-sensing (NAS) Toll-like receptors TLR3, TLR7, and TLR9, are highly susceptible to Toxoplasma gondii infection. However, none of the single or even the triple NAS-TLR-deficient animals recapitulated the 3d susceptible phenotype to experimental toxoplasmosis. Investigating this further, we found that while parasite RNA and DNA activate innate immune responses via TLR7 and TLR9, TLR11 and TLR12 working as heterodimers are required for sensing and responding to Toxoplasma profilin. Consequently, the triple TLR7/TLR9/TLR11-deficient mice are highly susceptible to T. gondii infection, recapitulating the phenotype of 3d mice. Humans lack functional TLR11 and TLR12 genes. Consistently, human cells produce high levels of proinflammatory cytokines in response to parasite-derived RNA and DNA, but not to Toxoplasma profilin, supporting a more critical role for NAS-TLRs in human toxoplasmosis.
PMID: 23290966 [PubMed - as supplied by publisher]
Combined Action of Nucleic Acid-Sensing Toll-like Receptors and TLR11/TLR12 Heterodimers Imparts Resistance to Toxoplasma gondii in Mice
Andrade WA, Souza MD, Ramos-Martinez E, Nagpal K, Dutra MS, Melo MB, Bartholomeu DC, Ghosh S, Golenbock DT, Gazzinelli RT.
University of Massachusetts Medical School, Worcester, MA 01605, USA; Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, MG, Brazil.
"Triple-defective" (3d) mice carrying a mutation in UNC93B1, a chaperone for the endosomal nucleic acid-sensing (NAS) Toll-like receptors TLR3, TLR7, and TLR9, are highly susceptible to Toxoplasma gondii infection. However, none of the single or even the triple NAS-TLR-deficient animals recapitulated the 3d susceptible phenotype to experimental toxoplasmosis. Investigating this further, we found that while parasite RNA and DNA activate innate immune responses via TLR7 and TLR9, TLR11 and TLR12 working as heterodimers are required for sensing and responding to Toxoplasma profilin. Consequently, the triple TLR7/TLR9/TLR11-deficient mice are highly susceptible to T. gondii infection, recapitulating the phenotype of 3d mice. Humans lack functional TLR11 and TLR12 genes. Consistently, human cells produce high levels of proinflammatory cytokines in response to parasite-derived RNA and DNA, but not to Toxoplasma profilin, supporting a more critical role for NAS-TLRs in human toxoplasmosis.
PMID: 23290966 [PubMed - as supplied by publisher]
Bradyzoite pseudokinase 1 is crucial for efficient oral infectivity of the Toxoplasma tissue cyst
Eukaryot Cell. 2013 Jan 4. [Epub ahead of print]
Bradyzoite pseudokinase 1 is crucial for efficient oral infectivity of the Toxoplasma tissue cyst
Buchholz KR, Bowyer PW, Boothroyd JC.
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, 94305, USA.
The tissue cyst formed by the bradyzoite stage of Toxoplasma gondii is essential for persistent infection of the host as well as oral transmission. Bradyzoite secreted pseudokinase 1 (BPK1) is a component of the cyst wall, but nothing has previously been known about its function. Here, we show that immunoprecipitation of BPK1 from in vitro bradyzoite cultures, 4 days post-infection, identifies at least four associating proteins: MAG1, MCP4, GRA8 and GRA9. To determine the role of BPK1, a strain of Toxoplasma was generated with the bpk1 locus deleted. This BPK1 knock-out strain (Δbpk1) was investigated in vitro and in vivo. No defect was found in terms of in vitro cyst formation and no difference in pathogenesis or cyst burden 4 weeks post-infection (wpi) was detected after intraperitoneal (i.p.) infection with Δbpk1 tachyzoites, although the Δbpk1 cysts were significantly smaller than parental or BPK1-complemented strains at 8 wpi. Pepsin-acid treatment of 4 wpi in vivo cysts revealed Δbpk1 parasites are significantly more sensitive to this treatment compared to the parental and complemented strains. Consistent with this, 4 wpi Δbpk1 cysts were reduced in the ability to cause oral infection compared to the parental and complemented strains. Together, these data reveal that BPK1 plays a crucial role in the in vivo development and infectivity of Toxoplasma cysts.
PMID: 23291621 [PubMed - as supplied by publisher]
Bradyzoite pseudokinase 1 is crucial for efficient oral infectivity of the Toxoplasma tissue cyst
Buchholz KR, Bowyer PW, Boothroyd JC.
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, 94305, USA.
The tissue cyst formed by the bradyzoite stage of Toxoplasma gondii is essential for persistent infection of the host as well as oral transmission. Bradyzoite secreted pseudokinase 1 (BPK1) is a component of the cyst wall, but nothing has previously been known about its function. Here, we show that immunoprecipitation of BPK1 from in vitro bradyzoite cultures, 4 days post-infection, identifies at least four associating proteins: MAG1, MCP4, GRA8 and GRA9. To determine the role of BPK1, a strain of Toxoplasma was generated with the bpk1 locus deleted. This BPK1 knock-out strain (Δbpk1) was investigated in vitro and in vivo. No defect was found in terms of in vitro cyst formation and no difference in pathogenesis or cyst burden 4 weeks post-infection (wpi) was detected after intraperitoneal (i.p.) infection with Δbpk1 tachyzoites, although the Δbpk1 cysts were significantly smaller than parental or BPK1-complemented strains at 8 wpi. Pepsin-acid treatment of 4 wpi in vivo cysts revealed Δbpk1 parasites are significantly more sensitive to this treatment compared to the parental and complemented strains. Consistent with this, 4 wpi Δbpk1 cysts were reduced in the ability to cause oral infection compared to the parental and complemented strains. Together, these data reveal that BPK1 plays a crucial role in the in vivo development and infectivity of Toxoplasma cysts.
PMID: 23291621 [PubMed - as supplied by publisher]
Thursday, January 03, 2013
The 2-methylcitrate cycle is implicated in the detoxification of propionate in Toxoplasma gondii
Mol Microbiol. 2012 Dec 25. doi: 10.1111/mmi.12139. [Epub ahead of print]
The 2-methylcitrate cycle is implicated in the detoxification of propionate in Toxoplasma gondii
Limenitakis J, Oppenheim RD, Creek DJ, Foth BJ, Barrett MP, Soldati-Favre D.
Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, CMU 1 Rue Michel Servet, 1211, Geneva, Switzerland.
Toxoplasma gondii belongs to the coccidian-subgroup of the Apicomplexa phylum. The Coccidia are obligate intracellular pathogens that establish infection in their mammalian host via the enteric route. These parasites lack a mitochondrial pyruvate dehydrogenase complex but have preserved the degradation of branched-chain amino acids (BCAA) as a possible pathway to generate acetyl-CoA. Importantly, degradation of leucine, isoleucine and valine could lead to concomitant accumulation of propionyl-CoA, a toxic metabolite that inhibits cell growth. Like fungi and bacteria, the Coccidia possess the complete set of enzymes necessary to metabolize and detoxify propionate by oxidation to pyruvate via the 2-methylcitrate cycle (2-MCC). Phylogenetic analysis provides evidence that the 2-MCC was acquired via horizontal gene transfer. In T. gondii tachyzoites, this pathway is split between the cytosol and the mitochondrion. Although the rate-limiting enzyme 2-methylisocitrate lyase is dispensable for parasite survival, its substrates accumulate in parasites deficient in the enzyme and its absence confers increased sensitivity to propionic acid. BCAA is also dispensable in tachyzoites, leaving unresolved the source of mitochondrial acetyl-CoA.
PMID: 23279335 [PubMed - as supplied by publisher]
The 2-methylcitrate cycle is implicated in the detoxification of propionate in Toxoplasma gondii
Limenitakis J, Oppenheim RD, Creek DJ, Foth BJ, Barrett MP, Soldati-Favre D.
Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, CMU 1 Rue Michel Servet, 1211, Geneva, Switzerland.
Toxoplasma gondii belongs to the coccidian-subgroup of the Apicomplexa phylum. The Coccidia are obligate intracellular pathogens that establish infection in their mammalian host via the enteric route. These parasites lack a mitochondrial pyruvate dehydrogenase complex but have preserved the degradation of branched-chain amino acids (BCAA) as a possible pathway to generate acetyl-CoA. Importantly, degradation of leucine, isoleucine and valine could lead to concomitant accumulation of propionyl-CoA, a toxic metabolite that inhibits cell growth. Like fungi and bacteria, the Coccidia possess the complete set of enzymes necessary to metabolize and detoxify propionate by oxidation to pyruvate via the 2-methylcitrate cycle (2-MCC). Phylogenetic analysis provides evidence that the 2-MCC was acquired via horizontal gene transfer. In T. gondii tachyzoites, this pathway is split between the cytosol and the mitochondrion. Although the rate-limiting enzyme 2-methylisocitrate lyase is dispensable for parasite survival, its substrates accumulate in parasites deficient in the enzyme and its absence confers increased sensitivity to propionic acid. BCAA is also dispensable in tachyzoites, leaving unresolved the source of mitochondrial acetyl-CoA.
PMID: 23279335 [PubMed - as supplied by publisher]
Tuesday, January 01, 2013
Disruption of the Expression of a Non-Coding RNA Significantly Impairs Cellular Differentiation in Toxoplasma gondii
Int J Mol Sci. 2012 Dec 28;14(1):611-24. doi: 10.3390/ijms14010611
Disruption of the Expression of a Non-Coding RNA Significantly Impairs Cellular Differentiation in Toxoplasma gondii
Patil V, Lescault PJ, Lirussi D, Thompson AB, Matrajt M.
Department of Microbiology and Molecular Genetics, University of Vermont, 95 Carrigan Dr. Burlington, VT 05405, USA. mmatrajt@uvm.edu.
The protozoan parasite Toxoplasma gondii is an important human and veterinary pathogen. Asexual replication of T. gondii in humans and intermediate hosts is characterized by two forms: rapidly growing "tachyzoites" and latent "bradyzoite" tissue cysts. Tachyzoites are responsible for acute illness and congenital neurological birth defects, while the more slowly dividing bradyzoite form can remain latent within the tissues for many years, representing a threat to immunocompromised patients. We have developed a genetic screen to identify regulatory genes that control parasite differentiation and have isolated mutants that fail to convert to bradyzoites. One of these mutants has an insertion disrupting a locus that encodes a developmentally regulated non-coding RNA transcript, named Tg-ncRNA-1. Microarray hybridizations suggest that Tg-ncRNA-1 is involved in the early steps of bradyzoite differentiation. Since Tg-ncRNA-1 does not contain an open reading frame, we used the algorithm Coding Potential Calculator (CPC) that evaluates the protein-coding potential of a transcript, to classify Tg-ncRNA-1. The CPC results strongly indicate that Tg-ncRNA-1 is a non-coding RNA (ncRNA). Interestingly, a previously generated mutant also contains an insertion in Tg-ncRNA-1. We show that both mutants have a decreased ability to form bradyzoites, and complementation of both mutants with wild-type Tg-ncRNA-1 restores the ability of the parasites to differentiate. It has been shown that an important part of bradyzoite differentiation is transcriptionally controlled, but this is the first time that a non-coding RNA is implicated in this process.
PMID: 23275028 [PubMed - in process]
Disruption of the Expression of a Non-Coding RNA Significantly Impairs Cellular Differentiation in Toxoplasma gondii
Patil V, Lescault PJ, Lirussi D, Thompson AB, Matrajt M.
Department of Microbiology and Molecular Genetics, University of Vermont, 95 Carrigan Dr. Burlington, VT 05405, USA. mmatrajt@uvm.edu.
The protozoan parasite Toxoplasma gondii is an important human and veterinary pathogen. Asexual replication of T. gondii in humans and intermediate hosts is characterized by two forms: rapidly growing "tachyzoites" and latent "bradyzoite" tissue cysts. Tachyzoites are responsible for acute illness and congenital neurological birth defects, while the more slowly dividing bradyzoite form can remain latent within the tissues for many years, representing a threat to immunocompromised patients. We have developed a genetic screen to identify regulatory genes that control parasite differentiation and have isolated mutants that fail to convert to bradyzoites. One of these mutants has an insertion disrupting a locus that encodes a developmentally regulated non-coding RNA transcript, named Tg-ncRNA-1. Microarray hybridizations suggest that Tg-ncRNA-1 is involved in the early steps of bradyzoite differentiation. Since Tg-ncRNA-1 does not contain an open reading frame, we used the algorithm Coding Potential Calculator (CPC) that evaluates the protein-coding potential of a transcript, to classify Tg-ncRNA-1. The CPC results strongly indicate that Tg-ncRNA-1 is a non-coding RNA (ncRNA). Interestingly, a previously generated mutant also contains an insertion in Tg-ncRNA-1. We show that both mutants have a decreased ability to form bradyzoites, and complementation of both mutants with wild-type Tg-ncRNA-1 restores the ability of the parasites to differentiate. It has been shown that an important part of bradyzoite differentiation is transcriptionally controlled, but this is the first time that a non-coding RNA is implicated in this process.
PMID: 23275028 [PubMed - in process]
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