Wednesday, January 27, 2016

Garcinol inhibits GCN5-mediated lysine acetyltransferase activity and prevents replication of the parasite Toxoplasma gondii

2016 Jan 25. pii: AAC.03059-15. [Epub ahead of print]


Lysine acetylation is a critical post-translational modification that influences protein activity, stability, and binding properties. The acetylation of histone proteins in particular is a well-characterized feature of gene expression regulation. In the protozoan parasite Toxoplasma gondii, a number of lysine acetyltransferases (KATs) contribute to gene expression and are essential for parasite viability. The natural product, garcinol, was recently reported to inhibit enzymatic activities of GCN5 and p300 family KATs in other species. Here we show that garcinol inhibits TgGCN5b, the only nuclear GCN5-family KAT known to be required for Toxoplasma tachyzoite replication. Treatment of tachyzoites with garcinol led to a reduction of global lysine acetylation, particularly on histone H3 and TgGCN5b itself. We also performed RNAseq analysis on intracellular tachyzoites treated with garcinol, which revealed increasing aberrant gene expression coincident with increasing concentrations of garcinol. The majority of the genes that were most significantly affected by garcinol were also associated with TgGCN5b in a previous ChIP-chip analysis. The dysregulated gene expression induced by garcinol significantly inhibits Toxoplasma tachyzoite replication, and the concentrations used exhibit no overt toxicity on human host cells. Garcinol also inhibits Plasmodium falciparum asexual replication with a similar IC50 as Toxoplasma. Together, these data support that pharmacological inhibition of TgGCN5b leads to a catastrophic failure in gene expression control that prevents parasite replication.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
PMID:
26810649
[PubMed - as supplied by publisher]

Wednesday, January 20, 2016

Infection of male rats with Toxoplasma gondii induces effort-aversion in a T-maze decision-making task

2016 Jan 16. pii: S0889-1591(16)30015-0. doi: 10.1016/j.bbi.2016.01.015. [Epub ahead of print]


Rats chronically infected with protozoan Toxoplasma gondii exhibit greater delay aversion in an inter-temporal task. Moreover Toxoplasma gondii infection also results in dendritic atrophy of basolateral amygdala neurons. Basolateral amygdala is reported to bias decision making towards greater effortful alternatives. In this context, we report that Toxoplasma gondii increases effort aversion in infected male rats. This host-parasite association has been widely studied in the context of loss of innate fear in the infected males. It is suggested that reduced fear towards predators reflects a parasitic behavioral manipulation to enhance trophic transmission of Toxoplasma gondii. Observations reported here extend this paradigm away from a monolithic change in fear and towards a multi-dimensional change in decision making.
Copyright © 2016. Published by Elsevier Inc.

KEYWORDS:

Behavioral manipulation; Decision making; Effort discounting; Operant learning; Parasites
PMID:
26783701
[PubMed - as supplied by publisher]

Monday, January 18, 2016

Toxoplasma gondii infection and testosterone congruently increase tolerance of male rats for risk of reward forfeiture

2016 Jan 13. pii: S0018-506X(16)30020-4. doi: 10.1016/j.yhbeh.2016.01.003. [Epub ahead of print]


Decision making under risk involves balancing the potential of gaining rewards with the possibility of loss and/or punishment. Tolerance to risk varies between individuals. Understanding the biological basis of risk tolerance is pertinent because excessive tolerance contributes to adverse health and safety outcomes. Yet, not much is known about biological factors mediating inter-individual variability in this regard. We investigate if latent Toxoplasma gondii infection can cause risk tolerance. Using a rodent model of the balloon analogous risk task, we show that latent T. gondii infection leads to a greater tolerance of reward forfeiture. Furthermore, effects of the infection on risk can be recapitulated with testosterone supplementation alone, demonstrating that greater testosterone synthesis by the host post-infection is sufficient to change risk tolerance. T. gondii is a frequent parasite of humans and animals. Thus, the infection status can potentially explain some of the inter-individual variability in the risky decision making.
Copyright © 2015. Published by Elsevier Inc.

KEYWORDS:

BART; Behavioral manipulation; Decision making; Impulsivity; Parasitism; Steroids
PMID:
26774464
[PubMed - as supplied by publisher]

Thursday, January 14, 2016

The Conoid Associated Motor MyoH Is Indispensable for Toxoplasma gondii Entry and Exit from Host Cells

 2016 Jan 13;12(1):e1005388. doi: 10.1371/journal.ppat.1005388. eCollection 2016.

Abstract

Many members of the phylum of Apicomplexa have adopted an obligate intracellular life style and critically depend on active invasion and egress from the infected cells to complete their lytic cycle. Toxoplasma gondii belongs to the coccidian subgroup of the Apicomplexa, and as such, the invasive tachyzoite contains an organelle termed the conoid at its extreme apex. This motile organelle consists of a unique polymer of tubulin fibres and protrudes in both gliding and invading parasites. The class XIV myosin A, which is conserved across the Apicomplexa phylum, is known to critically contribute to motility, invasion and egress from infected cells. The MyoA-glideosome is anchored to the inner membrane complex (IMC) and is assumed to translocate the components of the circular junction secreted by the micronemes and rhoptries, to the rear of the parasite. Here we comprehensively characterise the class XIV myosin H (MyoH) and its associated light chains. We show that the 3 alpha-tubulin suppressor domains, located in MyoH tail, are necessary to anchor this motor to the conoid. Despite the presence of an intact MyoA-glideosome, conditional disruption of TgMyoH severely compromises parasite motility, invasion and egress from infected cells. We demonstrate that MyoH is necessary for the translocation of the circular junction from the tip of the parasite, where secretory organelles exocytosis occurs, to the apical position where the IMC starts. This study attributes for the first time a direct function of the conoid in motility and invasion, and establishes the indispensable role of MyoH in initiating the first step of motility along this unique organelle, which is subsequently relayed by MyoA to enact effective gliding and invasion.
PMID:
 
26760042
 
[PubMed - as supplied by publisher]

Wednesday, January 13, 2016

Analysis of non-canonical calcium dependent protein kinases in Toxoplasma gondii by targeted gene deletion using CRISPR/Cas9

 2016 Jan 11. pii: IAI.01173-15. [Epub ahead of print]

Abstract

Calcium dependent protein kinases are expanded in apicomplexan parasites, especially in T. gondii where 14 separate genes encoding these enzymes are found. Although previous studies have shown a role for several CDPKs in controlling invasion, egress, and cell division in T. gondii, the role of most of these genes is unexplored. Here we developed a more efficient method for gene disruption using CRISRP/Cas9 that was modified to completely delete large, multi-exonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the function of non-canonical CPDKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growth in vitro or infection in the mouse model. The exception to this was CDPK6, loss of which lead to reduced plaquing, fitness defect in a competition assay, and reduced tissue cysts formation in chronically infected mice. Our findings highlight the utility of CRISPR/cas9 for rapid serial gene deletion, and also suggest that additional models are needed to reveal the function of many genes in T. gondii.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
PMID:
 
26755159
 
[PubMed - as supplied by publisher]

Tuesday, January 12, 2016

Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites

2016 Jan 11. doi: 10.1038/nm.4023. [Epub ahead of print]
 
 
Protozoan infections are a serious global health problem. Natural killer (NK) cells and cytolytic T lymphocytes (CTLs) eliminate pathogen-infected cells by releasing cytolytic granule contents-granzyme (Gzm) proteases and the pore-forming perforin (PFN)-into the infected cell. However, these cytotoxic molecules do not kill intracellular parasites. CD8+ CTLs protect against parasite infections in mice primarily by secreting interferon (IFN)-γ. However, human, but not rodent, cytotoxic granules contain the antimicrobial peptide granulysin (GNLY), which selectively destroys cholesterol-poor microbial membranes, and GNLY, PFN and Gzms rapidly kill intracellular bacteria. Here we show that GNLY delivers Gzms into three protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii and Leishmania major), in which the Gzms generate superoxide and inactivate oxidative defense enzymes to kill the parasite. PFN delivers GNLY and Gzms into infected cells, and GNLY then delivers Gzms to the intracellular parasites. Killer cell-mediated parasite death, which we term 'microbe-programmed cell death' or 'microptosis', is caspase independent but resembles mammalian apoptosis, causing mitochondrial swelling, transmembrane potential dissipation, membrane blebbing, phosphatidylserine exposure, DNA damage and chromatin condensation. GNLY-transgenic mice are protected against infection by T. cruzi and T. gondii, and survive infections that are lethal to wild-type mice. Thus, GNLY-, PFN- and Gzm-mediated elimination of intracellular protozoan parasites is an unappreciated immune defense mechanism.
PMID:
26752517
[PubMed - as supplied by publisher]

Saturday, January 09, 2016

Local admixture of amplified and diversified secreted pathogenesis determinants shapes mosaic Toxoplasma gondii genomes

 2016 Jan 7;7:10147. doi: 10.1038/ncomms10147.

Abstract

Toxoplasma gondii is among the most prevalent parasites worldwide, infecting many wild and domestic animals and causing zoonotic infections in humans. T. gondii differs substantially in its broad distribution from closely related parasites that typically have narrow, specialized host ranges. To elucidate the genetic basis for these differences, we compared the genomes of 62 globally distributed T. gondii isolates to several closely related coccidian parasites. Our findings reveal that tandem amplification and diversification of secretory pathogenesis determinants is the primary feature that distinguishes the closely related genomes of these biologically diverse parasites. We further show that the unusual population structure of T. gondii is characterized by clade-specific inheritance of large conserved haploblocks that are significantly enriched in tandemly clustered secretory pathogenesis determinants. The shared inheritance of these conserved haploblocks, which show a different ancestry than the genome as a whole, may thus influence transmission, host range and pathogenicity. 
PMID:
 
26738725
 
[PubMed - in process]

Thursday, January 07, 2016

Antigen Presentation of Vacuolated Apicomplexans - Two Gateways to a Vaccine Antigen

2015 Dec 27. pii: S1471-4922(15)00284-6. doi: 10.1016/j.pt.2015.12.011. [Epub ahead of print]
 
For parasites that sequester themselves within a vacuole, new rules governing antigen presentation are coming into focus. Components of the host's autophagy machinery and the parasite's membranous nanotubular network within the parasitophorous vacuole play a major role in determining antigenicity of Toxoplasma proteins. As such, both parasite and vaccinologist may exploit these pathways to regulate the ever important CD8 T cell response to apicomplexan parasites.
Copyright © 2015. Published by Elsevier Ltd.
PMID:
26733404
[PubMed - as supplied by publisher]

Immunity in the spleen and blood of mice immunized with irradiated Toxoplasma gondii tachyzoites

2016 Jan 5. [Epub ahead of print]
 
 
Toxoplasma gondii infection induces a strong and long-lasting immune response that is able to prevent most reinfections but allows tissue cysts. Irradiated, sterilized T. gondii tachyzoites are an interesting vaccine, and they induce immunity that is similar to infection, but without cysts. In this study, we evaluated the cellular immune response in the blood and spleen of mice immunized with this preparation by mouth (v.o.) or intraperitoneally (i.p.) and analyzed the protection after challenge with viable parasites. BALB/c mice were immunized with three i.p. or v.o. doses of irradiated T. gondii tachyzoites. Oral challenge with ten cysts of the ME-49 or VEG strain at 90 days after the last dose resulted in high levels of protection with low parasite burden in the immunized animals. There were higher levels of specific IgG, IgA and IgM antibodies in the serum, and the i.p. immunized mice had higher levels of the high-affinity IgG and IgM antibodies than the orally immunized mice, which had more high-affinity IgA antibodies. B cells (CD19+), plasma cells (CD138+) and the CD4+ and CD8+ T cell populations were increased in both the blood and spleen. Cells from the spleen of the i.p. immunized mice also showed antigen-induced production of interleukin-10 (IL-10), interferon gamma (IFN-γ) and interleukin 4 (IL-4). The CD4+ T cells, B cells and likely CD8+ T cells from the spleens of the i.p. immunized mice proliferated with a specific antigen. The protection was correlated with the spleen and blood CD8+ T cell, high-affinity IgG and IgM and antigen-induced IL-10 and IL-4 production. Immunization with irradiated T. gondii tachyzoites induces an immune response that is mediated by B cells and CD4+ and CD8+ T cells, with increased humoral and cellular immune responses that are necessary for host protection after infection. The vaccine is similar to natural infection, but free of tissue cysts; this immunity restrains infection at challenge and can be an attractive and efficient model for vaccine development in toxoplasmosis.

KEYWORDS:

B lymphocytes; CD4+ T lymphocytes; CD8+ T lymphocytes; Ionizing radiation; Toxoplasma gondii; Vaccine
PMID:
26732075
[PubMed - as supplied by publisher]

Tuesday, January 05, 2016

Antisense technologies in the studying of Toxoplasma gondii

2015 Dec 24. pii: S0167-7012(15)30136-6. doi: 10.1016/j.mimet.2015.12.013. [Epub ahead of print]
 
 
This review covers a brief history of antisense RNAs and its applications, and summarizes the current stage of antisense technologies used in Toxoplasma gondii, a fascinating model organism with a unique characteristic blend of genetic regulatory systems normally found in plants or animals. Based on the current knowledge of regulatory RNAs and non-coding RNA (ncRNA), the antisense technologies are reviewed according to the classification of ncRNAs, which are roughly categorized into small, ranging from ~20-200 nucleotides in length, and long >200 nucleotides. Techniques utilizing small regulatory RNAs such as siRNA, miRNA, antagomirs, ribozymes and morpholino oligomers are discussed along with long non-coding RNA (lncRNA) including antisense and double stranded. These antisense technologies can be used in forward and reverse genetics studies. The future of technologies is limitless, particularly by combining these technologies with conventional methods, and should allow for ever greater understanding of gene regulation of the organism and related pathogenic microorganisms.
Copyright © 2015. Published by Elsevier B.V.

KEYWORDS:

Antisense technologies; Genetic tools; Toxoplasma gondii
PMID:
26724749
[PubMed - as supplied by publisher]

Saturday, January 02, 2016

The E3-ubiquitin ligase adaptor protein Skp1 is glycosylated by an evolutionarily conserved pathway that regulates protist growth and development

J Biol Chem. 2015 Dec 30. pii: jbc.M115.703751. [Epub ahead of print]

Author information

  • 1University of Georgia, United States;
  • 2University at Buffalo, United States.
  • 3University of Georgia, United States; westcm@uga.edu.

Abstract

Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by 5 sugars via the action of three glycosyltransferases, Gnt1, PgtA and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.

KEYWORDS: 

E3 ubiquitin ligase; SCF-complex; Skp1; Toxoplasma gondii; cytoplasmic glycosylation; glycobiology; glycosyltransferase; parasitology
PMID:
 
26719340
 
[PubMed - as supplied by publisher]