toxplasma news feeds

Yeast two-hybrid screening service for Toxoplasma gondii

The Anti-Toxo Blog - 26 July 2017 - 1:01pm
We recently developed a yeast two-hybrid (Y2H) library with Hybrigenics to facilitate the discovery of protein-protein interactions. The library was made from Toxoplasma type I RH strain tachyzoites and is ready for use! --Bill Sullivan


Please find below a summary of Hybrigenics differentiating features regarding our Y2H screening services:

1. Bait design: Investigators have an option of expressing their “bait” either as a N- or C-terminal fusion protein relative to the DNA-binding domain (DBD).

2. Bait testing: An initial test screen will be performed to evaluate whether your “bait” autoactivates a HIS3 gene reporter.  The “bait” will be tested in the absence or presence of a dose-range of 3-aminotriazol (3-AT), which is used to minimize background levels. If your “bait” displays some degree of autoactivation, then the amount of 3-AT will be adjusted accordingly.
 If the “bait” exhibits some degree of toxicity in yeast, then your cDNA insert will be transferred into an inducible vector.

3. Library construction: We offer high complexity domain-enriched libraries. Inserts are between 800-1000 bp, which corresponds to an average size of a protein domain. Our random-primed libraries contain several independent fragments coding for the same protein.  I have included for you a link to a complete list of our cDNA libraries (90+) for Y2H screens: (http://www.hybrigenics-services.com/files/medias/hybrigenics-list-of-libraries-ultimate-screen.pdf).

The use of domains is one of the key features of our libraries. Why is this important? When expressed in yeast, full-length proteins, especially if they are large, can be misfolded, mislocalized or toxic. Construction of highly complex random-primed libraries overcomes these barriers. Random oligonucleotide priming generates overlapping fragments of a given protein domain thereby increasing the chances of proper folding. Moreover, overlapping fragments span the entire length of the protein sequence, which increases the likelihood of detecting a protein interaction. Importantly, fragments of membrane, secreted and toxic proteins are well represented in our libraries and are routinely identified in our Y2H screens. Lastly, the identification of several independent fragments in a Y2H screen allows us to easily determine a minimal interacting domain of a prey protein.

4. Y2H screening: Large-scale Y2H screens are performed using our LexA and Gal4 systems. We use a patented cell-to-cell yeast-mating assay in order to obtain and subsequently test on average 83 million interactions, which corresponds to a 8-fold in library coverage.

5. Turnaround time: Approximately 3 months

6. Bioinformatics analysis:  Included in our Y2H results package is a comprehensive bioinformatics analysis of protein interactors. Hybrigenics’ bioinformatics algorithms compute a numerical rank for each protein interaction, which is then assigned a statistical confidence score. This information helps guide investigators to identify the most relevant protein binding partners as well as to exclude false positives.

7. Deliverables: Results from your ULTImate Y2H screen are reported on 3 separate files:
a) Results Summary File: Includes the technical parameters of your screen, a Predicted Biological Score (PBS), which is computed to assess the reliability of each interaction and prey fragment analysis.
b) DomSight file: Compares the bait fragment and the Selected Interaction Domain (SID) of the prey proteins with the functional and structural domains (PFAM, SMART, TMHMM, SignalP, Coil algorithms) of these proteins.
c) Excel worksheet file: Contains raw data, in particular 5’ and 3’ experimental sequences of the preys and bioinformatics analysis.

8. Expertise and scientific assistance: You will be assigned a dedicated scientific project leader, who will be in charge of your project and will be working with you throughout the course of your Y2H screen. They will help you with the design of your “bait”, provide regular updates on your Y2H screen and thoroughly review all results with you.

For more information about getting Hybrigenics to complete your Y2H screen, contact Brent Passer.
Categories: toxoplasma news feeds

Toxoplasma gondii: Entry, association, and physiological influence on the central nervous system

The Anti-Toxo Blog - 21 July 2017 - 1:52pm
 2017 Jul 20;13(7):e1006351. doi: 10.1371/journal.ppat.1006351. eCollection 2017 Jul.
Mendez OA1,2, Koshy AA2,3,4.AbstractToxoplasma gondii is one of the world's most successful parasites, in part because of its ability to infect and persist in most warm-blooded animals. A unique characteristic of T. gondii is its ability to persist in the central nervous system (CNS) of a variety of hosts, including humans and rodents. How, what, and why T. gondii encysts in the CNS has been the topic of study for decades. In this review, we will discuss recent work on how T. gondii is able to traverse the unique barrier surrounding the CNS, what cells of the CNS play host to T. gondii, and finally, how T. gondii infection may influence global and cellular physiology of the CNS.
PMID:
 
28727854
 
DOI:
 
10.1371/journal.ppat.1006351
Categories: toxoplasma news feeds

A plant/fungal-type phosphoenolpyruvate carboxykinase located in the parasite mitochondrion ensures glucose-independent survival of Toxoplasma gondii

The Anti-Toxo Blog - 21 July 2017 - 1:51pm
 2017 Jul 18. pii: jbc.M117.802702. doi: 10.1074/jbc.M117.802702. [Epub ahead of print]
Nitzsche R1, Günay-Esiyok Ö1, Tischer M1, Zagoriy V2, Gupta N3.AbstractToxoplasma gondii is considered as one of the most successful intracellular pathogens, because it can reproduce in varied nutritional milieus, encountered in diverse host-cell types of essentially any warm-blooded organism. Our earlier work has demonstrated that the acute (tachyzoite) stage of T. gondii depends on cooperativity of glucose and glutamine catabolism to meet biosynthetic demands. Either of these two nutrients can sustain the parasite survival; however, what determines the metabolic plasticity has not been resolved yet. Here, we reveal two discrete phosphoenolpyruvate carboxykinase (PEPCK) enzymes in the parasite, one of which resides in the mitochondrion (TgPEPCKmt), whereas the other protein is not expressed in tachyzoites (TgPEPCKnet). Parasites with an intact glycolysis can tolerate genetic deletions of TgPEPCKmt as well as of TgPEPCKnet, indicating their nonessential roles for the tachyzoite survival. TgPEPCKnet can also be ablated in glycolysis-deficient mutant, whereas TgPEPCKmt is refractory to deletion. In accord, the lytic cycle of a conditional mutant of TgPEPCKmt in the glycolysis-impaired strain was aborted upon induced repression of the mitochondrial isoform, demonstrating its essential role for the glucose-independent survival of parasites. Isotope-resolved metabolomics of the conditional mutant revealed defective flux of glutamine-derived carbon into RNA-bound ribose sugar as well as metabolites associated with gluconeogenesis, entailing a critical nodal role of PEPCKmt in linking catabolism of glucose and glutamine with anabolic pathways. Our data also suggest a homeostatic function of TgPEPCKmt in cohesive operation of glycolysis and TCA cycle under normal glucose-replete milieu. Conversely, we found that otherwise-integrative enzyme pyruvate carboxylase (TgPyC) is dispensable not only in glycolysis-competent but also in glycolysis-deficient tachyzoites despite a mitochondrial localization. Last but not least, the observed physiology of T. gondii tachyzoites appears to phenocopy cancer cells, which holds promise for developing common therapeutics against both threats.Copyright © 2017, The American Society for Biochemistry and Molecular Biology.KEYWORDS: Toxoplasma gondii; cancer metabolism; gluconeogenesis; glutamine catabolism; glycolysis; intracellular parasitism; parasite metabolism; phosphoenolpyruvate carboxykinase; pyruvate carboxylase (PC); tricarboxylic acid cycle (TCA cycle) (Krebs cycle)
PMID:
 
28726641
 
DOI:
 
10.1074/jbc.M117.802702
Categories: toxoplasma news feeds

Impact of the host on Toxoplasma stage differentiation

The Anti-Toxo Blog - 15 July 2017 - 5:32pm
 2017 Jun 22;4(7):203-211. doi: 10.15698/mic2017.07.579.
Lüder CGK1, Rahman T1.AbstractThe unicellular parasite Toxoplasma gondii infects warm-blooded animals and humans, and it is highly prevalent throughout the world. Infection of immunocompetent hosts is usually asymptomatic or benign but leads to long-term parasite persistence mainly within neural and muscular tissues. The transition from acute primary infection towards chronic toxoplasmosis is accompanied by a developmental switch from fast replicating and metabolically highly active tachyzoites to slow replicating and largely dormant bradyzoites within tissue cysts. Such developmental differentiation is critical for T. gondii in order to complete its life cycle and for pathogenesis. Herein, we summarize accumulating evidence indicating a major impact of the host cell physiology on stage conversion between the tachyzoite and the bradyzoite stage of the parasite. Withdrawal from cell cycle progression, proinflammatory responses, reduced availability of nutrients and extracellular adenosine can indeed induce tachyzoite-to-bradyzoite differentiation and tissue cyst formation. In contrast, high glycolytic activity as indicated by increased lactate secretion can inhibit bradyzoite formation. These examples argue for the intriguing possibility that after dissemination within its host, T. gondii can sense its cellular microenvironment to initiate the developmental program towards the bradyzoite stage in distinct cells. This may also explain the predominant localization of T. gondii in neural and muscular tissues during chronic toxoplasmosis.KEYWORDS: Toxoplasma gondii; bradyzoite; cell cycle; host cell; immune response; metabolism; parasite host-interaction; stage conversion
PMID:
 
28706936
 
PMCID:
 
PMC5507683
 
DOI:
 
10.15698/mic2017.07.579
Categories: toxoplasma news feeds

Structural and Biochemical Characterization of Apicomplexan Inorganic Pyrophosphatases

The Anti-Toxo Blog - 14 July 2017 - 1:10pm
2017 Jul 12;7(1):5255. doi: 10.1038/s41598-017-05234-y.
Jamwal A1,2, Yogavel M1, Abdin MZ2, Jain SK2,3, Sharma A4.
Inorganic pyrophosphatases (PPase) participate in energy cycling and they are essential for growth and survival of organisms. Here we report extensive structural and functional characterization of soluble PPases from the human parasites Plasmodium falciparum (PfPPase) and Toxoplasma gondii (TgPPase). Our results show that PfPPase is a cytosolic enzyme whose gene expression is upregulated during parasite asexual stages. Cambialistic PfPPase actively hydrolyzes linear short chain polyphosphates like PPi, polyP3 and ATP in the presence of Zn2+. A remarkable new feature of PfPPase is the low complexity asparagine-rich N-terminal region that mediates its dimerization. Deletion of N-region has an unexpected and substantial effect on the stability of PfPPase domain, resulting in aggregation and significant loss of enzyme activity. Significantly, the crystal structures of PfPPase and TgPPase reveal unusual and unprecedented dimeric organizations and provide new fundamental insights into the variety of oligomeric assemblies possible in eukaryotic inorganic PPases.
PMID:
28701714
DOI:
10.1038/s41598-017-05234-y
Categories: toxoplasma news feeds

Biochemical characterization of aminopeptidase N2 from Toxoplasma gondii

The Anti-Toxo Blog - 14 July 2017 - 1:09pm
2017 Jul 13. doi: 10.1292/jvms.17-0119. [Epub ahead of print]
Li Q1, Jia H2, Cao S2, Zhang Z2, Zheng J2, Zhang Y1.
Aminopeptidase N (APN) is a member of the highly conserved M1 family of metalloproteases, and is considered to be a valuable target for the treatment of a variety of diseases, e.g., cancer, malaria, and coccidiosis. In this study, we identified an APN gene (TgAPN2) in the Toxoplasma gondii genome, and performed a biochemical characterization of the recombinant TgAPN2 (rTgAPN2) protein. Active rTgAPN2 was first produced and purified in Escherichia coli. The catalytic activity of the enzyme was verified using a specific fluorescent substrate, H-Ala-MCA; the rTgAPN2 was relatively active in the absence of added metal ions. The addition of some metal ions, especially Zn2+, inhibited the activity of the recombinant enzyme. The activity of rTgAPN2 was reduced in the presence of the EDTA chelator in the absence of added metal ions. The optimum pH for enzyme activity was 8.0; the enzyme was active in the 3-10 pH range. The substrate preference of rTgAPN2 was evaluated. The enzyme showed a preference for substrates containing N-terminal Ala and Arg residues. Finally, bestatin and amastatin were shown to inhibit the activity of the enzyme. In conclusion, rTgAPN2 shared general characteristics with the M1 family of aminopeptidases but also had some unique characteristics. This provides a basis for the function of aminopeptidases and the study of drug targets.KEYWORDS: Toxoplasma gondii; activity; aminopeptidase N (APN); enzyme
PMID:
28701624
DOI:
10.1292/jvms.17-0119
Categories: toxoplasma news feeds

Strategies for detecting toxoplasma immunity

The Anti-Toxo Blog - 14 July 2017 - 1:09pm
2003 Jan;60(2):105-108. doi: 10.1080/09674845.2003.11783684.
Ashburn D1, Evans R1, Chatterton JMW1, Joss AWL1, Ho-Yen DO1.
A strategy for identifying toxoplasma immunity in pregnancy must provide good evidence of immunity but not falsely reassure; that for immunocompromised patients should identify immunity and also the risk of reactivated toxoplasmosis. Using sera from both of these patient groups, the performance of an in-house IgG EIA and two commonly used commercial assays (Abbott AxSYM Toxo-G and Eiken latex test) were compared with the dye test. False-positive results were obtained using the IgG enzyme immunoassay (EIA) and AxSYM Toxo-G, and false negatives using all three screen tests. During pregnancy, positive results may falsely reassure, and patients should be tested for toxoplasma-specific IgM to differentiate between current infection and immunity. In immunocompromised patients, positive results indicate immunity but negative results do not exclude it; these should be tested by dye test. Despite these reservations, we have demonstrated that immunity screening can be performed within a district general hospital.KEYWORDS: Immunity; Immunocompromised host; Pregnancy; Toxoplasma
PMID:
28700887
DOI:
10.1080/09674845.2003.11783684
Categories: toxoplasma news feeds

TRIM21 is critical for survival of Toxoplasma gondii infection and localises to GBP-positive parasite vacuoles

The Anti-Toxo Blog - 14 July 2017 - 1:08pm
2017 Jul 12;7(1):5209. doi: 10.1038/s41598-017-05487-7.
Foltz C1, Napolitano A1, Khan R1, Clough B1, Hirst EM1, Frickel EM2.
Interferon gamma (IFNγ) is the major proinflammatory cytokine conferring resistance to the intracellular vacuolar pathogen Toxoplasma gondii by inducing the destruction of the parasitophorous vacuole (PV). We previously identified TRIM21 as an IFNγ-driven E3 ubiquitin ligase mediating the deposition of ubiquitin around pathogen inclusions. Here, we show that TRIM21 knockout mice were highly susceptible to Toxoplasma infection, exhibiting decreased levels of serum inflammatory cytokines and higher parasite burden in the peritoneum and brain. We demonstrate that IFNγ drives recruitment of TRIM21 to GBP1-positive Toxoplasma vacuoles, leading to Lys63-linked ubiquitination of the vacuole and restriction of parasite early replication without interfering with vacuolar disruption. As seen in vivo, TRIM21 impacted the secretion of inflammatory cytokines. This study identifies TRIM21 as a previously unknown modulator of Toxoplasma gondii resistance in vivo thereby extending host innate immune recognition of eukaryotic pathogens to include E3 ubiquitin ligases.
PMID:
28701773
DOI:
10.1038/s41598-017-05487-7
Categories: toxoplasma news feeds

Toxoplasmosis accelerates acquisition of epilepsy in rats undergoing chemical kindling

The Anti-Toxo Blog - 10 July 2017 - 3:25pm
2017 Jun 24;135:137-142. doi: 10.1016/j.eplepsyres.2017.06.012. [Epub ahead of print]
Babaie J1, Sayyah M2, Choopani S3, Asgari T3, Golkar M4, Gharagozli K5.
Epilepsy is one of the most common neurologic disorders worldwide with no distinguishable cause in 60% of patients. One-third of the world population has been infected with Toxoplasma gondii. This intracellular parasite has high tropism for excitable cells including neurons. We assessed impact of acute and chronic T. gondii infection on epileptogenesis in pentylenetetrazole (PTZ) kindling model in male rats. T. gondii cysts were administered to rats by intraperitoneal (i.p.) injection. The presence of T. gondii cysts in the brain of rats was verified by hematoxylin-eosin staining. One and eight weeks after cysts injection, as acute and chronic phases of infection, PTZ (30mg/kg, i.p.) was injected to the rats every other day until manifestation of generalized seizures. Histologic findings confirmed cerebral toxoplasmosis in rats. The rats with acute or chronic Toxoplasma infection became kindled by lower number of PTZ injections (14.8±1 and 13.6±1 injections, respectively) compared to corresponding uninfected rats (18.7±1 and 16.9±1 injections, p0.05). Toxoplasma infection increased the rate of kindling in rats. The chronically-infected rats achieved focal and also generalized seizures earlier than the rats with acute infection. Toxoplasmosis might be considered as a risk factor for acquisition of epilepsy.
Copyright © 2017 Elsevier B.V. All rights reserved.KEYWORDS: Epileptogenesis; Kindling; Pentylenetetrazole; Rat; Toxoplasma gondii
PMID:
28688333
DOI:
10.1016/j.eplepsyres.2017.06.012
Categories: toxoplasma news feeds

Thioredoxin reductase from Toxoplasma gondii: an essential virulence effector with antioxidant function

The Anti-Toxo Blog - 10 July 2017 - 3:25pm
2017 Jul 7. pii: fj.201700008R. doi: 10.1096/fj.201700008R. [Epub ahead of print]
Xue J1,2,3, Jiang W1, Chen Y1, Gong F2, Wang M1, Zeng P2, Xia C2, Wang Q4, Huang K5.
Thioredoxin reductase (TR) can help pathogens resist oxidative-burst injury from host immune cells by maintaining a thioredoxin-reduction state during NADPH consumption. TR is a necessary virulence factor that enables the persistent infection of some parasites. We performed bioinformatics analyses and biochemical assays to characterize the activity, subcellular localization, and genetic ablation of Toxoplasma gondii TR (TgTR), to shed light on its biologic function. We expressed the TgTR protein with an Escherichia coli expression system and analyzed its enzyme activity, reporting a Km for the recombinant TgTR of 11.47-15.57 μM, using NADPH as a substrate, and 130.48-151.09 μM with dithio-bis-nitrobenzoic acid as a substrate. The TgTR sequence shared homology with that of TR, but lacked a selenocysteine residue in the C-terminal region and was thought to contain 2 flavin adenine dinucleotide (FAD) domains and 1 NADPH domain. In addition, immunoelectron microscopy results showed that TgTR was widely dispersed in the cytoplasm, and we observed that parasite antioxidant capacity, invasion efficiency, and proliferation were decreased in TR-knockout (TR-KO) strains in vitro, although this strain still stimulated the release of reactive oxygen species release in mouse macrophages while being more sensitive to H2O2 toxicity in vitro Furthermore, our in vivo results revealed that the survival time of mice infected with the TR-KO strain was significantly prolonged relative to that of mice infected with the wild-type strain. These results suggest that TgTR plays an important role in resistance to oxidative damage and can be considered a virulence factor associated with T. gondii infection.-Xue, J., Jiang, W., Chen, Y., Gong, F., Wang, M., Zeng, P., Xia, C., Wang, Q., Huang, K. Thioredoxin reductase from Toxoplasma gondii: an essential virulence effector with antioxidant function.
© FASEB.KEYWORDS: enzyme activity; gene knockout; immune evasion; protein expression
PMID:
28687608
DOI:
10.1096/fj.201700008R
Categories: toxoplasma news feeds

Actin Nanobodies Uncover the Mystery of Actin Filament Dynamics in Toxoplasma gondii

The Anti-Toxo Blog - 10 July 2017 - 3:24pm
2017 Jul 4. pii: S1471-4922(17)30156-3. doi: 10.1016/j.pt.2017.06.007. [Epub ahead of print]

Tardieux I1.


While the intracellular parasite Toxoplasma relies on a divergent actomyosin motor to support unique speeds in directional movement, the dynamics and architecture of parasite actin filaments remain a much-discussed issue. Using actin chromobodies, Periz et al. started to unveil how networks of dynamic F-actin connect Toxoplasma progeny and expand in the replicative vacuole.
Copyright © 2017 Elsevier Ltd. All rights reserved.
PMID:
28687476
DOI:
10.1016/j.pt.2017.06.007
Categories: toxoplasma news feeds

Proteasomal Degradation of T. gondii ROP18 Requires Derlin2

The Anti-Toxo Blog - 4 July 2017 - 12:44pm
 2017 Jun 29. pii: S0001-706X(17)30351-0. doi: 10.1016/j.actatropica.2017.06.027. [Epub ahead of print]
Tang Y1, An R1, Chen L2, Gong L3, Cai H4, Liu K4, Yu L5, Shen J5, Du J6.AbstractT. gondii is an obligate intracellular parasite, belonging to the phylum Apicomplexa, infecting all warm-blooded animals including humans. During host cell invasion, specialized cytoskeletal and secretory organelles play a pivotal role. ROP18, as a member of the ROP2 family, has been identified as a key virulence factor mediating pathogenesis in T. gondii. Here, we identify an ER-resident protein, Derlin2, a factor implicated in the removal of misfolded proteins from the ER for cytosolic degradation, as a component of the machinery required for ER-associated protein degradation (ERAD). We identified Derlin2 interacting with ROP18 by yeast two-hybrid screening system. The interaction between ROP18 and Derlin2 was further confirmed through in vitro GST pull-down and in vivo immunoprecipitation assays. By immunofluorescence assay, we found that ROP18 co-localized with Derlin2 in the endoplasmic reticulum. Using overexpression and knockdown approaches, we demonstrated that Derlin2 was required for T.gondii ROP18 degradation. Consistently, cycloheximide chase experiments showed that the degradation of ROP18 relied on the Derlin2, but not Derlin1. These results indicate that interaction between Derlin2 and ROP18 is functionally relevant and leads ultimately to degradation of ROP18. The finding provides the basis for future studies on Derlin2-dependent ERAD of T. gondii ROP18 and subsequent antigen generation.Copyright © 2017 Elsevier B.V. All rights reserved.KEYWORDS: Derlin2; ERAD; ROP18; T. gondii; degradation
PMID:
 
28669563
 
DOI:
 
10.1016/j.actatropica.2017.06.027
Categories: toxoplasma news feeds

Checkpoints of apicomplexan cell division identified in Toxoplasma gondii

The Anti-Toxo Blog - 4 July 2017 - 12:43pm
 2017 Jul 3;13(7):e1006483. doi: 10.1371/journal.ppat.1006483. [Epub ahead of print]
Alvarez CA1, Suvorova ES1.AbstractThe unusual cell cycles of Apicomplexa parasites are remarkably flexible with the ability to complete cytokinesis and karyokinesis coordinately or postpone cytokinesis for several rounds of chromosome replication, and are well recognized. Despite this surprising biology, the molecular machinery required to achieve this flexibility is largely unknown. In this study, we provide comprehensive experimental evidence that apicomplexan parasites utilize multiple Cdk-related kinases (Crks) to coordinate cell division. We determined that Toxoplasma gondii encodes seven atypical P-, H-, Y- and L- type cyclins and ten Crks to regulate cellular processes. We generated and analyzed conditional tet-OFF mutants for seven TgCrks and four TgCyclins that are expressed in the tachyzoite stage. These experiments demonstrated that TgCrk1, TgCrk2, TgCrk4 and TgCrk6, were required or essential for tachyzoite growth revealing a remarkable number of Crk factors that are necessary for parasite replication. G1 phase arrest resulted from the loss of cytoplasmic TgCrk2 that interacted with a P-type cyclin demonstrating that an atypical mechanism controls half the T. gondii cell cycle. We showed that T. gondii employs at least three TgCrks to complete mitosis. Novel kinases, TgCrk6 and TgCrk4 were required for spindle function and centrosome duplication, respectively, while TgCrk1 and its partner TgCycL were essential for daughter bud assembly. Intriguingly, mitotic kinases TgCrk4 and TgCrk6 did not interact with any cyclin tested and were instead dynamically expressed during mitosis indicating they may not require a cyclin timing mechanism. Altogether, our findings demonstrate that apicomplexan parasites utilize distinctive and complex mechanisms to coordinate their novel replicative cycles.
PMID:
 
28671988
 
DOI:
 
10.1371/journal.ppat.1006483
Categories: toxoplasma news feeds

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

The Anti-Toxo Blog - 4 July 2017 - 12:43pm
 2017 Jun 22;(124). doi: 10.3791/55185.
Shen B1, Powell RH2, Behnke MS3.AbstractScientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.
PMID:
 
28671645
 
DOI:
 
10.3791/55185
Categories: toxoplasma news feeds

The Toxoplasma gondii inhibitor-2 regulates protein phosphatase 1 activity through multiple motifs

The Anti-Toxo Blog - 3 July 2017 - 6:35pm
2017 Jun 30. doi: 10.1007/s00436-017-5543-6. [Epub ahead of print]
Deveuve Q1, Lesage K1, Mouveaux T1, Gissot M2.
Toxoplasma gondii has a complex life cycle characterized by multiple differentiation steps that are essential for its survival in both human and definitive feline host. Several studies have demonstrated the importance of phosphorylations by protein kinases during the life cycle of T. gondii. However, very little is known about protein phosphatases and their regulators in the parasite. We report the molecular and functional characterization of the T. gondii ortholog of the inhibitor-2 protein, designated TgI2. We show that TgI2 encompasses conserved motifs involved in the interaction and modulation of the phosphatase activity of T. gondii protein phosphatase 1, named TgPP1. We show that a specific combination of motifs is involved in binding and/or inhibition of the TgPP1 activity. We show here that the TgI2 protein is a potent inhibitor of TgPP1 phosphatase activity. TgI2 SILK and RVxF motifs are critical for regulating the activity of TgPP1, a feature that is common with the higher eukaryotes inhibitor-2 protein.KEYWORDS: Cell-cycle; HYNE; Inhibitor-2; PP1; Phosphatase; Plasmodium; RVxF; SILK; Toxoplasma
PMID:
28667522
DOI:
10.1007/s00436-017-5543-6
Categories: toxoplasma news feeds

A Plastid Protein That Evolved from Ubiquitin and Is Required for Apicoplast Protein Import in Toxoplasma gondii

The Anti-Toxo Blog - 29 June 2017 - 1:13pm
2017 Jun 27;8(3). pii: e00950-17. doi: 10.1128/mBio.00950-17.
Fellows JD1, Cipriano MJ2, Agrawal S1, Striepen B3,2.
Apicomplexan parasites cause a variety of important infectious diseases, including malaria, toxoplasma encephalitis, and severe diarrhea due to Cryptosporidium Most apicomplexans depend on an organelle called the apicoplast which is derived from a red algal endosymbiont. The apicoplast is essential for the parasite as the compartment of fatty acid, heme, and isoprenoid biosynthesis. The majority of the approximate 500 apicoplast proteins are nucleus encoded and have to be imported across the four membranes that surround the apicoplast. Import across the second outermost membrane of the apicoplast, the periplastid membrane, depends on an apicoplast-specific endoplasmic reticulum-associated protein degradation (ERAD) complex and on enzymes of the associated ubiquitination cascade. However, identification of an apicoplast ubiquitin associated with this machinery has long been elusive. Here we identify a plastid ubiquitin-like protein (PUBL), an apicoplast protein that is derived from a ubiquitin ancestor but that has significantly changed in its primary sequence. PUBL is distinct from known ubiquitin-like proteins, and phylogenomic analyses suggest a clade specific to apicomplexans. We demonstrate that PUBL and the AAA ATPase CDC48AP both act to translocate apicoplast proteins across the periplastid membrane during protein import. Conditional null mutants and genetic complementation show that both proteins are critical for this process and for parasite survival. PUBL residues homologous to those that are required for ubiquitin conjugation onto target proteins are essential for this function, while those required for polyubiquitination and preprotein processing are dispensable. Our experiments provide a mechanistic understanding of the molecular machinery that drives protein import across the membranes of the apicoplast.
IMPORTANCE Apicomplexan parasites are responsible for important human diseases. There are no effective vaccines for use in humans, and drug treatment faces multiple challenges, including emerging resistance, lack of efficacy across the lifecycle, and adverse drug effects. The apicoplast is a promising target for novel treatments: this chloroplast-like organelle is derived from an algal symbiont, is absent from the host, and is essential for parasite growth and pathogenesis. We use Toxoplasma gondii as a model to study the apicoplast due to its strong genetic tools and established functional assays. We identify a plastid ubiquitin-like protein (PUBL) which is a novel ubiquitin-like protein and demonstrate its importance and that of the motor protein CDC48AP for apicoplast protein import. These findings broaden our understanding of the evolution and mechanistic workings of a unique parasite organelle and may lead to new opportunities for treatments against important human pathogens.
Copyright © 2017 Fellows et al.KEYWORDS: Toxoplasma; apicomplexan parasites; apicoplast; chloroplast; organelle protein import; ubiquitin
PMID:
28655825
DOI:
10.1128/mBio.00950-17
Categories: toxoplasma news feeds

Characterization of the Activities of Dinuclear Thiolato-Bridged Arene Ruthenium Complexes against Toxoplasma gondii

The Anti-Toxo Blog - 28 June 2017 - 2:46pm

2017 Jun 26. pii: AAC.01031-17. doi: 10.1128/AAC.01031-17. [Epub ahead of print]
Basto AP1, Müller J1, Rubbiani R2, Stibal D3, Giannini F4, Süss-Fink G3, Balmer V1, Hemphill A5, Gasser G6, Furrer J7.
The in vitro effects of 18 dinuclear-thiolato bridged arene ruthenium complexes, (1 mono-, 4 di- and 13-tri-thiolato compounds), originally designed as anti-cancer agents, were studied in the apicomplexan parasite Toxoplasma gondii grown in human foreskin fibroblast host cells (HFF). Some tri-thiolato compounds exhibited anti-parasitic efficacy at 250 nM and below. Among those, complex 1 and complex 2 inhibited T. gondii proliferation with IC50 values of 34 and 62 nM, respectively, and they did not affect HFF at dosages of 200 μM or above, resulting in selectivity indices of > 23' 000. The IC50 values of complex 9 were 1.2 nM for T. gondii and above 5 μM for HFF. TEM detected ultrastructural alterations in the matrix of the parasite mitochondria at the early stages of treatment, followed by more pronounced destruction of tachyzoites. However, all three compounds applied at 250 nM for 15 days were not parasiticidal. By affinity chromatography using complex 9 coupled to epoxy-activated sepharose followed by mass spectrometry, T. gondii translation elongation factor-1 alpha and two ribosomal proteins, RPS18, and RPL27 were identified as potential binding proteins. In conclusion, organometallic ruthenium complexes exhibit promising activities against Toxoplasma, and potential mechanisms of action of these compounds as well as their prospective applications for the treatment of toxoplasmosis are discussed.
Copyright © 2017 American Society for Microbiology.
PMID:
28652238
DOI:
10.1128/AAC.01031-17
Categories: toxoplasma news feeds

Proteomic profiling of extracellular vesicles secreted from Toxoplasma gondii

The Anti-Toxo Blog - 24 June 2017 - 6:56pm
 2017 Jun 23. doi: 10.1002/pmic.201600477. [Epub ahead of print]
Wowk PF1, Zardo ML1, Miot HT1, Goldenberg S1, Carvalho PC1, Mörking PA1.AbstractToxoplasma gondii infects a wide range of hosts worldwide, including humans and domesticated animals causing toxoplasmosis disease. Recently, exosomes, small extracellular vesicles (EV) that contain nucleic acids, proteins, and lipids derived from their original cells were linked with disease protection. The effect of EVs derived from T. gondii on the immune response and its relevance in a physiological context is unknown. Here we disclose the first proteomic profiling of T. gondii EVs compared to EVs isolated from a human foreskin fibroblast infected cell line cultured in a vesicle- free medium. Our results reveal a broad range of canonical exosomes proteins. Data are available via ProteomeXchange with identifier PXD004895. This article is protected by copyright. All rights reserved.This article is protected by copyright. All rights reserved.KEYWORDS: Extracellular vesicles; MS; Toxoplasma gondii; Toxoplasma-infected cell
PMID:
 
28643940
 
DOI:
 
10.1002/pmic.201600477
Categories: toxoplasma news feeds

Parasites lacking the micronemal protein MIC2 are deficient in surface attachment and host cell egress, but remain virulent in vivo

The Anti-Toxo Blog - 22 June 2017 - 2:27pm
 2017 May 19;2:32. doi: 10.12688/wellcomeopenres.11594.1. eCollection 2017.
Gras S1, Jackson A1, Woods S2, Pall G1, Whitelaw J1, Leung JM3,4, Ward GE4, Roberts CW2, Meissner M1.AbstractBackground: Micronemal proteins of the thrombospondin-related anonymous protein (TRAP) family are believed to play essential roles during gliding motility and host cell invasion by apicomplexan parasites, and currently represent major vaccine candidates against Plasmodium falciparum, the causative agent of malaria. However, recent evidence suggests that they play multiple and different roles than previously assumed. Here, we analyse a null mutant for MIC2, the TRAP homolog in Toxoplasma gondiiMethods: We performed a careful analysis of parasite motility in a 3D-environment, attachment under shear stress conditions, host cell invasion and in vivo virulence. Results: We verified the role of MIC2 in efficient surface attachment, but were unable to identify any direct function of MIC2 in sustaining gliding motility or host cell invasion once initiated. Furthermore, we find that deletion of mic2causes a slightly delayed infection in vivo, leading only to mild attenuation of virulence; like with wildtype parasites, inoculation with even low numbers of mic2 KO parasites causes lethal disease in mice. However, deletion of mic2 causes delayed host cell egress in vitro, possibly via disrupted signal transduction pathways. Conclusions: We confirm a critical role of MIC2 in parasite attachment to the surface, leading to reduced parasite motility and host cell invasion. However, MIC2 appears to not be critical for gliding motility or host cell invasion, since parasite speed during these processes is unaffected. Furthermore, deletion of MIC2 leads only to slight attenuation of the parasite.KEYWORDS: Gliding motility; Host cell invasion; MIC2; Microneme; Plasmodium; TRAP; Toxoplasma
PMID:
 
28630943
 
PMCID:
 
PMC5473411
 
DOI:
 
10.12688/wellcomeopenres.11594.1
Categories: toxoplasma news feeds

New molecular tools in Neospora caninum for studying apicomplexan parasite proteins

The Anti-Toxo Blog - 22 June 2017 - 2:27pm
 2017 Jun 19;7(1):3768. doi: 10.1038/s41598-017-03978-1.
Mota CM1, Chen AL2, Wang K2, Nadipuram S2, Vashisht AA3, Wohlschlegel JA3, Mineo TWP4, Bradley PJ5,6.AbstractThe development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. We have established a system specifically designed for Neospora caninum, and used this system as a heterologous platform for the expression of foreign genes. Plasmid constructs containing fluorescent proteins or targeted genes of Toxoplasma gondii, driven by N. caninum promoters, have yielded robust expression and correct trafficking of target gene products as assessed by immunofluorescence assays and Western blot analyses. Using this approach, we here demonstrated that N. caninum expressing T. gondii's GRA15 and ROP16 kinase are biologically active and induced immunological phenotypes consistent with T. gondii strains. N. caninum expressing TgGRA15 differentially disturbed the NF-κB pathway, inducing an increased IL-12 production. On the other hand, N. caninum expressing TgROP16 induced host STAT3 phosphorylation and consequent reduction of IL-12 synthesis. These results indicate that heterologous gene expression in N. caninum is a useful tool for the study of specific gene functions and may allow the identification of antigenic targets responsible for the phenotypic differences observed between these two closely related apicomplexan parasites. Additionally, these observations may prove to be useful for the development of vaccine protocols to control toxoplasmosis and/or neosporosis.
PMID:
 
28630403
 
DOI:
 
10.1038/s41598-017-03978-1
Categories: toxoplasma news feeds

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