Wednesday, July 26, 2017

Yeast two-hybrid screening service for Toxoplasma gondii

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: (

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.

Friday, July 21, 2017

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

 2017 Jul 20;13(7):e1006351. doi: 10.1371/journal.ppat.1006351. eCollection 2017 Jul.

Mendez OA1,2Koshy AA2,3,4.


Toxoplasma 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.

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

 2017 Jul 18. pii: jbc.M117.802702. doi: 10.1074/jbc.M117.802702. [Epub ahead of print]


Toxoplasma 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.


Toxoplasma gondii; cancer metabolism; gluconeogenesis; glutamine catabolism; glycolysis; intracellular parasitism; parasite metabolism; phosphoenolpyruvate carboxykinase; pyruvate carboxylase (PC); tricarboxylic acid cycle (TCA cycle) (Krebs cycle)

Saturday, July 15, 2017

Impact of the host on Toxoplasma stage differentiation

 2017 Jun 22;4(7):203-211. doi: 10.15698/mic2017.07.579.


The 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.


Toxoplasma gondii; bradyzoite; cell cycle; host cell; immune response; metabolism; parasite host-interaction; stage conversion

Friday, July 14, 2017

Structural and Biochemical Characterization of Apicomplexan Inorganic Pyrophosphatases

2017 Jul 12;7(1):5255. doi: 10.1038/s41598-017-05234-y.

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.

Biochemical characterization of aminopeptidase N2 from Toxoplasma gondii

2017 Jul 13. doi: 10.1292/jvms.17-0119. [Epub ahead of print]

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.


Toxoplasma gondii; activity; aminopeptidase N (APN); enzyme

Strategies for detecting toxoplasma immunity

2003 Jan;60(2):105-108. doi: 10.1080/09674845.2003.11783684.

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.


Immunity; Immunocompromised host; Pregnancy; Toxoplasma

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

2017 Jul 12;7(1):5209. doi: 10.1038/s41598-017-05487-7.

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.

Monday, July 10, 2017

Toxoplasmosis accelerates acquisition of epilepsy in rats undergoing chemical kindling

2017 Jun 24;135:137-142. doi: 10.1016/j.eplepsyres.2017.06.012. [Epub ahead of print]

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.


Epileptogenesis; Kindling; Pentylenetetrazole; Rat; Toxoplasma gondii

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

2017 Jul 7. pii: fj.201700008R. doi: 10.1096/fj.201700008R. [Epub ahead of print]

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.


enzyme activity; gene knockout; immune evasion; protein expression

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

2017 Jul 4. pii: S1471-4922(17)30156-3. doi: 10.1016/ [Epub ahead of print]

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.

Tuesday, July 04, 2017

Proteasomal Degradation of T. gondii ROP18 Requires Derlin2

 2017 Jun 29. pii: S0001-706X(17)30351-0. doi: 10.1016/j.actatropica.2017.06.027. [Epub ahead of print]

Tang Y1An R1Chen L2Gong L3Cai H4Liu K4Yu L5Shen J5Du J6.


T. 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.


Derlin2; ERAD; ROP18; T. gondii; degradation

Checkpoints of apicomplexan cell division identified in Toxoplasma gondii

 2017 Jul 3;13(7):e1006483. doi: 10.1371/journal.ppat.1006483. [Epub ahead of print]


The 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.

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

 2017 Jun 22;(124). doi: 10.3791/55185.


Scientific 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.

Monday, July 03, 2017

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

2017 Jun 30. doi: 10.1007/s00436-017-5543-6. [Epub ahead of print]

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.


Cell-cycle; HYNE; Inhibitor-2; PP1; Phosphatase; Plasmodium; RVxF; SILK; Toxoplasma