Toxoplasma Genome Research Articles

Serological and molecular detection of Toxoplasma gondii in ß. thalassemia patients.

Toxoplasma gondii is a worldwide opportunistic protozoan causing life-threatening infection in immunocompromised patients, while frequently asymptomatic in immunocompetent individuals. The current study aimed to detect T. gondii; serologically and molecularly in ß. thalassemia patients and evaluate the association of infection with some hematological parameters in these patients. Blood samples were collected from 100 ß. thalassemia patients. Serological diagnosis of T. gondii using ELISA for IgG and IgM antibodies was performed. Molecular diagnosis by Real-Time (RE) PCR was performed using specifically designed primers amplifying 389bp fragments of Toxoplasma genome. 45 patients (45%) had anti-Toxoplasma IgG antibodies with no detectable IgM antibodies while both anti-Toxoplasma IgM and IgG antibodies were noticed in 10 patients (10%). IgM only antibodies were discovered in two cases (2%). The total seropositivity rate among patients was 57%. RE PCR analysis revealed Toxoplasma DNA in 20% out of 100 patients. PCR and serological examination showed slight agreement. A statistically significant relation was observed between the results of IgG and IgM ELISA and PCR for the detection of T. gondii infection among patients with ß. thalassemia. None of the studied risk factors (age, gender, contact with cats, consumption of undercooked meat) or hematological parameters (ESR, anemia degree, ferritin level, type of blood transfusion, spleen status) showed statistically significant association with Toxoplasma infection. It can be concluded that patients with thalassemia have a high risk of infection with T. gondii. RE PCR should be used as a diagnostic method in association with serology especially in immunocompromised patients to increase sensitivity.

Identification of a Putative BAX inhibitor in Toxoplasma gondii (TgBI-1)

Cellular homeostasis is quintessential of regulated protein turnover machinery within a cell. Any form of interference with this highly orchestrated phenomenon of protein production in the Endoplasmic reticulum (ER) can induce cellular stress. The continued blockade of the ER with misfolded proteins can lead to structural abnormalities of the ER and the mitochondria, following which a cascade of cellular reactions is triggered, marked by the increase in the levels of cytosolic Ca++ and dissipation of mitochondrial membrane potential. BAX promotes the loss of membrane potential of the mitochondria causing the release of Cytochrome-c and to counter it, BAX-inhibitor (BI-1) plays a crucial role in protecting the cell from undergoing programmed cell death (PCD). A homolog of BI-1 from Plasmodium Spp. has been identified in the Toxoplasma genome (TgBI-1), which we intend to characterize by inducing stress on the parasite cell and then by reversibly modulating the levels of the concerned protein (putative BI-1) by using a tunable system like transactivator (Tet-off) to observe the repercussions in the cellular behaviour.

Development of a real-time recombinase-aided amplification (RT-RAA) molecular diagnosis assay for sensitive and rapid detection of Toxoplasma gondii

Toxoplasma gondii, a protozoan intracellular parasite, is present in a wide range of hosts, including virtually all species of warm-blooded vertebrates. Toxoplasmosis spreads to humans through a variety of pathways, including contaminated food or water, and close contact with various types of domestic animals. It poses a severe threat to human health, and contributes to important economic losses, not only in cost-of-illness but also in surveillance programs. It is thus necessary to develop a rapid point-of-care field diagnostic technology to control or prevent pathogen transmission to economically important livestock animals, domestic animals, and human beings. In this study, we develop a real-time isothermal amplification method capable of detecting the T. gondii genome in swine and feline blood samples. This method can detect toxoplasma genome with a lowest detection limit of 102 copies of per reaction under optimal reaction conditions of 36 °C for 25 min. The assay displayed advantages in sensitivity and specificity in comparison to traditional real-time PCR, and can be performed in a portable instrument.

Toxoplasma F-box protein 1 is required for daughter cell scaffold function during parasite replication.

By binding to the adaptor protein SKP1 and serving as substrate receptors for the SKP1 Cullin, F-box E3 ubiquitin ligase complex, F-box proteins regulate critical cellular processes including cell cycle progression and membrane trafficking. While F-box proteins are conserved throughout eukaryotes and are well studied in yeast, plants, and animals, studies in parasitic protozoa are lagging. We have identified eighteen putative F-box proteins in the Toxoplasma genome of which four have predicted homologs in Plasmodium. Two of the conserved F-box proteins were demonstrated to be important for Toxoplasma fitness and here we focus on an F-box protein, named TgFBXO1, because it is the most highly expressed by replicative tachyzoites and was also identified in an interactome screen as a Toxoplasma SKP1 binding protein. TgFBXO1 interacts with Toxoplasma SKP1 confirming it as a bona fide F-box protein. In interphase parasites, TgFBXO1 is a component of the Inner Membrane Complex (IMC), which is an organelle that underlies the plasma membrane. Early during replication, TgFBXO1 localizes to the developing daughter cell scaffold, which is the site where the daughter cell IMC and microtubules form and extend from. TgFBXO1 localization to the daughter cell scaffold required centrosome duplication but before kinetochore separation was completed. Daughter cell scaffold localization required TgFBXO1 N-myristoylation and was dependent on the small molecular weight GTPase, TgRab11b. Finally, we demonstrate that TgFBXO1 is required for parasite growth due to its function as a daughter cell scaffold effector. TgFBXO1 is the first F-box protein to be studied in apicomplexan parasites and represents the first protein demonstrated to be important for daughter cell scaffold function.

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

Infection with the protozoan parasite Toxoplasma gondii is a major health risk owing to birth defects, its chronic nature, ability to reactivate to cause blindness and encephalitis, and high prevalence in human populations. Unlike most eukaryotes, Toxoplasma propagates in intracellular parasitophorous vacuoles, but like nearly all other eukaryotes, Toxoplasma glycosylates many cellular proteins and lipids and assembles polysaccharides. Toxoplasma glycans resemble those of other eukaryotes, but species-specific variations have prohibited deeper investigations into their roles in parasite biology and virulence. The Toxoplasma genome encodes a suite of likely glycogenes expected to assemble N-glycans, O-glycans, a C-glycan, GPI-anchors, and polysaccharides, along with their precursors and membrane transporters. To investigate the roles of specific glycans in Toxoplasma, here we coupled genetic and glycomics approaches to map the connections between 67 glycogenes, their enzyme products, the glycans to which they contribute, and cellular functions. We applied a double-CRISPR/Cas9 strategy, in which two guide RNAs promote replacement of a candidate gene with a resistance gene; adapted MS-based glycomics workflows to test for effects on glycan formation; and infected fibroblast monolayers to assess cellular effects. By editing 17 glycogenes, we discovered novel Glc0-2-Man6-GlcNAc2-type N-glycans, a novel HexNAc-GalNAc-mucin-type O-glycan, and Tn-antigen; identified the glycosyltransferases for assembling novel nuclear O-Fuc-type and cell surface Glc-Fuc-type O-glycans; and showed that they are important for in vitro growth. The guide sequences, editing constructs, and mutant strains are freely available to researchers to investigate the roles of glycans in their favorite biological processes.

Translationally controlled tumor protein is required for the fast growth of Toxoplasma gondii and maintenance of its intracellular development.

Translationally controlled tumor protein (TCTP) is a highly conserved, multifunctional protein that has been implicated in a range of cell physiologic processes, especially cell growth and development. A TCTP-like gene has been identified in the Toxoplasma genome [ Toxoplasma gondii TCTP ( TgTCTP)], although its function remains unknown. The sequence analysis of TgTCTP indicated that it is a highly conserved protein in eukaryotes. We found that the expression level of TgTCTP in the virulent RH strain was significantly higher than that in the avirulent PLK strain. Indirect immunofluorescence showed that TgTCTP was expressed in the parasite cytoplasm. The localization of TgTCTP was unchanged during the replication of the parasite. We expressed a functional recombinant TgTCTP (r TgTCTP) protein in Escherichia coli and found that the recombinant protein could form a multimer. We then evaluated the function of TgTCTP using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 knockout (KO) system. Phenotypic analysis of the KO strain (Δ TgTCTP) revealed that TgTCTP is required for the robust growth of the parasites. TgTCTP deficiency also led to early egress of the parasites and subsequent impairment in their invasion and attachment abilities. We subsequently found that the multimer form of TgTCTP might not be necessary for the growth and replication of the parasite. Then the expression profiling of genes in the Δ TgTCTP and complement strains were analyzed. The results revealed that 988 genes were regulated in Δ TgTCTP compared with the complement strain. Overall, although not essential, TgTCTP is required for the fast growth of Tg and maintenance of its intracellular development.-Zheng, J., Chen, Y., Li, Z., Cao, S., Zhang, Z., Jia, H. Translationally controlled tumor protein is required for the fast growth of Toxoplasma gondii and maintenance of its intracellular development.

Metallopeptidases of Toxoplasma gondii: in silico identification and gene expression.

Metallopeptidases are a family of proteins with domains that remain highly conserved throughout evolution. These hydrolases require divalent metal cation(s) to activate the water molecule in order to carry out their catalytic action on peptide bonds by nucleophilic attack. Metallopeptidases from parasitic protozoa, including Toxoplasma, are investigated because of their crucial role in parasite biology. In the present study, we screened the T. gondii database using PFAM motifs specific for metallopeptidases in association with the MEROPS peptidase Database (release 10.0). In all, 49 genes encoding proteins with metallopeptidase signatures were identified in the Toxoplasma genome. An Interpro Search enabled us to uncover their domain/motif organization, and orthologs with the highest similarity by BLAST were used for annotation. These 49 Toxoplasma metallopeptidases clustered into 15 families described in the MEROPS database. Experimental expression analysis of their genes in the tachyzoite stage revealed transcription for all genes studied. Further research on the role of these peptidases should increase our knowledge of basic Toxoplasma biology and provide opportunities to identify novel therapeutic targets. This type of study would also open a path towards the comparative biology of apicomplexans.

Functional motif analysis for glycosyltransferases in apicomplexan genomes (608.5)

Parasitic protozoa of the Phylum Apicomplexa include the genus Plasmodium (causative agents of malaria) and Toxoplasma gondii which causes toxoplasmosis. Little is known about glycosylation pathways and their contributions to pathogenesis. Increased glycosylation is known to be a feature of the chronic infection with Toxoplasma defined by the formation of the tissue cyst. Lectin reactivity and biochemical identification indicate that Toxoplasma tissue cysts contain diverse glycan modifications that are hypothesized to originate from both the parasite and the host. In order to establish the potential extent of host derived glycan modifications we have undertaken a comprehensive survey of the encoded potential in the toxoplasma genome (www.toxodb.org) by conducting a rigorous bioinformatics survey for predicted glycosyltransferases (GT). In addition to the identification of a truncated pathway for the synthesis of N‐linked glycans predicting simple (ConA positive) but not complex (LCA positive) labeling we find a complete absence of any of the machinery to permit modification with sialic acid. The presence in tissue cysts of both complex N‐linked glycans and sialic acid (SNA, MALII, Limax agglutinin and anti Sia‐Tn antigen) implicates host cell involvement. In order to understand the full extent of the novel form of parasitism, the hijacking of host glycan modifying enzymes, we are now investigating whether the Toxoplasma genome encodes other classes of glycosyltransferases. We hypothesize that the absence of a specific GT encoded in the parasite genome coupled with its phenotypic detection implicates thievery from the host cell. The current interrogation is being extended to other parasitic protozoa for which the genomes are now available (www.eupathdb.org). Such a broad survey across Apicomplexa and other parasitic Phyla may reveal insights into the evolution of the glycosylation machinery in these early branching eukaryotes and provide insights into infection and immunity.Grant Funding Source: NIHR21 AI099509 (to APS)

Apoptosis-like cell death pathways in the unicellular parasite Toxoplasma gondii following treatment with apoptosis inducers and chemotherapeutic agents: a proof-of-concept study

Ancient pathways of an apoptosis-like cell death have been identified in unicellular eukaryotes including protozoan parasites. Here, we examined programmed cell death in the apicomplexan Toxoplasma gondii which is a common intracellular pathogen of humans and warm-blooded animals. Treatment of extracellular T. gondii with various pro-apoptotic stimuli significantly induced DNA strand breaks as revealed by TUNEL and flow cytometry. Using staurosporine or miltefosine as pro-apoptotic stimuli, parasites also presented a reduced cell size, i.e. pyknosis and externalized phosphatidylserine while the plasma membrane remained intact. Importantly, staurosporine also induced DNA strand breaks in intracellular T. gondii. Data mining of the Toxoplasma genome resource identified 17 putative cell death-associated genes encoding proteases, a nuclease and several apoptosis regulators. Staurosporine-treated parasites but not controls strongly up-regulated several of these genes in a time-dependent fashion with a putative PDCD2 protein being more than 100-fold up-regulated. However, the mitochondrial membrane potential (ΔΨ m) remained intact and caspase-like activity increased only slightly during staurosporine-triggered cell death. As compared to staurosporine, the transcriptional response of parasites to miltefosine was more restricted but PDCD2 was again strongly induced. Furthermore, T. gondii lost their ΔΨ m and rapidly presented strong caspase-like activity during miltefosine treatment. Consequently, protease inhibitors abrogated miltefosine-induced but not staurosporine-induced Toxoplasma cell death. Finally, toxoplasmacidal drugs triggered DNA strand breaks in extracellular T. gondii. Interestingly, clindamycin also induced markers of an apoptosis-like cell death in intracellular parasites. Together, the data indicate that T. gondii possesses ancient apoptosis-like cell death machinery which can be triggered by chemotherapeutic agents.

De novo reconstruction of the Toxoplasma gondii transcriptome improves on the current genome annotation and reveals alternatively spliced transcripts and putative long non-coding RNAs

BackgroundAccurate gene model predictions and annotation of alternative splicing events are imperative for genomic studies in organisms that contain genes with multiple exons. Currently most gene models for the intracellular parasite, Toxoplasma gondii, are based on computer model predictions without cDNA sequence verification. Additionally, the nature and extent of alternative splicing in Toxoplasma gondii is unknown. In this study, we used de novo transcript assembly and the published type II (ME49) genomic sequence to quantify the extent of alternative splicing in Toxoplasma and to improve the current Toxoplasma gene annotations.ResultsWe used high-throughput RNA-sequencing data to assemble full-length transcripts, independently of a reference genome, followed by gene annotation based on the ME49 genome. We assembled 13,533 transcripts overlapping with known ME49 genes in ToxoDB and then used this set to; a) improve the annotation in the untranslated regions of ToxoDB genes, b) identify novel exons within protein-coding ToxoDB genes, and c) report on 50 previously unidentified alternatively spliced transcripts. Additionally, we assembled a set of 2,930 transcripts not overlapping with any known ME49 genes in ToxoDB. From this set, we have identified 118 new ME49 genes, 18 novel Toxoplasma genes, and putative non-coding RNAs.ConclusionRNA-seq data and de novo transcript assembly provide a robust way to update incompletely annotated genomes, like the Toxoplasma genome. We have used RNA-seq to improve the annotation of several Toxoplasma genes, identify alternatively spliced genes, novel genes, novel exons, and putative non-coding RNAs.

Beyond the genome: recent advances in Toxoplasma gondii functional genomics

Recent years have witnessed an explosion in the amount of genomic information available for Toxoplasma gondii and other closely related pathogens. These data, many of which have been made publicly available prior to publication, have facilitated a wide variety of functional genomics studies. In this review, we provide a brief overview of existing database tools for querying the Toxoplasma genome and associated genome-wide data and review recent publications that have been facilitated by these data. Topics covered include strain comparisons and quantitative trait loci mapping, gene expression analyses during the cell cycle as well as during parasite differentiation, and proteomics.

Diagnosis of ocular toxoplasmosis by two polymerase chain reaction (PCR) examinations: qualitative multiplex and quantitative real-time

To establish a two-step polymerase chain reaction (PCR) diagnostic system for ocular toxoplasmosis. A total of 13 ocular fluid samples (11 aqueous humor and 2 vitreous fluid) were collected from 13 patients with clinically suspected ocular toxoplasmosis. Ten ocular samples from other uveitis patients and 20 samples from subjects without ocular inflammation were used as controls. Two polymerase chain reaction (PCR) methods, i.e., qualitative multiplex PCR and quantitative real-time PCR, were used to measure the toxoplasma genome (T. gondii B1 gene). Qualitative multiplex PCR detected T. gondii B1 gene in the ocular fluids of 11 out of 13 patients with clinically suspected ocular toxoplasmosis. In real-time PCR, we detected high copy numbers of T. gondii DNA (5.1×10(2)-2.1×10(6)copies/mL) in a total of 10 patients (10/13, 77%). Only ocular toxoplasmosis scar lesions were observed in the three real-time PCR-negative patients. PCR assay results for the samples from the two control groups were all negative. The two-step PCR examination to detect toxoplasma DNA is a useful tool for diagnosing ocular toxoplasmosis.

MYST family histone acetyltransferases in the protozoan parasite Toxoplasma gondii.

The restructuring of chromatin precedes tightly regulated events such as DNA transcription, replication, and repair. One type of chromatin remodeling involves the covalent modification of nucleosomes by histone acetyltransferase (HAT) complexes. The observation that apicidin exerts antiprotozoal activity by targeting a histone deacetyltransferase has prompted our search for more components of the histone modifying machinery in parasitic protozoa. We have previously identified GNAT family HATs in the opportunistic pathogen Toxoplasma gondii and now describe the first MYST (named for members MOZ, Ybf2/Sas3, Sas2, and Tip60) family HATs in apicomplexa (TgMYST-A and -B). The TgMYST-A genomic locus is singular and generates a approximately 3.5-kb transcript that can encode two proteins of 411 or 471 amino acids. TgMYST-B mRNA is approximately 7.0 kb and encodes a second MYST homologue. In addition to the canonical MYST HAT catalytic domain, both TgMYST-A and -B possess an atypical C2HC zinc finger and a chromodomain. Recombinant TgMYST-A exhibits a predilection to acetylate histone H4 in vitro at lysines 5, 8, 12, and 16. Antibody generated to TgMYST-A reveals that both the long and short (predominant) versions are present in the nucleus and are also plentiful in the cytoplasm. Moreover, both TgMYST-A forms are far more abundant in rapidly replicating parasites (tachyzoites) than encysted parasites (bradyzoites). A bioinformatics survey of the Toxoplasma genome reveals numerous homologues known to operate in native MYST complexes. The characterization of TgMYST HATs represents another important step toward understanding the regulation of gene expression in pathogenic protozoa and provides evolutionary insight into how these processes operate in eukaryotic cells in general.

The Opportunistic Pathogen Toxoplasma gondii Deploys a Diverse Legion of Invasion and Survival Proteins

Host cell invasion is an essential step during infection by Toxoplasma gondii, an intracellular protozoan that causes the severe opportunistic disease toxoplasmosis in humans. Recent evidence strongly suggests that proteins discharged from Toxoplasma apical secretory organelles (micronemes, dense granules, and rhoptries) play key roles in host cell invasion and survival during infection. However, to date, only a limited number of secretory proteins have been discovered, and the full spectrum of effector molecules involved in parasite invasion and survival remains unknown. To address these issues, we analyzed a large cohort of freely released Toxoplasma secretory proteins by using two complementary methodologies, two-dimensional electrophoresis/mass spectrometry and liquid chromatography/electrospray ionization-tandem mass spectrometry (MudPIT, shotgun proteomics). Visualization of Toxoplasma secretory products by two-dimensional electrophoresis revealed approximately 100 spots, most of which were successfully identified by protein microsequencing or matrix-assisted laser desorption ionization-mass spectrometry analysis. Many proteins were present in multiple species suggesting they are subjected to substantial post-translational modification. Shotgun proteomic analysis of the secretory fraction revealed several additional products, including novel putative adhesive proteins, proteases, and hypothetical secretory proteins similar to products expressed by other related parasites including Plasmodium, the etiologic agent of malaria. A subset of novel proteins were re-expressed as fusions to yellow fluorescent protein, and this initial screen revealed shared and distinct localizations within secretory compartments of T. gondii tachyzoites. These findings provided a uniquely broad view of Toxoplasma secretory proteins that participate in parasite survival and pathogenesis during infection.

Toxoplasma gondii: Identification of a putative nitric oxide synthase motif DNA sequence

Nitric oxide (NO) plays an important role as a mediator of the immune response to intracellular pathogens in mice; however discordant results were obtained in in vitro human cell lines experiments. Thus, we found that nitrite levels (nitric oxide derivatives) were increased in presence of Toxoplasma but not with IFNγ plus LPS treatment during Toxoplasma infection of a human monocyte cell line THP1 and Griess assays confirmed that Toxoplasma alone has a nitrite production that was surprisingly increased by the most common inhibitor of nitric oxide synthase (NOS) in mammals. To look for genomic sequences that code for NOS gene in Toxoplasma, which could explain this production of NO derivatives, specific NOS motifs were sougth by bioinformatics methods. A putative NOS motif sequence was found in one contig of the Toxoplasma genome ( TGG_994574). Specific primers amplified a segment of 270 bp in RT-PCR assay, indicating that it is a transcription gene in the tachyzoite stage. Our results are the first description of the existence and transcription of a putative NOS DNA sequence in a pathogenic protozoan.

Toxoplasma gondii is capable of exogenous folate transport : A likely expansion of the BT1 family of transmembrane proteins

Folates are key elements in eukaryotic biosynthetic processes. The protozoan parasite Toxoplasma gondii possesses the enzymes necessary for de novo folate synthesis and has been suggested to lack alternative mechanisms for folate acquisition. In this paper, we present a different view by providing evidence that Toxoplasma is capable of salvaging exogenous folates. By monitoring uptake of radiolabeled folates by parasites in axenic conditions, our studies revealed a common folate transporter that has a high affinity for folic acid. Transport of this compound across the parasite plasma membrane is rapid, biphasic, temperature dependent, bi-directional, concentration dependent and specific. In addition, morphological evidence demonstrates that fluorescent methotrexate, a folate analog, is internalized by Toxoplasma and shows localization reminiscent to the mitochondrion. The presence of putative folate transporter genes in the Toxoplasma genome, which are homologous to the BT1 family of proteins, suggests that Toxoplasma may encode proteins involved in folate transport. Interestingly, genome analysis suggests that the BT1 family of proteins exists not only in Toxoplasma, but in other Apicomplexan parasites as well. Altogether, our results not only have implications for current therapeutic regimens against T. gondii, but they also allude that the folate transport mechanism may represent a novel Apicomplexan target for the development of new drugs.

Structure analysis of two Toxoplasma gondii and Neospora caninum satellite DNA families and evolution of their common monomeric sequence.

A family of repetitive DNA elements of approximately 350 bp-Sat350-that are members of Toxoplasma gondii satellite DNA was further analyzed. Sequence analysis identified at least three distinct repeat types within this family, called types A, B, and C. B repeats were divided into the subtypes B1 and B2. A search for internal repetitions within this family permitted the identification of conserved regions and the design of PCR primers that amplify almost all these repetitive elements. These primers amplified the expected 350-bp repeats and a novel 680-bp repetitive element (Sat680) related to this family. Two additional tandemly repeated high-order structures corresponding to this satellite DNA family were found by searching the Toxoplasma genome database with these sequences. These studies were confirmed by sequence analysis and identified: (1). an arrangement of AB1CB2 350-bp repeats and (2). an arrangement of two 350-bp-like repeats, resulting in a 680-bp monomer. Sequence comparison and phylogenetic analysis indicated that both high-order structures may have originated from the same ancestral 350-bp repeat. PCR amplification, sequence analysis and Southern blot showed that similar high-order structures were also found in the Toxoplasma-sister taxon Neospora caninum. The Toxoplasma genome database (http://ToxoDB.org ) permitted the assembly of a contig harboring Sat350 elements at one end and a long nonrepetitive DNA sequence flanking this satellite DNA. The region bordering the Sat350 repeats contained two differentially expressed sequence-related regions and interstitial telomeric sequences.

Cloning and in vitro expression of TPK3, a Toxoplasma gondii homologue of shaggy/glycogen synthase kinase-3 kinases

As an initial effort to dissect the signaling pathways responsible for pathogenesis of Toxoplasma gondii infection, we report the cloning and in vitro functional studies of TPK3 ( Toxoplasma protein kinase-3), a homologue of shaggy/glycogen synthase kinase-3 (GSK-3) kinases. The shaggy/GSK-3 family of kinases are highly conserved protein kinases that play important roles in cell fate determination, nuclear signaling and hormonal regulation. The TPK3 gene was isolated by RT-PCR with degenerate primers corresponding to highly conserved regions of serine/threonine protein kinases. The complete sequences of genomic and cDNA clones indicated the open reading frame, 1185 bp in size, is interrupted by five introns. The predicted protein sequence of TPK3 shows 54% identity to shaggy/GSK-3 over the catalytic domains. Southern analysis revealed TPK3 is a single copy locus in the Toxoplasma genome. Antisera to other GSK-3 proteins from other species recognized GST-TPK3 and a protein of the predicted size in parasites lysates. In vitro kinase assays with GST-TPK3 indicated that TPK3 autophosphorylates and phosphorylates protein phosphatase inhibitor-2 (I-2), a specific substrate of GSK-3 kinase.

Development of a Stable Episomal Shuttle Vector for Toxoplasma gondii

The rapid developments in the molecular genetics of Toxoplasma gondii have far reaching implications in treatment and vaccination strategies for this as well as closely related pathogens such as Plasmodium. Although stable transformation of this parasite through homologous and illegitimate genomic integration has provided many of the tools necessary for genetic analysis, subsequent manipulations of the DNA have proven laborious. This report describes the selection and subsequent characterization of a Toxoplasma sequence that permits the episomal maintenance of bacterial plasmids in this parasite. This sequence was isolated from the Toxoplasma genome through selection for episomal stability of a pUC19-based library in the absence of a selectable marker. A 500-base pair fragment was determined to possess the stabilization activity. Transformations of Toxoplasma using vectors possessing this fragment, referred to as EMS (episomal maintenance sequence), demonstrated an elevated stable transformation frequency compared with the vector alone. Mutants deficient in hypoxanthine-xanthine-guanine phosphoribosyltransferase activity were used as a test to see if this gene could be selected from a genomic library using a vector containing the EMS. The success of this test demonstrates the utility of EMS-containing vectors in complementation strategies and the ability of such constructs bearing large fragments of the Toxoplasma genome to be maintained episomally.

Complementation of a Toxoplasma gondii ROP1 knock-out mutant using phleomycin selection

The ROP1 gene of Toxoplasma gondii encodes a rhoptry protein that has been implicated in host cell invasion by this obligate intracellular protozoan. To further explore the function of this protein, we created a ROP1 deletion mutant by transfection with a plasmid encoding the bacterial chloramphenicol acetyltransferase ( cat) gene flanked by ROP1 genomic sequences. Selection for chloramphenicol resistance yielded the desired ROP1-deleted or ‘knock-out’ mutant. Analysis of this mutant both in vitro and in vivo shows no significant alterations in growth rate, host specificity, invasiveness or virulence and thus the ROP1 gene is not obligatory for the RH strain, at least under the conditions tested. However, electron microscopy reveals that the mutant strain's rhoptries are altered in ultrastructure: they are thinner and homogeneously electron-dense compared with the thicker and normally mottled or honeycombed appearance of wild-type rhoptries. The knock-out mutant was rescued using co-transfection of a cosmid carrying the ROP1 gene together with a plasmid encoding a new selectable marker for T. gondii, the bleomycin resistance gene ( ble) from Streptoalloteichus. Southern blot analysis showed that both DNAs were stably integrated into the Toxoplasma genome, although not into the ROP1 locus. The resulting strain showed wild-type levels of ROP1 expression and rescue of the ultrastructural phenotype (i.e., the rhoptries returned to their normal, mottled appearance), thus establishing a cause/effect relationship between the absence of ROP1 and the electron-opacity. These results demonstrate the utility of the reverse genetic approach in the study of Toxoplasma gene function and provide a further selectable marker for such manipulations.