Toxoplasma gondii: characterization of temperature-sensitive tachyzoite cell cycle mutants

We mutagenized RH delta hxgprt strain tachyzoites of Toxoplasma gondii using N-nitroso-N-ethylurea and analyzed 40 clonal isolates (of 3680 ENU mutants) that were unable to grow in cell culture at 40 degrees C. These isolates grew normally at 34 degrees C, but showed variable growth at temperatures between 34 and 39 degrees C. The inability to grow at 40 degrees C was also correlated with a loss of virulence in mice for those mutants examined. We further characterized the temperature-sensitive (ts) isolates using flow cytometry and propidium iodide staining and identified three types of cell cycle-related mutations. Regardless of temperature, in the isolates ts1C12, ts7B4, and ts7B10, the distribution of parasites with a haploid DNA content was substantially higher (congruent with 85%) than that observed for RH delta hxgprt (congruent with 60%). Four other isolates, ts4F6, ts6C11, ts8G10, and ts11F5, contained G1-related mutations, and in each case, the DNA distribution among parasites at the permissive temperature was similar to that of the parental strain, but at 40 degrees C only a single population containing a 1N nuclear DNA complement was evident. Furthermore, there was no evidence of nuclear division or cytokinesis at 40 degrees C, and these parasites demonstrated a distended cytoplasm typical of G1 arrest in other cell types. Finally, parasites of the ts11C9 mutant arrested in two near-equal populations with either 1N or 2N complements of nuclear DNA. All arrested ts11C9 parasites contained a single nucleus, and a major subfraction of the 2N population contained abnormal and incompletely formed daughters-indicating that the initiation of daughter formation can occur in the absence of nuclear division.     

Actin depolymerizing factor controls actin turnover and gliding motility in Toxoplasma gondii

Apicomplexan parasites rely on actin-based gliding motility to move across the substratum, cross biological barriers, and invade their host cells. Gliding motility depends on polymerization of parasite actin filaments, yet ～98% of actin is nonfilamentous in resting parasites. Previous studies suggest that the lack of actin filaments in the parasite is due to inherent instability, leaving uncertain the role of actin-binding proteins in controlling dynamics. We have previously shown that the single allele of Toxoplasma gondii actin depolymerizing factor (TgADF) has strong actin monomer-sequestering and weak filament-severing activities in vitro. Here we used a conditional knockout strategy to investigate the role of TgADF in vivo. Suppression of TgADF led to accumulation of actin-rich filaments that were detected by immunofluorescence and electron microscopy. Parasites deficient in TgADF showed reduced speed of motility, increased aberrant patterns of motion, and inhibition of sustained helical gliding. Lack of TgADF also led to severe defects in entry and egress from host cells, thus blocking infection in vitro. These studies establish that the absence of stable actin structures in the parasite are not simply the result of intrinsic instability, but that TgADF is required for the rapid turnover of parasite actin filaments, gliding motility, and cell invasion.     

Identification of Toxoplasma gondii cAMP dependent protein kinase and its role in the tachyzoite growth

Background

cAMP-dependent protein kinase (PKA) has been implicated in the asexual stage of the Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary.

Methodology/Principal Finding

The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C) was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R)-overexpressing tachyzoites showed a significant growth defect.

Conclusions/Significance

Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.     

Toxoplasma gondii: ultrastructure and antigenicity of purified tachyzoite pellicle.

When purified Toxoplasma gondii tachyzoites were treated with hemolysin, DNase, and RNase, the organisms yielded a three-component system containing the outer membrane (pellicle), microtubules, and conoid in relatively normal morphological configuration. Further treatment of this preparation with protease digested all but the pellicle which was more collapsed in appearance. These two preparations were used in rabbit anti-toxoplasma and goat anti-rabbit ferritin labeling experiments. The three-component system showed ferritin label on the conoid and equal ferritin label on the outer and inner surfaces of the pellicle. The microtubules were unlabeled. The pellicle after protease treatment was labeled equally on its outer and inner surfaces, which indicated that the rabbit anti-toxoplasma serum contained antibodies against antigens on the outer and inner surfaces of the pellicle.     

Molecular characterization of tgd057, a novel gene from Toxoplasma gondii

The expressed sequence tag (EST) effort in Toxoplasma gondii has generated a substantial amount of gene information. To exploit this valuable resource, we chose to study tgd057, a novel gene identified by a large number of ESTs that otherwise show no significant match to known sequences in the database. Northern analysis showed that tgd057 is transcribed in this tachyzoite. The complete cDNA sequence of tgd057 is 1169 bp in length. Sequence analysis revealed that tgd057 possibly adopts two polyadenylation sites, utilizes the fourth in-frame ATG for translation initiation, and codes for a secretory protein. The longest open reading frame for the tgd057 gene was cloned and expressed as a recombinant protein (rd57) in Escherichia coli. Western analysis revealed that serum against rd57 recognized a molecule of ~21 kDa in the tachyzoite protein extract. This suggests that the tgd057 gene is expressed in vivo in the parasite.     

Molecular characterization of Toxoplasma gondii isolates from cats in Spain

Cats are important in the epidemiology of Toxoplasma gondii because felids are the only definitive hosts that can excrete environmentally resistant oocysts. Fresh samples of brain from 103 Spanish cats with antibodies to T. gondii were analyzed for T. gondii DNA using nested-PCR; 47 (45.5%) were found to be positive. Further characterization of DNA from 46 cats using RFLP-PCR at the 3' and 5' ends of the SAG2 locus revealed that 12 (26%) isolates were Type I and 34 (74%) were Type II; no Type III were found, and the 47th sample could not be classified to its genetic type. In addition, T. gondii was also isolated by bioassay in mice from 42 of 103 seropositive cats. This is the first report of T. gondii characterization from cats in Spain.     

Toxoplasma gondii: purification and characterization of an immunogenic metallopeptidase

A Toxoplasma gondii aminopeptidase specific for the fluorogenic substrate L-arginine 7-amino-4-methylcoumarin was identified in cell-free extract. This enzyme was purified by high-performance liquid chromatography using first size exclusion, then anion exchange, followed by a second size exclusion. The purified enzyme exhibited a pl of 4.7 by chromatofocusing and had an apparent molecular weight of 110 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The purification factor was 80.9 and the yield was 14%. The optimal activity was at pH 7.4 and was strongly inhibited by EDTA and o-phenanthroline. Antibodies against this T. gondii metallopeptidase were detected by immunoprecipitation and immunoblotting in human sera obtained from patients undergoing toxoplasmosis.     

Molecular cloning and characterization of mitogen-activated protein kinase 2 in Toxoplasma gondii

Mitogen-activated protein kinase (MAPK) pathways are major signal transduction systems by which eukaryotic cells convert environmental cues to intracellular events, such as cell proliferation and differentiation. Toxoplasma gondii is an obligate intracellular protozoan that is both a human and animal pathogen. This Apicomplexan causes significant morbidity and mortality in immune-competent and immune-compromised hosts. In humans, the most common manifestations of T. gondii infections are chorioretinitis in congenital infection and encephalitis in immune-compromised patients, such as patients with advanced AIDS. We have identified a T. gondii homolog of the MAPK family that we have called TgMAPK2. Sequence analyses demonstrated that TgMAPK2 has homology with lower eukaryotic ERK2 but has significant differences from mammalian ERK2. TgMAPK2 has an open reading frame of 2,037 bp, 678 amino acids, and its molecular weight is 73.1 kDa. It contains the typical 12 subdomains of a MAPK and has a TDY motif in the dual phosphorylation and activation subdomains. This suggests that TgMAPK2 may play an important role in stress response. recombinant TgMAPK2 was catalytically active and was not inhibited by a human ERK2 inhibitor, FR180204. A partial TgMAPK2 lacking the ATP-binding motifs GxGxxGxV was successfully regulated by a ligand-controlled destabilization domain (ddFKBP) expression vector system in T. gondii. Since TgMAPK2 is significantly different from its mammalian counterpart, it may be useful as a drug target. This work establishes a foundation for further study for this unique kinase.     

Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii

The intracellular protozoan parasite Toxoplasma gondii shares with other members of the Apicomplexa a common set of apical structures involved in host cell invasion. Micronemes are apical secretory organelles releasing their contents upon contact with host cells. We have identified a transmembrane micronemal protein MIC6, which functions as an escorter for the accurate targeting of two soluble proteins MIC1 and MIC4 to the micronemes. Disruption of MIC1, MIC4, and MIC6 genes allowed us to precisely dissect their contribution in sorting processes. We have mapped domains on these proteins that determine complex formation and targeting to the organelle. MIC6 carries a sorting signal(s) in its cytoplasmic tail whereas its association with MIC1 involves a lumenal EGF-like domain. MIC4 binds directly to MIC1 and behaves as a passive cargo molecule. In contrast, MIC1 is linked to a quality control system and is absolutely required for the complex to leave the early compartments of the secretory pathway. MIC1 and MIC4 bind to host cells, and the existence of such a complex provides a plausible mechanism explaining how soluble adhesins act. We hypothesize that during invasion, MIC6 along with adhesins establishes a bridge between the host cell and the parasite.     

Molecular and biological characterization of the CIBMUQ/HDC strain, a reference strain for Colombian Toxoplasma gondii

There are few reports about characterization strains of Toxoplasma gondii that analyze the differences between isolates from Europe or United States with those obtained in South America. The current study analyzes virulence data from the mouse model, the gene SAG2 polymorphism by PCR-RFLP and microsatellite analysis in a single Colombian isolate. The strain was isolated from blood of a child with congenital toxoplasmosis, living in Armenia, Colombia. Analysis of virulence in the mouse showed that this strain has an LD100 of 10 tachyzoites. Both methods of genetic characterization demonstrated that this strain belonged to the clonal type 1 and was called HOM/CTCO/2002/CIBMUQ/BL/HDC (brief name: CIBMUQ/HDC). The CIBMUQ/HDC strain is the first Colombian strain available as a reference strain for national and international researchers.     

Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.     

Attenuation of mouse-virulent Toxoplasma gondii parasites is associated with a decrease in interleukin-12-inducing tachyzoite activity and reduced expression of actin, catalase and excretory proteins

Determinants of Toxoplasma gondii virulence are still unknown, although genetic markers associated with T. gondii pathogenicity or host susceptibility to infection have been identified. To define indicator proteins of mouse virulence, type I strain parasites were attenuated by continuous passage in fibroblast culture and compared with the parental strain passaged in mice. The loss of acute virulence, evident by a 1000-fold higher pathogen dose causing 100% lethality in mice correlated with a less efficient infection of inflammatory cells at the site of inoculation, while parasite proliferation and invasiveness in vitro proved unimpaired. Infection with the attenuated parasites elicited earlier local interleukin-12 and strong interferon-gamma responses in vivo, although the activity that triggers interleukin-12 secretion in macrophages is reduced in the attenuated compared to the virulent strain variant. The interleukin-12-inducing T. gondii stimulus was identified as a protein(s) present in tachyzoite excretory products. Comparative proteome analysis combined with immunodetection and quantitation of a variety of T. gondii antigens indicated that the steady-state levels of actin, catalase, microneme protein 5, as well as dense granule proteins 1, 2, 3, 4, 5, 7, 8 and nucleoside triphosphate hydrolase 1 are decreased in the attenuated phenotype, whereas the surface antigen 1 and rhoptry protein 1 are produced at a similar level by virulent and attenuated parasites. In conclusion, these findings reveal a correlation between the efficient establishment of T. gondii infection in vivo and parasite synthesis of actin, catalase and several excretory proteins, and thus postulate a role for these molecules in acute virulence.     

The ARP2/3 complex: an actin nucleator comes of age

The cellular functions of the actin cytoskeleton require precise regulation of both the initiation of actin polymerization and the organization of the resulting filaments. The actin-related protein-2/3 (ARP2/3) complex is a central player in this regulation. A decade of study has begun to shed light on the molecular mechanisms by which this powerful machine controls the polymerization, organization and recycling of actin-filament networks, both in vitro and in the living cell.     

Dynamics of the formin for3p in actin cable assembly

BACKGROUND: Formins are a conserved family of actin nucleators responsible for the assembly of diverse actin structures such as cytokinetic rings and filopodia. In the fission yeast Schizosaccharomyces pombe, the formin for3p is necessary for the formation of actin cables, which are bundles of short parallel actin filaments that regulate cell polarity. These filaments are largely organized with their barbed ends facing the cell tip, where for3p is thought to function in their assembly. RESULTS: Here, using a functional for3p-3GFP fusion expressed at endogenous levels, we find that for3p localizes to small dots that appear transiently at cell tips and then move away on actin cables at a rate of 0.3 microm/s. These movements were dependent on the continuous assembly of actin in cables, on the ability of for3p to bind actin within its FH2 domain, and on profilin and bud6p, two formin binding proteins that promote formin activity. Bud6p transiently colocalizes with for3p at the cell tip and stays behind at the cell tip when for3p detaches. CONCLUSIONS: These findings suggest a new model for actin cable assembly: a for3p particle is activated and promotes the assembly of a short actin filament at the cell tip for only seconds. For3p and the actin filament may then be released from the cell tip and carried passively into the cell interior by retrograde flow of actin filaments in the cable. These studies reveal a complex and dynamic cycle of formin regulation and actin cable assembly in vivo.     

Deficiency of mDia, an actin nucleator, disrupts integrity of neuroepithelium and causes periventricular dysplasia

During development of the central nervous system, the apical-basal polarity of neuroepithelial cells is critical for homeostasis of proliferation and differentiation of neural stem cells. While adherens junctions at the apical surface of neuroepithelial cells are important for maintaining the polarity, the molecular mechanism regulating integrity of these adherens junctions remains largely unknown. Given the importance of actin cytoskeleton in adherens junctions, we have analyzed the role of mDia, an actin nucleator and a Rho effector, in the integrity of the apical adherens junction. Here we show that mDia1 and mDia3 are expressed in the developing brain, and that mDia3 is concentrated in the apical surface of neuroepithelium. Mice deficient in both mDia1 and mDia3 develop periventricular dysplastic mass widespread throughout the developing brain, where neuroepithelial cell polarity is impaired with attenuated apical actin belts and loss of apical adherens junctions. In addition, electron microscopic analysis revealed abnormal shrinkage and apical membrane bulging of neuroepithelial cells in the remaining areas. Furthermore, perturbation of Rho, but not that of ROCK, causes loss of the apical actin belt and adherens junctions similarly to mDia-deficient mice. These results suggest that actin cytoskeleton regulated by Rho-mDia pathway is critical for the integrity of the adherens junctions and the polarity of neuroepithelial cells, and that loss of this signaling induces aberrant, ectopic proliferation and differentiation of neural stem cells.     

Effect of extracellular ions on motility and cell entry in Toxoplasma gondii

Toxoplasma gondii tachyzoites were quiescent in mouse peritoneal fluid or in K2SO4 buffer at pH 8.2. They became consistently motile when K+ was replaced by other monovalent or divalent cations at a constant pH (pH = 8.2). They also became motile when Cl- was substituted for SO4(2-). Nitrate or SCN-, can also be substituted for Cl- to a certain extent. Tachyzoites showed independent movement for more than 15 min in KCl, and for about 5 min in the other buffers at pH 8.2 after which they were exhausted and stopped. These tachyzoites could not then be further stimulated to motility by renewal of the suspension buffer. Infection of monolayer cells was demonstrated only with parasites which were motile during inoculation. The highest infectivity was thus obtained either with freshly collected tachyzoites or with those preincubated in K2SO4 buffer for 30 min at 37 degrees C at alkaline pH and thus not yet exhausted for motility. Approximately 34 to 38% of these latter organisms were seen to enter cells when they were inoculated into cultures immediately after being resuspended in MEM for 30 min at 37 degrees C. Conversely, those whose motility had been exhausted by the preincubation in buffers other than K2SO4, pH 8.2 could not enter monolayer cells. Additionally, parasites were unable to enter cells when inoculated into cultures in K2SO4 buffer at alkaline pH; instead they remained quiescent on the surface of the monolayer cells, suggesting that Toxoplasma enters the host cells by active invasion.     

Identification and purification of actin from the subpellicular network of Toxoplasma gondii tachyzoites

Toxoplasma gondii infects cells through dynamic events dependent on actin. Although the presence of cortical actin has been widely suggested, visualisation and localisation of actin filaments has not been reported. The subpellicular cytoskeleton network is a recently described structure possibly involved in the dynamic events. Using non-ionic detergent extractions, the cortical cytoskeleton network was enriched and used for the isolation and identification of actin. Actin was detected by Western blots in extracts of cytoskeleton networks, and it was localised by gold staining in the network and in both the apical end and the posterior polar ring. Actin was isolated from subpellicular cytoskeleton extracts by binding to DNase I, and it polymerised in vitro as filaments that were gold-decorated by a monoclonal anti-actin antibody. Filaments bound the subfragment 1 of heavy meromyosin, although with atypical arrangements in comparison with the arrowheads observed in muscle actin filaments. Treatment with cytochalasin D and colchicine altered the structural organisation of the subpellicular network indicating the participation of actin filaments and microtubules in the maintenance of its structure. Actin filaments and microtubules, in the subpellicular network, participate reciprocally in the maintaining of the parasite's shape and the gliding motility.     

Laboratory passage and characterization of an isolate of Toxoplasma gondii from an ocular patient in Korea

Toxoplasma gondii tachyzoites were isolated from the blood of an ocular patient, and have been successfully passaged in the laboratory, for over a year, by peritoneal inoculation in mice. The isolated parasite was designated the Korean Isolate-1 (KI-1) and its characteristics were compared with those of the RH strain, a wellknown virulent strain originating from a child who suffered from encephalitis. The morphology, pathogenicity, infectivity and cell culture characteristics of the KI-1 were similar to those of the RH strain. Both RH and KI-1 antigens were detected by an anti-T. gondii monoclonal antibody (mAb), Tg563, against the major surface protein SAG1 (30 kDa), whereas no reaction was observed against an anti-Neospora caninum mAb, 12B4. The KI-1 was confirmed as an isolate of T. gondii. A long-term laboratory maintenance and characterization of a local T. gondii isolate is reported for the first time in the Republic of Korea.     

Effects of extracellular potassium on acid release and motility initiation in Toxoplasma gondii

The internal pH (pHi) of Toxoplasma gondii was estimated by measuring the accumulation of the weak base 9-aminoacridine in buffers with various ionic compositions. The pHi of the metabolizing parasite increased when the extracellular K+ was elevated in alkaline medium or when the external pH (pHe) was substantially increased in medium employing high external K+ (90 mM). The parasite in mouse peritoneal fluid, or in potassium sulfate buffer (pH 8.2), where the pHi was demonstrated to be increased to 7.9, became motile when acidic buffer was substituted for the original suspension medium. This acid-induced independent movement subsided within 5 min but was repeatedly induced if the pHe was serially lowered to 6.0. Basic buffers, on the other hand, abolished motility when applied to the moving parasites. Nigericin, which is known to collapse pH gradients across the membrane, also abolished motility.     

Expression,characterization and inhibition of Toxoplasma gondii 1-deoxy-d-xylulose-5-phosphate reductoisomerase

The apicomplexan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, is an important human pathogen. 1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) in the non-mevalonate isoprene biosynthesis pathway is essential to the organism and therefore a target for developing anti-toxoplasmosis drugs. In order to find potent inhibitors, we expressed and purified recombinant T. gondii DXR (TgDXR). Biochemical properties of this enzyme were characterized and an enzyme activity/inhibition assay was developed. A collection of 11 compounds with a broad structural diversity were tested against TgDXR and several potent inhibitors were identified with K_i values as low as 48 nM. Analysis of the results as well as those of Escherichia coli and Plasmodium falciparum DXR enzymes revealed a different structure–activity relationship profile for the inhibition of TgDXR.