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Calcium-regulated exocytosis is a ubiquitous process in eukaryotes, whereby secretory vesicles fuse with the plasma membrane and release their contents in response to an intracellular calcium surge (1). This process regulates various cellular functions such as plasma membrane repair in plants and animals (2,3), the discharge of defensive spikes in Paramecium (4), and the secretion of insulin from pancreatic cells, immune modulators from lymphocytes, and chemical transmitters from neurons (5). In animal cells, serine/threonine kinases including cAMP-dependent protein kinase, protein kinase C and calmodulin kinases have been implicated in calcium-signal transduction leading to regulated secretion (1,6,7). Although plants and protozoa also regulate secretion by means of intracellular calcium, the method by which these signals are relayed has not been explained. Here we show that the Toxoplasma gondii calcium-dependent protein kinase 1 (TgCDPK1) is an essential regulator of calcium-dependent exocytosis in this opportunistic human pathogen. Conditional suppression of TgCDPK1 revealed that it controls calcium-dependent secretion of specialized organelles called micronemes, resulting in a block of essential phenotypes including parasite motility, host-cell invasion, and egress. These phenotypes were recapitulated by using a chemical biology approach in which pyrazolopyrimidine-derived compounds specifically inhibited TgCDPK1 and disrupted the parasite's life cycle at stages dependent on microneme secretion. Inhibition was specific to TgCDPK1, because expression of a resistant mutant kinase reversed sensitivity to the inhibitor. TgCDPK1 is conserved among apicomplexans and belongs to a family of kinases shared with plants and ciliates (8), suggesting that related CDPKs may have a function in calcium-regulated secretion in other organisms. Because this kinase family is absent from mammalian hosts, it represents a validated target that may be exploitable for chemotherapy against T. gondii and related apicomplexans., The apicomplexan parasite T. gondii has been used as a model for the secretion of numerous proteins from specialized organelles, called micronemes, in response to an increase in intracellular calcium [...]

Toxoplasma gondii, as an obligate intracellular and promiscuous pathogen of mammalian cells, utilizes host sugars for energy and to generate glycoconjugates that are important to its survival and virulence. Here, we report that T. gondii glucose transporter (TgGT1) is proficient in transporting mannose, galactose, and fructose besides glucose, and serves as a major hexose transporter at its plasma membrane. Toxoplasma harbors 3 additional putative sugar transporters (TgST1-3), of which TgST2 is expressed at its surface, whereas TgST1 and TgST3 are intracellular. Surprisingly, TgGT1 and TgST2 are nonessential to the parasite as their ablations inflict only a 30% or no defect in its intracellular growth, respectively. Indeed, Toxoplasma can also tolerate the deletion of both genes while incurring no further growth phenotype. Unlike [DELTA]tgst2, the modest impairment in [DELTA]tggt1 and [DELTA]tggtl/[DELTA]tgst2 mutants is because of a minor delay in their intracellular replication, which is a direct consequence of the abolished import of glucose. The [DELTA]tggt1 displays an attenuated motility in defined minimal media that is rescued by glutamine. TgGT1-complemented parasites show an entirely restored growth, motility, and sugar import. The lack of exogenous glucose in [DELTA]tggt1 culture fails to accentuate its intrinsic growth defect and prompts it to procure glutamine to sustain its metabolism. Unexpectedly, in vivo virulence of [DELTA]tggt1 in mice remains unaffected. Taken together, our data demonstrate that glucose is nonessential for T. gondii tachyzoites, underscore glutamine is a complement substrate, and provide a basis for understanding the adaptation of T. gondii to diverse host cells. glucose transport | glutamine metabolism | genetic manipulation

The processes promoting disease in wild animal populations are highly complex, yet identifying these processes is critically important for conservation when disease is limiting a population. By combining field studies with epidemiologic tools, we evaluated the relationship between key factors impeding southern sea otter (Enhydra lutris nereis) population growth: disease and resource limitation. This threatened population has struggled to recover despite protection, so we followed radio-tagged sea otters and evaluated infection with 2 disease-causing protozoal pathogens, Toxoplasma gondii and Sarcocystis neurona, to reveal risks that increased the likelihood of pathogen exposure. We identified patterns of pathogen infection that are linked to individual animal behavior, prey choice, and habitat use. We detected a high-risk spatial cluster of S. neurona infections in otters with home ranges in southern Monterey Bay and a coastal segment near San Simeon and Cambria where otters had high levels of infection with T. gondii. We found that otters feeding on abalone, which is the preferred prey in a resource-abundant marine ecosystem, had a very low risk of infection with either pathogen, whereas otters consuming small marine snails were more likely to be infected with T. gondii. Individual dietary specialization in sea otters is an adaptive mechanism for coping with limited food resources along central coastal California. High levels of infection with protozoal pathogens may be an adverse consequence of dietary specialization in this threatened species, with both depleted resources and disease working synergistically to limit recovery. Enhydra lutris nereis | Sarcocystis neurona | Toxoplasma gondii | diet specialization | wildlife disease

Perforin-like proteins are expressed by many bacterial and protozoan pathogens, yet little is known about their function or mode of action. Here, we describe Toxoplasma perforin-like protein 1 (TgPLP1), a secreted perforin-like protein of the intracellular protozoan pathogen Toxoplasma gondii that displays structural features necessary for pore formation. After intracellular growth, TgPLP1-deficient parasites failed to exit normally, resulting in entrapment within host cells. We show that this defect is due to an inability to rapidly permeabilize the parasitophorous vacuole membrane and host plasma membrane during exit. TgPLP1 ablation had little effect on growth in culture but resulted in a reduction greater than five orders of magnitude of acute virulence in mice. Perforin-like proteins from other intracellular pathogens may play a similar role in microbial egress and virulence.

The protozoan parasite Toxoplasma gondii blocks the innate aversion of rats for cat urine, instead producing an attraction to the pheromone; this may increase the likelihood of a cat predating a rat. This is thought to reflect adaptive, behavioral manipulation by Toxoplasma in that the parasite, although capable of infecting rats, reproduces sexually only in the gut of the cat. The 'behavioral manipulation' hypothesis postulates that a parasite will specifically manipulate host behaviors essential for enhancing its own transmission. However, the neural circuits implicated in innate fear, anxiety, and learned fear all overlap considerably, raising the possibility that Toxoplasma may disrupt all of these nonspecifically. We investigated these conflicting predictions. In mice and rats, latent Toxoplasma infection converted the aversion to feline odors into attraction. Such loss of fear is remarkably specific, because infection did not diminish learned fear, anxiety-like behavior, olfaction, or nonaversive learning. These effects are associated with a tendency for parasite cysts to be more abundant in amygdalar structures than those found in other regions of the brain. By closely examining other types of behavioral patterns that were predicted to be altered we show that the behavioral effect of chronic Toxoplasma infection is highly specific. Overall, this study provides a strong argument in support of the behavioral manipulation hypothesis. Proximate mechanisms of such behavioral manipulations remain unknown, although a subtle tropism on part of the parasite remains a potent possibility. behavioral manipulation | fear | parasites | predator

The lead enzymes of polyamine biosynthesis, i.e. ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), were not detected in Toxoplasma gondii [the limit of detection for ODC and ADC was 5 pmol [min.sup.-1] [(mg protein).sup.-1]], indicating that T. gondii lacks a forward-directed polyamine biosynthetic pathway, and is therefore a polyamine auxotroph. The biochemical results were supported by results obtained from data-mining the T. gondii genome. However, it was possible to demonstrate the presence of a highly active backconversion pathway that formed spermidine from spermine, and putrescine from spermidine, via the combined action of spermidine/ spermine [N.sup.1]-acetyltransferase (SSAT) or spermidine [N.sup.1]-acetyltransferase (SAT) and polyamine oxidase (PAO). With spermine as the substrate, T. gondii SSAT had a specific activity of 1.84 nmol [min.sup.-1] [(mg protein).sup.-1], and an apparent [K.sub.m] for spermine of 180 mM; with spermidine as the substrate, the SAT had a specific activity of 3.95 nmol [min.sup.-1] [(mg protein).sup.-1], and a [K.sub.m] for spermidine of 240 mM. T. gondii PAO had a specific activity of 10.6 nmol [min.sup.-1] [(mg protein).sup.-1], and a [K.sub.m] for acetylspermine of 36 mM. Furthermore, the results demonstrated that T. gondii SSAT was 50 % inhibited by 30 mM di(ethyl)norspermine. The parasite actively transported arginine and ornithine, which were converted via the arginine dihydrolase pathway to citrulline and carbamoyl phosphate, resulting in the formation of ATP via carbamate kinase. The lack of polyamine biosynthesis by T. gondii is contrasted with polyamine metabolism by other apicomplexans.

Biochemical reconstitution and high-resolution crystal structures have shown that rhomboid proteins function in diverse processes including quorum sensing in bacteria, mitochondrial membrane fusion, apoptosis, and stem cell differentiation in eukaryotes. The analysis of these novel enzymes has revealed the evolutionary path of rhomboid protein function, which provides insights into the forces that drive the molecular evolution of regulatory mechanisms.

Toxoplasma gondii is a protozoan parasite that infects nearly all mammal and bird species worldwide. Usually asymptomatic, toxoplasmosis can be severe and even fatal to many hosts, including people. Elucidating the contribution of genetic variation among parasites to patterns of disease transmission and manifestations has been the goal of many studies. Focusing on the geographic component of this variation, we show that most genotypes are locale-specific, but some are found across continents and are closely related to each other, indicating a recent radiation of a pandemic genotype. Furthermore, we show that the geographic structure of T. gondii is extraordinary in having one population that is found in all continents except South America, whereas other populations are generally confined to South America, and yet another population is found worldwide. Our evidence suggests that South American and Eurasian populations have evolved separately until recently, when ships populated by rats, mice, and cats provided T. gondii with unprecedented migration opportunities, probably during the transatlantic slave trade. Our results explain several enigmatic features of the population structure of T. gondii and demonstrate how pervasive, prompt, and elusive the impact of human globalization is on nature. evolutionary history | migration | pandemic genotype | protozoan parasite | trade

Toxoplasma gondii, an obligate intracellular protozoan of the phylum Apicomplexa, is estimated to infect over a billion people worldwide as well as a great many other mammalian and avian hosts. Despite this ubiquity, the vast majority of human infections in Europe and North America are thought to be due to only three genotypes. Using a genome-wide analysis of single-nucleotide polymorphisms, we have constructed a genealogy for these three lines. The data indicate that types I and III are second- and first-generation offspring, respectively, of a cross between a type II strain and one of two ancestral strains. An extant T. gondii strain (P89) appears to be the modern descendant of the non-type II parent of type III, making the full genealogy of the type III clonotype known. The simplicity of this family tree demonstrates that even a single cross can lead to the emergence and dominance of a new clonal genotype that completely alters the population biology of a sexual pathogen. clonal population structure | genetic recombination | virulence

The city of Erechim, Brazil, has a 17% prevalence of ocular toxoplasmosis, and type 1 Toxoplasma gondii predominates. To examine risk factors for acute T. gondii infection in this area, [...]

Limited proteolysis is used to carry out an extensive study of the conformation of Toxoplasma gondii ferrodoxin-NADP(super +) reductase (FNR). It is found that two flexible polypeptide regions of Toxoplasma gondii FNR are involved in ferrodoxin (Fd) interaction but have opposite roles in modulating the binding affinity for the protein ligand.

Water was the suspected vehicle of Toxoplasma gondii dissemination in a toxoplasmosis outbreak in Brazil. A case-control study and geographic mapping of cases were performed. T. gondii was isolated directly [...]

Infection with Toxoplasma gondii can lead to congenital and acquired disease, resulting in loss of vision and neurologic illness. We tested sera collected in the National Health and Examination Survey [...]

Obligate intracellular apicomplexan parasites rely on gliding motion powered by their actomyosin system to disperse throughout tissues and to penetrate host cells. Toxoplasma gondii myosin A has been implicated in this process, but direct proof has been lacking. We designed a genetic screen to generate a tetracycline-inducible transactivator system in T. gondii. The MyoA gene was disrupted in the presence of a second regulatable copy of MyoA. Conditional removal of this myosin caused severe impairment in host cell invasion and parasite spreading in cultured cells, and unambiguously established the pathogenic function of this motor in an animal model., Among the many vital functions of an obligate intracellular parasite, host cell invasion is a prerequisite for survival and replication, and this process is dependent on the ability of T. [...]

Name a single-celled eukaryote that boasts a small genome size, is easily cultivated in haploid form, for which a wide variety of molecular genetic tools are available, and that exhibits a simple, polarized secretory apparatus with a well-defined endoplasmic reticulum and Golgi that can serve as a model for understanding secretion. Got it? Now name a cell with all these attributes that contains at least a dozen distinct and morphologically well-defined intracellular organelles, including three distinct types of secretory vesicles and two endosymbiotic organelles. Not so sure anymore?

Toxoplasma gondii is an obligatory intracellular parasite, an important human pathogen, and a convenient laboratory model for many other human and veterinary pathogens in the phylum Apicomplexa, such as Plasmodium, Eimeria, and Cryptosporidia. 22 subpellicular microtubules form a scaffold that defines the cell shape of T. gondii. Its cytoskeleton also includes an intricate apical structure consisting of the conoid, two intraconoid microtubules, and two polar rings. The conoid is a 380-nm diameter motile organelle, consisting of fibers wound into a spiral like a compressed spring. FRAP analysis of transgenic T. gondii expressing YFP-[alpha]-tubulin reveals that the conoid fibers are assembled by rapid incorporation of tubulin subunits during early, but not late, stages of cell division. Electron microscopic analysis shows that in the mature conoid, tubulin is arranged into a novel polymer form that is quite different from typical microtubules.

Toxoplasma gondii is an intracellular parasite that proliferates within most nucleated cells, an important human pathogen, and a model for the study of human and veterinary parasitic infections. We used a stable yellow fluorescent protein-[alpha]-tubulin transgenic line to determine the structure of the microtubule cytoskeleton in T. gondii. Imaging of living yellow fluorescent protein-[alpha]-tubulin parasites by laser-scanning confocal microscopy (LSCM) failed to resolve the 22 subpellicular microtubules characteristic of the parasite cytoskeleton. To understand this result, we analyzed sources of noise in the LSCM and identified illumination fluctuations on time scales from microseconds to hours that introduce significant amounts of noise. We confirmed that weakly fluorescent structures could not be imaged in LSCM by using fluorescent bead standards. By contrast, wide-field microscopy (WFM) did visualize weak fluorescent standards and the individual microtubules of the parasite cytoskeleton. We therefore measured the fluorescence per unit length of microtubule by using WFM and used this information to estimate the tubulin content of the conoid (a structure important for T. gondii infection) and in the mitotic spindle pole. The conoid contains sufficient tubulin for [approximately equal to] 10 microtubule segments of 0.5-/[micro]m length, indicating that tubulin forms the structural core of the organelle. We also show that the T. gondii mitotic spindle contains [approximately equal to] 1 microtubule per chromosome. This analysis expands the understanding of structures used for invasion and intracellular proliferation by an important human pathogen and shows the advantage of WFM combined with image deconvolution over LSCM for quantitative studies of weakly fluorescent structures in moderately thin living cells.

The mechanism by which the asexual stage of Toxoplasma gondii attaches to, penetrates, and reproduces in host cells is described. This protozoan is widespread and normally only causes severe disease in immunocompromised people.

Most intracellular pathogens avoid lysing their host cells during invasion by wrapping themselves in a vacuolar membrane. This parasitophorous vacuole membrane (PVM) is often retained, serving as a critical transport interface between the parasite and the host cell cytoplasm. To test whether the PVM formed by the parasite Toxoplasma gondii is derived from host cell membrane or from lipids secreted by the parasite, we used time-resolved capacitance measurements and video microscopy to assay host cell surface area during invasion. We observed no significant change in host cell surface area during PVM formation, demonstrating that the PVM consists primarily of invaginated host cell membrane. Pinching off of the PVM from the host cell membrane occurred after an unexpected delay (34-305 sec) and was seen as a 0.219 [+ or -] 0.006 pF drop in capacitance, which corresponds well to the predicted surface area of the entire PVM (30-33 [[[micro]meter].sup.2]). The formation and closure of a fission pore connecting the extracellular medium and the vacuolar space was detected as the PVM pinched off. This final stage of parasite entry was accomplished without any breach in cell membrane integrity.

Catalytically active Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TS) and the individual DHFR and TS regions are expressed in high levels in Escherichia coli. DHFR-TS contribute to 10% of the total soluble cell protein and are purified using methotrexate-Sepharose chromatography. The TS region is overexpressed as soluble proteins when the cells are grown at 24 degrees Celsius. The DHFR domains occur as insoluble bodies which are easily unfolded and refolded to get the active enzyme.

Toxoplasma gondii is an opportunistic intracellular parasite that causes congenital infections and severe complications in a number of vertebrates. A member of a diverse group of obligate protozoan parasites, Toxoplasma invades host cells through a mechanism that relies upon the parasite's actin cytoskeleton for motility. This mechanism was demonstrated using a combination of parasite and host cell mutants characterized by their resistance to cytochalasin. Results indicate that invasion is actively powered by Toxoplasma's actin-based contractile system.

Stable DNA transformation system for the intracellular parasite Toxoplasma gondii was developed to generate a system for molecular genetic evaluation. This system was based on tryptophan auxotrophy complementation which was achieved when the Escherichia coli TrPb gene was expressed. This parasite, which served as an opportunistic pathogen in humans, infected several vertebrate hosts.

Microinjection of polar fluorescent molecules into fibroblasts infected with the obligate intracellular protozoan parasite Toxoplasma gondii confirms its role as a molecular sieve for the parasitophorous vacuole membrane (PVM) surrounding the parasite. The PVM is the special membrane-bonded cytoplasmic compartment formed by the T. gondii, which enters it during mammalian host cell invasion.

The spectrum of lipid mediators released by human platelets after interaction with Toxoplasma gondii was observed. It was revealed that besides thromboxane, the interaction between human platelets and Toxoplasma gondii also released 13- and 19-hydroxyoctadecadienoic acids. The results show that Toxoplasma-stimulated platelets can release oxygenated products from arachidonic and linoleic acid. This fact may prove important in the host response to Toxoplasma gondii infection.

Experiments using peritoneal exudate cells show that heat shock proteins are produced following cell infection with a low-virulence Toxoplasma gondii strain, but are significantly absent when highly-virurlent strains are used. Results show that low-virulence strains have the ability to persist in host macrophages and thus have ample time to induce HSP production in the macrophages they infect. In contrast, highly-virulent strains rapidly induce host immunologic responses andtherefore, have insufficient time to cause HSP generation.

French Guiana is a French territory in South America; the Amazon rain forest covers 92% of the territory. Toxoplasmosis is a cosmopolitan parasitic disease. It is usually benign in patients [...]

Scientists use DNA to track ancient evolutionary lineages and genetic migrations for a range of animals--including modern humans. ARS zoologist Ben Rosenthal is tracing the family tree of one of [...]

Toxoplasmosis is a zoonotic disease in various species of animals caused by a sporozoan parasite, Toxoplasma gondii. The disease occurs throughout the world including Iran. This disease is a common infection of sheep, goats and it is recognized as one of major infection cause of reproductive failure. This survey was carried out to show the prevalence of this disease in cattle in Tabriz (Iran). 490 sera samples were collected from both native and industrial cattle. The Indirect Fluorescent Antibody Test (IFAT) was used to determine the rate of infection. The results of this survey are showed that the overall seroprevalence of antibody of Toxoplasma incattle in Tabriz is 15.91%. There was significant differences in infection to Toxoplasma gondii in the age group of cattle (P1 years old cattle. Also a significant differences was observed between rate of infection and sex in cattle (P Key words: Seroprevalence, toxoplasma, cattle, Tabriz, Iran, INTRODUCTION Toxoplasmosis is a zoonotic disease in various species of animals caused by a sporozoan parasite, Toxoplasma gondii, distributed throughout the world including Iran. Toxoplasma gondii is a heteroxenous coccidian [...]

Toxoplasma gondii, an obligate intracellular parasite, feeds on the host and it is evidenced that the parasite takes host cell endosomes and lysosomes hostage by sequestering them where the parasite resides within invaginations of the parasitophporous vacuole. This parasite can infect most warm-blooded animals and nucleated cells and is responsible for one of the most common parasitic infection in humans.

The global predominance of three donal Toxoplasma gondii lineages suggests that they are endowed with an exceptional trait responsible for their current parasitism of nearly all warm-blooded vertebrates. Genetic polymorphism analyses indicate that these clonal lineages emerged within the last 10,000 years after a single genetic cross. Comparison with ancient strains (~1 million years) suggests that the success of the clonal lineages resulted from the concurrent acquisition of direct oral infectivity. This key adaptation circumvented sexual recombination, simultaneously promoting transmission through successive hosts, hence leading to clonal expansion. Thus, changes in complex life cycles can occur rapidly and can profoundly influence pathogenicity., Toxoplasma gondii is a member of the phylum Apicomplexa, an ancient group of ~5000 species of parasitic protozoa that infect a wide range of vertebrates (1-3). Most closely related members [...]

A laboratory method for detecting Toxoplasma gondii oocysts in drinking water at the Humpback Reservoir in British Columbia was described. The case-control study was conducted in response to a community outbreak of toxoplasmosis in the area. An aeration technique was used to sporulate and render infectious any Toxoplasma gondii oocysts in the sediment. Results showed that the Humpback Reservoir was the source of the Toxoplasma gondii oocysts. It was concluded that contamination of the drinking water occurred during periods of high runoff at the reservoir.