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At least 991 records · Page 55Linked to original sources
PubMed2004

Ionophore-resistant mutant of Toxoplasma gondii reveals involvement of a sodium/hydrogen exchanger in calcium regulation.

Calcium is a critical mediator of many intracellular processes in eukaryotic cells. In the obligate intracellular parasite Toxoplasma gondii, for example, a rise in [Ca2+] is associated with significant morphological changes and rapid egress from host cells. To understand the mechanisms behind such dramatic effects, we isolated a mutant that is altered in its responses to the Ca2+ ionophore A23187 and found the affected gene encodes a homologue of Na+/H+ exchangers (NHEs) located on the parasite's plasma membrane. We show that in the absence of TgNHE1, Toxoplasma is resistant to ionophore-induced egress and extracellular death and amiloride-induced proton efflux inhibition. In addition, the mutant has increased levels of intracellular Ca2+, which explains its decreased sensitivity to A23187. These results provide direct genetic evidence of a role for NHE1 in Ca2+ homeostasis and important insight into how this ubiquitous pathogen senses and responds to changes in its environment.

Amiloride↗
PubMed2004

Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii.

Apicomplexan parasites exhibit a unique form of substrate-dependent motility, gliding motility, which is essential during their invasion of host cells and during their spread between host cells. This process is dependent on actin filaments and myosin that are both located between the plasma membrane and two underlying membranes of the inner membrane complex. We have identified a protein complex in the apicomplexan parasite Toxoplasma gondii that contains the class XIV myosin required for gliding motility, TgMyoA, its associated light chain, TgMLC1, and two novel proteins, TgGAP45 and TgGAP50. We have localized this complex to the inner membrane complex of Toxoplasma, where it is anchored in the membrane by TgGAP50, an integral membrane glycoprotein. Assembly of the protein complex is spatially controlled and occurs in two stages. These results provide the first molecular description of an integral membrane protein as a specific receptor for a myosin motor, and further our understanding of the motile apparatus underlying gliding motility in apicomplexan parasites.

Amino Acid Sequence↗
PubMed1999

Invasion by Toxoplasma gondii establishes a moving junction that selectively excludes host cell plasma membrane proteins on the basis of their membrane anchoring.

The protozoan parasite Toxoplasma gondii actively penetrates its host cell by squeezing through a moving junction that forms between the host cell plasma membrane and the parasite. During invasion, this junction selectively controls internalization of host cell plasma membrane components into the parasite-containing vacuole. Membrane lipids flowed past the junction, as shown by the presence of the glycosphingolipid G(M1) and the cationic lipid label 1. 1'-dihexadecyl-3-3'-3-3'-tetramethylindocarbocyanine (DiIC(16)). Glycosylphosphatidylinositol (GPI)-anchored surface proteins, such as Sca-1 and CD55, were also readily incorporated into the parasitophorous vacuole (PV). In contrast, host cell transmembrane proteins, including CD44, Na(+)/K(+) ATPase, and beta1-integrin, were excluded from the vacuole. To eliminate potential differences in sorting due to the extracellular domains, parasite invasion was examined in host cells transfected with recombinant forms of intercellular adhesion molecule 1 (ICAM-1, CD54) that differed in their mechanism of membrane anchoring. Wild-type ICAM-1, which contains a transmembrane domain, was excluded from the PV, whereas both GPI-anchored ICAM-1 and a mutant of ICAM-1 missing the cytoplasmic tail (ICAM-1-Cyt(-)) were readily incorporated into the PV membrane. Our results demonstrate that during host cell invasion, Toxoplasma selectively excludes host cell transmembrane proteins at the moving junction by a mechanism that depends on their anchoring in the membrane, thereby creating a nonfusigenic compartment.

Animals↗
PubMed2001

Antibodies to Toxoplasma gondii in individuals with first-episode schizophrenia.

We employed enzyme immunoassay (EIA) and Western blotting techniques to measure the level of antibodies to Toxoplasma gondii proteins in serum samples from 38 individuals undergoing their first episode of schizophrenia and from a group of matched control subjects. We found that the individuals with first-episode schizophrenia had significantly increased levels of IgG, IgM, and IgA class antibodies to Toxoplasma proteins, as compared with the control subjects.

Adult↗
PubMed2003

Serotyping of Toxoplasma gondii infections in humans using synthetic peptides.

To determine whether the characteristics of disease due to Toxoplasma gondii (toxoplasmosis) are dependent on the infecting strain, we have developed an enzyme-linked immunosorbent assay for typing strains that uses infection serum reacted against polymorphic peptides derived from Toxoplasma antigens SAG2A, GRA3, GRA6, and GRA7. Pilot studies with infected mice established the validity of the approach, which was then tested with human serum. In 8 patients who had Sabin-Feldman dye test titers >64 and for whom the infecting strain type was known, the peptides correctly distinguished type II from non-type II infections. ELISA analysis of a second group of 10 infected pregnant women from whom the parasite strain had not been isolated gave a clear prediction of the strain type causing infection. This method should allow statistically significant data to be obtained about whether different strain types cause disease with different characteristics.

Alleles↗
PubMed2002

Serologic survey of Toxoplasma gondii in noncarnivorous free-ranging neotropical mammals in French Guiana.

The aim of this study was to determine the frequency of Toxoplasma infection in wild mammals, particularly noncarnivorous species, and to identify possible sources of infection for humans. A serological study was conducted that included 310 animals representing 10 species of terrestrial mammals from the same region (primary tropical rain forest inhabited by humans). Toxoplasmosis was diagnosed using a direct agglutination test using formalin-treated Toxoplasma gondii. The prevalence of antibodies for toxoplasmosis differed greatly between species and ranged from 0% to 62%. The seroprevalence of toxoplasmosis was zero or very low for tree-dwelling animals, which included sloth, porcupine, and howler monkey. Conversely, the prevalence of infection was 46-62% among granivores, insectivores, and burrowing animals, such as peccary, paca, and armadillo. We conclude that wild mammals in French Guiana constitute a reservoir of T. gondii parasites. Given the absence of domestic cats from the forest, noncarnivorous species may be infected by the ingestion of oocysts that are eliminated into the soil or water in the stools of wild cats and then dispersed.

Agglutination Tests↗
PubMed1994

Human indoleamine 2,3-dioxygenase inhibits Toxoplasma gondii growth in fibroblast cells.

Interferon-gamma (IFN-gamma) is known to inhibit the growth of Toxoplasma gondii both in vivo and in vitro. The IFN-gamma induced anti-toxoplasma activity in human cells is strongly correlated with the degradation of the essential amino acid L-tryptophan in vitro. Destruction of L-tryptophan is due to an increased activity of indoleamine 2,3-dioxygenase (IDO), which is transcriptionally activated by IFN-gamma. To determine if indoleamine 2,3-dioxygenase alone is sufficient to block the T. gondii growth, we transfected human fibroblast cells with an IDO cDNA expression plasmid using a metallothionein-inducible promoter. We showed that IDO mRNA and its enzymatic activity are inducible in fibroblast cells transfected with right-orientation IDO cDNA upon addition of CdCl2 to culture medium. The elevated IDO enzyme activity is strongly correlated with an inhibition of T. gondii growth. No IDO mRNA nor enzyme activity is induced by CdCl2 in reverse orientation transfected cells, and no adverse effects were observed on T. gondii growth in cells transfected with the reverse IDO-construct or in control parent cells with or without supplementation of CdCl2. Our observations along with the recent report by Habara-Ohkubo et al. (Infect. Immun. 61, 1810-1813, 1993) suggest that IFN-gamma-induced antitoxoplasma activity is due at least in part to the activation of IDO gene.

Animals↗
PubMed1999

Transmembrane insertion of the Toxoplasma gondii GRA5 protein occurs after soluble secretion into the host cell.

The intracellular parasite Toxoplasma gondii resides within a specialized compartment, the parasitophorous vacuole (PV), that resists fusion with host cell endocytic and lysosomal compartments. The PV is extensively modified by secretion of parasite proteins, including the dense granule protein GRA5 that is specifically targeted to the delimiting membrane of the PV (PVM). We show here that GRA5 is present both in a soluble form and in hydrophobic aggregates. GRA5 is secreted as a soluble form into the PV after which it becomes stably associated with the PVM. Topological studies demonstrated that GRA5 was inserted into the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its C terminus in the vacuole lumen. Deletion of 8 of the 18 hydrophobic amino acids of the single predicted transmembrane domain resulted in the failure of GRA5 to associate with the PVM; yet it remained correctly packaged in the dense granules and was secreted as a soluble protein into the PV. Collectively, these studies demonstrate that the secretory pathway in Toxoplasma is unusual in two regards; it allows soluble export of proteins containing typical transmembrane domains and provides a mechanism for their insertion into a host cell membrane after secretion from the parasite.

3T3 Cells↗
PubMed2005

Unusual kinetic and structural properties control rapid assembly and turnover of actin in the parasite Toxoplasma gondii.

Toxoplasma is a protozoan parasite in the phylum Apicomplexa, which contains a number of medically important parasites that rely on a highly unusual form of motility termed gliding to actively penetrate their host cells. Parasite actin filaments regulate gliding motility, yet paradoxically filamentous actin is rarely detected in these parasites. To investigate the kinetics of this unusual parasite actin, we expressed TgACT1 in baculovirus and purified it to homogeneity. Biochemical analysis showed that Toxoplasma actin (TgACT1) rapidly polymerized into filaments at a critical concentration that was 3-4-fold lower than conventional actins, yet it failed to copolymerize with mammalian actin. Electron microscopic analysis revealed that TgACT1 filaments were 10 times shorter and less stable than rabbit actin. Phylogenetic comparison of actins revealed a limited number of apicomplexan-specific residues that likely govern the unusual behavior of parasite actin. Molecular modeling identified several key alterations that affect interactions between monomers and that are predicted to destabilize filaments. Our findings suggest that conserved molecular differences in parasite actin favor rapid cycles of assembly and disassembly that govern the unusual form of gliding motility utilized by apicomplexans.

Actin Cytoskeleton↗
PubMed2001

Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole.

Rapid discharge of secretory organelles called rhoptries is tightly coupled with host cell entry by the protozoan parasite Toxoplasma gondii. Rhoptry contents were deposited in clusters of vesicles within the host cell cytosol and within the parasitophorous vacuole. To examine the fate of these rhoptry-derived secretory vesicles, we utilized cytochalasin D to prevent invasion, leading to accumulation of protein-rich vesicles in the host cell cytosol. These vesicles lack an internal parasite and are hence termed evacuoles. Like the mature parasite-containing vacuole, evacuoles became intimately associated with host cell mitochondria and endoplasmic reticulum, while remaining completely resistant to fusion with host cell endosomes and lysosomes. In contrast, evacuoles were recruited to pre-existing, parasite-containing vacuoles and were capable of fusing and delivering their contents to these compartments. Our findings indicate that a two-step process involving direct rhoptry secretion into the host cell cytoplasm followed by incorporation into the vacuole generates the parasitophorous vacuole occupied by TOXOPLASMA: The characteristic properties of the mature vacuole are likely to be determined by this early delivery of rhoptry components.

Animals↗
PubMed2002

Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor.

Successful host cell invasion is a prerequisite for survival of the obligate intracellular apicomplexan parasites and establishment of infection. Toxoplasma gondii penetrates host cells by an active process involving its own actomyosin system and which is distinct from induced phagocytosis. Toxoplasma gondii myosin A (TgMyoA) is presumed to achieve power gliding motion and host cell penetration by the capping of apically released adhesins towards the rear of the parasite. We report here an extensive biochemical characterization of the functional TgMyoA motor complex. TgMyoA is anchored at the plasma membrane and binds a novel type of myosin light chain (TgMLC1). Despite some unusual features, the kinetic and mechanical properties of TgMyoA are unexpectedly similar to those of fast skeletal muscle myosins. Microneedle-laser trap and sliding velocity assays established that TgMyoA moves in unitary steps of 5.3 nm with a velocity of 5.2 microm/s towards the plus end of actin filaments. TgMyoA is the first fast, single-headed myosin and fulfils all the requirements for power parasite gliding.

Amino Acid Sequence↗
PubMed2003

Rapid invasion of host cells by Toxoplasma requires secretion of the MIC2-M2AP adhesive protein complex.

Vertebrate cells are highly susceptible to infection by obligate intracellular parasites such as Toxoplasma gondii, yet the mechanism by which these microbes breach the confines of their target cell is poorly understood. While it is thought that Toxoplasma actively invades by secreting adhesive proteins from internal organelles called micronemes, no genetic evidence is available to support this contention. Here, we report successful disruption of M2AP, a microneme protein tightly associated with an adhesive protein called MIC2. M2AP knockout parasites were >80% impaired in host cell entry. This invasion defect was likely due to defective expression of MIC2, which partially accumulated in the parasite endoplasmic reticulum and Golgi. M2AP knockout parasites were also unable to rapidly secrete MIC2, an event that normally accompanies parasite attachment to a target cell. These findings indicate a critical role for the MIC2-M2AP protein complex in parasite invasion.

Animals↗
PubMed1975

Detection of circulating antigen in acute experimental infections with Toxoplasma gondii.

Although many serologic tests are available for determination of antibody to Toxoplasma gondii, circulating antigen has not been studied in infections with this organism. Presence of circulating antigen was sought by immunologic methods in experimental infections (Rh strain) of mice and rabbits. In mice, which succumbed to infection within four days; circulating antigen was detectable in serum by counter-current immunoelectrophoresis and agar gel diffusion on days 2-4 of infection. In rabbits; which succumbed to infection within eight days; serum antigen could not be detected by countercurrent immunoelectrophoresis or agar gel diffusion; Affinity chromatography, with use of cyanogen bromide-activated Sepharose and binding of the antigen to hyperimmune rabbit antiserum to Toxoplasma, permitted isolation of serum antigen on days 3, 5, 7, and 8 of infectionmalthough infected micce and rabbits may have parasitemia of 10-2--10-4 organisms/ml of blood, this concentration did not produce precipitin reactions with antiserum that detected antigenemia. Preliminary characterizations of the column-extracted antigen revealed heat inactivation by 56 C for 30 min, complete inactivation by trypsin, and a molecular weight of greater than 100,000 daltons, as determined by chromatography on a Sephadex column.

Acute Disease↗
PubMed1977

Characterization of Toxoplasma gondii from the feces of naturally infected cats.

Feces of 1,000 cats from a humane shelter in Columbus, Ohio, were examined microscopically for oocysts of Toxoplasma gondii and by inoculation into mice. From the first 541 cats examined, oocysts of Toxoplasma were found in the feces of seven cats but in none of the remaining 459 cats. Results of the dye test in these seven cats showed titers of antibody of less than 1:2 in four cats, and of 1:8, 1:6, and 1:32 in the remaining three cats. The pathogenicity and infectivity of oocysts and cysts of all seven strains were compared in mice after oral and intraperitoneal inoculations. Oocysts and cysts were more pathogenic when administered by the oral route than by the intraperitoneal route. The cysts were less pathogenic than the oocysts. Excellent cross-immunity between six of these seven feline strains and the M-7741 strain was deomonstrated in cats by the fact that oocysts were not shed in feces of cats challenged with cysts of homologous or heterologous strains.

Animals↗
PubMed1980

An enzyme-linked immunosorbent assay for detection of IgM antibodies to Toxoplasma gondii: use for diagnosis of acute acquired toxoplasmosis.

In an enzyme-linked immunosorbent assay (ELISA) for detection of IgM antibodies to Toxoplasma gondii, sera from individuals with negative results in the Sabin-Feldman dye test (DT) and sera from individuals with chronic toxoplasma infection all yielded negative results, even when tested undiluted. In contrast, of sera obtained from individuals with recently acquired toxoplasmosis, all sera that gave positive results in both the DT and the IgM-immunofluorescent antibody (IgM-IFA) test and 92.8% of sera that gave negative results in the IgM-IFA test yielded strongly positive results in the IgM-ELISA. Thus, the IgM-ELISA is more sensitive than the IgM-IFA test in the diagnosis of recently acquired infection with T. gondii. Moreover, sera that gave negative results in the DT but that contained either antinuclear antibodies or rheumatoid factor and thus caused false-positive results in the IgM-IFA test all yielded negative results in the IgM-ELISA, probably because serum IgM and IgG fractions were separated during the initial step.

Acute Disease↗
PubMed1986

Analysis of Toxoplasma gondii antigens recognized by human sera obtained before and after acute infection.

The antibody response to Toxoplasma gondii was studied in human serum samples obtained before and during acute infection with T. gondii. Analysis was by the Sabin-Feldman dye test, double-sandwich IgM ELISA, agglutination test, and protein blots of toxoplasma antigens. Seventeen patients were studied: 12 pregnant women, 2 heart transplant recipients, and 3 symptomatic but immunologically normal individuals. Both IgM and IgG protein blots of the first serologically positive serum sample from each patient revealed new bands, as well as intensified staining of bands noted with the serologically negative serum samples. In each patient, bands corresponding to antigens of approximately 4 kilodaltons (kDa; IgM blots) and of approximately 35 kDa (IgG) were the most intensely stained. In one patient, IgM antibodies to the 4-kDa antigen(s) were demonstrable by protein blots before they were demonstrable by IgM ELISA. IgG protein blots of sera from individuals with chronic infection with T. gondii revealed weakly reactive antibodies to both the 4-kDa and 35-kDa antigens; these bands were not demonstrable in the IgM blots.

Adult↗
PubMed1988

In vitro assessment of antimicrobial agents against Toxoplasma gondii.

We have modified a method of quantitating growth of Toxoplasma organisms by measuring incorporation of [3H]uracil into Toxoplasma-infected, differentiated L6E9 rat myocytes and have found that low-dose pyrimethamine (0.1 microgram/ml) and sulfadiazine (25 micrograms/ml) are synergistic. Pyrimethamine at higher concentrations (0.5 and 1.0 micrograms/ml) inhibits uptake to the same degree as the low-dose pyrimethamine-sulfadiazine combination. Spiramycin was effective only at high concentrations (200 micrograms/ml) and with prolonged incubation of greater than 72 h. Clindamycin and several of its analogues, methotrexate and difluoromethylornithine, were all ineffective and showed no additive effect with either pyrimethamine or sulfadiazine. Spirogermanium, an experimental antineoplastic and antiprotozoan agent, was effective only at concentrations close to those toxic to the system. 5-Fluorouracil was effective even at 0.1 microgram/ml. At 0.01 microgram/ml it was synergistic with pyrimethamine (0.1 microgram/ml), and the combination was as effective as high-dose pyrimethamine (1.0 microgram/ml).

Animals↗
PubMed1995

Potential of the polymerase chain reaction in the diagnosis of active Toxoplasma infection by detection of parasite in blood.

Blood samples from 54 patients presenting with acute toxoplasmic lymphadenopathy were tested for the presence of Toxoplasma gondii DNA using a nested polymerase chain reaction (PCR). PCR test results of a single blood sample obtained 2-23 weeks after onset of illness were positive for 19 (35%) of the 54 patients. Nine (53%) of 17 patients were positive by PCR when the initial blood sample was collected within the first 5 weeks of illness. In 7 of the 19 patients found positive, further blood samples were available, and subsequent clearance of T. gondii DNA from the blood was demonstrated. On the basis of positive findings among patients with acute toxoplasmosis and the absence of positive findings among 10 uninfected persons and 43 with past Toxoplasma infection, a positive PCR result appears to be a helpful indicator of active disease. However, since only 53% of patients with lymphadenopathy persisting < or = 5 weeks were positive, a negative PCR result does not exclude recent infection.

Animals↗