Molecular cloning of a rhoptry protein (ROP6) secreted from Toxoplasma gondii

Monoclonal antibody (mAb) Tg786 against Toxoplasma gondii has been found to detect a 42-kDa rhoptry protein (ROP6) which showed protease activity and host cell binding characteristics after secretion. Using the mAb, a colony containing a 3o-UTR was probed in a T. gondii cDNA expression library. A full length cDNA sequence of the rhoptry protein was completed after 5o-RACE, which consisted of 1,908 bp with a 1,443 bp ORF. The deduced amino acid sequence of ROP6 consisted of a polypeptide of 480 amino acids without significant homology to any other known proteins. This sequence contains an amino terminal stop transfer sequence downstream of a short neutral sequence, hydrophilic middle sequence, and hydrophobic carboxy terminus. It is suggested that the ROP6 is inserted into the rhoptry membrane with both N- and C-termini.     

Detection and characterization of excretory/secretory proteins from Toxoplasma gondii by monoclonal antibodies

Excretory/secretory proteins (ESP) from Toxoplasma gondii were analyzed to define the function in the penetration process into host cells. Whole ESP obtained at 37 degrees C were composed of 15 bands with molecular mass of 110, 97, 86, 80, 70, 60, 54, 42, 40, 36, 30, 28, 26, 22, and 19 kDa. Five ESP of 86, 80, 42, 36, and 28 kDa were reacted with monoclonal antibodies (mAb), named as Tg386 (microneme), Tg485 (surface membrane), Tg786 (rhoptry), Tg378, and Tg556 (both dense granules), respectively. The ESP was released by a temperature-dependent/-independent manner and all at once whenever ready to pour out except Tg786. Each ESP was not exhausted within the parasite but the amount was limited. Tg786 was released continuously with increment, whereas Tg378 and Tg556 were ceased to release after 3 and 4 hr. Dense granular Tg378 and Tg556 were released spontaneously and constitutively before the entry into host cells also. The entry of T. gondii was inhibited by all the mAbs differentially. And the parasite deprived of ESP was inhibited to enter exponentially up to 90.1%. It is suggested that ESP play an essential function to provide appropriate environment for the entry of the parasite into host cells.     

Host cell binding of GRA10, a novel, constitutively secreted dense granular protein from Toxoplasma gondii

Monoclonal antibodies (mAbs) against Toxoplasma gondii, Tg378 and Tg556 clones, are specifically observed to localize to the dense granules of tachyzoites by immunofluorescence microscopy. mAb Tg556 is directed against GRA3, a previously described 30kDa dense granular protein. mAb Tg378 is directed against a novel 36kDa dense granular protein, which we refer to as GRA10. These are major proteins in the excretory/secretory proteins from T. gondii before the parasite's entry into host cells, and they are released into the parasitophorous vacuole (PV) during or shortly after invasion to be associated with the PV membrane. GRA10 binds to the membrane of the host cells regardless of its anchorage-dependence or -independence. The cDNA sequence encoding GRA10 was determined by screening a T. gondii cDNA expression library with mAb Tg378. The deduced amino acid sequence of GRA10 consists of a polypeptide of 364 amino acids, and it has no significant homology to any other known proteins. The sequence contains amino terminal signal peptides and two potential transmembrane domains in the middle of sequence that are not near the carboxy terminus. GRA10 has a RGD motif between the two potential transmembrane domains.     

Toxoplasma gondii: the model apicomplexan

Toxoplasma gondii is an obligate intracellular protozoan parasite which is a significant human and veterinary pathogen. Other members of the phylum Apicomplexa are also important pathogens including Plasmodium species (i.e. malaria), Eimeria species, Neospora, Babesia, Theileria and Cryptosporidium. Unlike most of these organisms, T. gondii is readily amenable to genetic manipulation in the laboratory. Cell biology studies are more readily performed in T. gondii due to the high efficiency of transient and stable transfection, the availability of many cell markers, and the relative ease with which the parasite can be studied using advanced microscopic techniques. Thus, for many experimental questions, T. gondii remains the best model system to study the biology of the Apicomplexa. Our understanding of the mechanisms of drug resistance, the biology of the apicoplast, and the process of host cell invasion has been advanced by studies in T. gondii. Heterologous expression of apicomplexan proteins in T. gondii has frequently facilitated further characterisation of proteins that could not be easily studied. Recent studies of Apicomplexa have been complemented by genome sequencing projects that have facilitated discovery of surprising differences in cell biology and metabolism between Apicomplexa. While results in T. gondii will not always be applicable to other Apicomplexa, T. gondii remains an important model system for understanding the biology of apicomplexan parasites.     

Incidence of adult brain cancers is higher in countries where the protozoan parasite Toxoplasma gondii is common

We explored associations between the common protozoan parasite Toxoplasma gondii and brain cancers in human populations. We predicted that T. gondii could increase the risk of brain cancer because it is a long-lived parasite that encysts in the brain, where it provokes inflammation and inhibits apoptosis. We used a medical geography approach based on the national incidence of brain cancers and seroprevalence of T. gondii. We corrected reports of incidence for national gross domestic product because wealth probably increases the ability to detect cancer. We also included gender, cell phone use and latitude as variables in our initial models. Prevalence of T. gondii explained 19 per cent of the residual variance in brain cancer incidence after controlling for the positive effects of gross domestic product and latitude among nations. Infection with T. gondii was associated with a 1.8-fold increase in the risk of brain cancers across the range of T. gondii prevalence in our dataset (4-67%). These results, though correlational, suggest that T. gondii should be investigated further as a possible oncogenic pathogen of humans.     

Pathogenicity of Five Strains of Toxoplasma gondii from Different Animals to Chickens

Toxoplasma gondii is a protozoan parasite with a broad range of intermediate hosts. Chickens as important food-producing animals can also serve as intermediate hosts. To date, experimental studies on the pathogenicity of T. gondii in broiler chickens were rarely reported. The objective of the present study was to compare the pathogenicity of 5 different T. gondii strains (RH, CN, JS, CAT2, and CAT3) from various host species origin in 10-day-old chickens. Each group of chickens was infected intraperitoneally with 5×10⁸, 1×10⁸, 1×10⁷, and 1×10⁶ tachyzoites of the 5 strains, respectively. The negative control group was mockly inoculated with PBS alone. After infection, clinical symptoms and rectal temperatures of all the chickens were checked daily. Dead chickens during acute phage of the infection were checked for T. gondii tachyzoites by microscope, while living cases were checked for T. gondii infection at day 53 post-inoculation (PI) by PCR method. Histopathological sections were used to observe the pathological changes in the dead chickens and the living animals at day 53 PI. No significant differences were found in survival periods, histopathological findings, and clinical symptoms among the chickens infected with the RH, CN, CAT2, and CAT3 strains. Histopathological findings and clinical symptoms of the JS (chicken origin) group were similar to the others. However, average survival times of infected chickens of the JS group inoculated with 5×10⁸ and 1×10⁸ tachyzoites were 30.0 and 188.4 hr, respectively, significantly shorter than those of the other 4 mammalian isolates. Chickens exposed to 10⁸ of T. gondii tachyzoites and higher showed acute signs of toxoplasmosis, and the lesions were relatively more severe than those exposed to lower doses. The results indicated that the pathogenicity of JS strain was comparatively stronger to the chicken, and the pathogenicity was dose-dependent.     

Recent Advances in Toxoplasma gondii Immunotherapeutics

Toxoplasmosis is an opportunistic infection caused by the protozoan parasite Toxoplasma gondii. T. gondii is widespread globally and causes severe diseases in individuals with impaired immune defences as well as congenitally infected infants. The high prevalence rate in some parts of the world such as South America and Africa, coupled with the current drug treatments that trigger hypersensitivity reactions, makes the development of immunotherapeutics intervention a highly important research priority. Immunotherapeutics strategies could either be a vaccine which would confer a pre-emptive immunity to infection, or passive immunization in cases of disease recrudescence or recurrent clinical diseases. As the severity of clinical manifestations is often greater in developing nations, the development of well-tolerated and safe immunotherapeutics becomes not only a scientific pursuit, but a humanitarian enterprise. In the last few years, much progress has been made in vaccine research with new antigens, novel adjuvants, and innovative vaccine delivery such as nanoparticles and antigen encapsulations. A literature search over the past 5 years showed that most experimental studies were focused on DNA vaccination at 52%, followed by protein vaccination which formed 36% of the studies, live attenuated vaccinations at 9%, and heterologous vaccination at 3%; while there were few on passive immunization. Recent progress in studies on vaccination, passive immunization, as well as insights gained from these immunotherapeutics is highlighted in this review.     

Sterculic Acid and Its Analogues Are Potent Inhibitors of Toxoplasma gondii

Toxoplasmosis is a serious disease caused by Toxoplasma gondii, one of the most widespread parasites in the world. Lipid metabolism is important in the intracellular stage of T. gondii. Stearoyl-CoA desaturase (SCD), a key enzyme for the synthesis of unsaturated fatty acid is predicted to exist in T. gondii. Sterculic acid has been shown to specifically inhibit SCD activity. Here, we examined whether sterculic acid and its methyl ester analogues exhibit anti-T. gondii effects in vitro. T. gondii-infected Vero cells were disintegrated at 36 hr because of the propagation and egress of intracellular tachyzoites. All test compounds inhibited tachyzoite propagation and egress, reducing the number of ruptured Vero cells by the parasites. Sterculic acid and the methyl esters also inhibited replication of intracellular tachyzoites in HFF cells. Among the test compounds, sterculic acid showed the most potent activity against T. gondii, with an EC₅₀ value of 36.2 μM, compared with EC₅₀ values of 248-428 μM for the methyl esters. Our study demonstrated that sterculic acid and its analogues are effective in inhibition of T. gondii growth in vitro, suggesting that these compounds or analogues targeting SCD could be effective agents for the treatment of toxoplasmosis.     

Genotype of Toxoplasma gondii from Blood of Stray Cats in Gyeonggi-do, Korea

Genotyping of Toxoplasma gondii has been performed in 23 PCR positive blood samples from stray cats in Korea. We used 2 separate PCR-restriction fragment length polymorphism (RFLP) patterns of SAG2 gene, amplifying the 5''and 3''ends of the locus. The results revealed that all samples belonged to the type I clonal lineage. Although T. gondii organisms were not isolated from the samples, the results of the present study represent that stray cats with T. gondii infection should be seriously concerned in our environment. Adequate and continuous control programs of stray cats are needed to reduce the risk of transmission of T. gondii as a zoonotic infection threatening the public health.     

Seroprevalence of Toxoplasma gondii Infection among HIV/AIDS Patients in Eastern China

Toxoplasmosis, a neglected tropical disease caused by the protozoan parasite Toxoplasma gondii, occurs throughout the world. Human T. gondii infection is asymptomatic in 80% of the population; however, the infection is life-threatening and causes substantial neurologic damage in immunocompromised patients such as HIV-infected persons. The major purpose of this study was to investigate the seroprevalence of T. gondii infection in subjects infected with HIV/AIDS in eastern China. Our findings showed 9.7% prevalence of anti-T. gondii IgG antibody in HIV/AIDS patients, which was higher than in intravenous drug users (2.2%) and healthy controls (4.7%), while no significant difference was observed in the seroprevalence of anti-Toxoplasma IgM antibody among all participants (P>0.05). Among all HIV/AIDS patients, 15 men (7.7%) and 10 women (15.9%) were positive for anti-T. gondii IgG antibody; however, no significant difference was detected in the seroprevalence of anti-Toxoplasma IgG antibody between males and females. The frequency of anti-Toxoplasma IgG antibody was 8.0%, 13.2%, 5.5%, and 0% in patients with normal immune function (CD4⁺ T-lymphocyte count ≥500 cells/ml), immunocompromised patients (cell count ≥200 and <500 cells/ml), severely immunocompromised patients (cell count ≥50 and <200 cells/ml), and advanced AIDS patients, respectively (cell count <50 cells/ml), while only 3 immunocompromised patients were positive for anti-T. gondii IgM antibody. The results indicate a high seroprevalence of T. gondii infection in HIV/AIDS patients in eastern China, and a preventive therapy for toxoplasmosis may be given to HIV/AIDS patients based on CD4⁺ T lymphocyte count.     

Toxoplasma gondii: Ultrastructure study of the entry of tachyzoites into mammalian cells

Toxoplama gondii (Apicomplexa: Coccidia), an obligatory intracellular parasite with a unique capacity to invade virtually all nucleated cell type from warm-blooded vertebrate hosts. Despite the efficiency with which Toxoplasma enters its host cell, it remains unresolved if invasion occurs by direct penetration of the parasite or through phagocytosis. In the present work, electron microscopic study was designed to examine the entry process of Toxoplasma (RH strain) into macrophages and non phagocytic-host cells (Hela cells) and to observe the ultrastructure changes associated with intracellular parasitism. The results showed that both active invasion and phagocytosis were occurred and revealed that invasion is an ordered process that initiates with binding of the parasite at its apical end followed by tight-fitting invagination of the host cell membrane and a prominent constriction in the parasite at the site of penetration. The process ended by the professional parasitophorous vacuole that is distinct at the outset from those formed by phagocytosis in which once Toxoplasma triggered, phagocytic uptake can proceed by capture of the parasite within a loose fitting vacuole formed by localized membrane ruffling. The cytopathic effects of the parasite on macrophages and Hela cells were demonstrated within 5–15 h post-inoculation in the form of degenerative mitochondria, swelling Golgi apparatus and widening of endoplasmic reticulum indicating intracellular oedema. These changes were exaggerated and several cells were found dead after 48–72 h.     

Molecular cloning and characterization of peroxiredoxin from Toxoplasma gondii.

A cDNA of 1.1 kb comprising the gene encoding the peroxiredoxin of Toxoplasma gondii (TgPrx) has been cloned. The open reading frame of 591 bp was translated into a protein of 196 amino acids with a molecular mass of 25 kDa. Conserved 2 cysteine domains of Phe-Val-Cys-Pro and Glu-Val-Cys-Pro indicated TgPrx belonged to 2-Cys Prx families. TgPrx showed the highest homology with that of Arabidopsis thaliana by 53.9% followed by Entamoeba histolytica with 39.5% by the amino acid sequence alignment. Polyclonal antibody against recombinant TgPrx detected 25 kDa band in T. gondii without binding to host cell proteins. TgPrx was located in the cytoplasm of T. gondii extracellularly or intracellularly by immunofluorescence assay. The expression of TgPrx was increased as early as 30 min after the treatment with artemisinin in the intracellular stage, while no changes in those of host Prx I and TgSOD. This result implies that TgPrx may function as an antioxidant protecting the cell from the attack of reactive oxygen intermediates. It is also suggested that TgPrx is a possible target of chemotherapy.     

Immunosuppression with cyclophosphamide favors reinfection with recombinant Toxoplasma gondii strains

The aim of this study was to verify the effect of immunosuppression by cyclophosphamide (Cy) on susceptibility of BALB/c mice subjected to challenge with recombinant strains of Toxoplasma gondii. Animals were prime infected with the D8 (recombinant I/III) or the ME49 (type II) non-virulent strains, weekly immunosuppressed with Cy and challenged with the CH3 or EGS virulent strains (I/III). Parasites recovered from surviving mice were submitted to PCR-RFLP analysis to confirm co-infection. Prime-infection with the D8 strain conferred more protection against challenge with the CH3 and EGS strains when compared with ME49 prime infection. Cy treatment caused significant leukopenia in the infected mice, what probably favors reinfection after challenge. Reinfection was associated with increased levels of IgA. Otherwise, Cy-treated mice presented significantly lower IgA levels after challenge, suggesting involvement of this immunoglobulin on protection against reinfection. In conclusion, BALB/c mice susceptibility to reinfection by T. gondii is related to genetic differences among the strains used for primary and challenge infections. Alteration of the host's immune integrity by Cy probably compromises the protection previously established by primary infection.     

High Toxoplasma gondii Seropositivity among Brain Tumor Patients in Korea

Toxoplasma gondii is an intracellular protozoan that can modulate the environment of the infected host. An unfavorable environment modulated by T. gondii in the brain includes tumor microenvironment. Literature has suggested that T. gondii infection is associated with development of brain tumors. However, in Korea, epidemiological data regarding this correlation have been scarce. In this study, in order to investigate the relationship between T. gondii infection and brain tumor development, we investigated the seroprevalence of T. gondii among 93 confirmed brain tumor patients (various histological types, including meningioma and astrocytoma) in Korea using ELISA. The results revealed that T. gondii seropositivity among brain tumor patients (18.3%) was significantly (P<0.05) higher compared with that of healthy controls (8.6%). The seropositivity of brain tumor patients showed a significant age-tendency, i.e., higher in younger age group, compared with age-matched healthy controls (P<0.05). In conclusion, this study supports the close relationship between T. gondii infection and incidence of brain tumors.     

Interactions between secreted GRA proteins and host cell proteins across the paratitophorous vacuolar membrane in the parasitism of Toxoplasma gondii

Interactions between GRA proteins of dense granules in Toxoplasma gondii and host cell proteins were analyzed by yeast two-hybrid technique. The cMyc-GRA fusion proteins expressed from pGBKT7 plasmid in Y187 yeast were bound to host cell proteins from pGADT7-Rec-HeLa cDNA library transformed to AH109 yeast by mating method. By the selection procedures, a total of 939 colonies of the SD/-AHLT culture, 348 colonies of the X-alpha-gal positive and PCR, 157 colonies of the X-beta-gal assay were chosen for sequencing the cDNA and finally 90 colonies containing ORF were selected to analyze the interactions. GRA proteins interacted with a variety of host cell proteins such as enzymes, structural and functional proteins of organellar proteins of broad spectrum. Several specific bindings of each GRA protein to host proteins were discussed presumptively the role of GRA proteins after secreting into the parasitophorous vacuoles (PV) and the PV membrane in the parasitism of this parasite.     

Toxoplasma gondii in wild and domestic animals from New Caledonia

Samples (serum or meat juice) collected from 205 animals in New Caledonia in April 2009 were tested for antibodies against Toxoplasma gondii by ELISA using the multi-species ID Screen^® Toxoplasmosis Indirect kit (IDVET, Montpellier). Antibodies to T. gondii were detected in 2% (1/49) of the pigs, in 3.3% (1/30) of the cattle, in 13.8% (4/29) of Rusa deers, in 16% (4/25) of the horses, in 32.8% (21/64) of the dogs, and in 50% (4/8) of cats. Statistically, no significant difference was observed between T. gondii seroprevalence and age or sex. No survey on the prevalence of T. gondii in animals has ever been conducted in New Caledonia and this is the first serological evidence of T. gondii in Rusa deer (Cervus timorensis russa). These results indicate an important circulation of T. gondii exists in the animal populations of New Caledonia. In view of humans being exposed, it is advisable to insist on sanitary education and on respect for good hygienic and food practice.     

Seropositivity and Serointensity of Toxoplasma gondii Antibodies and DNA among Patients with Schizophrenia

The aim of this cross sectional case control study was to examine the serofrequency and serointensity of Toxoplasma gondii (Tg) IgG, IgM, and DNA among patients with schizophrenia. A total of 101 patients with schizophrenia and 55 healthy controls from Sungai Buloh Hospital, Selangor, Malaysia and University Malaya Medical Center (UMMC) were included in this study. The diagnosis of schizophrenia was made based on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). The presence of Tg infection was examined using both indirect (ELISA) and direct (quantitative real-time PCR) detection methods by measuring Tg IgG and IgM and DNA, respectively. The serofrequency of Tg IgG antibodies (51.5%, 52/101) and DNA (32.67%, 33/101) among patients with schizophrenia was significantly higher than IgG (18.2%, 10/55) and DNA (3.64%, 2/55) of the controls (IgG, P=0.000, OD=4.8, CI=2.2-10.5; DNA, P=0.000, OD=12.9, CI=2.17-10.51). However, the Tg IgM antibody between patients with schizophrenia and controls was not significant (P>0.005). There was no significant difference (P>0.005) in both serointensity of Tg IgG and DNA between patients with schizophrenia and controls. These findings have further demonstrated the strong association between the active Tg infection and schizophrenia.     

Genotyping of a Korean isolate of Toxoplasma gondii by multilocus PCR-RFLP and microsatellite analysis

Although the Korean isolate KI-1 of Toxoplasma gondii has been considered to be a virulent type I lineage because of its virulent clinical manifestations, its genotype is unclear. In the present study, genotyping of the KI-1 was performed by multilocus PCR-RFLP and microsatellite sequencing. For 9 genetic markers (c22-8, c29-2, L358, PK1, SAG2, SAG3, GRA6, BTUB, and Apico), the KI-1 and RH strains exhibited typical PCR-RFLP patterns identical to the type I strains. DNA sequencing of tandem repeats in 5 microsatellite markers (B17, B18, TUB2, W35, and TgM-A) of the KI-1 also revealed patterns characteristic of the type I. These results provide strong genetic evidence that KI-1 is a type I lineage of T. gondii.     

Resistance to Toxoplasma gondii infection in mice treated with silk protein by enhanced immune responses

This study investigated whether elevated host immune capacity can inhibit T. gondii infection. For this purpose, we used silk protein extracted from Bombyx mori cocoons as a natural supplement to augment immune capacity. After silk protein administration to BALB/c mice for 6 weeks, ratios of T lymphocytes (CD4(+) and CD8(+) T-cells) and splenocyte proliferative capacities in response to Con A or T. gondii lysate antigen (TLA) were increased. Of various cytokines, which regulate immune systems, Th1 cytokines, such as IFN-γ, IL-2, and IL-12, were obviously increased in splenocyte primary cell cultures. Furthermore, the survival of T. gondii (RH strain)-infected mice increased from 2 days to 5 or more days. In a state of immunosuppression induced by methylprednisolone acetate, silk protein-administered mice were resistant to reduction in T-lymphocyte (CD4(+) and CD8(+) T-cells) numbers and the splenocyte proliferative capacity induced by Con A or TLA with a statistical significance. Taken together, our results suggest that silk protein augments immune capacity in mice and the increased cellular immunity by silk protein administration increases host protection against acute T. gondii infection.