Toxoplasma gondii: protection against colonization of the brain and muscles in a rat model

The objective was to test immune protection against the formation of Toxoplasma gondii tissue cysts in rats. It has been previously shown that 50 T. gondii tissue cysts of strain Me49 are not pathogenic for CF-1 mice, whereas 1 T. gondii tissue cyst of strain M-7741, can be lethal for mice 11-13 days after subcutaneous or oral administration. In the present study, ten rats were fed T. gondii oocysts of strain Me49 and after a further 30 days they were each orally challenged with T. gondii oocysts of strain M-7741. Thirty days after this, they were euthanased and brain and muscle samples inoculated subcutaneously or orally dosed, respectively, to mice for bioassay. None of the mice died, whereas all the mice that were inoculated with brain homogenates or were fed muscle samples from four non-immunized rats that had been inoculated with T. gondii oocysts of strain M-7741, died. These results encourage further research towards achieving vaccinal protection against the formation of T. gondii tissue cysts in meat animals and people.     

Microglia and macrophages as innate producers of interferon-gamma in the brain following infection with Toxoplasma gondii

We previously reported the requirement of interferon-gamma (IFN-gamma) expression by cells other than T and natural killer (NK) cells in the brain, in addition to T cells, for prevention of toxoplasmic encephalitis following infection with Toxoplasma gondii. In the present study, we analysed the identity of the IFN-gamma-producing non-T, non-NK cells in the brain using infected athymic nude and SCID mice that lack T cells but express IFN-gamma in their brains. Intracellular staining for IFN-gamma followed by flow cytometry revealed that approximately 45-60% of the cells expressing IFN-gamma in their brains were positive for CD11b or F4/80 on their surfaces. Smaller portions of the cells were positive for pan-NK marker. Further smaller portions were positive for CD11c, and these cells were less than 5% of the IFN-gamma-expressing cells in brains of infected SCID mice. In addition to IFN-gamma proteins, large amounts of mRNA for IFN-gamma were detected in CD11b+ cells purified from brains of infected mice, but it was not the case in the cells obtained from uninfected animals. In infected SCID mice depleted of NK cells by treatment with anti-asialo-GM1 antibody, cells expressing IFN-gamma in their brains were all positive for CD11b, and the IFN-gamma-producing cells were detected in both CD45low and CD45high populations. These results suggest that CD11b+ CD45low microglia and CD11b+ CD45high blood-derived macrophages are the major non-T, non-NK cells which express IFN-gamma in the brain of mice infected with T. gondii.     

Characterisation of hexokinase in Toxoplasma gondii tachyzoites

We have cloned the hexokinase [E.C. 2.7.1.1] gene of Toxoplasma gondii tachyzoite and obtained an active recombinant enzyme with a calculated molecular mass of 51,465Da and an isoelectric point of 5.82. Southern blot analysis indicated that the hexokinase gene existed as a single copy in the tachyzoites of T. gondii. The sequence of T. gondii hexokinase exhibited the highest identity (44%) to that of Plasmodium falciparum hexokinase and lower identity of less than 35% to those of hexokinases from other organisms. The specific activity of the homogeneously purified recombinant enzyme was 4.04 micromol/mg protein/min at 37 degrees C under optimal conditions. The enzyme could use glucose, fructose, and mannose as substrates, though it preferred glucose. Adenosine triphosphate was exclusively the most effective phosphorus donor, and pyrophosphate did not serve as a substrate. K(m) values for glucose and adenosine triphosphate were 8.0+/-0.8 microM and 1.05+/-0.25mM, respectively. No allosteric effect of substrates was observed, and the products, glucose 6-phosphate and adenosine diphosphate, had no inhibitory effect on T. gondii hexokinase activity. Other phosphorylated hexoses, fructose 6-phosphate, trehalose 6-phosphate which is an inhibitor of yeast hexokinase, and pyrophosphate, also did not affect T. gondii hexokinase activity. Native hexokinase activity was recovered in both the cytosol and membrane fractions of the whole lysate of T. gondii tachyzoites. This result suggests that T. gondii hexokinase weakly associates with the membrane or particulate fraction of the tachyzoite cell.     

Toxoplasma gondii and mucosal immunity

Toxoplasma gondii, an intracellular parasite infects the host through the oral route. Infection induces a cascade of immunological events that involve both the components of the innate and adaptative immune responses. Alteration of the homeostatic balance of infected intestine results in an acute inflammatory ileitis in certain strains of inbred mice. Both the infected enterocytes as well as the CD4 T cells from the lamina propria produce chemokines and cytokines that are necessary to clear the parasite whereas CD8 intraepithelial lymphocytes secrete transforming growth factor beta that reduces the inflammation. In this review, we describe the salient features of this complex network of interactions among the different components of the gut-associated lymphoid tissue cell population that are induced after oral infection with T. gondii.     

Toxoplasma gondii: Serological recognition of reinfection

Using murine chronic toxoplasmosis as an experimental model, we examined the utility of immunoenzymatic methods in recognizing reinfection in chronically infected individuals. Primary infection with avirulent Toxoplasma gondii DX strain (genotype II) induced strong immunity protecting the mice from mortality after inoculation with LD(100) of virulent BK strain (genotype I) and triggered highly expressed antibody production, within one new isotype detected by comparative immunoblots. The parasites multiplying at the site of reinfection were of BK origin as found by RAPD-PCR. The results revealed that the immunoblot assay seems to be a useful and reliable method for the monitoring of specific antibody profile in chronically infected individuals. In our opinion ELISA combined with immunoblot could enable the recognition of reinfection cases in humans, but earlier our experimental data should be verified in clinical laboratory.     

Evidence for de novo sphingolipid biosynthesis in Toxoplasma gondii

Glycolipids are important components of cellular membranes involved in various biological functions. In this report, we describe the identification of the de novo synthesis of glycosphingolipids by Toxoplasma gondii tachyzoites. Parasite-specific glycolipids were identified by metabolic labelling of parasites with tritiated serine and galactose. These glycolipids were characterised as sphingolipids based on the labelling protocol and their insensitivity towards alkaline treatment. Synthesis of parasite glycosphingolipids were inhibited by threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol and L-cycloserine, two well-established inhibitors of de novo sphingolipid biosynthesis. The identified glycolipids were insensitive towards treatment with endoglycoceramidase II indicating that they might belong to globo-type glycosphingolipids. Taken together, we provide evidence for the first time that T. gondii is capable of synthesising glycosphingolipids de novo.     

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.     

Toxoplasma gondii partially inhibits nitric oxide production of activated murine macrophages

Activated macrophages produce nitric oxide (NO) and as such are able to control the multiplication of Toxoplasma gondii. Until now, no reports have described a possible modulation of NO production of macrophages after T. gondii infection. To investigate this possibility, murine blood monocyte-derived and peritoneal macrophages were activated in vitro with interferon-gamma and lipopolysaccharide and infected with T. gondii and Trypanosoma cruzi, and NO production was evaluated. NO was produced by monocyte-derived macrophages only if cultured in the presence of macrophage-colony-stimulating factor. Monocyte-derived or peritoneal macrophages infected with T. gondii presented a significant reduction in NO production. NO production inhibition was not detected after T. cruzi infection. Macrophages infected with higher T. gondii/macrophage ratios presented lower NO production. Furthermore, only viable T. gondii could cause partial inhibition of NO production. In macrophages activated 24 h before the interaction, partial inhibition was detected after 3 h of infection and continued for 48 h. In macrophages activated immediately after the interaction, partial inhibition was not detected at 12 h, but was observed at 24 h. T. gondii-infected macrophages present lower inducible nitric oxide synthase expression as assayed by immunofluorescence. T. gondii did not develop in monocyte-derived macrophages producing NO, but were not totally eliminated. These results demonstrate that T. gondii infection partially inhibits NO production by murine macrophages, suggesting that a deactivating macrophage mechanism may be used for better survival into phagocytic cells.     

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.     

Toxoplasma gondii infection in humans and animals in the United States

This paper reviews clinical and asymptomatic Toxoplasma gondii infection in humans and other animals in the USA. Seroprevalence of T. gondii in humans and pigs is declining. Modes of transmission, epidemiology and environmental contamination with oocysts on land and sea are discussed.     

Transmission of Toxoplasma gondii in an urban population of domestic cats (Felis catus)

Toxoplasma gondii is a protozoan parasite that infects humans and animal species worldwide. The relative importance of each potential transmission route in the complex life cycle of this coccidia is largely unknown, due to the lack of studies taking into account all routes simultaneously. In this study, we analyzed the transmission of T. gondii in an urban population of stray cats captured between 1993 and 2004. Analyzing prevalence, our aim was to determine which factors influence transmission in this population. Specific anti-T. gondii IgG antibodies were detected using the modified agglutination test. Firstly, we analyzed the kinetics of antibody titers in cats captured several times, using mixed linear models and correspondence analysis. We showed that antibody titers did not vary significantly with time and that titer 40 was the best threshold to separate individuals into two serological groups. Overall, prevalence was only 18.6%, thus transmission of T. gondii is infrequent in this population. As expected, a highly significant association was detected between age and presence of IgG antibodies. Prevalence was lowest in kittens aged 3-4 months, suggesting that newborn kittens may carry maternal antibodies and that vertical transmission is rare. After taking into account the effect of age, logistic regression showed that antibody carriage was related to factors that possibly related to the survival of oocysts: localization in the study site, origin of the cats, maximal temperatures and rain. Our results suggest that in this population, vertical transmission is rare, low predation limits prevalence, and oocyst survival is a determining factor in the risk of infection. We discuss the more general importance of conditions determining oocyst survival in the life cycle of T. gondii.     

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.     

Toxoplasma gondii: an improved rat model of congenital infection

The objective of this study was to refine the rat model of congenital toxoplasmosis. In Fischer rats we found that visualization of spermatozoa in vaginal exudates and the detection of at least 6 g body weight increase between days 9 and 12 of pregnancy, allowed the diagnosis and timing of pregnancy with 60% specificity and 84% sensitivity. A dose of 10⁴Toxoplasma gondii bradyzoites or 10²T. gondii oocysts of the Prugniaud strain resulted in more than 50% of congenital infection of the rat litters. Transmission of T. gondii via lactation was not detected in rats inoculated with either bradyzoites or oocysts. Bioassays of 51 neonates born from mothers inoculated with bradyzoites (in tissue cysts) and 29 neonates from mothers inoculated with oocysts demonstrated that both liver and lungs can be used for the diagnosis of congenital transmission in this model.     

The immunobiology of the innate response to Toxoplasma gondii

Toxoplasma gondii is a unique intracellular parasite. It can infect a variety of cells in virtually all warm-blooded animals. It has a worldwide distribution and, overall, around one-third of people are seropositive for the parasite, with essentially the entire human population being at risk of infection. For most people, T. gondii causes asymptomatic infection but the parasite can cause serious disease in the immunocompromised and, if contracted for the first time during pregnancy, can cause spontaneous abortion or congenital defects, which have a substantial emotional, social and economic impact. Toxoplasma gondii provokes one of the most potent innate, pro-inflammatory responses of all infectious disease agents. It is also a supreme manipulator of the immune response so that innate immunity to T. gondii is a delicate balance between the parasite and its host involving a coordinated series of cellular interactions involving enterocytes, neutrophils, dendritic cells, macrophages and natural killer cells. Underpinning these interactions is the regulation of complex molecular reactions involving Toll-like receptors, activation of signalling pathways, cytokine production and activation of anti-microbial effector mechanisms including generation of reactive nitrogen and oxygen intermediates.     

Genetic diversity, clonality and sexuality in Toxoplasma gondii

The majority of Toxoplasma gondii strains from a variety of human and animal sources have been grouped into three highly clonal but closely related lineages. The low occurrence of nucleotide differences among the three predominant lineages and their unusual dimorphic allelic composition suggest that they have arisen from a recent common ancestry. Less than 1% of the previously studied strains contain unique genotypes and high divergence of DNA sequence, and therefore are considered 'exotic' or 'atypical' strains. The seemingly low genetic diversity in T. gondii may have been underestimated because most parasite strains in previous studies were collected from human patients and domestic animals in North America and Europe. To investigate the genetic diversity of T. gondii, we analysed parasite strains isolated from remote geographical regions by multilocus microsatellite sequencing and phylogenetic analysis. The genetic diversity indices, the molecular analysis of microsatellite genotypes and the constructed phylogram considered together suggest that the global T. gondii population is highly diversified and not characteristic of a clonal organism. The most parsimonious hypothesis is that T. gondii presents a complex population structure with a mix of clonal and sexual propagation as a function of the environmental conditions. The comparison between domestic strains data on one hand and wild strains data on the other hand is in favour of more frequent sexual recombinations in wild environment even though Toxoplasma subpopulation in human and domestic animals is largely clonal.     

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.     

Pyridinylimidazole p38 mitogen-activated protein kinase inhibitors block intracellular Toxoplasma gondii replication

Toxoplasma gondii is a medically important, obligate intracellular parasite. Little is known regarding factors that regulate its replication within cells. Such knowledge would further understanding of T. gondii pathogenesis, and might lead to novel therapeutic strategies. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes including proliferation and differentiation. We now show that treatment of T. gondii-infected cells with SB203580 or SB202190, substituted pyridinylimidazoles that are potent inhibitors of human p38 MAPK, inhibits intracellular T. gondii replication. Several independent experimental approaches suggest that the anti-proliferative effects of pyridinylimidazoles depend on direct action on tachyzoites, not the host cell: (i) selective inhibition of host p38 MAPK using recombinant adenoviruses had little effect on tachyzoite replication, (ii) pyridinylimidazole-treated tachyzoites developed abnormal morphology suggesting defective parasite division, and (iii) pyridinylimidazole-resistant mutant tachyzoites were developed through culture in progressively higher drug concentrations. We hypothesise that pyridinylimidazoles target a human p38 MAPK homologue in tachyzoites that regulates their replication. Phylogenetic data suggest that T. gondii likely encodes a p38 MAPK homologue, but such a homologue is absent from the incomplete Toxoplasma genomic data base. As all eukaryotic pathogens, including agents of malaria, leishmaniasis and trypanosomiasis encode endogenous MAPKs, drugs inhibiting endogenous MAPK activation may represent a novel, potentially broadly-acting class of anti-parasitic agents. Pyridinylimidazoles also represent tools to elucidate factors governing intracellular tachyzoite replication.     

The small ubiquitin-like modifier (SUMO)-conjugating system of Toxoplasma gondii

SUMOylation, the reversible covalent attachment of small ubiquitin-like modifier (SUMO) peptides has emerged as an important regulator of target protein function. Here we show, by characterization of the Toxoplasma gondii SUMO pathway, that the SUMO conjugation system operates in apicomplexan parasites. A gene encoding the SUMO tag was discovered as were genes encoding the various enzymes required for SUMO processing, ligation and release. Various SUMO conjugates were immuno-detected and by means of a global proteomic-based approach, we identified several T. gondii SUMOylated proteins that reveal many diverse cellular processes in which the modification plays a role. More specifically, SUMO conjugates were seen at the tachyzoite surface in response to signaling generated by host cell contact at the time of invasion. Also, under tissue culture conditions that stimulate bradyzoite differentiation (alkaline pH), we observed the conjugates at the parasitophorous vacuole membrane. The labeling was also at the surface of the mature cysts isolated from parasite-infected mouse brain. Overall, the SUMO conjugation system appears to be a complex and functionally heterogeneous pathway for protein modification in T. gondii with initial data indicating that it is likely to play a putative role in host cell invasion and cyst genesis.