Impact of foetus and mother on IFN-gamma-induced indoleamine 2,3-dioxygenase and inducible nitric oxide synthase expression in murine placenta following Toxoplasma gondii infection

IFN-gamma production is a hallmark of acute infection with the protozoan parasite Toxoplasma gondii. The tryptophan-catabolising enzyme indoleamine 2,3-dioxygenase (IDO), as well as inducible nitric oxide synthase (NOS2) are induced by IFN-gamma and can play extremely diverse roles in immune regulation, defence against pathogens and physiological homeostasis. We investigated the regulation of these two central enzymes in the placenta during acute infection of pregnant female mice. Using IFN-gamma receptor knockout (IFNgammaR-/-) mice, we showed that IDO is not constitutively expressed in term placentas. In contrast, NOS2 expression was observed, largely dependent on IFN-gamma signalling. Upon infection with the avirulent PRU strain of T. gondii, IDO mRNA expression was induced in an IFNgammaR-dependent manner. Surprisingly, NOS2 mRNA was severely suppressed. Importantly, we showed in crossing experiments of heterozygote (IFNgammaR+/-) mothers with IFNgammaR-/- males and vice versa that IDO expression largely depends on the presence of IFN-gamma receptors on foetal cells, and to a lesser extent on maternal cells. Immunohistochemical analysis localised foetal IDO production to invasive trophoblasts within the maternal part of the placenta. The placental vascular endothelium only stained positive when the mothers possessed functional IFN-gamma receptors. In contrast, placental NOS2 expression, but also its suppression following infection, seems to be largely dependent on IFN-gamma signalling in maternal cells. Neither factor appears to regulate placental T. gondii growth, as we observed no difference in parasite numbers between (+/-) and (-/-) foetuses. Taken together, our results demonstrate the crucial role of the foetus in placental IDO, but not NOS2, production following T. gondii infection.     

Proliferation of Toxoplasma gondii in inflammatory macrophages in vivo is associated with diminished oxygen radical production in the host cell

While reactive oxygen species (ROS) can kill Toxoplasma gondii in vitro the role these molecules play in vivo is not known. We used a flow cytometry-based assay to investigate the relationship between intracellular infection and ROS production during acute peritoneal toxoplasmosis in mice. A distinct population of ROS(+) inflammatory macrophages, detected by the oxidation of hydroethidine, was observed to increase progressively in frequency during the course of infection, and to be inversely correlated with the degree of cell parasitization. These data imply that either intracellular parasites inhibit ROS synthesis or, alternatively, ROS-producing cells contain anti-Toxoplasma activity. The latter interpretation was supported by the finding that uninfected ROS-producing inflammatory macrophages were resistant to infection in vivo. However, in the same animals, ROS-producing macrophages that had previously been parasitized could readily be infected with additional parasites, suggesting that the difference in ROS production between highly infected and less infected cells was not due to ROS-associated killing of parasites within these cells. In addition, macrophages infected with T. gondii in vitro and then briefly transferred to acutely infected mice upregulated ROS production in a manner that was again inversely correlated with the degree of intracellular parasitization. Taken together, these findings suggest that both ROS-associated anti-Toxoplasma activity and parasite-driven inhibition of ROS production underlie the observed pattern of ROS production. ROS function and parasite evasion of this function may contribute significantly to the balance between host defense and disease progression during acute infection.     

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.     

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.     

Suppressed production of pro-inflammatory cytokines by LPS-activated macrophages after treatment with Toxoplasma gondii lysate

During Toxoplasma gondii infection, macrophages, dendritic cells, and neutrophils are important sources of pro-inflammatory cytokines from the host. To counteract the pro-inflammatory activities, T. gondii is known to have several mechanisms inducing down-regulation of the host immunity. In the present study, we analyzed the production of proand anti-inflammatory cytokines from a human myelomonocytic cell line, THP-1 cells, in response to treatment with T. gondii lysate or lipopolysaccharide (LPS). Treatment of THP-1 cells with LPS induced production of IL-12, TNF-alpha, IL-8, and IL-10. Co-treatment of THP-1 cells with T. gondii lysate inhibited the LPS-induced IL-12, IL-8 and TNF-alpha expression, but increased the level of IL-10 synergistically. IL-12 and IL-10 production was down-regulated by anti-human toll-like receptor (TLR)-2 and TLR4 antibodies. T. gondii lysate triggered nuclear factor (NF)-kappaB-dependent IL-8 expression in HEK293 cells transfected with TLR2. It is suggested that immunosuppression induced by T. gondii lysate treatment might occur via TLR2-mediated NF-kappaB activation.     

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.     

Toxoplasma gondii inhibits granzyme B-mediated apoptosis by the inhibition of granzyme B function in host cells

Host defense to the apicomplexan parasite Toxoplasma gondii is critically dependent on CD8⁺ T cells, whose effector functions include the induction of apoptosis in target cells following the secretion of granzyme proteases. Here we demonstrate that T. gondii induces resistance of host cells to apoptosis induced by recombinant granzyme B. Granzyme B induction of caspase-independent cytochrome c release was blocked in T. gondii-infected cells. Prevention of apoptosis could not be attributed to altered expression of the Bcl-2 family of apoptotic regulatory proteins, but was instead associated with reduced granzyme B-mediated, caspase-independent cleavage of procaspase 3 to the p20 form in T. gondii-infected cells, as well as reduced granzyme B-mediated cleavage of the artificial granzyme B substrate, GranToxiLux. The reduction in granzyme B proteolytic function in T. gondii-infected cells could not be attributed to altered granzyme B uptake or reduced trafficking of granzyme B to the cytosol, implying a T. gondii-mediated inhibition of granzyme B activity. Apoptosis and GranToxiLux cleavage were similarly inhibited in T. gondii-infected cells exposed to the natural killer-like cell line YT-1. The endogenous granzyme B inhibitor PI-9 was not up-regulated in infected cells. We believe these findings represent the first demonstration of granzyme B inhibition by a cellular pathogen and indicate a new modality for host cell protection by T. gondii that may contribute to parasite immune evasion.     

Toxoplasma gondii: immunogenicity and protection by P30 peptides in a murine model

Vaccines are promising for the control of toxoplasmosis. Here, we evaluated the immunogenicity of 17 peptides derived from SAG1 surface protein of Toxoplasma gondii in CH3 mice. Only 8 of 16 peptides induced specific antibodies. After a lethal challenge, only the vaccination with 4 of 17 peptides that were from the carboxy terminal end of the protein conferred significant survival. Our work shows that vaccination with peptides from the carboxy-terminal positions of SAG1 major surface protein of Toxoplasma protects mice against a lethal challenge.     

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.     

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.     

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.     

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.     

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.     

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.     

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.