Seyha Seng

Expression of SAG-1 of Toxoplasma gondii in transgenic mice.

Abstract We describe the expression of SAG-1 cDNA in B6C3F1 mice by microinjecting a 3.3 kbp DNA fragment, consisting of the cytomegalovirus enhancer-chicken β-actin hybrid promoter and SAG-1 into the pronucleus of a fertilized egg at the one-cell stage. Offspring derived from this microinjection were analyzed for the integration and functional expression of the SAG-1 transgene. Steady-state expressions of both the mRNA for SAG-1 and SAG-1 protein product were detected in the brain, thymus, spleen and liver. Approximately 50% of F1 and F2 progeny inherited the SAG-1 transgene from SAG-1 transgenic mice in Mendelian fashion. These results indicated that SAG-1 transgenic lines were established. Transgenic mice harboring the SAG-1 gene will contribute a critical tool of defining the molecular mechanisms of SAG-1 in pathogenesis and host immune response.

SAG1 is a host-targeted antigen for protection against toxoplasma gondii infection.

We previously reported that SAG1 transgenic (tg) mice have an elevated susceptibility resulting from their inability to elicit strong Th1-based protection against Toxoplasma gondii infection. Here, we demonstrate that SAG1 tg mice were protected against T. gondii infection, characterized by a decline in IFN-γ levels, following administration of a lethal dose of T. gondii. Moreover, immunization with T. gondii homogenate conferred protection and induced production of IgG, with IgG1 and IgG2a subclasses driven by Th2 and Th1 responses, respectively, in both SAG1 tg and wild-type (wt) mice. IgG titers were significantly higher from day 10 after immunization in wt mice compared to those in SAG1 tg mice. There were no significant differences observed in levels of IgG1 in both groups. However, significantly lower IgG2a titers were measured in the sera from SAG1 tg mice on days 10, 15 and 20. IFN-γ levels in sera were significantly lower in SAG1 tg mice compared to those in wt mice on day 20 after immunization. When challenged with a lethal dose of the Beverley strain of T. gondii, 80 and 100% survival rates were observed in SAG1 tg and wt mice, respectively, indicating that SAG1 tg mice were protected to a lesser extent from challenge due to the decrease in protective immunity. These results suggest that SAG1 plays a critical role in eliciting protection, hence a target antigen for the development of protective Th1-based responses against T. gondii infection in mice.

Increased susceptibility to Toxoplasma gondii infection in SAG-1 transgenic mice.

SAG-1, one of the major surface proteins of Toxoplasma gondii, has been reported to play an important role in immune and pathogenic mechanisms of the parasites but its exact function is still unclear. We investigated the time courses of T. gondii infection in B6C3F1 transgenic mice carrying the SAG-1 gene. SAG-1 transgenic mice were infected intraperitoneally with a high virulent RH strain or a low virulent Beverley strain of T. gondii. When infected with RH strain tachyzoites, no significant differences in time courses of survivals between SAG-1 transgenic and wild-type mice were observed. Both groups succumbed to an acute infection within 8 days after infection. However, a lower survival rate (20%) was observed in SAG-1 transgenic mice than in wild-type (80%), when infected with Beverley strain cysts. This result indicates that SAG-1 transgenic mice are more susceptible to T. gondii infection as compared with their wild-type counterpart. ELISA using recombinant SAG-1 protein indicates that SAG-1 transgenic mice do not produce antibodies to the SAG-1 molecule. These findings may provide a critical tool for analysing the molecular mechanisms of pathogenesis and host immune responses during toxoplasmosis.

Unresponsiveness to Surface Antigen 1 Modifies Cytokine Profiles in Acute Toxoplasma gondii Infection

Resistance to Toxoplasma gondii involves the development of a highly polarized Th1-type cytokine expression. SAG1 transgenic mice are highly susceptible to T. gondii infection due to their non-reactivity to SAG1 of the protozoan parasite. Here we describe cytokine profiles during the acute phase of T. gondii infection, which are associated with the susceptibility of SAG1 transgenic mice. SAG1 transgenic mice showed a 4.5-fold increase in susceptibility upon inoculation with a sublethal dose of the Beverley strain of T. gondii compared to their wild-type counterparts (mortality: 81 vs. 18%, respectively). When analysis of the most important cytokines involved in the mediation of resistance to infection was carried out, SAG1 transgenic mice exhibited low production levels of IL-12, IFN-γ and TNF-α in sera during the acute phase of T. gondii infection. Antibody and T cells specific for SAG1 were not mounted upon SAG1 stimulation in SAG1 transgenic mice. Moreover, in vitro studies indicated that in SAG1 transgenic mice IFN-γ and IL-12 production was lower than in their wild-type counterparts, although levels of TNF-α increased in SAG1 transgenic mice on day 9 after infection. Low IgG2a levels were detected in SAG1 transgenic mouse sera. Unresponsiveness to SAG1 of T. gondii renders SAG1 transgenic mice unable to develop a strong Th1-based protection against T. gondii infection. These results provide evidence that SAG1 is a pivotal antigen involved in the induction of immune responses towards the development of Th1-protective immunity during T. gondii infection.