The function of gamma interferon-inducible GTP-binding protein IGTP in host resistance to Toxoplasma gondii is Stat1 dependent and requires expression in both hematopoietic and nonhematopoietic cellular compartments

Abstract

IGTP is a member of the 47-kDa family of gamma interferon (IFN-gamma)-induced GTPases. We have previously shown that IGTP is critical for host resistance to Toxoplasma gondii infection. In the present study, we demonstrate that T. gondii-induced IGTP expression in vivo and IFN-gamma-driven synthesis of the protein in vitro are dependent on Stat1. Consistent with this observation, Stat1-deficient animals succumbed to T. gondii infection with the same rapid kinetics as IGTP(-/-) mice. To ascertain the cellular levels at which IGTP functions in host control of acute infection, we constructed reciprocal bone marrow chimeras between IGTP-deficient and wild-type mice. Resistance to infection was observed only when IGTP was present in both hematopoietic and nonhematopoietic compartments. To assess the possible contribution of IGTP to the maintenance of parasite latency, partial chemotherapy was used to allow the establishment of chronic infection in IGTP-deficient animals. Upon cessation of drug treatment, these animals showed delayed mortality compared with similarly infected and treated IFN-gamma-deficient or inducible nitric oxide synthase-deficient mice, which succumbed rapidly. Parallel experiments performed with drug-treated bone marrow chimeras supported a role for the hematopoietic compartment in this NO-dependent, IGTP-independent control of chronic infection. Taken together, our findings demonstrate that host resistance mediated by IGTP is a Stat1-induced function which in the case of T. gondii acts predominantly to restrict acute as opposed to chronic infection. This effector mechanism requires expression of IGTP in cells of both hematopoietic and nonhematopoietic origin. In contrast, in latent infection, hematopoietically derived cells mediate resistance by means of a largely NO-dependent pathway.

FIG. 1.

Requirement for Stat1 in IFN-γ-dependent IGTP induction and in vivo control of T. gondii infection. (A) Production of IGTP in tissues from T. gondii-infected Stat1-deficient animals and WT control animals. Protein lysates were prepared from the spleen, liver, and lungs of individual uninfected (−) and infected (for 5 days) (+) mice and analyzed for the presence of IGTP by Western blotting. The bands shown had an approximate molecular mass of 47 kDa as determined by reference to molecular mass markers and were not seen in control samples from infected IGTP-deficient animals (data not shown). The background bands observed in uninfected WT as well as Stat1 KO mice are likely to represent basal expression of IGTP, since in a previous study, no reactivity was observed when comparable Western blots were probed with the preimmune serum for this antibody (35). (B) Expression of IGTP in IFN-γ-LPS-stimulated inflammatory macrophages from Stat1-deficient and WT control animals. Thioglycolate-elicited PC from individual uninfected WT or Stat1-deficient animals were cultured for 18 h in the presence (+) or absence (−) of IFN-γ plus LPS. Protein lysates were then prepared and subjected to immunoblotting with anti-IGTP antibody as described above. (C) Survival of WT (129/Sv/Ev), IGTP-deficient, and Stat1-deficient mice (n = 5 animals per group) following infection with 20 cysts of T. gondii strain ME49. The insert shows the percentage (mean and standard deviation) of tachyzoite-infected PC harvested from WT (n = 3) or Stat1 KO (n = 5) mice on day 5 after T. gondii exposure. The experiment shown is representative of two performed with nearly identical results.

FIG. 2.

Survival of IGTP KO and WT reciprocal bone marrow chimeric mice following infection with T. gondii. Chimeric animals were constructed by reconstituting lethally irradiated WT or IGTP KO mice with bone marrow from either of the two strains. The animals were infected intraperitoneally with 20 cysts of T. gondii strain ME49 8 to 12 weeks after reconstitution (WT to KO, n = 6; KO to WT, n = 7; KO to KO, n = 6; WT to WT, n = 6). The results shown were reproduced in a second experiment.

FIG. 3.

IGTP is expressed in brain tissue during chronic T. gondii infection. Protein extracts were prepared from brain homogenates of WT mice before (d0) and on days 14 (d14), 20 (d20), and 29 (d29) following T. gondii infection. The samples were then analyzed by immunoblotting with an anti-IGTP antibody for the presence of IGTP. Each lane represents an individual animal.

FIG. 4.

Limited role of IGTP in control of latent T. gondii infection. WT (C57BL/6 × 129Sv), IFN-γ receptor-deficient, and IGTP-deficient mice were infected intraperitoneally with 20 cysts of T. gondii strain ME49. Bactrim-containing water was administered daily beginning on day 3 (IFN-γ receptor-deficient and IGTP KO mice) or day 5 (WT animals) (Rx start) and withdrawn on day 20 (Rx stop) postinfection. Survival of the animals (WT, n = 5; IFN-γ receptor [R] KO, n = 6; IGTP KO, n = 12) was then monitored. The results shown are representative of four experiments performed.

FIG. 5.

The IGTP-independent functions responsible for control of chronic T. gondii infection are in part NOS2-dependent effector mechanisms. WT (C57BL/6 × 129Sv) and IGTP KO, IFN-γ KO, and NOS2 KO mice were infected intraperitoneally with 20 cysts of T. gondii strain ME49. Bactrim-containing water was supplied continuously beginning on day 3 (IFN-γ KO and IGTP KO mice) or day 5 (WT and NOS2 KO animals) (Rx start) and withdrawn on day 20 (Rx stop) postinfection. Beginning at day 20, the WT and IGTP KO animals received AG daily in their drinking water. Survival of the animals (WT, n = 12; WT plus AG, n = 11; IGTP KO, n = 14; IGTP KO plus AG, n = 10; IFN-γ KO, n = 8; NOS2 KO, n = 14) was then monitored. Data shown are representative of three experiments performed.

FIG. 6.

Role of cells of hematopoietic origin in the NO-dependent, IGTP-independent control of latent T. gondii infection. Bone marrow chimeras were constructed by reconstituting lethally irradiated WT animals with bone marrow from either WT or IGTP KO mice. The resulting animals were injected intraperitoneally with 20 cysts of T. gondii strain ME49 8 to 12 weeks after reconstitution. Bactrim-containing water was given daily starting on day 3 (IGTP KO-to-WT and IFN-γ KO mice) or day 5 (WT-to-WT and NOS2 KO animals) (Rx start), and antibiotic treatment was stopped at day 20 (Rx stop) postinfection. Beginning at day 20, AG was administered daily to the drinking water of the IGTP KO-to-WT chimeric mice. Cumulative mortality of the mice (WT to WT, n = 7; IGTP KO to WT plus AG, n = 7; IGTP KO to WT, n = 6; IFN-γ KO, n = 8; NOS2 KO, n = 8) was recorded daily.