The biological role of interferon y induced 65 kDa GBPs as effector molecules in host defense
Final Report Abstract
IFNγ orchestrates the host response against intracellular pathogens. Members of the guanylate binding proteins (GBP) comprise the most abundant IFNγ-induced transcriptional response. mGBPs are GTPases that are specifically up-regulated by IFNγ, other proinflammatory cytokines, toll-like receptor agonists, or activated in response to Listeria monocytogenes and Toxoplasma gondii infections. mGBP2 localizes at the parasitophorous vacuole (PV) of T. gondii. During the funding period, it could be demonstrated that mGBP2 is highly expressed in several cell types, including T and B cells after stimulation. Evidence was provided that the C-terminal domain of mGBP2 is sufficient and essential for recruitment to the T. gondii PV. The relationship between biochemistry and cellular host defense functions of mGBP2 in response to T. gondii was elucidated. The wild type protein exhibits low affinities to guanine nucleotides, self-assembles upon GTP binding, forming tetramers in the activated state, and stimulates the GTPase activity in a cooperative manner. The products of the two consecutive hydrolysis reactions are both GDP and GMP. The biochemical characterization of point mutants in the GTP-binding motifs of mGBP2 revealed amino acid residues that decrease the GTPase activity by orders of magnitude and strongly impair nucleotide binding and multimerization ability. Live cell imaging employing multiparameter fluorescence image spectroscopy (MFIS) using a Homo-FRET assay shows that the inducible multimerization of mGBP2 is dependent on a functional GTPase domain. These results indicate consistently that GTP binding, self-assembly, and stimulated hydrolysis activity are required for physiological localization of the protein in infected and uninfected cells. Ultimately, we demonstrated that the GTPase domain regulates efficient recruitment to the T. gondii PV in response to IFNγ. Functionally, mGBP2 reduces T. gondii proliferation because mGBP2-deficient cells display defects in the replication control of T. gondii. Ultimately, mGBP2-deficient mice revealed a marked immune susceptibility to T. gondii. Therefore, mGBP2 could be defined as an essential immune effector molecule mediating antiparasitic resistance. Furthermore, another member of the mGBP family, mGBP7, could be successfully inactivated by gene targeting. This mGBP7-deficient mouse line will provide the opportunity for the characterization of mGBP7 in the immune system.
Publications
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(See online at https://doi.org/10.1074/jbc.M111.251967) - (2012) The GTPase activity of murine guanylate-binding protein 2 (mGBP2) controls the intracellular localization and recruitment to the parasitophorous vacuole of Toxoplasma gondii. J Biol Chem. 287(33): 27452-27466
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(See online at https://doi.org/10.1074/jbc.M112.379636) - (2013) Antimicrobial effects of murine mesenchymal stromal cells directed against Toxoplasma gondii and Neospora caninum: role of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs). Med Microbiol Immunol. 202(3): 197-206
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(See online at https://doi.org/10.1007/s00430-012-0281-y) - (2013) Guanylate-binding protein 1 (Gbp1) contributes to cell-autonomous immunity against Toxoplasma gondii. PLoS Pathog. 9(4): e1003320
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(See online at https://doi.org/10.1371/journal.ppat.1003320) - (2013) Murine guanylate binding protein 2 (mGBP2) controls Toxoplasma gondii replication. Proc Natl Acad Sci U S A. 110(1): 294-299
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(See online at https://doi.org/10.1073/pnas.1205635110) - (2014) Guanylate binding proteins promote caspase-11-dependent pyroptosis in response to cytoplasmic LPS. Proc Natl Acad Sci USA. 111(16): 6046-6051
Pilla DM, Hagar JA, Haldar AK, Mason AK, Degrandi D, Pfeffer K, Ernst RK, Yamamoto M, Miao EA, Coers J
(See online at https://doi.org/10.1073/pnas.1321700111) - (2015) Guanylate-binding proteins promote activation of the AIM2 inflammasome during infection with Francisella novicida. Nat Immunol. 16(5): 476-484
Meunier E, Wallet P, Dreier RF, Costanzo S, Anton L, Rühl S, Dussurgey S, Dick MS, Kistner A, Rigard M, Degrandi D, Pfeffer K, Yamamoto M, Henry T, Broz P
(See online at https://doi.org/10.1038/ni.3119)
