Guanylate binding proteins (GBPs) are large interferon-inducible GTPases, executing essential host defense activities against Toxoplasma gondii, an invasive intracellular apicomplexan protozoan parasite of global importance. T. gondii establishes a parasitophorous vacuole (PV) which shields the parasite from the host’s intracellular defense mechanisms. Murine GBPs (mGBPs) recognize T. gondii PVs and assemble into supramolecular mGBP homo- and heterocomplexes that are required for the disruption of the membrane of PVs eventually resulting in the cell-autonomous immune control of vacuole-resident pathogens. We have previously shown that mGBP2 plays an important role in T. gondii immune control. Here, to unravel mGBP2 functions, we report Galectin-9 (Gal9) as a critical mGBP2 interaction partner engaged for immunity to T. gondii. Interestingly, Gal9 also accumulates and colocalizes with mGBP2 at the T. gondii PV. Furthermore, we could prove the requirement of Gal9 for growth control of T. gondii by CRISPR/Cas9 mediated gene editing. These discoveries clearly indicate that Gal9 is a crucial factor for the mGBP2-coordinated cell-autonomous host defense mechanism against T. gondii.

Guanylate binding proteins (GBPs) are large interferon-inducible GTPases, executing essential host defense activities against Toxoplasma gondii , an invasive intracellular apicomplexan protozoan parasite of global importance. T. gondii establishes a parasitophorous vacuole (PV) which shields the parasite from the host’s intracellular defense mechanisms. Murine GBPs (mGBPs) recognize T. gondii PVs and assemble into supramolecular mGBP homo- and heterocomplexes that are required for the disruption of the membrane of PVs eventually resulting in the cell-autonomous immune control of vacuole-resident pathogens. We have previously shown that mGBP2 plays an important role in T. gondii immune control. Here, in order to unravel mGBP2 functions, we report Galectin-9 (Gal9) and cytoskeleton-associated protein 4 (Ckap4) as critical mGBP2 interaction partners engaged for immunity to T. gondii . Interestingly, Gal9 and Ckap4 also accumulate and colocalize with mGBP2 at the T. gondii PV. Furthermore, we could prove the requirement of Gal9 and Ckap4 for growth control of T. gondii by CRISPR/Cas9 mediated gene editing. These discoveries clearly indicate that mGBP2 engages Gal9 and Ckap4 and that Gal9 and Ckap4 are critical factors for the mGBP2 coordinated cell autonomous host defense mechanism against T. gondii .Significance Statement We have previously shown that mGBP2 is an essential IFNγ induced protein for defense against Toxoplasma gondii infection. mGBP2 is rapidly recruited to the parasitophorous vacuole (PV) of intracellular T. gondii and is required for the rupture and/or permeabilization of the PV membrane. Subsequently mGBP2 attacks the parasite membrane. Up to now, no data about other mGBP2 interaction partners and their molecular requirement for T. gondii control has been made available.Here, we have identified Gal9 and Ckap4 as interacting proteins of mGBP2. Gal9 and Ckap4 are essential in the mGBP2 mediated attack of the T. gondii PV. Both proteins localize at the T. gondii PV in close association with mGBP2, as shown by state-of-the-art super resolution microscopy. In addition, T. gondii growth cannot be controlled anymore in Gal9 and Ckap4 deficient cell lines, comparable to mGBP2 deficient cells,.Thus, we have identified novel effector proteins in the intricate cell autonomous immune machinery against intracellular pathogens.### Competing Interest StatementThe authors have declared no competing interest.

Members of the murine guanylate-binding protein family (mGBP) are induced by interferon gamma (IFN-γ) and have been shown to be important factors in cell-autonomous immunity toward the intracellular pathogen Toxoplasma gondii Previously, we identified that mGBP2 mediates disruption of the parasitophorous vacuole membrane (PVM) and directly assaults the plasma membrane of the parasite. Here, we show that mGBP7-deficient mice are highly susceptible to T. gondii infection. This is demonstrated by the loss of parasite replication control, pronounced development of ascites, and death of the animals in the acute infection phase. Interestingly, live-cell microscopy revealed that mGBP7 recruitment to the PVM occurs after mGBP2 recruitment, followed by disruption of the PVM and T. gondii integrity and accumulation of mGBP7 inside the parasite. This study defines mGBP7 as a crucial effector protein in resistance to intracellular T. gondiiIMPORTANCE Guanylate-binding proteins (GBPs) are induced by the inflammatory cytokine interferon gamma (IFN-γ) and have been shown to be important factors in the defense of the intracellular pathogen Toxoplasma gondii In previous studies, we showed that members of the mouse GBP family, such as mGBP2 and mGBP7, accumulate at the parasitophorous vacuole of T. gondii, which is the replicatory niche of the parasite. In this study, we show that mice deficient in mGBP7 succumb early after infection with T. gondii, showing a complete failure of resistance to the pathogen. On a molecular level, mGBP7 is found directly at the parasite, likely mediating its destruction.

Chlamydia, the most common sexually transmitted pathogen, is an exquisitely adapted Gram-negative obligate intracellular bacterium. Intracellular Chlamydia trachomatis replicate in a specialized vacuole, termed inclusion, which shields the bacterium from antimicrobial immunity of the host cells and acts as a signalling interface. Previously it was shown that members of the interferon induced guanylate binding protein (mGBP) family, in particular murine GBP1 and mGBP2, were found to accumulate at the bacterial inclusions, similar to previously published recruitment of GBPs to the parasitophorous vacuole of Toxoplasma gondii. Here, we provide a wide comparison of mGBPs roles within the host cell in the context of Chlamydia and Toxoplasma infection. By confocal microscopy on fixed and living infected cells we show localization of mGBP3, mGBP6, mGBP7, mGBP9, and mGBP10, in addition to mGBP1 and mGBP2, at chlamydia inclusions. In time lapse videos using GFP expressing Chlamydia we show rapid and transient dynamics of mGBP9 accumulation onto chlamydia inclusions. Taken together this study reveals a broad activation of mGBP recruitment towards Chlamydia trachomatis inclusions after infection and provides evidence for time limited action of mGBP9 at the chlamydia inclusion.

GBPs are essential for immunity against intracellular pathogens, especially for Toxoplasma gondii control. Here, the molecular interactions of murine GBPs (mGBP1/2/3/5/6), homo- and hetero-multimerization properties of mGBP2 and its function in parasite killing were investigated by mutational, Multiparameter Fluorescence Image Spectroscopy, and live cell microscopy methodologies. Control of T. gondii replication by mGBP2 requires GTP hydrolysis and isoprenylation thus, enabling reversible oligomerization in vesicle-like structures. mGBP2 undergoes structural transitions between monomeric, dimeric and oligomeric states visualized by quantitative FRET analysis. mGBPs reside in at least two discrete subcellular reservoirs and attack the parasitophorous vacuole membrane (PVM) as orchestrated, supramolecular complexes forming large, densely packed multimers comprising up to several thousand monomers. This dramatic mGBP enrichment results in the loss of PVM integrity, followed by a direct assault of mGBP2 upon the plasma membrane of the parasite. These discoveries provide vital dynamic and molecular perceptions into cell-autonomous immunity.

IFNγ is an immunomodulatory cytokine that rapidly activates potent host cell effector mechanisms to confront a variety of intracellular pathogens. Some of the most abundantly IFNγ induced proteins are the 65-kDa guanylate-binding proteins (GBPs), which mediate cell-autonomous immunity. GBPs are related to the dynamin super family of GTPases and are highly conserved throughout the vertebrate lineage. The human genome harbors seven GBPs and at least one pseudogene, whereas the mouse genome contains 11 GBPs and two pseudogenes. The gene loci of murine GBPs (mGBPs) are tandemly organized in clusters on chromosomes 3 and 5. GBPs contain a conserved GTPase-domain which binds guanine nucleotides with low affinities. This induces nucleotide dependent GBP multimerization and cooperative hydrolysis of GTP via GDP to GMP. Some GBPs are isoprenylated, endowing them with the ability to associate with intracellular membranous compartments. Murine GBPs (mGBPs) exert a major impact on cell-autonomous restriction of Toxoplasma gondii. T. gondii is an apicomplexan protozoan parasite with a broad host range, is distributed worldwide and causes serious and often fatal infections in immunocompromised hosts. T. gondii infection experiments in mice deficient for mGBP1 or mGBP2 prove that mGBPs are essential immune effector molecules mediating antiparasitic resistance. In several cell types distinct mGBPs accumulate at the parasitophorous vacuole membrane (PVM) of T. gondii. The translocation of mGBPs to the PVM is accompanied by depletion of cytosolic vesicle like structures (VLS), formation of large multimers at the PVM, subsequent loss of PVM integrity, followed by a direct assault of mGBP2 upon the plasma membrane of the parasite. These findings provide important dynamic and molecular insights into the function of cell-autonomous immunity.

IFN-γ orchestrates the host response against intracellular pathogens. Members of the guanylate binding proteins (GBP) comprise the most abundant IFN-γ–induced transcriptional response. mGBPs are GTPases that are specifically up-regulated by IFN-γ, other proinflammatory cytokines, toll-like receptor agonists, as well as in response toListeria monocytogenesandToxoplasma gondiiinfection. mGBP2 localizes at the parasitophorous vacuole (PV) ofT. gondii; however, the molecular function of mGBP2 and its domains inT. gondiiinfection is not known. Here, we show that mGBP2 is highly expressed in several cell types, including T and B cells after stimulation. We provide evidence that the C-terminal domain is sufficient and essential for recruitment to theT. gondiiPV. Functionally, mGBP2 reducesT. gondiiproliferation because mGBP2-deficient cells display defects in the replication control ofT. gondii. Ultimately, mGBP2-deficient mice reveal a marked immune susceptibility toT. gondii. Taken together, mGBP2 is an essential immune effector molecule mediating antiparasitic resistance.

One of the most abundantly IFN-γ-induced protein families in different cell types is the 65-kDa guanylate-binding protein family that is recruited to the parasitophorous vacuole of the intracellular parasite Toxoplasma gondii. Here, we elucidate the relationship between biochemistry and cellular host defense functions of mGBP2 in response to Toxoplasma gondii. 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. The consistent results indicate that GTP binding, self-assembly, and stimulated hydrolysis activity are required for physiological localization of the protein in infected and uninfected cells. Ultimately, we show that the GTPase domain regulates efficient recruitment to T. gondii in response to IFN-γ.