Essential Role of mGBP7 for Survival of Toxoplasma gondii Infection.

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.

Murine guanylate binding protein 2 (mGBP2) controls Toxoplasma gondii replication.

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 to Listeria monocytogenes and Toxoplasma gondii infection. mGBP2 localizes at the parasitophorous vacuole (PV) of T. gondii; however, the molecular function of mGBP2 and its domains in T. gondii infection 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 the T. gondii PV. Functionally, mGBP2 reduces T. gondii proliferation because mGBP2-deficient cells display defects in the replication control of T. gondii. Ultimately, mGBP2-deficient mice reveal a marked immune susceptibility to T. gondii. Taken together, mGBP2 is an essential immune effector molecule mediating antiparasitic resistance.

SLy2 targets the nuclear SAP30/HDAC1 complex.

The adapter protein SLy2 (SH3 protein expressed in lymphocytes 2), also named HACS1, NASH1 or SAMSN1, is expressed in hematopoietic tissues, muscle, heart, brain, lung, pancreas, endothelial cells and myelomas. Endogenous SLy2 expression was shown to be upregulated in primary B cells upon differentiation and proliferation-inducing stimuli, and transduction experiments suggest a stimulatory role for SLy2 in B cell differentiation to plasma cells. However the signalling pathways regulated by SLy2 remain unknown. In this study we identify novel interaction partners of SLy2 providing a molecular framework for its function. We show that phosphorylated SLy2 directly interacts with 14-3-3 proteins via a previously unrecognized phosphorylation site. Furthermore, we demonstrate that 14-3-3 proteins control nucleo-cytoplasmic shuttling of SLy2 by retaining phosphorylated SLy2 in the cytoplasm. In the nucleus, SLy2 interacts with the SAP30/HDAC1 complex and regulates the activity of HDAC1. Thus, our findings unravel a novel mechanism how SLy2 localization is controlled and implicate SLy2 in the epigenetic control of gene expression.

The proinflammatory cytokine-induced IRG1 protein associates with mitochondria.

Interferon-gamma (IFN-gamma) and tumor necrosis factor (TNF) are essential cytokines for successful clearance of microbial infections. Activation of macrophages by synergistic effects of these cytokines leads to induction of antimicrobial effector systems like reactive oxygen and reactive nitrogen intermediates. Strikingly, IFN-gammaR(-/-) and TNFRp55(-/-) mice are considerably more susceptible to infections than inducible nitric oxide synthase(-/-) and p47phox(-/-) mice. Thus we applied transcriptome-profiling studies to identify genes synergistically upregulated by IFN-gamma and TNF in macrophages which are potentially involved in the defense against intracellular pathogens. From a total of 234 regulated genes we found 35 genes that were upregulated by combined effects of IFN-gamma and TNF and were at least 2-fold induced. The majority of these genes are involved in signal transduction and transcriptional regulation. However, we found several genes were poorly characterized with regard to immunological functions. As a prototypic TNF- and IFN-gamma-coregulated gene we characterized the expression and the subcellular localization of immunoresponsive gene 1 (IRG1) in murine macrophages. IRG1 is highly upregulated in murine ANA-1 macrophages by several proinflammatory cytokines and Toll-like receptor (TLR) agonists, as well as in spleen and lung of Listeria monocytogenes or Toxoplasma gondii infected mice, respectively. Furthermore, this study identifies 35 genes that constitute the IFN-gamma/TNF-triggered effector program in innate immunity.

Analyses of murine GBP homology clusters based on in silico, in vitro and in vivo studies.

The interactions between pathogens and hosts lead to a massive upregulation of antimicrobial host effector molecules. Among these, the 65 kDa guanylate binding proteins (GBPs) are interesting candidates as intricate components of the host effector molecule repertoire. Members of the GBP family are highly conserved in vertebrates. Previous reports indicate an antiviral activity of human GBP1 (hGBP1) and murine GBP2 (mGBP2). We recently demonstrated that distinct murine GBP (mGBP) family members are highly upregulated upon Toxoplasma gondii infection and localize around the intracellular protozoa T. gondii. Moreover, we characterised five new mGBP family members within the murine 65 kDa GBP family. Here, we identified a new mGBP locus named mGbp11. Based on bacterial artificial chromosome (BAC), expressed sequence tag (EST), and RT-PCR analyses this study provides a detailed insight into the genomic localization and organization of the mGBPs. These analyses revealed a 166-kb spanning region on chromosome 3 harboring five transcribed mGBPs (mGbp1, mGbp2, mGbp3, mGbp5, and mGbp7) and one pseudogene (pseudomGbp1), as well as a 332-kb spanning region on chromosome 5 consisting of six transcribed mGBPs (mGbp4, mGbp6, mGbp8, mGbp9, mGbp10, and mGbp11), and one pseudogene (pseudomgbp2). Besides the strikingly high homology of 65% to 98% within the coding sequences, the mGBPs on chromosome 5 cluster also exhibit a highly homologous exon-intron structure whereas the mGBP on chromosome 3 reveals a more divergent exon-intron structure. This study details the comprehensive genomic organization of mGBPs and suggests that a continuously changing microbial environment has exerted evolutionary pressure on this gene family leading to multiple gene amplifications. A list of links for this article can be found in the Availability and requirements section.

Extensive characterization of IFN-induced GTPases mGBP1 to mGBP10 involved in host defense.

IFN-gamma orchestrates a potent antimicrobial host response. However, the underlying molecular basis for this immunological defense system is largely unknown. In a systematic approach to identify IFN-gamma-regulated host effector molecules, a notable number of transcripts with consensus GTP-binding motives were obtained. Further extensive transcriptome and genome analyses identified five novel family members of murine guanylate-binding proteins (mGBPs) now designated mGBP6, 7, 8, 9, and 10. Moreover, in this study, all 10 mGBP members (mGBP1-10) were extensively characterized. mGBPs are selectively up-regulated in vitro by a set of proinflammatory cytokines and TLR agonists as well as in vivo after Listeria monocytogenes and Toxoplasma gondii infection. After IFN-gamma stimulation, mGBP1, 2, 3, 6, 7, and 9 are associated with intracellular Toxoplasma parasites and, interestingly, virulent Toxoplasma interfere with mGBP recruitment. Taken together, mGBPs comprise an important set of host defense molecules.

In silico and in vitro characterization of mGBP4 splice variants.

In a systematic approach to identify interferon-gamma (IFN-gamma)-regulated host effector molecules, we found several members of the 65 kDa guanylate-binding proteins (GBPs) highly upregulated. During extensive characterization of these guanosine triphosphatases (GTPases), we identified discrepancies between the cloned and published sequences of the murine GTPase mGBP4. Two splice variants of mGBP4 could be detected. One variant led to a premature stop codon after 312 bp. The second variant resulted in a transcript with a disrupted G2 domain and was deposited as mGBP4.1 to the GenBank. Interestingly, only mGBP4, not mGBP4.1 mRNA, was highly upregulated in mice after infection with Listeria monocytogenes.