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PIM1 controls GBP1 activity to limit self-damage and guard against pathogen infection

ABSTRACT: Pathogen virulence factors interfere with various cellular activities, a disruption that can trigger innate immune responses. Interferon-gamma (IFNγ)-inducible antimicrobial factors, such as the guanylate binding proteins (GBPs), promote cell-intrinsic defense against intracellular pathogens by directly attacking intracellular pathogens and by committing the infected host cell to programmed cell death. GBPs are large GTPases that traffic to pathogens in the cytosol or that reside within membrane-bound compartments and restrict microbial growth. We discovered that ectopic expression of the prototypical IFNγ-inducible GTPase, Guanylate Binding Protein 1 (GBP1), in the absence of IFNγ kills human macrophages, accompanied by fragmentation of the Golgi apparatus. Exposure to IFNγ improved host cell survival via the activity of PIM1 kinase. PIM1 phosphorylates GBP1 and imposes its sequestration by 14-3-3σ to prevent membrane association of GBP1. Analysis by cryo-EM showed that a 14-3-3σ-dimer trapped GBP1 in an inactive monomeric state. This control mechanism becomes evident during Toxoplasma gondii infection, where the effector protein TgIST interferes with IFNγ-signaling and thereby unintentionally depletes the short-lived kinase PIM1. This, in turn, increases GBP1-driven control of the pathogen. Uninfected bystander cells are spared during inflammation, while the infected cells restrain the pathogen in a rapid GBP1-dependent manner. Our work establishes PIM1 as a bait for pathogen virulence factors, thus safeguarding the integrity of IFNγ-signaling. This work presents a new paradigm of IFNγ-dependent protection of uninfected cells against self-inflicted innate immune damage.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Toxoplasma Gondii Pru

TISSUE(S): Macrophage

DISEASE(S): Toxoplasmosis

SUBMITTER: Daniel Fisch

LAB HEAD: Bram Snijders

PROVIDER: PXD029463 | Pride | 2023-07-26

REPOSITORIES: Pride

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Project description:Background: In previous studies we showed that IL-27 shares several effects with IFN-γ in human cancer cells. To identify novel extracellular mediators, potentially involved in epithelial ovarian cancer (EOC) biology, we analyzed the effect of IL-27 or IFN-γ on the secretome of EOC cells in culture. Methods: The secretome of cytokine-treated or untreated SKOV3 cells was analyzed by nano-UHPLC-MS/MS and eluting peptides by an Orbitrap Fusion Tribrid mass spectrometer. Extracellular GBP1 was studied by ELISA, immunoprecipitation, GTP-agarose pull-down, and Western blotting. GBP and STAT proteins were analysed on cell lysates by Western blot. GBP1 was transfected using lipofectamine reagent and cell viability measured by MTT assay. Results: IL-27 and IFN-γ modulate the extracellular release of a limited fraction of proteins that were also induced in the whole cell. Among these proteins we focused our attention on GBP1, a guanylate-binding protein and GTPase, which is a mediator of several biological activities of IFNs. Interestingly GBP1, 2, and 5 were induced by cytokine treatment in EOC cells, but only GBP1 was secreted. ELISA and immuno-blotting analyses showed that cytokine-stimulated EOC cells release full-length GBP1 molecule in vitro, through non-classical pathways, not involving microvesicles. Moreover, soluble, full-length GBP1 accumulates in the ascites of most EOC patients, suggesting a potential role of extracellular GBP1 in the tumor environment. EOC cells enriched from ascites showed constitutive tyrosine-phosphorylated STAT1 and STAT3 proteins and GBP1 expression, supporting a role of STAT-activating cytokines in GBP1 induction in vivo. Data from the TCGA dataset of EOC indicate that high GBP1 gene expression correlates with a better overall survival. Accordingly GBP1 transfection reduced SKOV3 cell proliferation, suggesting a potential anti-tumor activity of GBP1. Conclusions: Our data show for the first time that soluble GBP1 is released by cytokine-stimulated EOC cells in vitro and accumulates in EOC patients’ ascites. GBP1 expression may have anti-tumor effects, in EOC, as suggested by TCGA dataset analysis and in vitro transfection experiments. Further studies to identify the biological role of soluble GBP1 in the tumor environment of EOC are warranted.

Project description:The gut microbiota is increasingly recognized for playing a critical role in human health and disease, especially in conferring resistance to both virulent pathogens such as Salmonella, which infects 1.2 million people in the United States every year [1], and opportunistic pathogens like Candida, which causes an estimated 46,000 cases of invasive candidiasis each year in the United States [2]. The dynamics of pathogen-microbiome interactions and the metabolites involved in this process remain largely unknown. We use gnotobiotic mice infected with the virulent pathogen Salmonella enterica serovar Typhimurium or the opportunistic pathogen Candida albicans in combination with metagenomics and discovery metabolomics to identify changes in the community and metabolome during infection. To isolate the role of the microbiota in response to pathogens, we compared mice monocolonized with the pathogen, uninfected mice 'humanized' with a synthetic human microbiome, or infected humanized mice. We observe that changes in the community and in biosynthetic gene cluster potential occur within 3 days for the virulent Salmonella enterica serovar Typhimurium, but there are minimal changes with a poorly colonizing Candida albicans. In addition, the metabolome shifts depending on infection status, including changes in glutathione metabolites in response to Salmonella infection. The LC-MS metabolomic fingerprint of the cecum differed between mice monocolonized with either pathogen and humanized infected mice. Specifically, we identified an increase in glutathione disulfide, glutathione cysteine disulfide, inosine 5'-monophosphate, and hydroxybutyrylcarnitine in mice infected with Salmonella in contrast to uninfected mice and mice monocolonized with Salmonella. These metabolites potentially play a role in pathogen-induced oxidative stress. These results provide insight into how the microbiota community members interact with each other and with pathogens on a metabolic level. Ref:[1] Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, Jones JL, and Griffin PM. Foodborne Illness Acquired in the United States—Major Pathogens. Emerg Infect Dis 2011;17:7-15. doi.org/10.3201/eid1701.P11101[2] Centers for Disease Control and Prevention, Antibiotic Resistance Threats in the United States, 2013

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PIM1 controls GBP1 activity to limit self-damage and guard against pathogen infection

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