Homeostatic maintenance of pathogen-containing vacuoles requires TBK1-dependent regulation of aquaporin-1

Membranes are an integral component of many cellular functions and serve as a barrier to keep pathogenic bacteria from entering the nutrient-rich host cytosol. TANK-binding-kinase-1 (TBK1), a kinase of the IkappaB kinase family, is required for maintaining integrity of pathogen-containing vacuoles (PCV) upon bacterial invasion of host cells. Here we investigate how vacuolar integrity is maintained during bacterial infection, even in the presence of bacterial membrane damaging agents. We found that Aquaporin-1 (AQP1), a water channel that regulates swelling of secretory vesicles, associated with PCV. AQP1 levels were elevated in TBK1-deficient cells, and overexpression of AQP1 in wild-type cells led to PCV destabilization, similar to that observed in tbk1(-/-) cells. Inhibition of physiological levels of AQP1 in multiple cell types also led to increased instability of PCV, demonstrating a need for tightly regulated AQP1 function to maintain vacuole homeostasis during bacterial infection. AQP1-dependent modulation of PCV was triggered by bacterially induced membrane damage and ion flux. These results highlight the contribution of water channels to promoting PCV membrane integrity, and reveal an unexpected role for TBK1 and AQP1 in restricting bacterial pathogens to the vacuolar compartment.     

PPM1B negatively regulates antiviral response via dephosphorylating TBK1

The production of type I interferon must be tightly regulated and aberrant production of type I interferon is harmful or even fatal to the host. TBK1 phosphorylation at Ser172 plays an essential role in TBK1-mediated antiviral response. However, how TBK1 activity is negatively regulated remains poorly understood. Using a functional genomics approach, we have identified PPM1B as a TBK1 phosphatase. PPM1B dephosphorylates TBK1 in vivo and in vitro. PPM1B wild-type but not its phosphatase-deficient R179G mutant inhibits TBK1-mediated antiviral response and facilitates VSV replication in the cells. Viral infection induces association of PPM1B with TBK1 in a transient fashion in the cells. Conversely, suppression of PPM1B expression enhances virus-induced IRF3 phosphorylation and IFNβ production. Our study identifies a previously unrecognized role for PPM1B in the negative regulation of antiviral response by acting as a TBK1 phosphatase.     

Negative regulation of virus-triggered IFN-beta signaling pathway by alternative splicing of TBK1

Induction of Type I IFNs is a central event in antiviral responses and must be tightly controlled. The protein kinase TBK1 is critically involved in virus-triggered type I IFN signaling. In this study, we identify an alternatively spliced isoform of TBK1, termed TBK1s, which lacks exons 3-6. Upon Sendai virus (SeV) infection, TBK1s is induced in both human and mouse cells and binds to RIG-1, disrupting the interaction between RIG-I and VISA. Consistent with that result, overexpression of TBK1s inhibits IRF3 nuclear translocation and leads to a shutdown of SeV-triggered IFN-beta production. Taken together, our data indicate that TBK1s plays an inhibitory role in virus-triggered IFN-beta signaling pathways.     

Identification of nucleoporin 93 (Nup93) that mediates antiviral innate immune responses

RIG-I-like receptors (RLRs) are cytoplasmic sensors for viral RNA that elicit antiviral innate immune responses. RLR signaling culminates in the activation of the protein kinase TBK1, which mediates phosphorylation and nuclear translocation of IRF3 that regulates expression of type I interferon genes. Here, we found that Nucleoporin 93 (Nup93), components of nuclear pore complex (NPC), plays an important role in RLR-mediated antiviral responses. Nup93-deficient RAW264.7 macrophage cells exhibited decreased expression of Ifnb1 and Cxcl10 genes after treatment with a synthetic RLR agonist stimulation as well as Newcastle Disease Virus infection. Silencing Nup93 in murine primary macrophages and embryonic fibroblasts also resulted in reduced expression of these genes. IRF3 nuclear translocation during RLR signaling was impaired in Nup93-deficient RAW264.7 cells. Notably, the activation of TBK1 during RLR signaling was also decreased in Nup93-deficient cells. We found that Nup93 formed a complex with TBK1, and Nup93 overexpression enhanced TBK1-mediated IFNβ promoter activation. Taken together, our findings suggest that Nup93 regulates antiviral innate immunity by enhancing TBK1 activity and IRF3 nuclear translocation.     

Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that causes disease in a variety of hosts. S. Typhimurium actively invade host cells and typically reside within a membrane-bound compartment called the Salmonella-containing vacuole (SCV). The bacteria modify the fate of the SCV using two independent type III secretion systems (TTSS). TTSS are known to damage eukaryotic cell membranes and S. Typhimurium has been suggested to damage the SCV using its Salmonella pathogenicity island (SPI)-1 encoded TTSS. Here we show that this damage gives rise to an intracellular bacterial population targeted by the autophagy system during in vitro infection. Approximately 20% of intracellular S. Typhimurium colocalized with the autophagy marker GFP-LC3 at 1 h postinfection. Autophagy of S. Typhimurium was dependent upon the SPI-1 TTSS and bacterial protein synthesis. Bacteria targeted by the autophagy system were often associated with ubiquitinated proteins, indicating their exposure to the cytosol. Surprisingly, these bacteria also colocalized with SCV markers. Autophagy-deficient (atg5-/-) cells were more permissive for intracellular growth by S. Typhimurium than normal cells, allowing increased bacterial growth in the cytosol. We propose a model in which the host autophagy system targets bacteria in SCVs damaged by the SPI-1 TTSS. This serves to retain intracellular S. Typhimurium within vacuoles early after infection to protect the cytosol from bacterial colonization. Our findings support a role for autophagy in innate immunity and demonstrate that Salmonella infection is a powerful model to study the autophagy process.     

RKIP and TBK1 form a positive feedback loop to promote type I interferon production in innate immunity

TANK‐binding kinase 1 (TBK1) activation is a central event in type I interferon production in anti‐virus innate immunity. However, the regulatory mechanism underlying TBK1 activation remains unclear. Here we report that Raf kinase inhibitory protein (RKIP) is essential for TBK1 activation and type I interferon production triggered by viral infection. Upon viral infection, RKIP is phosphorylated at serine 109 (S109) by TBK1. Phosphorylation of RKIP enhances its interaction with TBK1 and in turn promotes TBK1 autophosphorylation. Mutation of RKIP S109 to alanine abrogates the interaction between RKIP and TBK1, and the anti‐viral function of RKIP. RKIP deficiency inhibits intracellular double‐stranded RNA‐ or DNA‐induced type I interferon production. Consistently, RKIP deficiency renders the mice more susceptible to vesicular stomatitis virus (VSV) and herpes simplex virus (HSV) infections. This study reveals a previously unrecognized positive feedback loop between RKIP and TBK1 that is essential for type I interferon production in anti‐viral innate immunity.     

How cells deploy ubiquitin and autophagy to defend their cytosol from bacterial invasion

Autophagy serves as a cell-autonomous effector mechanism of innate immunity in the cytosol. Autophagy restricts bacterial proliferation by separating bacteria from the nutrient-rich cytosol and delivering them into bactericidal autolysosomes. Autophagy also restricts inflammation by enclosing the membrane remnants of vacuoles from which bacteria have escaped. In contrast to starvation-induced autophagy, which engulfs cytosol nonspecifically, antibacterial autophagy is receptor-mediated and selective. Several distinct pathways of antibacterial autophagy have been identified recently, which can be triggered by either bacterial PAMPs, host-mediated modifications of bacteria-containing vacuoles, or cytosolic bacteria that have become decorated with ubiquitin. Ubiquitin-coated bacteria are sensed by p62, a promiscuous autophagy receptor required for the uptake of a variety of ubiquitin-marked autophagy substrates, and by NDP52, an autophagy receptor that, by associating with the immuno-regulatory kinase TBK1, may serve a dedicated function in cytosolic immunity.     

How cells deploy ubiquitin and autophagy to defend their cytosol from bacterial invasion

Autophagy serves as a cell-autonomous effector mechanism of innate immunity in the cytosol. Autophagy restricts bacterial proliferation by separating bacteria from the nutrient-rich cytosol and delivering them into bactericidal autolysosomes. Autophagy also restricts inflammation by enclosing the membrane remnants of vacuoles from which bacteria have escaped. In contrast to starvation-induced autophagy, which engulfs cytosol nonspecifically, antibacterial autophagy is receptor-mediated and selective. Several distinct pathways of antibacterial autophagy have been identified recently, which can be triggered by either bacterial PAMPs, host-mediated modifications of bacteria-containing vacuoles, or cytosolic bacteria that have become decorated with ubiquitin. Ubiquitin-coated bacteria are sensed by p62, a promiscuous autophagy receptor required for the uptake of a variety of ubiquitin-marked autophagy substrates, and by NDP52, an autophagy receptor that, by associating with the immunoregulatory kinase TBK1, may serve a dedicated function in cytosolic immunity.     

TBK1-associated Protein in Endolysosomes (TAPE)/CC2D1A Is a Key Regulator Linking RIG-I-like Receptors to Antiviral Immunity

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are key RNA viral sensors for triggering antiviral immunity. The underlying mechanisms for RLRs to trigger antiviral immunity have yet to be explored. Here we report the identification of TAPE (TBK1-associated protein in endolysosomes) as a novel regulator of the RLR pathways. TAPE functionally and physically interacts with RIG-I, MDA5, and IPS-1 to activate the IFN-β promoter. TAPE knockdown impairs IFN-β activation induced by RLRs but not IPS-1. TAPE-deficient cells are defective in cytokine production upon RLR ligand stimulation. During RNA virus infection, TAPE knockdown or deficiency diminishes cytokine production and antiviral responses. Our data demonstrate a critical role for TAPE in linking RLRs to antiviral immunity.     

Inhibition of TANK binding kinase 1 by herpes simplex virus 1 facilitates productive infection

The γ(1)34.5 protein of herpes simplex viruses (HSV) is essential for viral pathogenesis, where it precludes translational arrest mediated by double-stranded-RNA-dependent protein kinase (PKR). Paradoxically, inhibition of PKR alone is not sufficient for HSV to exhibit viral virulence. Here we report that γ(1)34.5 inhibits TANK binding kinase 1 (TBK1) through its amino-terminal sequences, which facilitates viral replication and neuroinvasion. Compared to wild-type virus, the γ(1)34.5 mutant lacking the amino terminus induces stronger antiviral immunity. This parallels a defect of γ(1)34.5 for interacting with TBK1 and reducing phosphorylation of interferon (IFN) regulatory factor 3. This activity is independent of PKR. Although resistant to IFN treatment, the γ(1)34.5 amino-terminal deletion mutant replicates at an intermediate level between replication of wild-type virus and that of the γ(1)34.5 null mutant in TBK1(+/+) cells. However, such impaired viral growth is not observed in TBK1(-/-) cells, indicating that the interaction of γ(1)34.5 with TBK1 dictates HSV infection. Upon corneal infection, this mutant replicates transiently but barely invades the trigeminal ganglia or brain, which is a difference from wild-type virus and the γ(1)34.5 null mutant. Therefore, in addition to PKR, γ(1)34.5 negatively regulates TBK1, which contributes viral replication and spread in vivo.     

Immune activation of type I IFNs by Listeria monocytogenes occurs independently of TLR4, TLR2, and receptor interacting protein 2 but involves TNFR-associated NF kappa B kinase-binding kinase 1

Type I IFNs are well established antiviral cytokines that have also been shown to be induced by bacteria. However, the signaling mechanisms regulating the activation of these cytokines during bacterial infections remain poorly defined. We show that although Gram-negative bacteria can activate the type I IFN pathway through TLR4, the intracellular Gram-positive bacterium Listeria monocytogenes (LM) can do so independently of TLR4 and TLR2. Furthermore, experiments using genetic mutants and chemical inhibitors suggest that LM-induced type I IFN activation occurs by an intracellular pathway involving the serine-threonine kinase TNFR-associated NF-kappaB kinase (TANK)-binding kinase 1 (TBK1). Interestingly, receptor-interacting protein 2, a component of the recently discovered nucleotide-binding oligomerization domain-dependent intracellular detection pathway, was not involved. Taken together, our data describe a novel signal transduction pathway involving TBK1 that is used by LM to activate type I IFNs. Additionally, we provide evidence that both the LM- and TLR-dependent pathways converge at TBK1 to activate type I IFNs, highlighting the central role of this molecule in modulating type I IFNs in host defense and disease.     

Mechanism of inhibiting type I interferon induction by hepatitis B virus X protein

Hepatitis B virus (HBV) is regarded as a stealth virus, invading and replicating efficiently in human liver undetected by host innate antiviral immunity. Here, we show that type I interferon (IFN) induction but not its downstream signaling is blocked by HBV replication in HepG2.2.15 cells. This effect may be partially due to HBV X protein (HBx), which impairs IFNβ promoter activation by both Sendai virus (SeV) and components implicated in signaling by viral sensors. As a deubiquitinating enzyme (DUB), HBx cleaves Lys63-linked polyubiquitin chains from many proteins except TANK-binding kinase 1 (TBK1). It binds and deconjugates retinoic acid-inducible gene I (RIG I) and TNF receptor-associated factor 3 (TRAF3), causing their dissociation from the downstream adaptor CARDIF or TBK1 kinase. In addition to RIG I and TRAF3, HBx also interacts with CARDIF, TRIF, NEMO, TBK1, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IKKi) and interferon regulatory factor 3 (IRF3). Our data indicate that multiple points of signaling pathways can be targeted by HBx to negatively regulate production of type I IFN.     

Type I interferon promotes cell‐to‐cell spread of Listeria monocytogenes

Type I interferons (IFNs) play a critical role in antiviral immune responses, but can be deleterious to the host during some bacterial infections. Listeria monocytogenes (Lm) induces a type I IFN response by activating cytosolic antiviral surveillance pathways. This is beneficial to the bacteria as mice lacking the type I IFN receptor (IFNAR1^?/?) are resistant to systemic infection by Lm. The mechanisms by which type I IFNs promote Lm infection are unclear. Here, we show that IFNAR1 is required for dissemination of Lm within infection foci in livers of infected mice and for efficient cell‐to‐cell spread in vitro in macrophages. IFNAR1 promotes ActA polarization and actin‐based motility in the cytosol of host cells. Our studies suggest type I IFNs directly impact the intracellular life cycle of Lm and provide new insight into the mechanisms used by bacterial pathogens to exploit the type I IFN response.     

The Timing of IFNβ Production Affects Early Innate Responses to Listeria monocytogenes and Determines the Overall Outcome of Lethal Infection

Dendritic cells (DCs) and natural killer (NK) cells are essential components of the innate immunity and play a crucial role in the first phase of host defense against infections and tumors. Listeria monocytogenes (Lm) is an intracellular pathogen that colonizes the cytosol of eukaryotic cells. Recent findings have shown Lm specifically in splenic CD8a(+) DCs shortly after intravenous infection. We examined gene expression profiles of mouse DCs exposed to Lm to elucidate the molecular mechanisms underlying DCs interaction with Lm. Using a functional genomics approach, we found that Lm infection induced a cluster of late response genes including type I IFNs and interferon responsive genes (IRGs) in DCs. Type I INFs were produced at the maximal level only at 24 h post infection indicating that the regulation of IFNs in the context of Lm infection is delayed compared to the rapid response observed with viral pathogens. We showed that during Lm infection, IFNγ production and cytotoxic activity were severely impaired in NK cells compared to E. coli infection. These defects were restored by providing an exogenous source of IFNβ during the initial phase of bacterial challenge. Moreover, when treated with IFNβ during early infection, NK cells were able to reduce bacterial titer in the spleen and significantly improve survival of infected mice. These findings show that the timing of IFNβ production is fundamental to the efficient control of the bacterium during the early innate phase of Lm infection.     

Tumor Progression Locus 2 Promotes Induction of IFNλ, Interferon Stimulated Genes and Antigen-Specific CD8+ T Cell Responses and Protects against Influenza Virus

Mitogen-activated protein kinase (MAP) cascades are important in antiviral immunity through their regulation of interferon (IFN) production as well as virus replication. Although the serine-threonine MAP kinase tumor progression locus 2 (Tpl2/MAP3K8) has been implicated as a key regulator of Type I (IFNα/β) and Type II (IFNγ) IFNs, remarkably little is known about how Tpl2 might contribute to host defense against viruses. Herein, we investigated the role of Tpl2 in antiviral immune responses against influenza virus. We demonstrate that Tpl2 is an integral component of multiple virus sensing pathways, differentially regulating the induction of IFNα/β and IFNλ in a cell-type specific manner. Although Tpl2 is important in the regulation of both IFNα/β and IFNλ, only IFNλ required Tpl2 for its induction during influenza virus infection both in vitro and in vivo. Further studies revealed an unanticipated function for Tpl2 in transducing Type I IFN signals and promoting expression of interferon-stimulated genes (ISGs). Importantly, Tpl2 signaling in nonhematopoietic cells is necessary to limit early virus replication. In addition to early innate alterations, impaired expansion of virus-specific CD8⁺ T cells accompanied delayed viral clearance in Tpl2^-/- mice at late time points. Consistent with its critical role in facilitating both innate and adaptive antiviral responses, Tpl2 is required for restricting morbidity and mortality associated with influenza virus infection. Collectively, these findings establish an essential role for Tpl2 in antiviral host defense mechanisms.     

Functional dissection of the TBK1 molecular network

TANK-binding kinase 1 (TBK1) and inducible IκB-kinase (IKK-i) are central regulators of type-I interferon induction. They are associated with three adaptor proteins called TANK, Sintbad (or TBKBP1) and NAP1 (or TBKBP2, AZI2) whose functional relationship to TBK1 and IKK-i is poorly understood. We performed a systematic affinity purification-mass spectrometry approach to derive a comprehensive TBK1/IKK-i molecular network. The most salient feature of the network is the mutual exclusive interaction of the adaptors with the kinases, suggesting distinct alternative complexes. Immunofluorescence data indicated that the individual adaptors reside in different subcellular locations. TANK, Sintbad and NAP1 competed for binding of TBK1. The binding site for all three adaptors was mapped to the C-terminal coiled-coil 2 region of TBK1. Point mutants that affect binding of individual adaptors were used to reconstitute TBK1/IKK-i-deficient cells and dissect the functional relevance of the individual kinase-adaptor edges within the network. Using a microarray-derived gene expression signature of TBK1 in response virus infection or poly(I∶C) stimulation, we found that TBK1 activation was strictly dependent on the integrity of the TBK1/TANK interaction.     

Crystal structure of the ubiquitin-like domain of human TBK1

TANK-binding kinase 1 (TBK1) is an important enzyme in the regulation of cellular antiviral effects. TBK1 regulates the activity of the interferon regulatory factors IRF3 and IRF7, thereby playing a key role in type I interferon (IFN) signaling pathways. The structure of TBK1 consists of an N-terminal kinase domain, a middle ubiquitin-like domain (ULD), and a C-terminal elongated helical domain. It has been reported that the ULD of TBK1 regulates kinase activity, playing an important role in signaling and mediating interactions with other molecules in the IFN pathway. In this study, we present the crystal structure of the ULD of human TBK1 and identify several conserved residues by multiple sequence alignment. We found that a hydrophobic patch in TBK1, containing residues Leu316, Ile353, and Val382, corresponding to the “Ile44 hydrophobic patch” observed in ubiquitin, was conserved in TBK1, IκB kinase epsilon (IKK?/IKKi), IκB kinase alpha (IKKα), and IκB kinase beta (IKKβ). In comparison with the structure of the IKKβ ULD domain of Xenopus laevis, we speculate that the Ile44 hydrophobic patch of TBK1 is present in an intramolecular binding surface between ULD and the C-terminal elongated helices. The varying surface charge distributions in the ULD domains of IKK and IKK-related kinases may be relevant to their specificity for specific partners.     

抗病毒天然免疫通路中IRF-3调控新机制

干扰素调节因子-3(interferon regulatory factor-3,IRF-3)是IRF家族中重要 转录因子之一,在调控干扰素(interferon, IFN)基因表达和抗病毒天然免疫反应中具有重要作 用. 最新发现的MITA (mediator of IRF-3 activation, 又称STING/ERIS)蛋白是宿主抗病 毒天然免疫反应中的一种重要调节分子. 病毒侵染时,MITA与IRF-3相互作用,特异性激活 IRF-3,并募集TANK结合激酶1（TANK binding kinase 1, TBK1）与IFN通路中的线粒体抗 病毒信号蛋白MAVS(mitochondrial anti-viral signaling protein)形成复合物,且MITA可 被TBK1磷酸化,诱导Ⅰ型IFN及IFN刺激基因(interferon stimulate genes, ISG)的表达 ,诱发抗病毒天然免疫反应. 同时还发现,泛素连接酶RNF5（ring finger protein 5）可对MITA 发生泛素化修饰从而抑制其对IRF-3活化,实现对宿主抗病毒天然免疫反应负调节作用. 本 室研究发现,严重性急性呼吸系统综合症冠状病毒(severe acute respiratory syndrome co ronavirus, SARS-CoV）和人类新型冠状病毒（human coronavirus NL63, HCoV-NL63）的 木瓜样蛋白酶（papain-like protease, PLP）利用其特有的去泛素化酶（deubiquitinase, DUB）活性,通过宿主细胞泛素-蛋白酶体信号系统对IRF-3的泛素化等翻译后修饰进行调节 ,从而成为该种病毒逃逸机体抗病毒防御系统主要手段之一.