RESUMEN
The innate immune system senses infection by detecting either evolutionarily conserved molecules essential for the survival of microbes or the abnormal location of molecules. Here we demonstrate the existence of a previously unknown innate detection mechanism induced by fusion between viral envelopes and target cells. Virus-cell fusion specifically stimulated a type I interferon response with expression of interferon-stimulated genes, in vivo recruitment of leukocytes and potentiation of signaling via Toll-like receptor 7 (TLR7) and TLR9. The fusion-dependent response was dependent on the stimulator of interferon genes STING but was independent of DNA, RNA and viral capsid. We suggest that membrane fusion is sensed as a danger signal with potential implications for defense against enveloped viruses and various conditions of giant-cell formation.
Asunto(s)
Fusión Celular , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/fisiología , Inmunidad Innata , Interferón Tipo I/biosíntesis , Fusión de Membrana , Proteínas de la Membrana/metabolismo , Animales , Quimiocina CXCL10/metabolismo , Células HEK293 , Células HeLa , Humanos , Leucocitos/inmunología , Leucocitos/metabolismo , Activación de Linfocitos , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de Señal , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 9/metabolismo , Internalización del VirusRESUMEN
The detection of intracellular microbial DNA is critical to appropriate innate immune responses; however, knowledge of how such DNA is sensed is limited. Here we identify IFI16, a PYHIN protein, as an intracellular DNA sensor that mediates the induction of interferon-ß (IFN-ß). IFI16 directly associated with IFN-ß-inducing viral DNA motifs. STING, a critical mediator of IFN-ß responses to DNA, was recruited to IFI16 after DNA stimulation. Lowering the expression of IFI16 or its mouse ortholog p204 by RNA-mediated interference inhibited gene induction and activation of the transcription factors IRF3 and NF-κB induced by DNA and herpes simplex virus type 1 (HSV-1). IFI16 (p204) is the first PYHIN protein to our knowledge shown to be involved in IFN-ß induction. Thus, the PYHIN proteins IFI16 and AIM2 form a new family of innate DNA sensors we call 'AIM2-like receptors' (ALRs).
Asunto(s)
ADN Viral/inmunología , Inmunidad Innata , Espacio Intracelular/inmunología , Proteínas Nucleares/inmunología , Fosfoproteínas/inmunología , Animales , Línea Celular , Proteínas de Unión al ADN , Herpesvirus Humano 1/inmunología , Humanos , Interferón beta/inmunología , Interferón beta/metabolismo , Proteínas de la Membrana/inmunología , Ratones , Monocitos/inmunología , Transducción de SeñalRESUMEN
In Australia, cases of shigellosis usually occur in returned travelers from regions of shigellosis endemicity or in men who have sex with men. Resistance to multiple antibiotics has significantly increased in Shigella sonnei isolates and represents a significant public health concern. We investigate an outbreak of multidrug-resistant S. sonnei in Victoria, Australia. We undertook whole-genome sequencing of 54 extended-spectrum-beta-lactamase (ESBL)-producing S. sonnei isolates received at the Microbiological Diagnostic Unit Public Health Laboratory between January 2019 and March 2020. The population structure and antimicrobial resistance profiles were identified by genomic analyses, with 73 previously characterized Australian S. sonnei isolates providing context. Epidemiological data, including age and sex of the shigellosis cases, were also collected. There was a significant increase in cases of ESBL S. sonnei from July 2019. Most of the ESBL S. sonnei isolates (65%) fell within a single cluster that was predominantly comprised of male cases that were characterized by the presence of the blaCTX-M-27 gene conferring resistance to extended-spectrum cephalosporins. These isolates were also multidrug resistant, including resistance to azithromycin and co-trimoxazole and reduced susceptibility to ciprofloxacin. Our data uncovered a prolonged clonal outbreak of ESBL S. sonnei infection that was likely first introduced by returned travelers and has subsequently been circulating locally in Australia. The emergence of a local outbreak of ESBL S. sonnei with a multidrug-resistant profile, including reduced susceptibility to ciprofloxacin, represents a significant public health threat.
Asunto(s)
Disentería Bacilar , Minorías Sexuales y de Género , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Brotes de Enfermedades , Disentería Bacilar/tratamiento farmacológico , Disentería Bacilar/epidemiología , Homosexualidad Masculina , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Shigella sonnei/genética , Victoria/epidemiología , beta-Lactamasas/genéticaRESUMEN
Listeria monocytogenes is a gram-positive facultative intracellular bacterium, which replicates in the cytoplasm of myeloid cells. Interferon ß (IFNß) has been reported to play an important role in the mechanisms underlying Listeria disease. Although studies in murine cells have proposed the bacteria-derived cyclic-di-AMP to be the key bacterial immunostimulatory molecule, the mechanism for IFNß expression during L. monocytogenes infection in human myeloid cells remains unknown. Here we report that in human macrophages, Listeria DNA rather than cyclic-di-AMP is stimulating the IFN response via a pathway dependent on the DNA sensors IFI16 and cGAS as well as the signalling adaptor molecule STING. Thus, Listeria DNA is a major trigger of IFNß expression in human myeloid cells and is sensed to activate a pathway dependent on IFI16, cGAS and STING.
Asunto(s)
Interacciones Huésped-Patógeno , Interferón beta/metabolismo , Listeria monocytogenes/patogenicidad , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/metabolismo , Fosfoproteínas/metabolismo , Animales , Células Cultivadas , Citosol/metabolismo , ADN Bacteriano/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Listeriosis/metabolismo , Listeriosis/microbiología , Macrófagos/metabolismo , Macrófagos/microbiología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/genética , Nucleotidiltransferasas/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de SeñalRESUMEN
Herpesviruses are DNA viruses harboring the capacity to establish lifelong latent-recurrent infections. There is limited knowledge about viruses targeting the innate DNA-sensing pathway, as well as how the innate system impacts on the latent reservoir of herpesvirus infections. In this article, we report that murine gammaherpesvirus 68 (MHV68), in contrast to α- and ß-herpesviruses, induces very limited innate immune responses through DNA-stimulated pathways, which correspondingly played only a minor role in the control of MHV68 infections in vivo. Similarly, Kaposi's sarcoma-associated herpesvirus also did not stimulate immune signaling through the DNA-sensing pathways. Interestingly, an MHV68 mutant lacking deubiquitinase (DUB) activity, embedded within the large tegument protein open reading frame (ORF)64, gained the capacity to stimulate the DNA-activated stimulator of IFN genes (STING) pathway. We found that ORF64 targeted a step in the DNA-activated pathways upstream of the bifurcation into the STING and absent in melanoma 2 pathways, and lack of the ORF64 DUB was associated with impaired delivery of viral DNA to the nucleus, which, instead, localized to the cytoplasm. Correspondingly, the ORF64 DUB active site mutant virus exhibited impaired ability to establish latent infection in wild-type, but not STING-deficient, mice. Thus, gammaherpesviruses evade immune activation by the cytosolic DNA-sensing pathway, which, in the MHV68 model, facilitates establishment of infections.
Asunto(s)
ADN Viral/inmunología , Gammaherpesvirinae/inmunología , Infecciones por Herpesviridae/inmunología , Inmunidad Innata/inmunología , Latencia del Virus/inmunología , Animales , Citosol/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Macrófagos/inmunología , Macrófagos/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The innate immune system is important for control of infections, including herpesvirus infections. Intracellular DNA potently stimulates antiviral IFN responses. It is known that plasmacytoid dendritic cells sense herpesvirus DNA in endosomes via TLR9 and that nonimmune tissue cells can sense herpesvirus DNA in the nucleus. However, it remains unknown how and where myeloid cells, such as macrophages and conventional dendritic cells, detect infections with herpesviruses. In this study, we demonstrate that the HSV-1 capsid was ubiquitinated in the cytosol and degraded by the proteasome, hence releasing genomic DNA into the cytoplasm for detection by DNA sensors. In this context, the DNA sensor IFN-γ-inducible 16 is important for induction of IFN-ß in human macrophages postinfection with HSV-1 and CMV. Viral DNA localized to the same cytoplasmic regions as did IFN-γ-inducible 16, with DNA sensing being independent of viral nuclear entry. Thus, proteasomal degradation of herpesvirus capsids releases DNA to the cytoplasm for recognition by DNA sensors.
Asunto(s)
Cápside/metabolismo , Citomegalovirus/metabolismo , ADN Viral/genética , Herpesvirus Humano 1/metabolismo , Macrófagos/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Línea Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citomegalovirus/genética , Citosol/metabolismo , ADN Viral/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/virología , Silenciador del Gen , Herpesvirus Humano 1/genética , Humanos , Interferón beta/biosíntesis , Interferón beta/inmunología , Macrófagos/virología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/inmunología , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/inmunología , ARN Interferente Pequeño/genética , Ubiquitinación , Células VeroRESUMEN
The innate immune response constitutes the first line of defense against infections. Pattern recognition receptors recognize pathogen structures and trigger intracellular signaling pathways leading to cytokine and chemokine expression. Reactive oxygen species (ROS) are emerging as an important regulator of some of these pathways. ROS directly interact with signaling components or induce other post-translational modifications such as S-glutathionylation, thereby altering target function. Applying live microscopy, we have demonstrated that herpes simplex virus (HSV) infection induces early production of ROS that are required for the activation of NF-κB and IRF-3 pathways and the production of type I IFNs and ISGs. All the known receptors involved in the recognition of HSV were shown to be dependent on the cellular redox levels for successful signaling. In addition, we provide biochemical evidence suggesting S-glutathionylation of TRAF family proteins to be important. In particular, by performing mutational studies we show that S-glutathionylation of a conserved cysteine residue of TRAF3 and TRAF6 is important for ROS-dependent activation of innate immune pathways. In conclusion, these findings demonstrate that ROS are essential for effective activation of signaling pathways leading to a successful innate immune response against HSV infection.
Asunto(s)
Herpes Simple/inmunología , Herpes Simple/metabolismo , Inmunidad Innata , Especies Reactivas de Oxígeno/metabolismo , Receptores de Reconocimiento de Patrones/metabolismo , Simplexvirus/inmunología , Factor 3 Asociado a Receptor de TNF/metabolismo , Animales , Quimiocinas/metabolismo , Citocinas/metabolismo , Glutatión/metabolismo , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/inmunología , Transducción de Señal , Simplexvirus/patogenicidad , Factor 3 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/metabolismoRESUMEN
Autophagy has been established as a player in host defense against viruses. The mechanisms by which the host induces autophagy during infection are diverse. In the case of HSV type 1 (HSV-1), dsRNA-dependent protein kinase is essential for induction of autophagy in fibroblasts through phosphorylation of eukaryotic initiation factor 2α (eIF2α). HSV-1 counteracts autophagy via ICP34.5, which dephosphorylates eIF2α and inhibits Beclin 1. Investigation of autophagy during HSV-1 infection has largely been conducted in permissive cells, but recent work suggests the existence of a eIF2α-independent autophagy-inducing pathway in nonpermissive cells. To clarify and further characterize the existence of a novel autophagy-inducing pathway in nonpermissive cells, we examined different HSV and cellular components in murine myeloid cells for their role in autophagy. We demonstrate that HSV-1-induced autophagy does not correlate with phosphorylation of eIF2α, is independent of functional dsRNA-dependent protein kinase, and is not antagonized by ICP34.5. Autophagy was activated independent of viral gene expression, but required viral entry. Importantly, we found that the presence of genomic DNA in the virion was essential for induction of autophagy and, conversely, that transfection of HSV-derived DNA induced microtubule-associated protein 1 L chain II formation, a marker of autophagy. This occurred through a mechanism dependent on stimulator of IFN genes, an essential component for the IFN response to intracellular DNA. Finally, we observed that HSV-1 DNA was present in the cytosol devoid of capsid material following HSV-1 infection of dendritic cells. Thus, our data suggest that HSV-1 genomic DNA induces autophagy in nonpermissive cells in a stimulator of IFN gene-dependent manner.
Asunto(s)
Autofagia/inmunología , Citosol/virología , ADN Viral , Herpesvirus Humano 1/inmunología , Proteínas de la Membrana/fisiología , Células Mieloides/inmunología , Células Mieloides/virología , Animales , Autofagia/genética , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/virología , Línea Celular , Citosol/inmunología , ADN Viral/genética , Células Dendríticas/inmunología , Células Dendríticas/virología , Femenino , Proteínas de la Membrana/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Células Mieloides/citologíaRESUMEN
Realising the promise of genomics to revolutionise identification and surveillance of antimicrobial resistance (AMR) has been a long-standing challenge in clinical and public health microbiology. Here, we report the creation and validation of abritAMR, an ISO-certified bioinformatics platform for genomics-based bacterial AMR gene detection. The abritAMR platform utilises NCBI's AMRFinderPlus, as well as additional features that classify AMR determinants into antibiotic classes and provide customised reports. We validate abritAMR by comparing with PCR or reference genomes, representing 1500 different bacteria and 415 resistance alleles. In these analyses, abritAMR displays 99.9% accuracy, 97.9% sensitivity and 100% specificity. We also compared genomic predictions of phenotype for 864 Salmonella spp. against agar dilution results, showing 98.9% accuracy. The implementation of abritAMR in our institution has resulted in streamlined bioinformatics and reporting pathways, and has been readily updated and re-verified. The abritAMR tool and validation datasets are publicly available to assist laboratories everywhere harness the power of AMR genomics in professional practice.
Asunto(s)
Antibacterianos , Farmacorresistencia Bacteriana , Antibacterianos/farmacología , Flujo de Trabajo , Farmacorresistencia Bacteriana/genética , Genómica , Biología Computacional , Pruebas de Sensibilidad MicrobianaRESUMEN
Reactive oxygen species (ROS) are crucial secondary messengers of signaling pathways. Redox-dependent signaling events have been previously described in the innate immune response. However, the mechanism by which ROS modulates anti-viral innate immune signaling is not fully clarified. Here, we report that mitochondria-derived ROS differentially regulate the innate response to DNA and RNA viruses (herpes simplex virus (HSV) and Sendai virus (SeV), respectively), with the cytokine response to HSV being negatively regulated by mitochondrial ROS. Importantly, specific activation of Toll-like receptors (TLRs) and DNA receptors (DNARs) but not retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), led to signaling cascades that were inhibited by mitochondrial ROS production. Thus, localized mitochondrial ROS exerts negative modulation of innate immune responses to the DNA virus HSV-2 but not the RNA virus SeV.
Asunto(s)
Herpesvirus Humano 2/inmunología , Inmunidad Innata , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Virus Sendai/inmunología , Animales , Citocinas/biosíntesis , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , ADN Viral/inmunología , Factor 3 Regulador del Interferón/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , ARN Viral/inmunología , Receptores de Superficie Celular/metabolismo , Transducción de SeñalRESUMEN
Innate recognition of viruses is mediated by pattern recognition receptors (PRRs) triggering expression of antiviral interferons (IFNs) and proinflammatory cytokines. In mice, Toll-like receptor 2 (TLR2) and TLR9 as well as intracellular nucleotide-sensing pathways have been shown to recognize herpes simplex virus (HSV). Here, we describe how human primary macrophages recognize early HSV infection via intracellular pathways. A number of inflammatory cytokines, IFNs, and IFN-stimulated genes were upregulated after HSV infection. We show that early recognition of HSV and induction of IFNs and inflammatory cytokines are independent of TLR2 and TLR9, since inhibition of TLR2 using TLR2 neutralizing antibodies did not affect virus-induced responses and the macrophages were unresponsive to TLR9 stimulation. Instead, HSV recognition involves intracellular recognition systems, since induction of tumor necrosis factor alpha (TNF-α) and IFNs was dependent on virus entry and replication. Importantly, expression of IFNs was strongly inhibited by small interfering RNA (siRNA) knockdown of MAVS, but this MAVS-dependent IFN induction occurred independently of the recently discovered polymerase III (Pol III)/RIG-I DNA sensing system. In contrast, induction of TNF-α was largely independent of MAVS, suggesting that induction of inflammatory cytokines during HSV infection proceeds via a novel pathway. Transfection with ODN2006, a broad inhibitor of intracellular nucleotide recognition, revealed that nucleotide-sensing systems are employed to induce both IFNs and TNF-α. Finally, using siRNA knockdown, we found that MDA5, but not RIG-I, was the primary mediator of HSV recognition. Thus, innate recognition of HSV by human primary macrophages occurs via two distinct intracellular nucleotide-sensing pathways responsible for induction of IFNs and inflammatory cytokine expression, respectively.
Asunto(s)
ARN Helicasas DEAD-box/inmunología , Inmunidad Innata , Macrófagos/virología , Simplexvirus/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Cultivadas , Citocinas/biosíntesis , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , Humanos , Helicasa Inducida por Interferón IFIH1 , Interferones/biosíntesis , ARN Polimerasa III/metabolismo , Receptores Inmunológicos , Transducción de Señal/inmunologíaRESUMEN
The 2'-5' oligoadenylate synthetase (OAS) proteins are traditionally considered intracellular antiviral proteins. However, several studies demonstrate a correlation between the concentration of freely circulating OAS protein in sera from hepatitis C patients and their clinical prognosis. Here we demonstrate that extracellular OAS1 enters into cells and possesses a strong antiviral activity, both in vitro and in vivo, which is independent of RNase L. The OAS protein directly inhibits viral proliferation and does not require the activation of known antiviral signaling pathways. We propose that OAS produced by cells infected with viruses is released to the extracellular space, where it acts as a paracrine antiviral agent. Thus, the OAS protein represents the first direct antiviral compound released by virus-infected cells.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/inmunología , Antivirales/inmunología , Endorribonucleasas/inmunología , Espacio Extracelular/enzimología , Interacciones Huésped-Patógeno , Virosis/enzimología , Virosis/inmunología , Virus/inmunología , 2',5'-Oligoadenilato Sintetasa/genética , Animales , Línea Celular , Endorribonucleasas/genética , Espacio Extracelular/inmunología , Humanos , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Virosis/virología , Fenómenos Fisiológicos de los VirusRESUMEN
Akt is a crucial phosphoinositide 3-kinase (PI(3)K) effector that regulates cell proliferation and survival. PI(3)K-generated signals, PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2), direct Akt plasma membrane engagement. Pathological Akt plasma membrane association promotes oncogenesis. PtdIns(3,4)P(2) is degraded by inositol polyphosphate 4-phosphatase-1 (4-ptase-1) forming PtdIns(3)P; however, the role of 4-ptase-1 in regulating the activation and function of Akt is unclear. In mouse embryonic fibroblasts lacking 4-ptase-1 ((-/-)MEFs), the Akt-pleckstrin homology (PH) domain was constitutively membrane-associated both in serum-starved and agonist-stimulated cells, in contrast to (+/+)MEFs, in which it was detected only at the plasma membrane following serum stimulation. Epidermal growth factor (EGF) stimulation resulted in increased Ser(473) and Thr(308)-Akt phosphorylation and activation of Akt-dependent signalling in (-/-)MEFs, relative to (+/+)MEFs. Significantly, loss of 4-ptase-1 resulted in increased cell proliferation and decreased apoptosis. SV40-transformed (-/-)MEFs showed increased anchorage-independent cell growth and formed tumours in nude mice. This study provides the first evidence, to our knowledge, that 4-ptase-1 controls the activation of Akt and thereby cell proliferation, survival and tumorigenesis.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Monoéster Fosfórico Hidrolasas/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cicloheximida/farmacología , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos , Ratones , Monoéster Fosfórico Hidrolasas/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Estaurosporina/farmacologíaRESUMEN
Phosphoinositides are membrane-bound signalling molecules that regulate cell proliferation and survival, cytoskeletal reorganization and vesicular trafficking by recruiting effector proteins to cellular membranes. Growth factor or insulin stimulation induces a canonical cascade resulting in the transient phosphorylation of PtdIns(4,5)P(2) by PI3K (phosphoinositide 3-kinase) to form PtdIns(3,4,5)P(3), which is rapidly dephosphorylated either by PTEN (phosphatase and tensin homologue deleted on chromosome 10) back to PtdIns(4,5)P(2), or by the 5-ptases (inositol polyphosphate 5-phosphatases), generating PtdIns(3,4)P(2). The 5-ptases also hydrolyse PtdIns(4,5)P(2), forming PtdIns4P. Ten mammalian 5-ptases have been identified, which share a catalytic mechanism similar to that of the apurinic/apyrimidinic endonucleases. Gene-targeted deletion of 5-ptases in mice has revealed that these enzymes regulate haemopoietic cell proliferation, synaptic vesicle recycling, insulin signalling, endocytosis, vesicular trafficking and actin polymerization. Several studies have revealed that the molecular basis of Lowe's syndrome is due to mutations in the 5-ptase OCRL (oculocerebrorenal syndrome of Lowe). Futhermore, the 5-ptases SHIP [SH2 (Src homology 2)-domain-containing inositol phosphatase] 2, SKIP (skeletal muscle- and kidney-enriched inositol phosphatase) and 72-5ptase (72 kDa 5-ptase)/Type IV/Inpp5e (inositol polyphosphate 5-phosphatase E) are implicated in negatively regulating insulin signalling and glucose homoeostasis in specific tissues. SHIP2 polymorphisms are associated with a predisposition to insulin resistance. Gene profiling studies have identified changes in the expression of various 5-ptases in specific cancers. In addition, 5-ptases such as SHIP1, SHIP2 and 72-5ptase/Type IV/Inpp5e regulate macrophage phagocytosis, and SHIP1 also controls haemopoietic cell proliferation. Therefore the 5-ptases are a significant family of signal-modulating enzymes that govern a plethora of cellular functions by regulating the levels of specific phosphoinositides. Emerging studies have implicated their loss or gain of function in human disease.
Asunto(s)
Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Humanos , Inositol Polifosfato 5-Fosfatasas , Neoplasias/genética , Neoplasias/metabolismo , Síndrome Oculocerebrorrenal/genética , Síndrome Oculocerebrorrenal/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Fosfatidilinositoles/metabolismo , Monoéster Fosfórico Hidrolasas/fisiologíaRESUMEN
Complete genomes of microbial pathogens are essential for the phylogenomic analyses that increasingly underpin core public health laboratory activities. Here, we announce a BioProject (PRJNA556438) dedicated to sharing complete genomes chosen to represent a range of pathogenic bacteria with regional importance to Australia and the Southwest Pacific; enriching the catalogue of globally available complete genomes for public health while providing valuable strains to regional public health microbiology laboratories. In this first step, we present 26 complete high-quality bacterial genomes. Additionally, we describe here a framework for reconstructing complete microbial genomes and highlight some of the challenges and considerations for accurate and reproducible genome reconstruction.
Asunto(s)
Bacterias/clasificación , Genoma Bacteriano , Secuenciación Completa del Genoma/métodos , Australia , Bacterias/genética , Bases de Datos Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Filogenia , Salud PúblicaRESUMEN
Exogenous delivery of carrier-linked phosphatidylinositol 3-phosphate [PtdIns(3)P] to adipocytes promotes the trafficking, but not the insertion, of the glucose transporter GLUT4 into the plasma membrane. However, it is yet to be demonstrated if endogenous PtdIns(3)P regulates GLUT4 trafficking and, in addition, the metabolic pathways mediating plasma membrane PtdIns(3)P synthesis are uncharacterized. In unstimulated 3T3-L1 adipocytes, conditions under which PtdIns(3,4,5)P3 was not synthesized, ectopic expression of wild-type, but not catalytically inactive 72-kDa inositol polyphosphate 5-phosphatase (72-5ptase), generated PtdIns(3)P at the plasma membrane. Immunoprecipitated 72-5ptase from adipocytes hydrolyzed PtdIns(3,5)P2, forming PtdIns(3)P. Overexpression of the 72-5ptase was used to functionally dissect the role of endogenous PtdIns(3)P in GLUT4 translocation and/or plasma membrane insertion. In unstimulated adipocytes wild type, but not catalytically inactive, 72-5ptase, promoted GLUT4 translocation and insertion into the plasma membrane but not glucose uptake. Overexpression of FLAG-2xFYVE/Hrs, which binds and sequesters PtdIns(3)P, blocked 72-5ptase-induced GLUT4 translocation. Actin monomer binding, using latrunculin A treatment, also blocked 72-5ptase-stimulated GLUT4 translocation. 72-5ptase expression promoted GLUT4 trafficking via a Rab11-dependent pathway but not by Rab5-mediated endocytosis. Therefore, endogenous PtdIns(3)P at the plasma membrane promotes GLUT4 translocation.
Asunto(s)
Membrana Celular/enzimología , Transportador de Glucosa de Tipo 4/metabolismo , Fosfatos de Fosfatidilinositol/biosíntesis , Monoéster Fosfórico Hidrolasas/metabolismo , Células 3T3-L1 , Actinas/metabolismo , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/enzimología , Animales , Diferenciación Celular , Células Cultivadas , Expresión Génica , Hidrólisis/efectos de los fármacos , Inositol Polifosfato 5-Fosfatasas , Insulina/farmacología , Ratones , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Phosphoinositide signals regulate cell proliferation, differentiation, cytoskeletal rearrangement and intracellular trafficking. Hydrolysis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3, by inositol polyphosphate 5-phosphatases regulates synaptic vesicle recycling (synaptojanin-1), hematopoietic cell function [SHIP1(SH2-containing inositol polyphosphate 5-phosphatase-1)], renal cell function [OCRL (oculocerebrorenal syndrome of Lowe)] and insulin signalling (SHIP2). We present here a detailed review of the characteristics of the ten mammalian 5-phosphatases. Knockout mouse phenotypes and underexpression studies are associated with significant phenotypic changes, indicating non-redundant roles, despite, in many cases, overlapping substrate specificity and tissue expression. The extraordinary complexity in the control of phosphoinositide signalling continues to be revealed.
Asunto(s)
Monoéster Fosfórico Hidrolasas/fisiología , Animales , Humanos , Inositol Polifosfato 5-Fosfatasas , Insulina/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiología , Modelos Biológicos , Síndrome Oculocerebrorrenal/enzimología , Síndrome Oculocerebrorrenal/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Estructura Terciaria de Proteína , Vesículas Sinápticas/metabolismoRESUMEN
Human ciliopathies, including Joubert syndrome (JBTS), arise from cilia dysfunction. The inositol polyphosphate 5-phosphatase INPP5E localizes to cilia and is mutated in JBTS. Murine Inpp5e ablation is embryonically lethal and recapitulates JBTS, including neural tube defects and polydactyly; however, the underlying defects in cilia signaling and the function of INPP5E at cilia are still emerging. We report Inpp5e-/- embryos exhibit aberrant Hedgehog-dependent patterning with reduced Hedgehog signaling. Using mouse genetics, we show increasing Hedgehog signaling via Smoothened M2 expression rescues some Inpp5e-/- ciliopathy phenotypes and "normalizes" Hedgehog signaling. INPP5E's phosphoinositide substrates PI(4,5)P2 and PI(3,4,5)P3 accumulated at the transition zone (TZ) in Hedgehog-stimulated Inpp5e-/- cells, which was associated with reduced recruitment of TZ scaffolding proteins and reduced Smoothened levels at cilia. Expression of wild-type, but not 5-phosphatase-dead, INPP5E restored TZ molecular organization and Smoothened accumulation at cilia. Therefore, we identify INPP5E as an essential point of convergence between Hedgehog and phosphoinositide signaling at cilia that maintains TZ function and Hedgehog-dependent embryonic development.
Asunto(s)
Anomalías Múltiples/enzimología , Cerebelo/anomalías , Cilios/enzimología , Embrión de Mamíferos/enzimología , Anomalías del Ojo/enzimología , Enfermedades Renales Quísticas/enzimología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Retina/anomalías , Epitelio Pigmentado de la Retina/enzimología , Sistemas de Mensajero Secundario , Anomalías Múltiples/genética , Animales , Línea Celular , Cerebelo/enzimología , Modelos Animales de Enfermedad , Anomalías del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Predisposición Genética a la Enfermedad , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Enfermedades Renales Quísticas/genética , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Monoéster Fosfórico Hidrolasas/deficiencia , Monoéster Fosfórico Hidrolasas/genética , Retina/enzimología , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Factores de Tiempo , Transfección , Proteína Gli2 con Dedos de ZincRESUMEN
Oncogenic mutations in PIK3CA lead to an increase in intrinsic phosphoinositide kinase activity, but it is thought that increased access of PI3Kα (phosphoinositide 3-kinase α) to its PM (plasma membrane) localized substrate is also required for increased levels of downstream PIP3/Akt [phosphoinositide-3,4,5-trisphosphate/also called PKB (protein kinase B)] signalling. We have studied the subcellular localization of wild-type and the two most common oncogenic mutants of PI3Kα in cells maintained in growth media, and starved or stimulated cells using a novel method in which PI3Kα is pre-formed as a 1:1 p110α:p85α complex in vitro then introduced into live cells by microinjection. Oncogenic E545K and H1047R mutants did not constitutively interact with membrane lipids in vitro or in cells maintained in 10% (v/v) FBS. Following stimulation of RTKs (receptor tyrosine kinases), microinjected PI3Kα was recruited to the PM, but oncogenic forms of PI3Kα were not recruited to the PM to a greater extent and did not reside at the PM longer than the wild-type PI3Kα. Instead, the E545K mutant specifically bound activated Cdc42 in vitro and microinjection of E545K was associated with the formation of cellular protrusions, providing some preliminary evidence that changes in protein-protein interactions may play a role in the oncogenicity of the E545K mutant in addition to the well-known changes in lipid kinase activity.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Mutación Missense , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Sustitución de Aminoácidos , Animales , Membrana Celular/enzimología , Membrana Celular/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Perros , Humanos , Células de Riñón Canino Madin Darby , Lípidos de la Membrana/genética , Lípidos de la Membrana/metabolismo , Transporte de Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteína de Unión al GTP cdc42/genéticaRESUMEN
BACKGROUND: Innate immune responses have recently been appreciated to play an important role in the pathogenesis of HIV infection. Whereas inadequate innate immune sensing of HIV during acute infection may contribute to failure to control and eradicate infection, persistent inflammatory responses later during infection contribute in driving chronic immune activation and development of immunodeficiency. However, knowledge on specific HIV PAMPs and cellular PRRs responsible for inducing innate immune responses remains sparse. METHODS/PRINCIPAL FINDINGS: Here we demonstrate a major role for RIG-I and the adaptor protein MAVS in induction of innate immune responses to HIV genomic RNA. We found that secondary structured HIV-derived RNAs induced a response similar to genomic RNA. In primary human peripheral blood mononuclear cells and primary human macrophages, HIV RNA induced expression of IFN-stimulated genes, whereas only low levels of type I IFN and tumor necrosis factor α were produced. Furthermore, secondary structured HIV-derived RNA activated pathways to NF-κB, MAP kinases, and IRF3 and co-localized with peroxisomes, suggesting a role for this organelle in RIG-I-mediated innate immune sensing of HIV RNA. CONCLUSIONS/SIGNIFICANCE: These results establish RIG-I as an innate immune sensor of cytosolic HIV genomic RNA with secondary structure, thereby expanding current knowledge on HIV molecules capable of stimulating the innate immune system.