RESUMEN
Allergies are considered to represent mal-directed type 2 immune responses against mostly innocuous exogenous compounds. Immunoglobulin E (IgE) antibodies are a characteristic feature of allergies and mediate hypersensitivity against allergens through activation of effector cells, particularly mast cells (MCs). Although the physiological functions of this dangerous branch of immunity have remained enigmatic, recent evidence shows that allergic immune reactions can help to protect against the toxicity of venoms. Because bacteria are a potent alternative source of toxins, we assessed the possible role of allergy-like type 2 immunity in antibacterial host defense. We discovered that the adaptive immune response against Staphylococcus aureus (SA) skin infection substantially improved systemic host defense against secondary SA infections in mice. Moreover, this acquired protection depended on IgE effector mechanisms and MCs. Importantly, our results reveal a previously unknown physiological function of allergic immune responses, IgE antibodies, and MCs in host defense against a pathogenic bacterium.
Asunto(s)
Inmunidad Adaptativa/inmunología , Inmunoglobulina E/inmunología , Mastocitos/inmunología , Infecciones Estafilocócicas/inmunología , Infecciones Cutáneas Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Alérgenos/inmunología , Animales , Femenino , Hipersensibilidad/inmunología , Hipersensibilidad/microbiología , Mastocitos/microbiología , Ratones , Ratones Endogámicos C57BL , Piel/inmunología , Piel/microbiología , Infecciones Estafilocócicas/microbiología , Infecciones Cutáneas Estafilocócicas/microbiologíaRESUMEN
Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.
Asunto(s)
Hepacivirus/genética , Hepacivirus/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Sistemas CRISPR-Cas , Transferencia Resonante de Energía de Fluorescencia , Técnicas de Inactivación de Genes , Células HeLa , Hepacivirus/patogenicidad , Interacciones Huésped-Patógeno , Humanos , Cinética , Datos de Secuencia Molecular , O(6)-Metilguanina-ADN Metiltransferasa/genética , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Ribosómicas/antagonistas & inhibidores , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismoRESUMEN
Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7(-/-) mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections.
Asunto(s)
Uniones Adherentes/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/metabolismo , Staphylococcus aureus Resistente a Meticilina/metabolismo , Infecciones Estafilocócicas/metabolismo , Vasculitis/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Uniones Adherentes/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Toxinas Bacterianas/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Proteínas Hemolisinas/genética , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Ratones Noqueados , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/patología , Vasculitis/genética , Vasculitis/microbiología , Vasculitis/patologíaRESUMEN
Whether or not autophagy has a role in defence against Mycobacterium tuberculosis infection remains unresolved. Previously, conditional knockdown of the core autophagy component ATG5 in myeloid cells was reported to confer extreme susceptibility to M. tuberculosis in mice, whereas depletion of other autophagy factors had no effect on infection. We show that doubling cre gene dosage to more robustly deplete ATG16L1 or ATG7 resulted in increased M. tuberculosis growth and host susceptibility in mice, although ATG5-depleted mice are more sensitive than ATG16L1- or ATG7-depleted mice. We imaged individual macrophages infected with M. tuberculosis and identified a shift from apoptosis to rapid necrosis in autophagy-depleted cells. This effect was dependent on phagosome permeabilization by M. tuberculosis. We monitored infected cells by electron microscopy, showing that autophagy protects the host macrophage by partially reducing mycobacterial access to the cytosol. We conclude that autophagy has an important role in defence against M. tuberculosis in mammals.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Ratones , Animales , Tuberculosis/microbiología , Autofagia/genética , Macrófagos/microbiología , Proteína 5 Relacionada con la Autofagia/genética , MamíferosRESUMEN
Macrophages are highly plastic cells with critical roles in immunity, cancer, and tissue homeostasis, but how these distinct cellular fates are triggered by environmental cues is poorly understood. To uncover how primary murine macrophages respond to bacterial pathogens, we globally assessed changes in post-translational modifications of proteins during infection with Mycobacterium tuberculosis, a notorious intracellular pathogen. We identified hundreds of dynamically regulated phosphorylation and ubiquitylation sites, indicating that dramatic remodeling of multiple host pathways, both expected and unexpected, occurred during infection. Most of these cellular changes were not captured by mRNA profiling, and included activation of ubiquitin-mediated autophagy, an evolutionarily ancient cellular antimicrobial system. This analysis also revealed that a particular autophagy receptor, TAX1BP1, mediates clearance of ubiquitylated Mtb and targets bacteria to LC3-positive phagophores. These studies provide a new resource for understanding how macrophages shape their proteome to meet the challenge of infection.
Asunto(s)
Macrófagos/microbiología , Mycobacterium tuberculosis/patogenicidad , Procesamiento Proteico-Postraduccional , Tuberculosis/metabolismo , Animales , Autofagia/inmunología , Proteínas Bacterianas/metabolismo , Humanos , Macrófagos/inmunología , Ratones , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/metabolismo , Fosforilación , Proteoma , Tuberculosis/inmunología , Tuberculosis/microbiología , UbiquitinaciónRESUMEN
Macrophages play critical roles in immunity, development, tissue repair, and cancer, but studies of their function have been hampered by poorly-differentiated tumor cell lines and genetically-intractable primary cells. Here we report a facile system for genome editing in non-transformed macrophages by differentiating ER-Hoxb8 myeloid progenitors from Cas9-expressing transgenic mice. These conditionally immortalized macrophages (CIMs) retain characteristics of primary macrophages derived from the bone marrow yet allow for easy genetic manipulation and a virtually unlimited supply of cells. We demonstrate the utility of this system for dissection of host genetics during intracellular bacterial infection using two important human pathogens: Listeria monocytogenes and Mycobacterium tuberculosis.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Listeria monocytogenes/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Mycobacterium tuberculosis/inmunología , Animales , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/microbiología , Línea Celular , Células Cultivadas , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Proteínas de Homeodominio/metabolismo , Interacciones Huésped-Patógeno/inmunología , Humanos , Listeria monocytogenes/fisiología , Macrófagos/microbiología , Ratones Transgénicos , Mycobacterium tuberculosis/fisiología , Células Madre/inmunología , Células Madre/metabolismoRESUMEN
We previously identified PLEKHA7 and other junctional proteins as host factors mediating death by S. aureus α-toxin, but the mechanism through which junctions promote toxicity was unclear. Using cell biological and biochemical methods, we now show that ADAM10 is docked to junctions by its transmembrane partner Tspan33, whose cytoplasmic C terminus binds to the WW domain of PLEKHA7 in the presence of PDZD11. ADAM10 is locked at junctions through binding of its cytoplasmic C terminus to afadin. Junctionally clustered ADAM10 supports the efficient formation of stable toxin pores. Instead, disruption of the PLEKHA7-PDZD11 complex inhibits ADAM10 and toxin junctional clustering. This promotes toxin pore removal from the cell surface through an actin- and macropinocytosis-dependent process, resulting in cell recovery from initial injury and survival. These results uncover a dock-and-lock molecular mechanism to target ADAM10 to junctions and provide a paradigm for how junctions regulate transmembrane receptors through their clustering.
Asunto(s)
Proteína ADAM10/metabolismo , Toxinas Bacterianas/toxicidad , Proteínas Hemolisinas/toxicidad , Uniones Intercelulares/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Humanos , Uniones Intercelulares/efectos de los fármacos , Proteínas de Microfilamentos/metabolismo , Pinocitosis/efectos de los fármacos , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Tetraspaninas/metabolismoRESUMEN
The innate immune system tightly regulates activation of interferon-stimulated genes (ISGs) to avoid inappropriate expression. Pathological ISG activation resulting from aberrant nucleic acid metabolism has been implicated in autoimmune disease; however, the mechanisms governing ISG suppression are unknown. Through a genome-wide genetic screen, we identified DEAD-box helicase 6 (DDX6) as a suppressor of ISGs. Genetic ablation of DDX6 induced global upregulation of ISGs and other immune genes. ISG upregulation proved cell intrinsic, imposing an antiviral state and making cells refractory to divergent families of RNA viruses. Epistatic analysis revealed that ISG activation could not be overcome by deletion of canonical RNA sensors. However, DDX6 deficiency was suppressed by disrupting LSM1, a core component of mRNA degradation machinery, suggesting that dysregulation of RNA processing underlies ISG activation in the DDX6 mutant. DDX6 is distinct among DExD/H helicases that regulate the antiviral response in its singular ability to negatively regulate immunity.