RESUMEN
Arboviruses are a diverse group of insect-transmitted pathogens that pose global public health challenges. Identifying evolutionarily conserved host factors that combat arbovirus replication in disparate eukaryotic hosts is important as they may tip the balance between productive and abortive viral replication, and thus determine virus host range. Here, we exploit naturally abortive arbovirus infections that we identified in lepidopteran cells and use bacterial effector proteins to uncover host factors restricting arbovirus replication. Bacterial effectors are proteins secreted by pathogenic bacteria into eukaryotic hosts cells that can inhibit antimicrobial defenses. Since bacteria and viruses can encounter common host defenses, we hypothesized that some bacterial effectors may inhibit host factors that restrict arbovirus replication in lepidopteran cells. Thus, we used bacterial effectors as molecular tools to identify host factors that restrict four distinct arboviruses in lepidopteran cells. By screening 210 effectors encoded by seven different bacterial pathogens, we identify several effectors that individually rescue the replication of all four arboviruses. We show that these effectors encode diverse enzymatic activities that are required to break arbovirus restriction. We further characterize Shigella flexneri-encoded IpaH4 as an E3 ubiquitin ligase that directly ubiquitinates two evolutionarily conserved proteins, SHOC2 and PSMC1, promoting their degradation in insect and human cells. We show that depletion of either SHOC2 or PSMC1 in insect or human cells promotes arbovirus replication, indicating that these are ancient virus restriction factors conserved across invertebrate and vertebrate hosts. Collectively, our study reveals a novel pathogen-guided approach to identify conserved antimicrobial machinery, new effector functions, and conserved roles for SHOC2 and PSMC1 in virus restriction.
Asunto(s)
Proteínas Bacterianas , Interacciones Huésped-Patógeno , Replicación Viral , Animales , Replicación Viral/fisiología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Humanos , Arbovirus , Shigella flexneri/patogenicidad , Infecciones por Arbovirus/virología , Línea CelularRESUMEN
The human facilitates chromatin transcription (FACT) complex is a chromatin remodeller composed of human suppressor of Ty 16 homologue (hSpt16) and structure-specific recognition protein-1 subunits that regulates cellular gene expression. Whether FACT regulates host responses to infection remained unclear. We identify a FACT-mediated, interferon-independent, antiviral pathway that restricts poxvirus replication. Cell culture and bioinformatics approaches suggest that early viral gene expression triggers nuclear accumulation of SUMOylated hSpt16 subunits required for the expression of E26 transformation-specific sequence-1 (ETS-1)-a transcription factor that activates virus restriction programs. However, biochemical studies show that poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting structure-specific recognition protein-1 binding to SUMOylated hSpt16 and by tethering SUMOylated hSpt16 to microtubules. Furthermore, A51R antagonism of FACT enhances poxvirus replication in human cells and virulence in mice. Finally, we show that FACT also restricts rhabdoviruses, flaviviruses and orthomyxoviruses, suggesting broad roles for FACT in antiviral immunity. Our study reveals the FACT-ETS-1 antiviral response (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.
Asunto(s)
Evasión Inmune , Interferones , Humanos , Animales , Ratones , CromatinaRESUMEN
Monkeypox virus (MPXV) infections in humans cause neurological disorders while studies of MPXV-infected animals indicate that the virus penetrates the brain. Pyroptosis is an inflammatory type of regulated cell death, resulting from plasma membrane rupture (PMR) due to oligomerization of cleaved gasdermins to cause membrane pore formation. Herein, we investigated the human neural cell tropism of MPXV compared to another orthopoxvirus, vaccinia virus (VACV), as well as its effects on immune responses and cell death. Astrocytes were most permissive to MPXV (and VACV) infections, followed by microglia and oligodendrocytes, with minimal infection of neurons based on plaque assays. Aberrant morphological changes were evident in MPXV-infected astrocytes that were accompanied with viral protein (I3) immunolabelling and detection of over 125 MPXV-encoded proteins in cell lysates by mass spectrometry. MPXV- and VACV-infected astrocytes showed increased expression of immune gene transcripts (IL12, IRF3, IL1B, TNFA, CASP1, and GSDMB). However, MPXV infection of astrocytes specifically induced proteolytic cleavage of gasdermin B (GSDMB) (50 kDa), evident by the appearance of cleaved N-terminal-GSDMB (30 kDa) and C-terminal- GSDMB (18 kDa) fragments. GSDMB cleavage was associated with release of lactate dehydrogenase and increased cellular nucleic acid staining, indicative of PMR. Pre-treatment with dimethyl fumarate reduced cleavage of GSDMB and associated PMR in MPXV-infected astrocytes. Human astrocytes support productive MPXV infection, resulting in inflammatory gene induction with accompanying GSDMB-mediated pyroptosis. These findings clarify the recently recognized neuropathogenic effects of MPXV in humans while also offering potential therapeutic options.
Asunto(s)
Monkeypox virus , Mpox , Animales , Humanos , Monkeypox virus/fisiología , Piroptosis , Astrocitos , GasderminasRESUMEN
Arboviruses are a diverse group of insect-transmitted pathogens that pose global public health challenges. Identifying evolutionarily conserved host factors that combat arbovirus replication in disparate eukaryotic hosts is important as they may tip the balance between productive and abortive viral replication, and thus determine virus host range. Here, we exploit naturally abortive arbovirus infections that we identified in lepidopteran cells and use bacterial effector proteins to uncover host factors restricting arbovirus replication. Bacterial effectors are proteins secreted by pathogenic bacteria into eukaryotic hosts cells that can inhibit antimicrobial defenses. Since bacteria and viruses can encounter common host defenses, we hypothesized that some bacterial effectors may inhibit host factors that restrict arbovirus replication in lepidopteran cells. Thus, we used bacterial effectors as molecular tools to identify host factors that restrict four distinct arboviruses in lepidopteran cells. By screening 210 effectors encoded by seven different bacterial pathogens, we identify six effectors that individually rescue the replication of all four arboviruses. We show that these effectors encode diverse enzymatic activities that are required to break arbovirus restriction. We further characterize Shigella flexneri-encoded IpaH4 as an E3 ubiquitin ligase that directly ubiquitinates two evolutionarily conserved proteins, SHOC2 and PSMC1, promoting their degradation in insect and human cells. We show that depletion of either SHOC2 or PSMC1 in insect or human cells promotes arbovirus replication, indicating that these are ancient virus restriction factors conserved across invertebrate and vertebrate hosts. Collectively, our study reveals a novel pathogen-guided approach to identify conserved antimicrobial machinery, new effector functions, and conserved roles for SHOC2 and PSMC1 in virus restriction.
RESUMEN
The FACT complex is an ancient chromatin remodeling factor comprised of Spt16 and SSRP1 subunits that regulates specific eukaryotic gene expression programs. However, whether FACT regulates host immune responses to infection was unclear. Here, we identify an antiviral pathway mediated by FACT, distinct from the interferon response, that restricts poxvirus replication. We show that early viral gene expression triggers nuclear accumulation of specialized, SUMOylated Spt16 subunits of FACT required for expression of ETS-1, a downstream transcription factor that activates a virus restriction program. However, poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting SSRP1 for binding to SUMOylated Spt16 in the cytosol and by tethering SUMOylated Spt16 to microtubules. Moreover, we show that A51R antagonism of FACT enhances both poxvirus replication in human cells and viral virulence in mice. Finally, we demonstrate that FACT also restricts unrelated RNA viruses, suggesting a broad role for FACT in antiviral immunity. Our study reveals the F ACT- E TS-1 A ntiviral R esponse (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.
RESUMEN
The multifunctional GSDMB protein is an important molecule in human immunity. The pyroptotic and bactericidal activity of GSDMB is a host response to infection by the bacterial pathogen Shigella flexneri, which employs the virulence effector IpaH7.8 to ubiquitinate and target GSDMB for proteasome-dependent degradation. Furthermore, IpaH7.8 selectively targets human but not mouse GSDMD, suggesting a non-canonical mechanism of substrate selection. Here, we report the crystal structure of GSDMB in complex with IpaH7.8. Together with biochemical and functional studies, we identify the potential membrane engagement sites of GSDMB, revealing general and unique features of gasdermin proteins in membrane recognition. We further illuminate how IpaH7.8 interacts with GSDMB, and delineate the mechanism by which IpaH7.8 ubiquitinates and suppresses GSDMB. Notably, guided by our structural model, we demonstrate that two residues in the α1-α2 loop make the mouse GSDMD invulnerable to IpaH7.8-mediated degradation. These findings provide insights into the versatile functions of GSDMB, which could open new avenues for therapeutic interventions for diseases, including cancers and bacterial infections.
Asunto(s)
Gasderminas , Piroptosis , Shigella flexneri , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/fisiología , Muerte Celular , Gasderminas/metabolismo , Gasderminas/fisiología , Proteínas Citotóxicas Formadoras de Poros , Shigella flexneri/metabolismo , Shigella flexneri/patogenicidadRESUMEN
Most of the cholesterol in the plasma membranes (PMs) of animal cells is sequestered through interactions with phospholipids and transmembrane domains of proteins. However, as cholesterol concentration rises above the PM's sequestration capacity, a new pool of cholesterol, called accessible cholesterol, emerges. The transport of accessible cholesterol between the PM and the endoplasmic reticulum (ER) is critical to maintain cholesterol homeostasis. This pathway has also been implicated in the suppression of both bacterial and viral pathogens by immunomodulatory oxysterols. Here, we describe a mechanism of depletion of accessible cholesterol from PMs by the oxysterol 25-hydroxycholesterol (25HC). We show that 25HC-mediated activation of acyl coenzyme A: cholesterol acyltransferase (ACAT) in the ER creates an imbalance in the equilibrium distribution of accessible cholesterol between the ER and PM. This imbalance triggers the rapid internalization of accessible cholesterol from the PM, and this depletion is sustained for long periods of time through 25HC-mediated suppression of SREBPs and continued activation of ACAT. In support of a physiological role for this mechanism, 25HC failed to suppress Zika virus and human coronavirus infection in ACAT-deficient cells, and Listeria monocytogenes infection in ACAT-deficient cells and mice. We propose that selective depletion of accessible PM cholesterol triggered by ACAT activation and sustained through SREBP suppression underpins the immunological activities of 25HC and a functionally related class of oxysterols.
Asunto(s)
Oxiesteroles , Infección por el Virus Zika , Virus Zika , Animales , Humanos , Ratones , Oxiesteroles/metabolismo , Aciltransferasas/metabolismo , Colesterol/metabolismo , Membrana Celular/metabolismo , Bacterias/metabolismoRESUMEN
Immune stimulation fuels cell signaling-transcriptional programs inducing biological responses to eliminate virus-infected cells. Yet, retroviruses that integrate into host cell chromatin, such as HIV-1, co-opt these programs to switch between latent and reactivated states; however, the regulatory mechanisms are still unfolding. Here, we implemented a functional screen leveraging HIV-1's dependence on CD4+ T cell signaling-transcriptional programs and discovered ADAP1 is an undescribed modulator of HIV-1 proviral fate. Specifically, we report ADAP1 (ArfGAP with dual PH domain-containing protein 1), a previously thought neuronal-restricted factor, is an amplifier of select T cell signaling programs. Using complementary biochemical and cellular assays, we demonstrate ADAP1 inducibly interacts with the immune signalosome to directly stimulate KRAS GTPase activity thereby augmenting T cell signaling through targeted activation of the ERK-AP-1 axis. Single cell transcriptomics analysis revealed loss of ADAP1 function blunts gene programs upon T cell stimulation consequently dampening latent HIV-1 reactivation. Our combined experimental approach defines ADAP1 as an unexpected tuner of T cell programs facilitating HIV-1 latency escape.
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Proteínas Adaptadoras Transductoras de Señales , Infecciones por VIH , VIH-1 , Sistema de Señalización de MAP Quinasas , Proteínas del Tejido Nervioso , Proteínas Proto-Oncogénicas p21(ras) , Linfocitos T , Factor de Transcripción AP-1 , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Linfocitos T CD4-Positivos , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Humanos , Proteínas del Tejido Nervioso/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Factor de Transcripción AP-1/metabolismo , Activación Viral , Latencia del VirusRESUMEN
Toxoplasma gondii is an important human pathogen infecting an estimated one in three people worldwide. The cytokine interferon gamma (IFNγ) is induced during infection and is critical for restricting T. gondii growth in human cells. Growth restriction is presumed to be due to the induction of interferon-stimulated genes (ISGs) that are upregulated to protect the host from infection. Although there are hundreds of ISGs induced by IFNγ, their individual roles in restricting parasite growth in human cells remain somewhat elusive. To address this deficiency, we screened a library of 414 IFNγ induced ISGs to identify factors that impact T. gondii infection in human cells. In addition to IRF1, which likely acts through the induction of numerous downstream genes, we identified RARRES3 as a single factor that restricts T. gondii infection by inducing premature egress of the parasite in multiple human cell lines. Overall, while we successfully identified a novel IFNγ induced factor restricting T. gondii infection, the limited number of ISGs capable of restricting T. gondii infection when individually expressed suggests that IFNγ-mediated immunity to T. gondii infection is a complex, multifactorial process.
Asunto(s)
Expresión Génica , Interacciones Huésped-Parásitos , Interferón gamma/inmunología , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/inmunología , Toxoplasma/inmunología , Células A549 , Biblioteca de Genes , Células HEK293 , Células HeLa , Interacciones Huésped-Parásitos/genética , Interacciones Huésped-Parásitos/inmunología , Humanos , Inmunidad Innata , Interferón gamma/genética , Interferón gamma/farmacologíaRESUMEN
The minimal genetic requirements for microbes to survive within multiorganism communities, including host-pathogen interactions, remain poorly understood. Here, we combined targeted gene mutagenesis with phenotype-guided genetic reassembly to identify a cooperative network of SPI-2 T3SS effector genes that are sufficient for Salmonella Typhimurium (STm) to cause disease in a natural host organism. Five SPI-2 effector genes support pathogen survival within the host cell cytoplasm by coordinating bacterial replication with Salmonella-containing vacuole (SCV) division. Unexpectedly, this minimal genetic repertoire does not support STm systemic infection of mice. In vivo screening revealed a second effector-gene network, encoded by the spv operon, that expands the life cycle of STm from growth in cells to deep-tissue colonization in a murine model of typhoid fever. Comparison between Salmonella infection models suggests how cooperation between effector genes drives tissue tropism in a pathogen group.
Asunto(s)
Proteínas Bacterianas/genética , Redes Reguladoras de Genes , Infecciones por Salmonella/microbiología , Salmonella typhimurium/genética , Animales , Proteínas Bacterianas/metabolismo , Citoplasma/microbiología , Femenino , Islas Genómicas , Interacciones Huésped-Patógeno , Humanos , Ratones , Ratones Endogámicos C57BL , Viabilidad Microbiana , Operón , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/patogenicidad , Salmonella typhimurium/fisiología , Tropismo , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/metabolismo , VirulenciaRESUMEN
Gasdermin B (GSDMB) belongs to a large family of pore-forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to human disease, its function in the immunological response to pathogens remains poorly understood. Here, we report a dynamic host-pathogen conflict between GSDMB and the IpaH7.8 effector protein secreted by enteroinvasive Shigella flexneri. We show that IpaH7.8 ubiquitinates and targets GSDMB for 26S proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of natural killer cells by suppressing granzyme-A-mediated activation of GSDMB. In contrast to the canonical function of most gasdermin family members, GSDMB does not inhibit Shigella by lysing host cells. Rather, it exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveal a mechanism employed by pathogens to counteract this host defense system.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Interacciones Huésped-Patógeno , Células Asesinas Naturales/inmunología , Proteínas de Neoplasias/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Shigella flexneri/fisiología , Ubiquitinación , Animales , Proteínas Bacterianas/metabolismo , Cardiolipinas/metabolismo , Línea Celular , Membrana Celular/metabolismo , Femenino , Granzimas/metabolismo , Humanos , Lípido A/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Viabilidad Microbiana , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteolisis , Especificidad por SustratoRESUMEN
The toxins that some bacteria secrete to kill off rival species can also generate mutations that help toxin-resistant populations adapt to new environments.
Asunto(s)
Toxinas Bacterianas , Adaptación Fisiológica/genética , Bacterias , Toxinas Bacterianas/genética , MutaciónRESUMEN
The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure. At its full capacity, each dynamin helix captures 12-16 actin filaments on the outer rim of the helix. GTP hydrolysis by dynamin triggers disassembly of fully assembled dynamin helices, releasing free dynamin dimers/tetramers and facilitating Arp2/3-mediated branched actin polymerization. The assembly/disassembly cycles of dynamin promote continuous actin bundling to generate mechanically stiff actin super-bundles. Super-resolution and immunogold platinum replica electron microscopy revealed dynamin along actin bundles at the fusogenic synapse. These findings implicate dynamin as a unique multifilament actin-bundling protein that regulates the dynamics and mechanical strength of the actin cytoskeletal network.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Comunicación Celular , Drosophila melanogaster/metabolismo , Dinaminas/metabolismo , Endocitosis , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/genética , Secuencia de Aminoácidos , Animales , Drosophila melanogaster/genética , Dinaminas/genética , Femenino , Guanosina Trifosfato/metabolismo , Masculino , Mioblastos/citología , Mioblastos/metabolismo , Unión Proteica , Homología de SecuenciaRESUMEN
Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene that converts cholesterol to the oxysterol 25-hydroxycholesterol (25HC). Circulating 25HC modulates essential immunological processes including antiviral immunity, inflammasome activation and antibody class switching; and dysregulation of CH25H may contribute to chronic inflammatory disease and cancer. Although 25HC is a potent regulator of cholesterol storage, uptake, efflux and biosynthesis, how these metabolic activities reprogram the immunological state of target cells remains poorly understood. Here, we used recently designed toxin-based biosensors that discriminate between distinct pools of plasma membrane cholesterol to elucidate how 25HC prevents Listeria monocytogenes from traversing the plasma membrane of infected host cells. The 25HC-mediated activation of acyl-CoA:cholesterol acyltransferase (ACAT) triggered rapid internalization of a biochemically defined fraction of cholesterol, termed 'accessible' cholesterol, from the plasma membrane while having little effect on cholesterol in complexes with sphingomyelin. We show that evolutionarily distinct bacterial species, L. monocytogenes and Shigella flexneri, exploit the accessible pool of cholesterol for infection and that acute mobilization of this pool by oxysterols confers immunity to these pathogens. The significance of this signal-mediated membrane remodelling pathway probably extends beyond host defence systems, as several other biologically active oxysterols also mobilize accessible cholesterol through an ACAT-dependent mechanism.
Asunto(s)
Bacterias/inmunología , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/microbiología , Membrana Celular/metabolismo , Colesterol/metabolismo , Inmunidad Innata/efectos de los fármacos , Oxiesteroles/farmacología , Infecciones Bacterianas/tratamiento farmacológico , Colesterol/química , Citocinas/metabolismo , Células Epiteliales/microbiología , Humanos , Interferones/metabolismo , Listeria/efectos de los fármacos , Listeria/inmunología , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Oxiesteroles/química , Oxiesteroles/metabolismo , Shigella/efectos de los fármacos , Shigella/inmunología , Esterol O-Aciltransferasa/metabolismo , Relación Estructura-ActividadRESUMEN
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the most successful human pathogens. One reason for its success is that Mtb can reside within host macrophages, a cell type that normally functions to phagocytose and destroy infectious bacteria. However, Mtb is able to evade macrophage defenses in order to survive for prolonged periods of time. Many intracellular pathogens secrete virulence factors targeting host membranes and organelles to remodel their intracellular environmental niche. We hypothesized that Mtb secreted proteins that target host membranes are vital for Mtb to adapt to and manipulate the host environment for survival. Thus, we characterized 200 secreted proteins from Mtb for their ability to associate with eukaryotic membranes using a unique temperature-sensitive yeast screen and to manipulate host trafficking pathways using a modified inducible secretion screen. We identified five Mtb secreted proteins that both associated with eukaryotic membranes and altered the host secretory pathway. One of these secreted proteins, Mpt64, localized to the endoplasmic reticulum during Mtb infection of murine and human macrophages and impaired the unfolded protein response in macrophages. These data highlight the importance of secreted proteins in Mtb pathogenesis and provide a basis for further investigation into their molecular mechanisms.IMPORTANCE Advances have been made to identify secreted proteins of Mycobacterium tuberculosis during animal infections. These data, combined with transposon screens identifying genes important for M. tuberculosis virulence, have generated a vast resource of potential M. tuberculosis virulence proteins. However, the function of many of these proteins in M. tuberculosis pathogenesis remains elusive. We have integrated three cell biological screens to characterize nearly 200 M. tuberculosis secreted proteins for eukaryotic membrane binding, host subcellular localization, and interactions with host vesicular trafficking. In addition, we observed the localization of one secreted protein, Mpt64, to the endoplasmic reticulum (ER) during M. tuberculosis infection of macrophages. Interestingly, although Mpt64 is exported by the Sec pathway, its delivery into host cells was dependent upon the action of the type VII secretion system. Finally, we observed that Mpt64 impairs the ER-mediated unfolded protein response in macrophages.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno , Mycobacterium tuberculosis/metabolismo , Factores de Virulencia/metabolismo , Animales , Antígenos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Membrana Celular/metabolismo , Células Cultivadas , Retículo Endoplásmico/metabolismo , Femenino , Células HeLa , Humanos , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Células RAW 264.7 , Tuberculosis/microbiologíaRESUMEN
The non-canonical NF-κB signalling cascade is essential for lymphoid organogenesis, B cell maturation, osteoclast differentiation, and inflammation in mammals1,2; dysfunction of this system is associated with human diseases, including immunological disorders and cancer3-6. Although expression of NF-κB-inducing kinase (NIK, also known as MAP3K14) is the rate-limiting step in non-canonical NF-κB pathway activation2,7, the mechanisms by which transcriptional responses are regulated remain largely unknown. Here we show that the sine oculis homeobox (SIX) homologue family transcription factors SIX1 and SIX2 are integral components of the non-canonical NF-κB signalling cascade. The developmentally silenced SIX proteins are reactivated in differentiated macrophages by NIK-mediated suppression of the ubiquitin proteasome pathway. Consequently, SIX1 and SIX2 target a subset of inflammatory gene promoters and directly inhibit the trans-activation function of the transcription factors RELA and RELB in a negative feedback circuit. In support of a physiologically pivotal role for SIX proteins in host immunity, a human SIX1 transgene suppressed inflammation and promoted the recovery of mice from endotoxic shock. In addition, SIX1 and SIX2 protected RAS/P53-driven non-small-cell lung carcinomas from inflammatory cell death induced by SMAC-mimetic chemotherapeutic agents (small-molecule activators of the non-canonical NF-κB pathway). Our findings identify a NIK-SIX signalling axis that fine-tunes inflammatory gene expression programs under both physiological and pathological conditions.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Inflamación/metabolismo , FN-kappa B/deficiencia , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Animales , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Femenino , Fibroblastos , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Proteínas de Homeodominio/inmunología , Humanos , Inflamación/genética , Listeria monocytogenes/inmunología , Masculino , Ratones , FN-kappa B/genética , Proteínas del Tejido Nervioso/inmunología , Regiones Promotoras Genéticas , Shigella flexneri/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor de Transcripción ReIA/metabolismo , Factor de Transcripción ReIB/metabolismo , Quinasa de Factor Nuclear kappa BRESUMEN
The enteric attaching and effacing (A/E) pathogens enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) and the invasive pathogens enteroinvasive E. coli (EIEC) and Shigella encode type III secretion systems (T3SS) used to inject effector proteins into human host cells during infection. Among these are a group of effectors required for NF-κB-mediated host immune evasion. Recent studies have identified several effector proteins from A/E pathogens and EIEC/Shigella that are involved in suppression of NF-κB and have uncovered their cellular and molecular functions. A novel mechanism among these effectors from both groups of pathogens is to coordinate effector function during infection. This cooperativity among effector proteins explains how bacterial pathogens are able to effectively suppress innate immune defense mechanisms in response to diverse classes of immune receptor signaling complexes (RSCs) stimulated during infection.
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Proteínas Bacterianas/inmunología , Proteínas de Escherichia coli/inmunología , Escherichia coli/fisiología , Interacciones Huésped-Patógeno/inmunología , Inmunomodulación , Shigella/fisiología , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Núcleo Celular/metabolismo , Disentería Bacilar/inmunología , Disentería Bacilar/metabolismo , Disentería Bacilar/microbiología , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Humanos , FN-kappa B/metabolismo , Transporte de Proteínas , Transducción de Señal , Sistemas de Secreción Tipo III , Ubiquitina/metabolismoRESUMEN
Membrane-bound organelles serve as platforms for the assembly of multi-protein complexes that function as hubs of signal transduction in eukaryotic cells. Microbial pathogens have evolved virulence factors that reprogram these host signaling responses, but the underlying molecular mechanisms are poorly understood. Here we test the ability of ~200 type III and type IV effector proteins from six Gram-negative bacterial species to interact with the eukaryotic plasma membrane and intracellular organelles. We show that over 30% of the effectors localize to yeast and mammalian cell membranes, including a subset of previously uncharacterized Legionella effectors that appear to be able to regulate yeast vacuolar fusion. A combined genetic, cellular, and biochemical approach supports that some of the tested bacterial effectors can bind to membrane phospholipids and may regulate membrane trafficking. Finally, we show that the type III effector IpgB1 from Shigella flexneri may bind to acidic phospholipids and regulate actin filament dynamics.Microbial pathogens secrete effector proteins into host cells to affect cellular functions. Here, the authors use a yeast-based screen to study around 200 effectors from six bacterial species, showing that over 30% of them interact with the eukaryotic plasma membrane or intracellular organelles.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Interacciones Huésped-Patógeno/fisiología , Proteínas Bacterianas/genética , Células HEK293 , Células HeLa , Humanos , Legionella pneumophila/metabolismo , Legionella pneumophila/patogenicidad , Lípidos de la Membrana/metabolismo , Microscopía Fluorescente , Fosfolípidos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Shigella flexneri/metabolismo , Shigella flexneri/patogenicidad , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The type I interferon (IFN) activated transcriptional response is a critical antiviral defense mechanism, yet its role in bacterial pathogenesis remains less well characterized. Using an intracellular pathogen Listeria monocytogenes (Lm) as a model bacterial pathogen, we sought to identify the roles of individual interferon-stimulated genes (ISGs) in context of bacterial infection. Previously, IFN has been implicated in both restricting and promoting Lm growth and immune stimulatory functions in vivo. Here we adapted a gain-of-function flow cytometry based approach to screen a library of more than 350 human ISGs for inhibitors and enhancers of Lm infection. We identify 6 genes, including UNC93B1, MYD88, AQP9, and TRIM14 that potently inhibit Lm infection. These inhibitors act through both transcription-mediated (MYD88) and non-transcriptional mechanisms (TRIM14). Further, we identify and characterize the human high affinity immunoglobulin receptor FcγRIa as an enhancer of Lm internalization. Our results reveal that FcγRIa promotes Lm uptake in the absence of known host Lm internalization receptors (E-cadherin and c-Met) as well as bacterial surface internalins (InlA and InlB). Additionally, FcγRIa-mediated uptake occurs independently of Lm opsonization or canonical FcγRIa signaling. Finally, we established the contribution of FcγRIa to Lm infection in phagocytic cells, thus potentially linking the IFN response to a novel bacterial uptake pathway. Together, these studies provide an experimental and conceptual basis for deciphering the role of IFN in bacterial defense and virulence at single-gene resolution.