RESUMEN
The innate immune response constitutes the first line of defense against pathogens. It involves the recognition of pathogen-associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs), the production of inflammatory cytokines and the recruitment of immune cells to infection sites. Recently, ADP-heptose, a soluble intermediate of the lipopolysaccharide biosynthetic pathway in Gram-negative bacteria, has been identified by several research groups as a PAMP. Here, we recapitulate the evidence that led to this identification and discuss the controversy over the immunogenic properties of heptose 1,7-bisphosphate (HBP), another bacterial heptose previously defined as an activator of innate immunity. Then, we describe the mechanism of ADP-heptose sensing by alpha-protein kinase 1 (ALPK1) and its downstream signaling pathway that involves the proteins TIFA and TRAF6 and induces the activation of NF-κB and the secretion of inflammatory cytokines. Finally, we discuss possible delivery mechanisms of ADP-heptose in cells during infection, and propose new lines of thinking to further explore the roles of the ADP-heptose/ALPK1/TIFA axis in infections and its potential implication in the control of intestinal homeostasis.
Asunto(s)
Heptosas/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Proteínas Quinasas/metabolismo , Citocinas/metabolismo , Bacterias Gramnegativas/inmunología , Bacterias Gramnegativas/metabolismo , Humanos , Inmunidad Innata , Lipopolisacáridos/biosíntesis , Lipopolisacáridos/química , FN-kappa B/metabolismo , Transducción de SeñalRESUMEN
The mouse pathogen Citrobacter rodentium is used to model infections with enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC). Pathogenesis is commonly modelled in mice developing mild disease (e.g., C57BL/6). However, little is known about host responses in mice exhibiting severe colitis (e.g., C3H/HeN), which arguably provide a more clinically relevant model for human paediatric enteric infection. Infection of C3H/HeN mice with C. rodentium results in rapid colonic colonisation, coinciding with induction of key inflammatory signatures and colonic crypt hyperplasia. Infection also induces dramatic changes to bioenergetics in intestinal epithelial cells, with transition from oxidative phosphorylation (OXPHOS) to aerobic glycolysis and higher abundance of SGLT4, LDHA, and MCT4. Concomitantly, mitochondrial proteins involved in the TCA cycle and OXPHOS were in lower abundance. Similar to observations in C57BL/6 mice, we detected simultaneous activation of cholesterol biogenesis, import, and efflux. Distinctly, however, the pattern recognition receptors NLRP3 and ALPK1 were specifically induced in C3H/HeN. Using cell-based assays revealed that C. rodentium activates the ALPK1/TIFA axis, which is dependent on the ADP-heptose biosynthesis pathway but independent of the Type III secretion system. This study reveals for the first time the unfolding intestinal epithelial cells' responses during severe infectious colitis, which resemble EPEC human infections.
Asunto(s)
Citrobacter rodentium/inmunología , Infecciones por Enterobacteriaceae/inmunología , Interacciones Microbiota-Huesped , Inflamación/microbiología , Mucosa Intestinal/microbiología , Animales , Citrobacter rodentium/patogenicidad , Colitis/inmunología , Colitis/microbiología , Infecciones por Enterobacteriaceae/metabolismo , Femenino , Microbioma Gastrointestinal , Células HeLa , Humanos , Mucosa Intestinal/inmunología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Proteómica , Organismos Libres de Patógenos EspecíficosRESUMEN
During an infection, the detection of pathogens is mediated through the interactions between pathogen-associated molecular patterns (PAMPs) and pathogen recognition receptors. ß-Heptose 1,7-bisphosphate (ßHBP), an intermediate of the lipopolysaccharide (LPS) biosynthesis pathway, was recently identified as a bacterial PAMP. It was reported that ßHBP sensing leads to oligomerization of TIFA proteins, a mechanism controlling NF-κB activation and pro-inflammatory gene expression. Here, we compare the ability of chemically synthesized ßHBP and Shigella flexneri lysate to induce TIFA oligomerization in epithelial cells. We find that, unlike bacterial lysate, ßHBP fails to initiate rapid TIFA oligomerization. It only induces delayed signaling, suggesting that ßHBP must be processed intracellularly to trigger inflammation. Gene deletion and complementation analysis of the LPS biosynthesis pathway revealed that ADP-heptose is the bacterial metabolite responsible for rapid TIFA oligomerization. ADP-heptose sensing occurs down to 10-10 M. During S. flexneri infection, it results in cytokine production, a process dependent on the kinase ALPK1. Altogether, our results rule out a major role of ßHBP in S. flexneri infection and identify ADP-heptose as a new bacterial PAMP.
Asunto(s)
Adenosina Difosfato/metabolismo , Heptosas/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Shigella flexneri/metabolismo , Células HeLa , Heptosas/síntesis química , Heptosas/química , Humanos , Neisseria , Proteínas Quinasas/metabolismo , Multimerización de Proteína , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
During infection by invasive bacteria, epithelial cells contribute to innate immunity via the local secretion of inflammatory cytokines. These are directly produced by infected cells or by uninfected bystanders via connexin-dependent cell-cell communication. However, the cellular pathways underlying this process remain largely unknown. Here we perform a genome-wide RNA interference screen and identify TIFA and TRAF6 as central players of Shigella flexneri and Salmonella typhimurium-induced interleukin-8 expression. We show that threonine 9 and the forkhead-associated domain of TIFA are necessary for the oligomerization of TIFA in both infected and bystander cells. Subsequently, this process triggers TRAF6 oligomerization and NF-κB activation. We demonstrate that TIFA/TRAF6-dependent cytokine expression is induced by the bacterial metabolite heptose-1,7-bisphosphate (HBP). In addition, we identify alpha-kinase 1 (ALPK1) as the critical kinase responsible for TIFA oligomerization and IL-8 expression in response to infection with S. flexneri and S. typhimurium but also to Neisseria meningitidis. Altogether, these results clearly show that ALPK1 is a master regulator of innate immunity against both invasive and extracellular gram-negative bacteria.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Inmunidad Innata/inmunología , Factor 6 Asociado a Receptor de TNF/inmunología , Quimiocinas/biosíntesis , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/inmunología , Técnica del Anticuerpo Fluorescente , Bacterias Gramnegativas/inmunología , Células HEK293 , Células HeLa , Heptosas/inmunología , Humanos , Procesamiento de Imagen Asistido por Computador , Immunoblotting , Inmunoprecipitación , Neisseria meningitidis/inmunología , Salmonella typhimurium/inmunología , Shigella flexneri/inmunologíaRESUMEN
The enteroinvasive bacterium Shigella flexneri uses multiple secreted effector proteins to downregulate interleukin-8 (IL-8) expression in infected epithelial cells. Yet, massive IL-8 secretion is observed in Shigellosis. Here we report a host mechanism of cell-cell communication that circumvents the effector proteins and strongly amplifies IL-8 expression during bacterial infection. By monitoring proinflammatory signals at the single-cell level, we found that the activation of the transcription factor NF-κB and the MAP kinases JNK, ERK, and p38 rapidly propagated from infected to uninfected adjacent cells, leading to IL-8 production by uninfected bystander cells. Bystander IL-8 production was also observed during Listeria monocytogenes and Salmonella typhimurium infection. This response could be triggered by recognition of peptidoglycan and is mediated by gap junctions. Thus, we have identified a mechanism of cell-cell communication that amplifies innate immunity against bacterial infection by rapidly spreading proinflammatory signals via gap junctions to yet uninfected cells.
Asunto(s)
Disentería Bacilar/inmunología , Inmunidad Innata , Sistema de Señalización de MAP Quinasas/inmunología , Proteínas Quinasas Activadas por Mitógenos/inmunología , FN-kappa B/inmunología , Shigella flexneri/inmunología , Células CACO-2 , Comunicación Celular/inmunología , Proliferación Celular , Disentería Bacilar/enzimología , Uniones Comunicantes/inmunología , Uniones Comunicantes/microbiología , Células HeLa , Humanos , Interleucina-8/análisis , Interleucina-8/inmunología , Listeria monocytogenes/inmunología , Listeriosis/enzimología , Listeriosis/inmunología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Peptidoglicano/inmunología , Shigella flexneri/enzimologíaRESUMEN
The enteroinvasive bacterium Shigella flexneri invades the intestinal epithelium of humans. During infection, several injected effector proteins promote bacterial internalization, and interfere with multiple host cell responses. To obtain a systems-level overview of host signaling during infection, we analyzed the global dynamics of protein phosphorylation by liquid chromatography-tandem MS and identified several hundred of proteins undergoing a phosphorylation change during the first hours of infection. Functional bioinformatic analysis revealed that they were mostly related to the cytoskeleton, transcription, signal transduction, and cell cycle. Fuzzy c-means clustering identified six temporal profiles of phosphorylation and a functional module composed of ATM-phosphorylated proteins related to genotoxic stress. Pathway enrichment analysis defined mTOR as the most overrepresented pathway. We showed that mTOR complex 1 and 2 were required for S6 kinase and AKT activation, respectively. Comparison with a published phosphoproteome of Salmonella typhimurium-infected cells revealed a large subset of coregulated phosphoproteins. Finally, we showed that S. flexneri effector OspF affected the phosphorylation of several hundred proteins, thereby demonstrating the wide-reaching impact of a single bacterial effector on the host signaling network.
Asunto(s)
Disentería Bacilar/metabolismo , Interacciones Huésped-Patógeno/fisiología , Fosfoproteínas/metabolismo , Shigella flexneri/fisiología , Animales , Proteínas Bacterianas/metabolismo , Línea Celular , Células HeLa , Humanos , Ratones , Fosforilación , Proteómica/métodos , Salmonella typhi/metabolismoRESUMEN
BACKGROUND: Large-scale RNAi screening has become an important technology for identifying genes involved in biological processes of interest. However, the quality of large-scale RNAi screening is often deteriorated by off-targets effects. In order to find statistically significant effector genes for pathogen entry, we systematically analyzed entry pathways in human host cells for eight pathogens using image-based kinome-wide siRNA screens with siRNAs from three vendors. We propose a Parallel Mixed Model (PMM) approach that simultaneously analyzes several non-identical screens performed with the same RNAi libraries. RESULTS: We show that PMM gains statistical power for hit detection due to parallel screening. PMM allows incorporating siRNA weights that can be assigned according to available information on RNAi quality. Moreover, PMM is able to estimate a sharedness score that can be used to focus follow-up efforts on generic or specific gene regulators. By fitting a PMM model to our data, we found several novel hit genes for most of the pathogens studied. CONCLUSIONS: Our results show parallel RNAi screening can improve the results of individual screens. This is currently particularly interesting when large-scale parallel datasets are becoming more and more publicly available. Our comprehensive siRNA dataset provides a public, freely available resource for further statistical and biological analyses in the high-content, high-throughput siRNA screening field.
Asunto(s)
Genómica/métodos , Interferencia de ARN , ARN Interferente Pequeño/genética , Línea Celular , Biblioteca de Genes , Genómica/normas , Ensayos Analíticos de Alto Rendimiento , Interacciones Huésped-Patógeno/genética , Humanos , Curva ROC , Reproducibilidad de los ResultadosRESUMEN
The anaerobic bacterium Fusobacterium nucleatum is significantly associated with human colorectal cancer (CRC) and is considered a significant contributor to the disease. The mechanisms underlying the promotion of intestinal tumor formation by F. nucleatum have only been partially uncovered. Here, we showed that F. nucleatum releases a metabolite into the microenvironment that strongly activates NF-κB in intestinal epithelial cells via the ALPK1/TIFA/TRAF6 pathway. Furthermore, we showed that the released molecule had the biological characteristics of ADP-heptose. We observed that F. nucleatum induction of this pathway increased the expression of the inflammatory cytokine IL-8 and two anti-apoptotic genes known to be implicated in CRC, BIRC3 and TNFAIP3. Finally, it promoted the survival of CRC cells and reduced 5-fluorouracil chemosensitivity in vitro. Taken together, our results emphasize the importance of the ALPK1/TIFA pathway in Fusobacterium induced-CRC pathogenesis, and identify the role of ADP-H in this process.
Asunto(s)
Neoplasias Colorrectales , Microbioma Gastrointestinal , Humanos , Fusobacterium nucleatum/metabolismo , Composición de Base , Filogenia , ARN Ribosómico 16S , Análisis de Secuencia de ADN , Neoplasias Colorrectales/patología , Heptosas/metabolismo , Microambiente TumoralRESUMEN
Alpha-protein kinase 1 (ALPK1) is a pathogen recognition receptor that detects ADP-heptose (ADPH), a lipopolysaccharide biosynthesis intermediate, recently described as a pathogen-associated molecular pattern in Gram-negative bacteria. ADPH binding to ALPK1 activates its kinase domain and triggers TIFA phosphorylation on threonine 9. This leads to the assembly of large TIFA oligomers called TIFAsomes, activation of NF-κB and pro-inflammatory gene expression. Furthermore, mutations in ALPK1 are associated with inflammatory syndromes and cancers. While this kinase is of increasing medical interest, its activity in infectious or non-infectious diseases remains poorly characterized. Here, we use a non-radioactive ALPK1 in vitro kinase assay based on the use of ATPγS and protein thiophosphorylation. We confirm that ALPK1 phosphorylates TIFA T9 and show that T2, T12 and T19 are also weakly phosphorylated by ALPK1. Interestingly, we find that ALPK1 itself is phosphorylated in response to ADPH recognition during Shigella flexneri and Helicobacter pylori infection and that disease-associated ALPK1 mutants exhibit altered kinase activity. In particular, T237M and V1092A mutations associated with ROSAH syndrome and spiradenoma/spiradenocarcinoma respectively, exhibit enhanced ADPH-induced kinase activity and constitutive assembly of TIFAsomes. Altogether, this study provides new insights into the ADPH sensing pathway and disease-associated ALPK1 mutants.
Asunto(s)
Infecciones por Helicobacter , Helicobacter pylori , Humanos , Fosforilación , Infecciones por Helicobacter/microbiología , Inmunidad Innata , Helicobacter pylori/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Heptosas/química , Heptosas/metabolismoRESUMEN
During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , GMP Cíclico/análogos & derivados , Proteínas Periplasmáticas/genética , Liasas de Fósforo-Oxígeno/genética , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Caenorhabditis elegans , Células Cultivadas , GMP Cíclico/metabolismo , Elementos Transponibles de ADN/genética , Proteínas de Escherichia coli , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Mutagénesis/fisiología , Operón/genética , Proteínas Periplasmáticas/metabolismo , Fagocitosis/fisiología , Fenotipo , Liasas de Fósforo-Oxígeno/metabolismo , Neumonía Bacteriana/enzimología , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/microbiología , Infecciones por Pseudomonas/enzimología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/metabolismo , Sistemas de Mensajero Secundario/fisiologíaRESUMEN
The commensal bacteria that make up the gut microbiota impact the health of their host on multiple levels. In particular, the interactions taking place between the microbe-associated molecule patterns (MAMPs) and pattern recognition receptors (PRRs), expressed by intestinal epithelial cells (IECs), are crucial for maintaining intestinal homeostasis. While numerous studies showed that TLRs and NLRs are involved in the control of gut homeostasis by commensal bacteria, the role of additional innate immune receptors remains unclear. Here, we seek for novel MAMP-PRR interactions involved in the beneficial effect of the commensal bacterium Akkermansia muciniphila on intestinal homeostasis. We show that A. muciniphila strongly activates NF-κB in IECs by releasing one or more potent activating metabolites into the microenvironment. By using drugs, chemical and gene-editing tools, we found that the released metabolite(s) enter(s) epithelial cells and activate(s) NF-κB via an ALPK1, TIFA and TRAF6-dependent pathway. Furthermore, we show that the released molecule has the biological characteristics of the ALPK1 ligand ADP-heptose. Finally, we show that A. muciniphila induces the expression of the MUC2, BIRC3 and TNFAIP3 genes involved in the maintenance of the intestinal barrier function and that this process is dependent on TIFA. Altogether, our data strongly suggest that the commensal A. muciniphila promotes intestinal homeostasis by activating the ALPK1/TIFA/TRAF6 axis, an innate immune pathway exclusively described so far in the context of Gram-negative bacterial infections.
Asunto(s)
Microbioma Gastrointestinal , FN-kappa B , Adenosina Difosfato , Akkermansia , Heptosas , Inmunidad Innata , Factor 6 Asociado a Receptor de TNF , VerrucomicrobiaRESUMEN
Chemotaxis is a cellular sensing mechanism that guides immune cells to sites of infection and leads fibroblasts to sites of injury. Here, we show in migrating primary dendritic cells and fibroblasts that the leading edge is not a uniform signaling entity, but instead consists of independent coupling units in which transient activation of PI3-kinase links to local lamellipod extension and small discrete turns in the direction of migration. These findings led to a model in which global cell polarization is independent from the chemotaxis mechanism. In this model, chemotaxis does not require spatial integration but is instead a stochastic process in which each receptor binding event within the leading edge triggers a local lamellipod extension and a small turn in the direction of migration. We show that this model and a derived "compass parameter" are sufficient to simulate the observed random migration, biased random walk, and persistent chemotactic behaviors of eukaryotic cells.
Asunto(s)
Quimiotaxis/fisiología , Modelos Biológicos , Animales , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Quimiotaxis/inmunología , Complemento C5a/administración & dosificación , Complemento C5a/fisiología , Simulación por Computador , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/fisiología , Células Eucariotas , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Fibroblastos/fisiología , Humanos , Ratones , Células 3T3 NIH , Fosfatidilinositol 3-Quinasas/fisiología , Fosfatos de Fosfatidilinositol/metabolismo , Factor de Crecimiento Derivado de Plaquetas/administración & dosificación , Factor de Crecimiento Derivado de Plaquetas/fisiología , Seudópodos/efectos de los fármacos , Seudópodos/fisiología , Transducción de Señal , Procesos EstocásticosRESUMEN
Intestinal epithelial cells (IECs) act as sentinels for incoming pathogens. Cytosol-invasive bacteria, such as Shigella flexneri, trigger a robust pro-inflammatory nuclear factor κB (NF-κB) response from IECs that is believed to depend entirely on the peptidoglycan sensor NOD1. We found that, during Shigella infection, the TRAF-interacting forkhead-associated protein A (TIFA)-dependent cytosolic surveillance pathway, which senses the bacterial metabolite heptose-1,7-bisphosphate (HBP), functions after NOD1 to detect bacteria replicating free in the host cytosol. Whereas NOD1 mediated a transient burst of NF-κB activation during bacterial entry, TIFA sensed HBP released during bacterial replication, assembling into large signaling complexes to drive a dynamic inflammatory response that reflected the rate of intracellular bacterial proliferation. Strikingly, IECs lacking TIFA were unable to discriminate between proliferating and stagnant intracellular bacteria, despite the NOD1/2 pathways being intact. Our results define TIFA as a rheostat for intracellular bacterial replication, escalating the immune response to invasive Gram-negative bacteria that exploit the host cytosol for growth.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Citosol/metabolismo , Inmunidad Innata , Espacio Intracelular/microbiología , Shigella flexneri/crecimiento & desarrollo , Transducción de Señal , Células HeLa , Humanos , Proteína Adaptadora de Señalización NOD1/metabolismo , Fosfatos/metabolismo , Vacuolas/metabolismoRESUMEN
Methods enabling the delivery of proteins into eukaryotic cells are essential to address protein functions. Here we propose broad applications to cell biology for a protein delivery tool based on bacterial type III secretion (T3S). We show that bacterial, viral, and human proteins, fused to the N-terminal fragment of the Yersinia enterocolitica T3S substrate YopE, are effectively delivered into target cells in a fast and controllable manner via the injectisome of extracellular bacteria. This method enables functional interaction studies by the simultaneous injection of multiple proteins and allows the targeting of proteins to different subcellular locations by use of nanobody-fusion proteins. After delivery, proteins can be freed from the YopE fragment by a T3S-translocated viral protease or fusion to ubiquitin and cleavage by endogenous ubiquitin proteases. Finally, we show that this delivery tool is suitable to inject proteins in living animals and combine it with phosphoproteomics to characterize the systems-level impact of proapoptotic human truncated BID on the cellular network.
Asunto(s)
Sistemas de Secreción Tipo III/farmacología , Células 3T3 , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/fisiología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Permeabilidad de la Membrana Celular , Sistemas de Liberación de Medicamentos , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteoma/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Pez CebraRESUMEN
Recognition of the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, by the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) complex is essential for the control of bacterial infection. A pro-inflammatory signaling cascade is initiated upon binding of membrane-associated portion of LPS, a glycophospholipid Lipid A, by a coreceptor protein MD-2, which results in a protective host innate immune response. However, activation of TLR4 signaling by LPS may lead to the dysregulated immune response resulting in a variety of inflammatory conditions including sepsis syndrome. Understanding of structural requirements for Lipid A endotoxicity would ensure the development of effective anti-inflammatory medications. Herein, we report on design, synthesis, and biological activities of a series of conformationally confined Lipid A mimetics based on ß,α-trehalose-type scaffold. Replacement of the flexible three-bond ß(1â6) linkage in diglucosamine backbone of Lipid A by a two-bond ß,α(1â1) glycosidic linkage afforded novel potent TLR4 antagonists. Synthetic tetraacylated bisphosphorylated Lipid A mimetics based on a ß-GlcN(1â1)α-GlcN scaffold selectively block the LPS binding site on both human and murine MD-2 and completely abolish lipopolysaccharide-induced pro-inflammatory signaling, thereby serving as antisepsis drug candidates. In contrast to their natural counterpart lipid IVa, conformationally constrained Lipid A mimetics do not activate mouse TLR4. The structural basis for high antagonistic activity of novel Lipid A mimetics was confirmed by molecular dynamics simulation. Our findings suggest that besides the chemical structure, also the three-dimensional arrangement of the diglucosamine backbone of MD-2-bound Lipid A determines endotoxic effects on TLR4.
Asunto(s)
Biomimética , Lípido A/química , Modelos Biológicos , Péptidos/química , Receptor Toll-Like 4/química , Animales , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Escherichia coli/química , Células HEK293 , Humanos , Interleucina-6/antagonistas & inhibidores , Interleucina-6/metabolismo , Lipopolisacáridos/inmunología , Masculino , Ratones , Conformación Molecular , Péptidos/antagonistas & inhibidores , Unión Proteica , Transducción de Señal , Receptor Toll-Like 4/antagonistas & inhibidoresAsunto(s)
Proteínas Bacterianas/genética , Técnicas Citológicas/métodos , Sistemas de Secreción Tipo III , Yersinia enterocolitica/genética , Aspartato-Semialdehído Deshidrogenasa/genética , Microinyecciones , Nanotecnología , Transporte de Proteínas , Proteínas Recombinantes de Fusión , Transfección , Yersinia enterocolitica/químicaRESUMEN
BACKGROUND: Nuclear factor-κB (NF-κB) is a transcription factor that regulates the transcription of genes involved in a variety of biological processes, including innate and adaptive immunity, stress responses and cell proliferation. Constitutive or excessive NF-κB activity has been associated with inflammatory disorders and higher risk of cancer. In contrast to the mechanisms controlling inducible activation, the regulation of basal NF-κB activation is not well understood. Here we test whether clathrin heavy chain (CHC) contributes to the regulation of basal NF-κB activity in epithelial cells. METHODOLOGY: Using RNA interference to reduce endogenous CHC expression, we found that CHC is required to prevent constitutive activation of NF-κB and gene expression. Immunofluorescence staining showed constitutive nuclear localization of the NF-κB subunit p65 in absence of stimulation after CHC knockdown. Elevated basal p65 nuclear localization is caused by constitutive phosphorylation and degradation of inhibitor of NF-κB alpha (IκBα) through an IκB kinase α (IKKα)-dependent mechanism. The role of CHC in NF-κB signaling is functionally relevant as constitutive expression of the proinflammatory chemokine interleukin-8 (IL-8), whose expression is regulated by NF-κB, was found after CHC knockdown. Disruption of clathrin-mediated endocytosis by chemical inhibition or depletion of the µ2-subunit of the endocytosis adaptor protein AP-2, and knockdown of clathrin light chain a (CHLa), failed to induce constitutive NF-κB activation and IL-8 expression, showing that CHC acts on NF-κB independently of endocytosis and CLCa. CONCLUSIONS: We conclude that CHC functions as a built-in molecular brake that ensures a tight control of basal NF-κB activation and gene expression in unstimulated cells. Furthermore, our data suggest a potential link between a defect in CHC expression and chronic inflammation disorder and cancer.
Asunto(s)
Cadenas Pesadas de Clatrina/metabolismo , Cadenas Pesadas de Clatrina/fisiología , Endocitosis/fisiología , FN-kappa B/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cadenas Pesadas de Clatrina/antagonistas & inhibidores , Cadenas Pesadas de Clatrina/genética , Endocitosis/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Células HeLa , Humanos , Quinasa I-kappa B/metabolismo , Quinasa I-kappa B/fisiología , Interleucina-8/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Células Tumorales CultivadasRESUMEN
Shigella flexneri type III secreted effector OspF harbors a phosphothreonine lyase activity that irreversibly dephosphorylates MAP kinases (MAPKs) p38 and ERK in infected epithelial cells and thereby, dampens innate immunity. Whereas this activity has been well characterized, the impact of OspF on other host signaling pathways that control inflammation was unknown. Here we report that OspF potentiates the activation of the MAPK JNK and the transcription factor NF-κB during S. flexneri infection. This unexpected effect of OspF was dependent on the phosphothreonine lyase activity of OspF on p38, and resulted from the disruption of a negative feedback loop regulation between p38 and TGF-beta activated kinase 1 (TAK1), mediated via the phosphorylation of TAK1-binding protein 1. Interestingly, potentiated JNK activation was not associated with enhanced c-Jun signaling as OspF also inhibits c-Jun expression at the transcriptional level. Altogether, our data reveal the impact of OspF on the activation of NF-κB, JNK and c-Jun, and demonstrate the existence of a negative feedback loop regulation between p38 and TAK1 during S. flexneri infection. Furthermore, this study validates the use of bacterial effectors as molecular tools to identify the crosstalks that connect important host signaling pathways induced upon bacterial infection.
Asunto(s)
Proteínas Bacterianas/metabolismo , Disentería Bacilar/metabolismo , Mediadores de Inflamación/metabolismo , Proteínas Recombinantes/metabolismo , Shigella flexneri , Animales , Línea Celular , Disentería Bacilar/inmunología , Activación Enzimática , Retroalimentación Fisiológica , Humanos , Inflamación/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Fosforilasas/metabolismo , Fosforilación , Transducción de Señal , Factor de Transcripción ReIA/metabolismo , Transcripción Genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND: During pathogen infection, innate immunity is initiated via the recognition of microbial products by pattern recognition receptors and the subsequent activation of transcription factors that upregulate proinflammatory genes. By controlling the expression of cytokines, chemokines, anti-bacterial peptides and adhesion molecules, the transcription factor nuclear factor-kappa B (NF-κB) has a central function in this process. In a typical model of NF-κB activation, the recognition of pathogen associated molecules triggers the canonical NF-κB pathway that depends on the phosphorylation of Inhibitor of NF-κB (IκB) by the catalytic subunit IκB kinase ß (IKKß), its degradation and the nuclear translocation of NF-κB dimers. METHODOLOGY: Here, we performed an RNA interference (RNAi) screen on Shigella flexneri-induced NF-κB activation to identify new factors involved in the regulation of NF-κB following infection of epithelial cells by invasive bacteria. By targeting a subset of the human signaling proteome, we found that the catalytic subunit IKKα is also required for complete NF-κB activation during infection. Depletion of IKKα by RNAi strongly reduces the nuclear translocation of NF-κB p65 during S. flexneri infection as well as the expression of the proinflammatory chemokine interleukin-8. Similar to IKKß, IKKα contributes to the phosphorylation of IκBα on serines 32 and 36, and to its degradation. Experiments performed with the synthetic Nod1 ligand L-Ala-D-γ-Glu-meso-diaminopimelic acid confirmed that IKKα is involved in NF-κB activation triggered downstream of Nod1-mediated peptidoglycan recognition. CONCLUSIONS: Taken together, these results demonstrate the unexpected role of IKKα in the canonical NF-κB pathway triggered by peptidoglycan recognition during bacterial infection. In addition, they suggest that IKKα may be an important drug target for the development of treatments that aim at limiting inflammation in bacterial infection.
Asunto(s)
Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Proteína Adaptadora de Señalización NOD1/metabolismo , Peptidoglicano/metabolismo , Secuencia de Bases , Western Blotting , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Células HeLa , Humanos , Interferencia de ARNRESUMEN
Cyclic AMP (cAMP) and Ca(2+) are key regulators of exocytosis in many cells, including insulin-secreting beta cells. Glucose-stimulated insulin secretion from beta cells is pulsatile and involves oscillations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), but little is known about the detailed kinetics of cAMP signaling. Using evanescent-wave fluorescence imaging we found that glucose induces pronounced oscillations of cAMP in the submembrane space of single MIN6 cells and primary mouse beta cells. These oscillations were preceded and enhanced by elevations of [Ca(2+)](i). However, conditions raising cytoplasmic ATP could trigger cAMP elevations without accompanying [Ca(2+)](i) rise, indicating that adenylyl cyclase activity may be controlled also by the substrate concentration. The cAMP oscillations correlated with pulsatile insulin release. Whereas elevation of cAMP enhanced secretion, inhibition of adenylyl cyclases suppressed both cAMP oscillations and pulsatile insulin release. We conclude that cell metabolism directly controls cAMP and that glucose-induced cAMP oscillations regulate the magnitude and kinetics of insulin exocytosis.