RESUMEN
Listeria can escape host autophagy defense pathways through mechanisms that remain poorly understood. We show here that in epithelial cells, Listeriolysin (LLO)-dependent cytosolic escape of Listeria triggered a transient amino-acid starvation host response characterized by GCN2 phosphorylation, ATF3 induction and mTOR inhibition, the latter favouring a pro-autophagic cellular environment. Surprisingly, rapid recovery of mTOR signalling was neither sufficient nor necessary for Listeria avoidance of autophagic targeting. Instead, we observed that Listeria phospholipases PlcA and PlcB reduced autophagic flux and phosphatidylinositol 3-phosphate (PI3P) levels, causing pre-autophagosomal structure stalling and preventing efficient targeting of cytosolic bacteria. In co-infection experiments, wild-type Listeria protected PlcA/B-deficient bacteria from autophagy-mediated clearance. Thus, our results uncover a critical role for Listeria phospholipases C in the inhibition of autophagic flux, favouring bacterial escape from host autophagic defense.
Asunto(s)
Autofagia , Listeria monocytogenes/enzimología , Listeriosis/patología , Fagosomas/patología , Fosfolipasas/metabolismo , Factor de Transcripción Activador 3/genética , Factor de Transcripción Activador 3/metabolismo , Animales , Toxinas Bacterianas/farmacología , Western Blotting , Proliferación Celular , Células Cultivadas , Citosol/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/microbiología , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/microbiología , Técnica del Anticuerpo Fluorescente , Células HeLa , Proteínas de Choque Térmico/farmacología , Proteínas Hemolisinas/farmacología , Humanos , Listeriosis/metabolismo , Listeriosis/microbiología , Ratones , Fagosomas/metabolismo , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipasas/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
To subvert host immunity, influenza A virus (IAV) induces early apoptosis in innate immune cells by disrupting mitochondria membrane potential via its polymerase basic protein 1-frame 2 (PB1-F2) accessory protein. Whether immune cells have mechanisms to counteract PB1-F2-mediated apoptosis is currently unknown. Herein, we define that the host mitochondrial protein nucleotide-binding oligomerization domain-like receptor (NLR)X1 binds to viral protein PB1-F2, preventing IAV-induced macrophage apoptosis and promoting both macrophage survival and type I IFN signaling. We initially observed that Nlrx1-deficient mice infected with IAV exhibited increased pulmonary viral replication, as well as enhanced inflammatory-associated pulmonary dysfunction and morbidity. Analysis of the lungs of IAV-infected mice revealed markedly enhanced leukocyte recruitment but impaired production of type I IFN in Nlrx1(-/-) mice. Impaired type I IFN production and enhanced viral replication was recapitulated in Nlrx1(-/-) macrophages and was associated with increased mitochondrial mediated apoptosis. Through gain- and loss-of-function strategies for protein interaction, we identified that NLRX1 directly bound PB1-F2 in the mitochondria of macrophages. Using a recombinant virus lacking PB1-F2, we confirmed that deletion of PB1-F2 abrogated NLRX1-dependent macrophage type I IFN production and apoptosis. Thus, our results demonstrate that NLRX1 acts as a mitochondrial sentinel protecting macrophages from PB1-F2-induced apoptosis and preserving their antiviral function. We further propose that NLRX1 is critical for macrophage immunity against IAV infection by sensing the extent of viral replication and maintaining a protective balance between antiviral immunity and excessive inflammation within the lungs.
Asunto(s)
Apoptosis , Virus de la Influenza A/inmunología , Macrófagos/inmunología , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Virales/metabolismo , Animales , Línea Celular Tumoral , Humanos , Inflamación , Virus de la Influenza A/fisiología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Mitocondriales/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Replicación ViralRESUMEN
Invasive bacterial pathogens induce an amino acid starvation (AAS) response in infected host cells that controls host defense in part by promoting autophagy. However, whether AAS has additional significant effects on the host response to intracellular bacteria remains poorly characterized. Here we showed that Shigella, Salmonella, and Listeria interfere with spliceosomal U snRNA maturation in the cytosol. Bacterial infection resulted in the rerouting of U snRNAs and their cytoplasmic escort, the survival motor neuron (SMN) complex, to processing bodies, thus forming U snRNA bodies (U bodies). This process likely contributes to the decline in the cytosolic levels of U snRNAs and of the SMN complex proteins SMN and DDX20 that we observed in infected cells. U body formation was triggered by membrane damage in infected cells and was associated with the induction of metabolic stresses, such as AAS or endoplasmic reticulum stress. Mechanistically, targeting of U snRNAs to U bodies was regulated by translation initiation inhibition and the ATF4/ATF3 pathway, and U bodies rapidly disappeared upon removal of the stress, suggesting that their accumulation represented an adaptive response to metabolic stress. Importantly, this process likely contributed to shape the host response to invasive bacteria because down-regulation of DDX20 expression using short hairpin RNA (shRNA) amplified ATF3- and NF-κB-dependent signaling. Together, these results identify a critical role for metabolic stress and invasive bacterial pathogens in U body formation and suggest that this process contributes to host defense.
Asunto(s)
Interacciones Huésped-Patógeno/genética , Listeria monocytogenes/metabolismo , ARN Nuclear Pequeño/metabolismo , Salmonella typhimurium/metabolismo , Shigella flexneri/metabolismo , Estrés Fisiológico/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Factor de Transcripción Activador 3/genética , Factor de Transcripción Activador 3/metabolismo , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citoplasma/microbiología , Proteína 20 DEAD-Box/antagonistas & inhibidores , Proteína 20 DEAD-Box/genética , Proteína 20 DEAD-Box/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Listeria monocytogenes/patogenicidad , FN-kappa B/genética , FN-kappa B/metabolismo , Iniciación de la Cadena Peptídica Traduccional , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/ultraestructura , Salmonella typhimurium/patogenicidad , Shigella flexneri/patogenicidad , Transducción de Señal , Empalmosomas/metabolismo , Empalmosomas/microbiología , Proteína 1 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
BACKGROUND: The intestinal epithelium plays a critical role in nutrient absorption and innate immune defense. Recent studies showed that metabolic stress pathways, in particular the integrated stress response (ISR), control intestinal epithelial cell fate and function. Here, we used RNA-seq to analyze the global transcript level and alternative splicing responses of primary murine enteroids undergoing two distinct ISR-triggering stresses, endoplasmic reticulum (ER) stress and nutrient starvation. RESULTS: Our results reveal the core transcript level response to ISR-associated stress in murine enteroids, which includes induction of stress transcription factors, as well as genes associated with chemotaxis and inflammation. We also identified the transcript expression signatures that are unique to each ISR stress. Among these, we observed that ER stress and nutrient starvation had opposite effects on intestinal stem cell (ISC) transcriptional reprogramming. In agreement, ER stress decreased EdU incorporation, a marker of cell proliferation, in primary murine enteroids, while nutrient starvation had an opposite effect. We also analyzed the impact of these cellular stresses on mRNA splicing regulation. Splicing events commonly regulated by both stresses affected genes regulating splicing and were associated with nonsense-mediated decay (NMD), suggesting that splicing is modulated by an auto-regulatory feedback loop during stress. In addition, we also identified a number of genes displaying stress-specific splicing regulation. We suggest that functional gene expression diversity may arise during stress by the coordination of alternative splicing and alternative translation, and that this diversity might contribute to the cellular response to stress. CONCLUSIONS: Together, these results provide novel understanding of the importance of metabolic stress pathways in the intestinal epithelium. Specifically, the importance of cellular stresses in the regulation of immune and defense function, metabolism, proliferation and ISC activity in the intestinal epithelium is highlighted. Furthermore, this work highlights an under-appreciated role played by alternative splicing in shaping the response to stress and reveals a potential mechanism for gene regulation involving coupling of AS and alternative translation start sites.
Asunto(s)
Estrés del Retículo Endoplásmico/genética , Mucosa Intestinal/metabolismo , Empalme del ARN , Inanición/genética , Transcriptoma , Animales , Biología Computacional/métodos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Intrones , Ratones , Organoides , Células Madre/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
NLRX1 is a mitochondrial Nod-like receptor (NLR) protein whose function remains enigmatic. Here, we observed that NLRX1 expression was glucose-regulated and blunted by SV40 transformation. In transformed but not primary murine embryonic fibroblasts, NLRX1 expression mediated resistance to an extrinsic apoptotic signal, whereas conferring susceptibility to intrinsic apoptotic signals, such as glycolysis inhibition, increased cytosolic calcium and endoplasmic reticulum stress. In a murine model of colorectal cancer induced by azoxymethane, NLRX1-/- mice developed fewer tumors than wild type mice. In contrast, in a colitis-associated cancer model combining azoxymethane and dextran sulfate sodium, NLRX1-/- mice developed a more severe pathology likely due to the increased sensitivity to dextran sulfate sodium colitis. Together, these results identify NLRX1 as a critical mitochondrial protein implicated in the regulation of apoptosis in cancer cells. The unique capacity of NLRX1 to regulate the cellular sensitivity toward intrinsic versus extrinsic apoptotic signals suggests a critical role for this protein in numerous physiological processes and pathological conditions.
Asunto(s)
Apoptosis , Colitis/metabolismo , Neoplasias del Colon/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Línea Celular Transformada , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Sulfato de Dextran/toxicidad , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genética , Proteínas de Neoplasias/genéticaRESUMEN
The cyclic dinucleotides 3'-5'diadenylate (c-diAMP) and 3'-5' diguanylate (c-diGMP) are important bacterial second messengers that have recently been shown to stimulate the secretion of type I Interferons (IFN-Is) through the c-diGMP-binding protein MPYS/STING. Here, we show that physiologically relevant levels of cyclic dinucleotides also stimulate a robust secretion of IL-1ß through the NLRP3 inflammasome. Intriguingly, this response is independent of MPYS/STING. Consistent with most NLRP3 inflammasome activators, the response to c-diGMP is dependent on the mobilization of potassium and calcium ions. However, in contrast to other NLRP3 inflammasome activators, this response is not associated with significant changes in mitochondrial potential or the generation of mitochondrial reactive oxygen species. Thus, cyclic dinucleotides activate the NLRP3 inflammasome through a unique pathway that could have evolved to detect pervasive bacterial pathogen-associated molecular patterns associated with intracellular infections.
Asunto(s)
Proteínas Portadoras/metabolismo , GMP Cíclico/análogos & derivados , Fosfatos de Dinucleósidos/farmacología , Inflamasomas/metabolismo , Animales , Calcio/metabolismo , Proteínas Portadoras/genética , Línea Celular Tumoral , GMP Cíclico/farmacología , Humanos , Interleucina-1beta/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Potencial de la Membrana Mitocondrial , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR , Potasio/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: The natural history and pathophysiology of diverticular disease (DD) are still uncertain. An ex-vivo human complicated DD (cDD) model has recently shown a predominant transmural oxidative imbalance. The present study aims to evaluate whether the previously described alterations may precede the symptomatic form of the disease. METHODS: Colonic surgical samples obtained from patients with asymptomatic diverticulosis (DIV), complicated DD, and controls were systematically and detailed morphologically and molecularly analyzed. Therefore, histologic, histomorphometric, immunohistochemical evaluation, and gene and protein expression analysis were performed to characterize colonic muscle changes and evaluate chronic inflammation, oxidative imbalance, and hypoxia. Functional muscle activity was tested on strips and isolated cells in response to contractile and relaxant agents. KEY RESULTS: Compared with controls, DD showed a marketed increase in muscle layer thickness, smooth muscle cell syncytium disarray, and increased interstitial fibrosis; moreover, the observed features were more evident in the cDD group. These changes mainly affected longitudinal muscle and were associated with altered contraction-relaxation dynamics and fibrogenic switch of smooth muscle cells. Chronic lymphoplasmacytic inflammation was primarily evident in the mucosa and spared the muscle. A transmural increase in carbonylated and nitrated proteins, with loss of antioxidant molecules, characterized both stages of DD, suggesting early oxidative stress probably triggered by recurrent ischemic events, more pronounced in cDD, where HIF-1 was detected in both muscle and mucosa. CONCLUSION & INFERENCES: The different DD clinical scenarios are part of a progressive process, with oxidative imbalance representing a new target in the management of DD.
Asunto(s)
Progresión de la Enfermedad , Músculo Liso , Estrés Oxidativo , Humanos , Masculino , Femenino , Persona de Mediana Edad , Anciano , Estrés Oxidativo/fisiología , Músculo Liso/metabolismo , Músculo Liso/patología , Enfermedades Diverticulares/metabolismo , Diverticulosis del Colon/metabolismo , Diverticulosis del Colon/patología , Colon/patología , Colon/metabolismo , Contracción Muscular/fisiologíaRESUMEN
Luciferase reporter assays (LRAs) are widely used to assess the activity of specific signal transduction pathways. Although powerful, rapid and convenient, this technique can also generate artifactual results, as revealed for instance in the case of high throughput screens of inhibitory molecules. Here we demonstrate that the previously reported inhibitory effect of the Nod-like receptor (NLR) protein NLRX1 on NF-κB- and type I interferon-dependent pathways in LRAs was a nonspecific consequence of the overexpression of the NLRX1 leucine-rich repeat (LRR) domain. By comparing luciferase activity and luciferase gene expression using quantitative PCR from the same samples, we showed that NLRX1 inhibited LRAs in a post-transcriptional manner. In agreement, NLRX1 also repressed LRAs if luciferase was expressed under the control of a constitutive promoter, although the degree of inhibition by NLRX1 seemed to correlate with the dynamic inducibility of luciferase reporter constructs. Similarly, we observed that overexpression of another NLR protein, NLRC3, also resulted in artifactual inhibition of LRAs; thus suggesting that the capacity to inhibit LRAs at a post-transcriptional level is not unique to NLRX1. Finally, we demonstrate that host type I interferon response to Sendai virus infection was normal in NLRX1-silenced human HEK293T cells. Our results thus highlight the fact that LRAs are not a reliable technique to assess the inhibitory function of NLRs, and possibly other overexpressed proteins, on signal transduction pathways.
Asunto(s)
Genes Reporteros , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Luciferasas/biosíntesis , Proteínas Mitocondriales/metabolismo , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Luciferasas/genética , Proteínas Mitocondriales/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transducción de Señal/genéticaRESUMEN
Mitochondria are cellular organelles involved in host-cell metabolic processes and the control of programmed cell death. A direct link between mitochondria and innate immune signalling was first highlighted with the identification of MAVS-a crucial adaptor for RIGI-like receptor signalling-as a mitochondria-anchored protein. Recently, other innate immune molecules, such as NLRX1, TRAF6, NLRP3 and IRGM have been functionally associated with mitochondria. Furthermore, mitochondrial alarmins-such as mitochondrial DNA and formyl peptides-can be released by damaged mitochondria and trigger inflammation. Therefore, mitochondria emerge as a fundamental hub for innate immune signalling.
Asunto(s)
Inmunidad Innata , Mitocondrias/inmunología , Mitocondrias/metabolismo , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis , Humanos , Mitocondrias/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/inmunología , Proteínas Mitocondriales/metabolismoRESUMEN
Mitochondria have been long recognized for their key role in the modulation of cell death pathways. Thus, it is therefore not surprising that this organelle represents a recurrent target for pathogenic microbes, aiming to manipulate the fate of the infected host cell. More recently, mitochondria have been shown to serve as a crucial platform for innate immune signaling, as illustrated by the identification of MAVS (also known as IPS-1, VISA and Cardif), NLRX1 and STING as mitochondrial proteins. This review discusses the tight interplay between microbial infection, innate immune signaling and mitochondria.
Asunto(s)
Infecciones Bacterianas/inmunología , Mitocondrias/inmunología , Virosis/inmunología , Animales , Infecciones Bacterianas/metabolismo , Infecciones Bacterianas/patología , Muerte Celular , Humanos , Inmunidad Innata , Mitocondrias/metabolismo , Transducción de Señal , Virosis/metabolismo , Virosis/patologíaRESUMEN
AIM: To test the activities of culture-extracted or commercially available toll-like receptors (TLRs) ligands to establish their direct impact on target gastrointestinal motor cells. METHODS: Short-term and long-term effects of Shigella flexneri M90T and Escherichia coli K-2 strains-extracted lipopolysaccharides (LPS), commercially highly purified LPS (E. coli O111:B4 and EH100), and Pam2CSK4 and Pam3CSK4, which bind TLR2/6 and TLR1/2 heterodimers, respectively, have been assessed on pure primary cultures of colonic human smooth muscle cells (HSMC). RESULTS: Pathogenic Shigella-LPS and nonpathogenic E. coli K-2-LPS induced a time-dependent decrease of resting cell length and acetylcholine-induced contraction, with both alterations occurring rapidly and being more pronounced in response to the former. However, their effects differed, prolonging HSMC exposure with Shigella-LPS effects maintained throughout the 4 hours of observation compared with E. coli K-2-LPS, which disappeared after 60 minutes of incubation. Similar differences in magnitude and time dependency of myogenic effects were observed between pure TLR4 and TLR2/1 or TLR2/6 ligands. The specific activation of TLR4 with LPS from pathogen or nonpathogen E. coli, O111:B4 and EH100, respectively, induced smooth muscle alterations that progressively increased, prolonging incubation, whereas TLR2 ligands induced short-term alterations, of a lesser magnitude, which decreased over time. The real-time polymerase chain reaction analysis showed that HSMC express mRNA for TLR1, 2, 4, and 6, substantiating a direct effect of TLR ligands on human colonic smooth muscle. CONCLUSIONS: This study highlights that bacterial products can directly affect gastrointestinal motility and that TLRs subtypes may differ in their cellular activity.
Asunto(s)
Escherichia coli/inmunología , Motilidad Gastrointestinal/efectos de los fármacos , Lipopolisacáridos/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Shigella flexneri/inmunología , Receptores Toll-Like/metabolismo , Células Cultivadas , Colon/citología , Colon/inmunología , Colon/metabolismo , Colon/fisiopatología , Escherichia coli/metabolismo , Motilidad Gastrointestinal/fisiología , Humanos , Inmunidad Innata , Ligandos , Metagenoma , Contracción Muscular , Miocitos del Músculo Liso/inmunología , Miocitos del Músculo Liso/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Shigella flexneri/metabolismo , Receptor Toll-Like 2/efectos de los fármacos , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/efectos de los fármacos , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Receptores Toll-Like/efectos de los fármacos , Receptores Toll-Like/genéticaRESUMEN
During obesity and high fat-diet (HFD) feeding in mice, sustained low-grade inflammation includes not only increased pro-inflammatory macrophages in the expanding adipose tissue, but also bone marrow (BM) production of invasive Ly6Chigh monocytes. As BM adiposity also accrues with HFD, we explored the relationship between the gains in BM white adipocytes and invasive Ly6Chigh monocytes by in vivo and ex vivo paradigms. We find a temporal and causal link between BM adipocyte whitening and the Ly6Chigh monocyte surge, preceding the adipose tissue macrophage rise during HFD in mice. Phenocopying this, ex vivo treatment of BM cells with conditioned media from BM adipocytes or bona fide white adipocytes favoured Ly6Chigh monocyte preponderance. Notably, Ly6Chigh skewing was preceded by monocyte metabolic reprogramming towards glycolysis, reduced oxidative potential and increased mitochondrial fission. In sum, short-term HFD changes BM cellularity, resulting in local adipocyte whitening driving a gradual increase and activation of invasive Ly6Chigh monocytes.
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Médula Ósea , Monocitos , Adipocitos , Animales , Medios de Cultivo Condicionados , Ratones , Ratones Endogámicos C57BL , Monocitos/metabolismo , Obesidad/metabolismoRESUMEN
Chlamydia trachomatis infections cause severe and irreversible damage that can lead to infertility and blindness in both males and females. Following infection of epithelial cells, Chlamydia induces production of reactive oxygen species (ROS). Unconventionally, Chlamydiae use ROS to their advantage by activating caspase-1, which contributes to chlamydial growth. NLRX1, a member of the Nod-like receptor family that translocates to the mitochondria, can augment ROS production from the mitochondria following Shigella flexneri infections. However, in general, ROS can also be produced by membrane-bound NADPH oxidases. Given the importance of ROS-induced caspase-1 activation in growth of the chlamydial vacuole, we investigated the sources of ROS production in epithelial cells following infection with C. trachomatis. In this study, we provide evidence that basal levels of ROS are generated during chlamydial infection by NADPH oxidase, but ROS levels, regardless of their source, are enhanced by an NLRX1-dependent mechanism. Significantly, the presence of NLRX1 is required for optimal chlamydial growth.
Asunto(s)
Chlamydia trachomatis/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/química , Especies Reactivas de Oxígeno , Animales , Caspasa 1/metabolismo , Células HeLa , Humanos , Inmunidad Innata , Lentivirus/metabolismo , Ratones , Ratones Transgénicos , NADPH Oxidasas/química , ARN Interferente Pequeño/metabolismo , Shigella flexneri/metabolismoRESUMEN
NLRX1 is the only member of the Nod-like receptor (NLR) family that is targeted to the mitochondria, and its overexpression induces the generation of reactive oxygen species (ROS), thus impacting on NFkappaB- and JNK-dependent signaling cascades. In addition, NLRX1 has been shown to interact with MAVS (also known as IPS-1, VISA and Cardif) at the mitochondrial outer membrane and to modulate antiviral responses. Here we report that NLRX1 has a functional leader sequence and fully translocates to the mitochondrial matrix via a mechanism requiring the mitochondrial inner-membrane potential, DeltaPsim. Importantly, we failed to detect NLRX1 at the mitochondrial outer membrane. We also show that the leader sequence of NLRX1 is removed, which generates a mature protein lacking the first 39 amino acids through a maturation process that is common for mitochondrial-matrix proteins. Finally, we identified UQCRC2, a matrix-facing protein of the respiratory chain complex III, as an NLRX1-interacting molecule, thus providing a molecular basis for the role of NLRX1 in ROS generation. These results provide the first identification of a protein belonging to the NLR family that is targeted to the mitochondrial matrix.
Asunto(s)
Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Secuencias de Aminoácidos , Células HeLa , Humanos , Potencial de la Membrana Mitocondrial , Membranas Mitocondriales/química , Proteínas Mitocondriales/genética , Señales de Clasificación de Proteína , Transporte de ProteínasRESUMEN
The neurodegenerative disease spinal muscular atrophy (SMA) is caused by deficiency in the survival motor neuron (SMN) protein. Currently approved SMA treatments aim to restore SMN, but the potential for SMN expression beyond physiological levels is a unique feature of adeno-associated virus serotype 9 (AAV9)-SMN gene therapy. Here, we show that long-term AAV9-mediated SMN overexpression in mouse models induces dose-dependent, late-onset motor dysfunction associated with loss of proprioceptive synapses and neurodegeneration. Mechanistically, aggregation of overexpressed SMN in the cytoplasm of motor circuit neurons sequesters components of small nuclear ribonucleoproteins, leading to splicing dysregulation and widespread transcriptome abnormalities with prominent signatures of neuroinflammation and the innate immune response. Thus, long-term SMN overexpression interferes with RNA regulation and triggers SMA-like pathogenic events through toxic gain-of-function mechanisms. These unanticipated, SMN-dependent and neuron-specific liabilities warrant caution on the long-term safety of treating individuals with SMA with AAV9-SMN and the risks of uncontrolled protein expression by gene therapy.
Asunto(s)
Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Degeneración Nerviosa , Proteína 1 para la Supervivencia de la Neurona Motora/toxicidad , Animales , Dependovirus , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Técnicas de Transferencia de Gen , Terapia Genética/efectos adversos , Vectores Genéticos , Inyecciones Intraventriculares , Ratones , Trastornos Motores/genética , Trastornos Motores/metabolismo , Trastornos Motores/patología , Degeneración Nerviosa/genética , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Proteína 1 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
Pioneering studies from the early 1980s suggested that bacterial peptidoglycan-derived muramyl peptides (MPs) could exert either stimulatory or immunosuppressive functions depending, in part, on chronicity of exposure. However, this Janus-faced property of MPs remains largely unexplored. Here, we demonstrate the immunosuppressive potential of Nod1, the bacterial sensor of diaminopimelic acid (DAP)-containing MPs. Using a model of self-limiting peritonitis, we show that systemic Nod1 activation promotes an autophagy-dependent reprogramming of macrophages toward an alternative phenotype. Moreover, Nod1 stimulation induces the expansion of myeloid-derived suppressor cells (MDSCs) and maintains their immunosuppressive potential via arginase-1 activity. Supporting the role of MDSCs and tumor-associated macrophages in cancer, we demonstrate that myeloid-intrinsic Nod1 expression sustains intra-tumoral arginase-1 levels to foster an immunosuppressive and tumor-permissive microenvironment during colorectal cancer (CRC) development. Our findings support the notion that bacterial products, via Nod1 detection, modulate the immunosuppressive activity of myeloid cells and fuel tumor progression in CRC.
Asunto(s)
Neoplasias Colorrectales/inmunología , Células Supresoras de Origen Mieloide/inmunología , Proteína Adaptadora de Señalización NOD1/inmunología , Animales , Carcinogénesis/inmunología , Neoplasias Colorrectales/patología , Femenino , Humanos , Masculino , Ratones , Microambiente Tumoral/inmunologíaRESUMEN
Bacterial infections trigger the activation of innate immunity through the interaction of pathogen-associated molecular patterns (PAMPs) with pattern recognition molecules (PRMs). The nucleotide-binding oligomerization domain (Nod) proteins are intracellular PRMs that recognize muramylpeptides contained in peptidoglycan (PGN) of bacteria. It is still unclear how Nod1 physically interacts with PGN, a structure internal to the Gram-negative bacterial envelope. To contribute to the understanding of this process, we demonstrate that, like Escherichia coli, Bordetella pertussis and Neisseria gonorrheae, the Gram-negative pathogen Shigella spontaneously releases PGN fragments and that this process can be increased by inactivating either ampG or mppA, genes involved in PGN recycling. Both Shigella mutants, but especially the strain carrying the mppA deletion, trigger Nod1-mediated NF-kappaB activation to a greater extent than the wild-type strain. Likewise, muramylpeptides spontaneously shed by Shigella are able per se to trigger a Nod1-mediated response consistent with the relative amount. Finally, we found that qualitative changes in muramylpeptide shedding can alter in vivo host responses to Shigella infection. Our findings support the idea that muramylpeptides released by pathogens during infection could modulate the immune response through Nod proteins and thereby influence the outcome of disease.
Asunto(s)
Peptidoglicano/metabolismo , Shigella flexneri/inmunología , Shigella flexneri/metabolismo , Animales , Fusión Artificial Génica , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Recuento de Colonia Microbiana , Citocinas/análisis , Disentería Bacilar , Femenino , Eliminación de Gen , Silenciador del Gen , Genes Reporteros , Humanos , Hígado/microbiología , Hígado/patología , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Pulmón/química , Pulmón/microbiología , Pulmón/patología , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Endogámicos BALB C , FN-kappa B/inmunología , FN-kappa B/metabolismo , Proteína Adaptadora de Señalización NOD1/antagonistas & inhibidores , Proteína Adaptadora de Señalización NOD1/inmunología , Proteína Adaptadora de Señalización NOD1/metabolismo , Bazo/microbiología , Bazo/patologíaRESUMEN
Infected epithelial cells, which act as a first barrier against pathogens, seldom undergo apoptosis. Rather, infected epithelial cells undergo a slow cell death that displays hallmarks of necrosis. Here, we demonstrate that rapid intracellular lysis of Shigella flexneri, provoked by either the use of a diaminopimelic acid auxotroph mutant or treatment of infected cells with antibiotics of the beta-lactam family, resulted in a massive and rapid induction of apoptotic cell death. This intracellular bacteriolysis-mediated apoptotic death (IBAD) was characterized by the specific involvement of the mitochondrial-dependent cytochrome c/Apaf-1 axis that resulted in the activation of caspases-3, -6 and -9. Importantly, Bcl-2 family members and the NF-kappaB pathway seemed to be critical modulators of IBAD. Finally, we identified that IBAD was also triggered by Salmonella enterica serovar Typhimurium but not by the Gram-positive bacteria, Listeria monocytogenes. Together, our results demonstrate that, contrary to previous findings, epithelial cells are intrinsically able to mount an efficient apoptotic cell death response following infection. Indeed, apoptosis in normal circumstances is masked by powerful anti-apoptotic mechanisms, which are overcome in IBAD. Our results also uncover an unexpected consequence of the treatment of infected cells with certain classes of antibiotics.
Asunto(s)
Apoptosis , Bacteriólisis , Células Epiteliales/citología , Células Epiteliales/microbiología , Salmonella typhimurium/fisiología , Shigella flexneri/fisiología , Antibacterianos/farmacología , Caspasa 3/metabolismo , Caspasa 6/metabolismo , Caspasa 9/metabolismo , Supervivencia Celular , Citocromos c/metabolismo , Células Epiteliales/metabolismo , Células HeLa , Humanos , Etiquetado Corte-Fin in Situ , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Salmonella typhimurium/efectos de los fármacos , Shigella flexneri/efectos de los fármacosRESUMEN
Nod-like receptor (NLR)X1 is an NLR family protein that localizes to the mitochondrial matrix and modulates reactive oxygen species production, possibly by directly interacting with the electron transport chain. Recent work demonstrated that cells lacking NLRX1 have higher oxygen consumption but lower levels of adenosine triphosphate, suggesting that NLRX1 might prevent uncoupling of oxidative phosphorylation. We therefore hypothesized that NLRX1 might regulate whole-body energy metabolism through its effect on mitochondria. Male NLRX1 whole-body knockout (KO) mice and wild-type (WT) C57BL/6N controls were fed a low-fat or a high-fat (HF) diet for 16 weeks from weaning. Contrary to this hypothesis, there were no differences in body weight, adiposity, energy intake, or energy expenditure between HF-fed KO and WT mice, but instead HF KO mice were partially protected from the development of diet-induced hyperglycemia. Additionally, HF KO mice did not present with hyperinsulinemia during the glucose tolerance test, as did HF WT mice. There were no genotype differences in insulin tolerance, which led us to consider a pancreatic phenotype. Histology revealed that KO mice were protected from HF-induced pancreatic lipid accumulation, suggesting a potential role for NLRX1 in pancreatic dysfunction during the development diet-induced type 2 diabetes mellitus. Hence, NLRX1 depletion partially protects against postabsorptive hyperglycemia in obesity that may be linked to the prevention of pancreatic lipid accumulation. Although the actual mechanisms restoring glucose and insulin dynamics remain unknown, NLRX1 emerges as a potentially interesting target to inhibit for the prevention of type 2 diabetes mellitus.
RESUMEN
In physiological settings, the complement protein C3 is deposited on all bacteria, including invasive pathogens. However, because experimental host-bacteria systems typically use decomplemented serum to avoid the lytic action of complement, the impact of C3 coating on epithelial cell responses to invasive bacteria remains unexplored. Here, we demonstrate that following invasion, intracellular C3-positive Listeria monocytogenes is targeted by autophagy through a direct C3/ATG16L1 interaction, resulting in autophagy-dependent bacterial growth restriction. In contrast, Shigella flexneri and Salmonella Typhimurium escape autophagy-mediated growth restriction in part through the action of bacterial outer membrane proteases that cleave bound C3. Upon oral infection with Listeria, C3-deficient mice displayed defective clearance at the intestinal mucosa. Together, these results demonstrate an intracellular role of complement in triggering antibacterial autophagy and immunity against intracellular pathogens. Since C3 indiscriminately associates with foreign surfaces, the C3-ATG16L1 interaction may provide a universal mechanism of xenophagy initiation.