RESUMEN
The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1-deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte-associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function.
Asunto(s)
MicroARNs/metabolismo , Linfocitos T Reguladores/metabolismo , Animales , Citometría de Flujo , Ratones , Ratones Noqueados , MicroARNs/genética , Microscopía Confocal , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Timocitos/metabolismoRESUMEN
The interdependence of posttranscriptional gene regulation via miRNA and transcriptional regulatory networks in lymphocyte development is poorly understood. Here, we identified miR-191 as direct upstream modulator of a transcriptional module comprising the transcription factors Foxp1, E2A, and Egr1. Deletion as well as ectopic expression of miR-191 resulted in developmental arrest in B lineage cells, indicating that fine tuning of the combined expression levels of Foxp1, E2A, and Egr1, which in turn control somatic recombination and cytokine-driven expansion, constitutes a prerequisite for efficient B-cell development. In conclusion, we propose that miR-191 acts as a rheostat in B-cell development by fine tuning a key transcriptional program.
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Linfocitos B/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Factores de Transcripción Forkhead/metabolismo , MicroARNs/genética , Proteínas Represoras/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular , Células Cultivadas , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Factores de Transcripción Forkhead/genética , Redes Reguladoras de Genes , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Interferente Pequeño/genética , Recombinación Genética , Proteínas Represoras/genética , Transcripción Genética , Transgenes/genéticaRESUMEN
BACKGROUND: Myb-like, SWIRM, and MPN domains 1 (MYSM1) is a transcriptional regulator mediating histone deubiquitination. Its role in human immunity and hematopoiesis is poorly understood. OBJECTIVES: We sought to investigate the clinical, cellular, and molecular features in 2 siblings presenting with progressive bone marrow failure (BMF), immunodeficiency, and developmental aberrations. METHODS: We performed genome-wide homozygosity mapping, whole-exome and Sanger sequencing, immunophenotyping studies, and analysis of genotoxic stress responses. p38 activation, reactive oxygen species levels, rate of apoptosis and clonogenic survival, and growth in immune and nonimmune cells were assessed. The outcome of allogeneic hematopoietic stem cell transplantation (HSCT) was monitored. RESULTS: We report 2 patients with progressive BMF associated with myelodysplastic features, immunodeficiency affecting B cells and neutrophil granulocytes, and complex developmental aberrations, including mild skeletal anomalies, neurocognitive developmental delay, and cataracts. Whole-exome sequencing revealed a homozygous premature stop codon mutation in the gene encoding MYSM1. MYSM1-deficient cells are characterized by increased sensitivity to genotoxic stress associated with sustained induction of phosphorylated p38 protein, increased reactive oxygen species production, and decreased survival following UV light-induced DNA damage. Both patients were successfully treated with allogeneic HSCT with sustained reconstitution of hematopoietic defects. CONCLUSIONS: Here we show that MYSM1 deficiency is associated with developmental aberrations, progressive BMF with myelodysplastic features, and increased susceptibility to genotoxic stress. HSCT represents a curative therapy for patients with MYSM1 deficiency.
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Enfermedades de la Médula Ósea/inmunología , Daño del ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Discapacidades del Desarrollo/inmunología , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Síndromes de Inmunodeficiencia/inmunología , Factores de Transcripción/metabolismo , Células Cultivadas , Consanguinidad , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Estudio de Asociación del Genoma Completo , Genotipo , Histonas/metabolismo , Humanos , Linaje , Eliminación de Secuencia/genética , Transactivadores , Factores de Transcripción/genética , Proteasas Ubiquitina-Específicas , Ubiquitinación , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The microbiome and the phage meta-genome within the human gut are influenced by antibiotic treatments. Identifying a novel mechanism, here we demonstrate that bacteria use the universal communication molecule AI-2 to induce virulence genes and transfer them via phage release. High concentrations (i.e. 100 µM) of AI-2 promote dispersal of bacteria from already established biofilms, and is associated with release of phages capable of infecting other bacteria. Enterococcus faecalis V583ΔABC harbours 7 prophages in its genome, and a mutant deficient in one of these prophages (i.e. prophage 5) showed a greatly reduced dispersal of biofilm. Infection of a probiotic E. faecalis strain without lytic prophages with prophage 5 resulted in increased biofilm formation and also in biofilm dispersal upon induction with AI-2. Infection of the probiotic E. faecalis strain with phage-containing supernatants released through AI-2 from E. faecalis V583ΔABC resulted in a strong increase in pathogenicity of this strain. The polylysogenic probiotic strain was also more virulent in a mouse sepsis model and a rat endocarditis model. Both AI-2 and ciprofloxacin lead to phage release, indicating that conditions in the gastrointestinal tract of hospitalized patients treated with antibiotics might lead to distribution of virulence genes to apathogenic enterococci and possibly also to other commensals or even to beneficial probiotic strains.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Endocarditis Bacteriana/microbiología , Enterococcus faecalis , Profagos/fisiología , Percepción de Quorum , Sepsis/microbiología , Factores de Virulencia/metabolismo , Liberación del Virus/fisiología , Animales , Biopelículas/efectos de los fármacos , Células CACO-2 , Ciprofloxacina/farmacología , Endocarditis Bacteriana/patología , Enterococcus faecalis/patogenicidad , Enterococcus faecalis/fisiología , Enterococcus faecalis/virología , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Wistar , Sepsis/patología , Liberación del Virus/efectos de los fármacosRESUMEN
The origins of dendritic cells (DCs) and other myeloid cells in the thymus have remained controversial. In this study, we assessed developmental relationships between thymic dendritic cells and thymocytes, employing retrovirus-based cellular barcoding and reporter mice, as well as intrathymic transfers coupled with DC depletion. We demonstrated that a subset of early T-lineage progenitors expressed CX3CR1, a bona fide marker for DC progenitors. However, intrathymic transfers into nonmanipulated mice, as well as retroviral barcoding, indicated that thymic dendritic cells and thymocytes were largely of distinct developmental origin. In contrast, intrathymic transfers after in vivo depletion of DCs resulted in intrathymic development of non-T-lineage cells. In conclusion, our data support a model in which the adoption of T-lineage fate by noncommitted progenitors at steady state is enforced by signals from the thymic microenvironment unless niches promoting alternative lineage fates become available.
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Células Dendríticas/inmunología , Células Mieloides/inmunología , Nicho de Células Madre/inmunología , Células Madre/inmunología , Linfocitos T/inmunología , Timo/inmunología , Animales , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Células Dendríticas/citología , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Células Mieloides/citología , Células Madre/citología , Linfocitos T/citología , Timo/citologíaRESUMEN
BACKGROUND: Neutrophils are the predominant phagocytes that provide protection against bacterial and fungal infections. Genetically determined neutrophil disorders confer a predisposition to severe infections and reveal novel mechanisms that control vesicular trafficking, hematopoiesis, and innate immunity. METHODS: We clinically evaluated seven children from five families who had neutropenia, neutrophil dysfunction, bone marrow fibrosis, and nephromegaly. To identify the causative gene, we performed homozygosity mapping using single-nucleotide polymorphism arrays, whole-exome sequencing, immunoblotting, immunofluorescence, electron microscopy, a real-time quantitative polymerase-chain-reaction assay, immunohistochemistry, flow cytometry, fibroblast motility assays, measurements of apoptosis, and zebrafish models. Correction experiments were performed by transfecting mutant fibroblasts with the nonmutated gene. RESULTS: All seven affected children had homozygous mutations (Thr224Asn or Glu238Lys, depending on the child's ethnic origin) in VPS45, which encodes a protein that regulates membrane trafficking through the endosomal system. The level of VPS45 protein was reduced, as were the VPS45 binding partners rabenosyn-5 and syntaxin-16. The level of ß1 integrin was reduced on the surface of VPS45-deficient neutrophils and fibroblasts. VPS45-deficient fibroblasts were characterized by impaired motility and increased apoptosis. A zebrafish model of vps45 deficiency showed a marked paucity of myeloperoxidase-positive cells (i.e., neutrophils). Transfection of patient cells with nonmutated VPS45 corrected the migration defect and decreased apoptosis. CONCLUSIONS: Defective endosomal intracellular protein trafficking due to biallelic mutations in VPS45 underlies a new immunodeficiency syndrome involving impaired neutrophil function. (Funded by the National Human Genome Research Institute and others.).
Asunto(s)
Síndromes de Inmunodeficiencia/genética , Neutropenia/congénito , Proteínas de Transporte Vesicular/genética , Animales , Niño , Endosomas/metabolismo , Homocigoto , Humanos , Síndromes de Inmunodeficiencia/congénito , Síndromes de Inmunodeficiencia/inmunología , Mutación , Neutropenia/genética , Neutrófilos/fisiología , Fenotipo , Transporte de Proteínas , Proteínas de Transporte Vesicular/metabolismo , Pez CebraRESUMEN
Severe congenital neutropenia (SCN) is characterized by low numbers of peripheral neutrophil granulocytes and a predisposition to life-threatening bacterial infections. We describe a novel genetic SCN type in 2 unrelated families associated with recessively inherited loss-of-function mutations in CSF3R, encoding the granulocyte colony-stimulating factor (G-CSF) receptor. Family A, with 3 affected children, carried a homozygous missense mutation (NM_000760.3:c.922C>T, NP_000751.1:p.Arg308Cys), which resulted in perturbed N-glycosylation and aberrant localization to the cell surface. Family B, with 1 affected infant, carried compound heterozygous deletions provoking frameshifts and premature stop codons (NM_000760.3:c.948_963del, NP_000751.1:p.Gly316fsTer322 and NM_000760.3:c.1245del, NP_000751.1:p.Gly415fsTer432). Despite peripheral SCN, all patients had morphologic evidence of full myeloid cell maturation in bone marrow. None of the patients responded to treatment with recombinant human G-CSF. Our study highlights the genetic and morphologic SCN variability and provides evidence both for functional importance and redundancy of G-CSF receptor-mediated signaling in human granulopoiesis.
Asunto(s)
Mutación Missense , Neutropenia/congénito , Receptores del Factor Estimulante de Colonias/genética , Secuencia de Bases , Niño , Preescolar , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Femenino , Células HeLa , Homocigoto , Humanos , Lactante , Recién Nacido , Masculino , Modelos Moleculares , Neutropenia/genética , Linaje , Receptores del Factor Estimulante de Colonias/químicaRESUMEN
Nature and physiological status of antigen-presenting cells, such as dendritic cells DCs, are decisive for the immune reactions elicited. Multiple factors and cell interactions have been described that affect maturation of DCs. Here, we show that DCs arising in the absence of immunoglobulins (Ig) in vivo are impaired in cross-presentation of soluble antigen. This deficiency was due to aberrant cellular targeting of antigen to lysosomes and its rapid degradation. Function of DCs could be restored by transfer of Ig irrespective of antigen specificity and isotype. Modulation of cross-presentation by Ig was inhibited by coapplication of mannan and, thus, likely to be mediated by C-type lectin receptors. This unexpected dependency of splenic DCs on Ig to cross-present antigen provides insights into the interplay between cellular and humoral immunity and the immunomodulatory capacity of Ig.
Asunto(s)
Reactividad Cruzada , Células Dendríticas/inmunología , Inmunoglobulinas/metabolismo , Animales , Linfocitos B/inmunología , Diferenciación Celular , Células Dendríticas/citología , Células Dendríticas/metabolismo , Femenino , Lectinas Tipo C/metabolismo , Linfopenia/inmunología , Linfopenia/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Bazo/citología , Bazo/inmunología , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Pseudomonas aeruginosa is an environmentally ubiquitous Gram-negative bacterium and important opportunistic human pathogen, causing severe chronic respiratory infections in patients with underlying conditions such as cystic fibrosis (CF) or bronchiectasis. In order to identify mechanisms responsible for adaptation during bronchiectasis infections, a bronchiectasis isolate, PAHM4, was phenotypically and genotypically characterized. RESULTS: This strain displays phenotypes that have been associated with chronic respiratory infections in CF including alginate over-production, rough lipopolysaccharide, quorum-sensing deficiency, loss of motility, decreased protease secretion, and hypermutation. Hypermutation is a key adaptation of this bacterium during the course of chronic respiratory infections and analysis indicates that PAHM4 encodes a mutated mutS gene responsible for a ~1,000-fold increase in mutation rate compared to wild-type laboratory strain P. aeruginosa PAO1. Antibiotic resistance profiles and sequence data indicate that this strain acquired numerous mutations associated with increased resistance levels to ß-lactams, aminoglycosides, and fluoroquinolones when compared to PAO1. Sequencing of PAHM4 revealed a 6.38 Mbp genome, 5.9 % of which were unrecognized in previously reported P. aeruginosa genome sequences. Transcriptome analysis suggests a general down-regulation of virulence factors, while metabolism of amino acids and lipids is up-regulated when compared to PAO1 and metabolic modeling identified further potential differences between PAO1 and PAHM4. CONCLUSIONS: This work provides insights into the potential differential adaptation of this bacterium to the lung of patients with bronchiectasis compared to other clinical settings such as cystic fibrosis, findings that should aid the development of disease-appropriate treatment strategies for P. aeruginosa infections.
Asunto(s)
Bronquiectasia/microbiología , Fibrosis Quística/complicaciones , Genotipo , Fenotipo , Infecciones por Pseudomonas/etiología , Pseudomonas aeruginosa/fisiología , Adaptación Biológica/genética , Alelos , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas , Enfermedad Crónica , Biología Computacional , Farmacorresistencia Bacteriana , Perfilación de la Expresión Génica , Orden Génico , Genoma Bacteriano , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutación , Tasa de Mutación , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/patogenicidad , Percepción de Quorum/genética , Metabolismo Secundario , Transcriptoma , Virulencia/genéticaRESUMEN
PURPOSE: Loss-of-function mutations in IL10 and IL10R cause very early onset inflammatory bowel disease (VEO-IBD). Here, we investigated the molecular pathomechanism of a novel intronic IL10RA mutation and describe a new therapeutic approach of T cell replete haploidentical hematopoietic stem cell transplantation (HSCT). METHODS: Clinical data were collected by chart review. Genotypes of IL10 and IL10R genes were determined by Sanger sequencing. Expression and function of mutated IL-10R1 were assessed by quantitative PCR, Western blot analysis, enzyme-linked immunosorbent assays, confocal microscopy, and flow cytometry. RESULTS: We identified a novel homozygous point mutation in intron 3 of the IL10RA (c.368-10C > G) in three related children with VEO-IBD. Bioinformatical analysis predicted an additional 3' splice site created by the mutation. Quantitative PCR analysis showed normal mRNA expression of mutated IL10RA. Sequencing of the patient's cDNA revealed an insertion of the last nine nucleotides of intron 3 as a result of aberrant splicing. Structure-based modeling suggested misfolding of mutated IL-10R1. Western blot analysis demonstrated a different N-linked glycosylation pattern of mutated protein. Immunofluorescence and FACS analysis revealed impaired expression of mutated IL-10R1 at the plasma membrane. In the absence of HLA-identical donors, T cell replete haploidentical HSCT was successfully performed in two patients. CONCLUSIONS: Our findings expand the spectrum of IL10R mutations in VEO-IBD and emphasize the need for genetic diagnosis of mutations in conserved non-coding sequences of candidate genes. Transplantation of haploidentical stem cells represents a curative therapy in IL-10R-deficient patients, but may be complicated by non-engraftment.
Asunto(s)
Trasplante de Médula Ósea , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/terapia , Subunidad alfa del Receptor de Interleucina-10/metabolismo , Edad de Inicio , Empalme Alternativo , Secuencia de Aminoácidos , Línea Celular , Membrana Celular/metabolismo , Niño , Preescolar , Consanguinidad , Análisis Mutacional de ADN , Femenino , Genotipo , Glicosilación , Trasplante de Células Madre Hematopoyéticas , Humanos , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/inmunología , Subunidad alfa del Receptor de Interleucina-10/química , Subunidad alfa del Receptor de Interleucina-10/genética , Intrones , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Linaje , Fenotipo , Conformación Proteica , Transporte de Proteínas , Alineación de Secuencia , Transducción de Señal , Linfocitos T/inmunología , Resultado del TratamientoRESUMEN
Mycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint-based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time-dependent changes, albeit using a static model. By performing an in silico knock-out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms.
Asunto(s)
Metabolismo Energético/genética , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/metabolismo , Simulación por Computador , Redes y Vías Metabólicas/genética , Modelos Biológicos , MutaciónRESUMEN
BACKGROUND & AIMS: Homozygous loss of function mutations in interleukin-10 (IL10) and interleukin-10 receptors (IL10R) cause severe infantile (very early onset) inflammatory bowel disease (IBD). Allogeneic hematopoietic stem cell transplantation (HSCT) was reported to induce sustained remission in 1 patient with IL-10R deficiency. We investigated heterogeneity among patients with very early onset IBD, its mechanisms, and the use of allogeneic HSCT to treat this disorder. METHODS: We analyzed 66 patients with early onset IBD (younger than 5 years of age) for mutations in the genes encoding IL-10, IL-10R1, and IL-10R2. IL-10R deficiency was confirmed by functional assays on patients' peripheral blood mononuclear cells (immunoblot and enzyme-linked immunosorbent assay analyses). We assessed the therapeutic effects of standardized allogeneic HSCT. RESULTS: Using a candidate gene sequencing approach, we identified 16 patients with IL-10 or IL-10R deficiency: 3 patients had mutations in IL-10, 5 had mutations in IL-10R1, and 8 had mutations in IL-10R2. Refractory colitis became manifest in all patients within the first 3 months of life and was associated with perianal disease (16 of 16 patients). Extraintestinal symptoms included folliculitis (11 of 16) and arthritis (4 of 16). Allogeneic HSCT was performed in 5 patients and induced sustained clinical remission with a median follow-up time of 2 years. In vitro experiments confirmed reconstitution of IL-10R-mediated signaling in all patients who received the transplant. CONCLUSIONS: We identified loss of function mutations in IL-10 and IL-10R in patients with very early onset IBD. These findings indicate that infantile IBD patients with perianal disease should be screened for IL-10 and IL-10R deficiency and that allogeneic HSCT can induce remission in those with IL-10R deficiency.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Enfermedades Inflamatorias del Intestino , Subunidad alfa del Receptor de Interleucina-10/genética , Subunidad beta del Receptor de Interleucina-10/genética , Interleucina-10/genética , Western Blotting , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática , Femenino , Marcadores Genéticos , Humanos , Lactante , Enfermedades Inflamatorias del Intestino/diagnóstico , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/cirugía , Interleucina-10/deficiencia , Subunidad alfa del Receptor de Interleucina-10/deficiencia , Subunidad beta del Receptor de Interleucina-10/deficiencia , Masculino , Mutación , Análisis de Secuencia de ADN , Resultado del TratamientoRESUMEN
Many Gram-negative bacteria employ cell-to-cell communication mediated by N-acyl homoserine lactones (quorum sensing) to control expression of a wide range of genes including, but not limited to, genes encoding virulence factors. Outside the laboratory, the bacteria live in complex communities where signals may be perceived across species. We here present a newly found natural quorum sensing inhibitor, produced by the pseudomonads Pseudomonas sp. B13 and Pseudomonas reinekei MT1 as a blind end in the biodegradation of organochloride xenobiotics, which inhibits quorum sensing in P. aeruginosa in naturally occurring concentrations. This catabolite, 4-methylenebut-2-en-4-olide, also known as protoanemonin, has been reported to possess antibacterial properties, but seems to have dual functions. Using transcriptomics and proteomics, we found that protoanemonin significantly reduced expression of genes and secretion of proteins known to be under control of quorum sensing in P. aeruginosa. Moreover, we found activation of genes and gene products involved in iron starvation response. It is thus likely that inhibition of quorum sensing, as the production of antibiotics, is a phenomenon found in complex bacterial communities.
Asunto(s)
Furanos/metabolismo , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismo , Percepción de Quorum/genética , Antibacterianos/farmacología , Furanos/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Proteoma/efectos de los fármacos , Pseudomonas/efectos de los fármacos , Estrés Fisiológico/fisiologíaRESUMEN
The opportunistic bacterium Pseudomonas aeruginosa is a major nosocomial pathogen causing both devastating acute and chronic persistent infections. During the course of an infection, P. aeruginosa rapidly adapts to the specific conditions within the host. In the present study, we aimed at the identification of genes that are highly expressed during biofilm infections such as in chronically infected lungs of patients with cystic fibrosis (CF), burn wounds and subcutaneous mouse tumours. We found a common subset of differentially regulated genes in all three in vivo habitats and evaluated whether their inactivation impacts on the bacterial capability to form biofilms in vitro and to establish biofilm-associated infections in a murine model. Additive effects on biofilm formation and host colonization were discovered by the combined inactivation of several highly expressed genes. However, even combined inactivation was not sufficient to abolish the establishment of an infection completely. These findings can be interpreted as evidence that either redundant traits encode functions that are essential for in vivo survival and chronic biofilm infections and/or bacterial adaptation is considerably achieved independently of transcription levels. Supplemental screens, will have to be applied in order to identify the minimal set of key genes essential for the establishment of chronic infectious diseases.
Asunto(s)
Adaptación Fisiológica/genética , Fibrosis Quística/complicaciones , Fibrosis Quística/microbiología , Neoplasias , Infecciones por Pseudomonas/complicaciones , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Animales , Biopelículas , Ecosistema , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Silenciador del Gen , Genes Reguladores , Humanos , Pulmón/microbiología , Ratones , Neoplasias/complicaciones , Neoplasias/microbiología , Pseudomonas aeruginosa/genéticaRESUMEN
BACKGROUND: Genome scale metabolic reconstructions are developed to efficiently engineer biocatalysts and bioprocesses based on a rational approach. However, in most reconstructions, due to the lack of appropriate measurements, experimentally determined growth parameters are simply taken from literature including other organisms, which reduces the usefulness and suitability of these models. Pseudomonas putida KT2440 is an outstanding biocatalyst given its versatile metabolism, its ability to generate sufficient energy and turnover of NADH and NAD. To apply this strain optimally in industrial production, a previously developed genome-scale metabolic model (iJP815) was experimentally assessed and streamlined to enable accurate predictions of the outcome of metabolic engineering approaches. RESULTS: To substantially improve the accuracy of the genome scale model (iJP815), continuous bioreactor cultures on a mineral medium with glucose as a sole carbon source were carried out at different dilution rates, which covered pulling analysis of the macromolecular composition of the biomass. Besides, the maximum biomass yield (on substrate) of 0.397 gDCW · gglc-1, the maintenance coefficient of 0.037 gglc · gDCW-1 · h-1 and the maximum specific growth rate of 0.59 h-1 were determined. Only the DNA fraction increased with the specific growth rate. This resulted in reliable estimation for the Growth-Associated Maintenance (GAM) of 85 mmolATP · gDCW-1 and the Non Growth-Associated Maintenance (NGAM) of 3.96 mmolATP · gDCW-1 · h-1. Both values were found significantly different from previous assignment as a consequence of a lower yield and higher maintenance coefficient than originally assumed. Contrasting already published 13C flux measurements and the improved model allowed for constraining the solution space, by eliminating futile cycles. Furthermore, the model predictions were compared with transcriptomic data at overall good consistency, which helped to identify missing links. CONCLUSIONS: By careful interpretation of growth stoichiometry and kinetics when grown in the presence of glucose, this work reports on an accurate genome scale metabolic model of Pseudomonas putida, providing a solid basis for its use in designing superior strains for biocatalysis. By consideration of substrate specific variation in stoichiometry and kinetics, it can be extended to other substrates and new mutants.
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Reactores Biológicos , Microbiología Industrial , Pseudomonas putida/crecimiento & desarrollo , Biocatálisis , Biomasa , Carbono/metabolismo , Medios de Cultivo/química , Glucosa/metabolismo , Ingeniería Metabólica , Modelos Moleculares , TranscriptomaAsunto(s)
Agammaglobulinemia/diagnóstico , Síndromes de Inmunodeficiencia/diagnóstico , Síndromes de Inmunodeficiencia/inmunología , Mutación/genética , Patología Molecular/métodos , Receptores CXCR4/genética , Verrugas/diagnóstico , Verrugas/inmunología , Agammaglobulinemia/genética , Preescolar , Femenino , Humanos , Inmunoglobulinas Intravenosas/uso terapéutico , Masculino , Enfermedades de Inmunodeficiencia PrimariaRESUMEN
In the past decade, over 50 genome-scale metabolic reconstructions have been built for a variety of single- and multi- cellular organisms. These reconstructions have enabled a host of computational methods to be leveraged for systems-analysis of metabolism, leading to greater understanding of observed phenotypes. These methods have been sparsely applied to comparisons between multiple organisms, however, due mainly to the existence of differences between reconstructions that are inherited from the respective reconstruction processes of the organisms to be compared. To circumvent this obstacle, we developed a novel process, termed metabolic network reconciliation, whereby non-biological differences are removed from genome-scale reconstructions while keeping the reconstructions as true as possible to the underlying biological data on which they are based. This process was applied to two organisms of great importance to disease and biotechnological applications, Pseudomonas aeruginosa and Pseudomonas putida, respectively. The result is a pair of revised genome-scale reconstructions for these organisms that can be analyzed at a systems level with confidence that differences are indicative of true biological differences (to the degree that is currently known), rather than artifacts of the reconstruction process. The reconstructions were re-validated with various experimental data after reconciliation. With the reconciled and validated reconstructions, we performed a genome-wide comparison of metabolic flexibility between P. aeruginosa and P. putida that generated significant new insight into the underlying biology of these important organisms. Through this work, we provide a novel methodology for reconciling models, present new genome-scale reconstructions of P. aeruginosa and P. putida that can be directly compared at a network level, and perform a network-wide comparison of the two species. These reconstructions provide fresh insights into the metabolic similarities and differences between these important Pseudomonads, and pave the way towards full comparative analysis of genome-scale metabolic reconstructions of multiple species.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/fisiología , Genoma , Algoritmos , Biotecnología/métodos , Biología Computacional/métodos , Simulación por Computador , Bases de Datos Factuales , Genoma Bacteriano , Redes y Vías Metabólicas/genética , Fenotipo , Pseudomonas/genética , Reproducibilidad de los Resultados , Programas Informáticos , Especificidad de la EspecieRESUMEN
BACKGROUND: Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures. RESULTS: We found that 26, 62, and 81% of the cell dry weight consist of PHA under conditions of carbon, dual, and nitrogen limitation, respectively. Under nitrogen limitation a specific PHA production rate of 0.43 (g·(g·h)-1) was obtained. The residual biomass was not constant for dual- and strict nitrogen-limiting growth, showing a different feature in comparison to other P. putida strains. Dual limitation resulted in patterns of gene expression, protein level, and metabolite concentrations that substantially differ from those observed under exclusive carbon or nitrogen limitation. The most pronounced differences were found in the energy metabolism, fatty acid metabolism, as well as stress proteins and enzymes belonging to the transport system. CONCLUSION: This is the first study where the interrelationship between nutrient limitations and PHA synthesis has been investigated under well-controlled conditions using a system level approach. The knowledge generated will be of great assistance for the development of bioprocesses and further metabolic engineering work in this versatile organism to both enhance and diversify the industrial production of PHAs.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Polihidroxialcanoatos/biosíntesis , Pseudomonas putida/metabolismo , Biomasa , Carbono/metabolismo , Metabolismo Energético , Ácidos Grasos/metabolismo , Metabolómica , Nitrógeno/metabolismo , Proteómica , Pseudomonas putida/crecimiento & desarrollo , TranscriptomaRESUMEN
Type I IFNs play a key role in linking the innate and adaptive arms of the immune system. Although produced rapidly in response to pathogens, IFNs are also produced at low levels in the absence of infection. In the present study, we demonstrate that constitutively produced IFNs are necessary in vivo to maintain dendritic cells in an "Ag presentation-competent" state. Conventional dendritic cells (cDCs) isolated from spleens of IFN-beta or IFNAR-deficient mice exhibit a highly impaired ability to present Ag and activate naive T cells. Microarray analysis of mRNA isolated from IFN-beta(-/-) and IFNAR(-/-) cDCs revealed diminished expression of two genes that encoded members of the heat shock protein 70 (Hsp70) family. Consistent with this observation, pharmacological inhibition of Hsp70 in cDCs from wild-type mice impaired their T cell stimulatory capacity. Similarly, the Ag presentation ability of splenic cDCs isolated from Hsp70.1/3(-/-) mice was also severely impaired in comparison to wild-type cDCs. Thus, constitutive IFN-beta expression regulates Hsp70 levels to help maintain dendritic cells in a competent state for efficient priming of effector T cells in vivo.
Asunto(s)
Presentación de Antígeno , Células Dendríticas/inmunología , Regulación hacia Abajo , Interferón beta/fisiología , Animales , Proteínas HSP70 de Choque Térmico , Factores Inmunológicos , Interferón beta/deficiencia , Ratones , Ratones Noqueados , Receptor de Interferón alfa y beta/deficiencia , Bazo/citología , Linfocitos T/inmunologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.