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Congenital heart malformations include mitral valve defects, which remain largely unexplained. During embryogenesis, a restricted population of endocardial cells within the atrioventricular canal undergoes an endothelial-to-mesenchymal transition to give rise to mitral valvular cells. However, the identity and fate decisions of these progenitors as well as the behavior and distribution of their derivatives in valve leaflets remain unknown. We used single-cell RNA sequencing (scRNA-seq) of genetically labeled endocardial cells and microdissected mouse embryonic and postnatal mitral valves to characterize the developmental road. We defined the metabolic processes underlying the specification of the progenitors and their contributions to subtypes of valvular cells. Using retrospective multicolor clonal analysis, we describe specific modes of growth and behavior of endocardial cell-derived clones, which build up, in a proper manner, functional valve leaflets. Our data identify how both genetic and metabolic mechanisms specifically drive the fate of a subset of endocardial cells toward their distinct clonal contribution to the formation of the valve.
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Desarrollo Embrionario , Válvula Mitral , Animales , Ratones , Válvula Mitral/anomalías , Válvula Mitral/metabolismo , Estudios Retrospectivos , Diferenciación CelularRESUMEN
Flow cytometry is the method of choice for immunophenotyping in the context of clinical, translational, and systems immunology studies. Among the latter, the Milieu Intérieur (MI) project aims at defining the boundaries of a healthy immune response to identify determinants of immune response variation. MI used immunophenotyping of a 1000 healthy donor cohort by flow cytometry as a principal outcome for immune variance at steady state. New generation spectral cytometers now enable high-dimensional immune cell characterization from small sample volumes. Therefore, for the MI 10-year follow up study, we have developed two high-dimensional spectral flow cytometry panels for deep characterization of innate and adaptive whole blood immune cells (35 and 34 fluorescent markers, respectively). We have standardized the protocol for sample handling, staining, acquisition, and data analysis. This approach enables the reproducible quantification of over 182 immune cell phenotypes at a single site. We have applied the protocol to discern minor differences between healthy and patient samples and validated its value for application in immunomonitoring studies. Our protocol is currently used for characterization of the impact of age and environmental factors on peripheral blood immune phenotypes of >400 donors from the initial MI cohort.
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Estudios de Seguimiento , Humanos , Inmunofenotipificación , Fenotipo , Citometría de Flujo/métodosRESUMEN
Human induced pluripotent stem cells (hiPSCs) have been used extensively in vitro to model early events in neurodevelopment. Because of a number of shortcomings, previous work has established a potential to use these cells in vivo after transplantation into the mouse brain. Here, we describe a systematic approach for the analysis of transplanted hiPSC-derived neurons and glial cells over time in the mouse brain. Using functional two-photon imaging of GCaMP6f- expressing human neural cells, we define and quantify the embryonic-like features of their spontaneous activity. This is substantiated by detailed electron microscopy (EM) of the graft. We relate this to the synaptic development the neurons undergo up to 7 months in vivo. This system can now be used further for the genetic or experimental manipulation of developing hiPSC-derived cells addressing neurodevelopmental diseases like schizophrenia or Autism Spectrum Disorder.
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Single-cell RNA-sequencing (scRNAseq) experiments are becoming a standard tool for bench-scientists to explore the cellular diversity present in all tissues. Data produced by scRNAseq is technically complex and requires analytical workflows that are an active field of bioinformatics research, whereas a wealth of biological background knowledge is needed to guide the investigation. Thus, there is an increasing need to develop applications geared towards bench-scientists to help them abstract the technical challenges of the analysis so that they can focus on the science at play. It is also expected that such applications should support closer collaboration between bioinformaticians and bench-scientists by providing reproducible science tools. We present SCHNAPPs, a Graphical User Interface (GUI), designed to enable bench-scientists to autonomously explore and interpret scRNAseq data and associated annotations. The R/Shiny-based application allows following different steps of scRNAseq analysis workflows from Seurat or Scran packages: performing quality control on cells and genes, normalizing the expression matrix, integrating different samples, dimension reduction, clustering, and differential gene expression analysis. Visualization tools for exploring each step of the process include violin plots, 2D projections, Box-plots, alluvial plots, and histograms. An R-markdown report can be generated that tracks modifications and selected visualizations. The modular design of the tool allows it to easily integrate new visualizations and analyses by bioinformaticians. We illustrate the main features of the tool by applying it to the characterization of T cells in a scRNAseq and Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) experiment of two healthy individuals.
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Leucocitos Mononucleares/citología , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Programas Informáticos , Humanos , Leucocitos Mononucleares/inmunologíaRESUMEN
Cell plasticity plays a key role in embryos by maintaining the differentiation potential of progenitors. Whether postnatal somatic cells revert to an embryonic-like naïve state regaining plasticity and redifferentiate into a cell type leading to a disease remains intriguing. Using genetic lineage tracing and single-cell RNA sequencing, we reveal that Oct4 is induced by nuclear factor κB (NFκB) at embyronic day 9.5 in a subset of mouse endocardial cells originating from the anterior heart forming field at the onset of endocardial-to-mesenchymal transition. These cells acquired a chondro-osteogenic fate. OCT4 in adult valvular aortic cells leads to calcification of mouse and human valves. These calcifying cells originate from the Oct4 embryonic lineage. Genetic deletion of Pou5f1 (Pit-Oct-Unc, OCT4) in the endocardial cell lineage prevents aortic stenosis and calcification of ApoE−/− mouse valve. We established previously unidentified self-cell reprogramming NFκB- and OCT4-mediated inflammatory pathway triggering a dose-dependent mechanism of valve calcification.
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OBJECTIVE: Primary Sjögren's syndrome (pSS) is characterised by chronic hyperactivation of B lymphocytes. Salivary gland epithelial cells (SGECs) could play a role in promoting B-lymphocyte activation within the target tissue. We aimed to study the interactions between SGECs from patients with pSS or controls and B lymphocytes. METHODS: Patients had pSS according to 2016 European League Against Rheumatism/American College of Rheumatology criteria. Gene expression analysis of SGECs and B lymphocytes from pSS and controls isolated from salivary gland biopsies and blood was performed by RNA-seq. SGECs from pSS and controls were cocultured with B-lymphocytes sorted from healthy donor blood and were stimulated. Transwell and inhibition experiments were performed. RESULTS: Gene expression analysis of SGECs identified an upregulation of interferon signalling pathway and genes involved in immune responses (HLA-DRA, IL-7 and B-cell activating factor receptor) in pSS. Activation genes CD40 and CD48 were upregulated in salivary gland sorted B lymphocytes from patients with pSS. SGECs induced an increase in B-lymphocyte survival, which was higher for SGECs from patients with pSS than controls. Moreover, when stimulated with poly(I:C), SGECs from patients with pSS induced higher activation of B-lymphocytes than those from controls. This effect depended on soluble factors. Inhibition with anti-B-cell activating factor, anti-A proliferation-inducing ligand, anti-interleukin-6-R antibodies, JAK1/3 inhibitor or hydroxychloroquine had no effect, conversely to leflunomide, Bruton's tyrosine kinase (BTK) or phosphatidyl-inositol 3-kinase (PI3K) inhibitors. CONCLUSIONS: SGECs from patients with pSS had better ability than those from controls to induce survival and activation of B lymphocytes. Targeting a single cytokine did not inhibit this effect, whereas leflunomide, BTK or PI3K inhibitors partially decreased B-lymphocyte viability in this model. This gives indications for future therapeutic options in pSS.
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Linfocitos B/inmunología , Células Epiteliales/inmunología , Activación de Linfocitos/inmunología , Glándulas Salivales/inmunología , Síndrome de Sjögren/inmunología , Anciano , Linfocitos B/metabolismo , Supervivencia Celular , Células Cultivadas , Técnicas de Cocultivo , Células Epiteliales/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Glándulas Salivales/metabolismo , TranscriptomaRESUMEN
RATIONALE: Fibro-fatty infiltration of subepicardial layers of the atrial wall has been shown to contribute to the substrate of atrial fibrillation. OBJECTIVE: Here, we examined if the epicardium that contains multipotent cells is involved in this remodeling process. METHODS AND RESULTS: One hundred nine human surgical right atrial specimens were evaluated. There was a relatively greater extent of epicardial thickening and dense fibro-fatty infiltrates in atrial tissue sections from patients aged over 70 years who had mitral valve disease or atrial fibrillation when compared with patients aged less than 70 years with ischemic cardiomyopathy as indicated using logistic regression adjusted for age and gender. Cells coexpressing markers of epicardial progenitors and fibroblasts were detected in fibro-fatty infiltrates. Such epicardial remodeling was reproduced in an experimental model of atrial cardiomyopathy in rat and in Wilms tumor 1 (WT1)CreERT2/+;ROSA-tdT+/- mice. In the latter, genetic lineage tracing demonstrated the epicardial origin of fibroblasts within fibro-fatty infiltrates. A subpopulation of human adult epicardial-derived cells expressing PDGFR (platelet-derived growth factor receptor)-α were isolated and differentiated into myofibroblasts in the presence of Ang II (angiotensin II). Furthermore, single-cell RNA-sequencing analysis identified several clusters of adult epicardial-derived cells and revealed their specification from adipogenic to fibrogenic cells in the rat model of atrial cardiomyopathy. CONCLUSIONS: Epicardium is reactivated during the formation of the atrial cardiomyopathy. Subsets of adult epicardial-derived cells, preprogrammed towards a specific cell fate, contribute to fibro-fatty infiltration of subepicardium of diseased atria. Our study reveals the biological basis for chronic atrial myocardial remodeling that paves the way of atrial fibrillation.
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Tejido Adiposo/patología , Fibrilación Atrial/etiología , Remodelación Atrial , Cardiomiopatías/complicaciones , Atrios Cardíacos/patología , Miocardio/patología , Pericardio/patología , Potenciales de Acción , Adipocitos/metabolismo , Adipocitos/patología , Tejido Adiposo/metabolismo , Anciano , Animales , Fibrilación Atrial/metabolismo , Fibrilación Atrial/patología , Fibrilación Atrial/fisiopatología , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Cardiomiopatías/fisiopatología , Linaje de la Célula , Modelos Animales de Enfermedad , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis , Atrios Cardíacos/metabolismo , Atrios Cardíacos/fisiopatología , Frecuencia Cardíaca , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocardio/metabolismo , Pericardio/metabolismo , Pericardio/fisiopatología , Ratas Wistar , Células Madre/metabolismo , Células Madre/patología , Proteínas WT1/genética , Proteínas WT1/metabolismoRESUMEN
Identifying the factors that shape protein expression variability in complex multi-cellular organisms has primarily focused on promoter architecture and regulation of single-cell expression in cis. However, this targeted approach has to date been unable to identify major regulators of cell-to-cell gene expression variability in humans. To address this, we have combined single-cell protein expression measurements in the human immune system using flow cytometry with a quantitative genetics analysis. For the majority of proteins whose variability in expression has a heritable component, we find that genetic variants act in trans, with notably fewer variants acting in cis. Furthermore, we highlight using Mendelian Randomization that these variability-Quantitative Trait Loci might be driven by the cis regulation of upstream genes. This indicates that natural selection may balance the impact of gene regulation in cis with downstream impacts on expression variability in trans.
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Regulación de la Expresión Génica/genética , Expresión Génica/genética , Alelos , Bases de Datos Genéticas , Femenino , Perfilación de la Expresión Génica/métodos , Pruebas Genéticas/métodos , Estudio de Asociación del Genoma Completo/métodos , Humanos , Sistema Inmunológico/metabolismo , Inmunidad/genética , Masculino , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo/genética , Selección Genética/genéticaRESUMEN
Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.
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Células Endoteliales/patología , Proteínas Hedgehog/genética , Prolapso de la Válvula Mitral/patología , Válvula Mitral/patología , Células Madre Pluripotentes/patología , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Proteína Morfogenética Ósea 2/farmacología , Proteínas Relacionadas con las Cadherinas , Cadherinas/genética , Cadherinas/metabolismo , Diferenciación Celular/efectos de los fármacos , Embrión de Mamíferos , Endocardio/metabolismo , Endocardio/patología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/trasplante , Transición Epitelial-Mesenquimal/efectos de los fármacos , Factor de Transcripción GATA5/genética , Factor de Transcripción GATA5/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Atrios Cardíacos/metabolismo , Atrios Cardíacos/patología , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Válvula Mitral/metabolismo , Prolapso de la Válvula Mitral/genética , Prolapso de la Válvula Mitral/metabolismo , Prolapso de la Válvula Mitral/terapia , Modelos Biológicos , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Cultivo Primario de Células , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Proteína Wnt3A/farmacologíaRESUMEN
BACKGROUND: The SOS response is an almost ubiquitous response of cells to genotoxic stresses. The full complement of genes in the SOS regulon for Vibrio species has only been addressed through bioinformatic analyses predicting LexA binding box consensus and in vitro validation. Here, we perform whole transcriptome sequencing from Vibrio cholerae treated with mitomycin C as an SOS inducer to characterize the SOS regulon and other pathways affected by this treatment. RESULTS: Comprehensive transcriptional profiling allowed us to define the full landscape of promoters and transcripts active in V. cholerae. We performed extensive transcription start site (TSS) mapping as well as detection/quantification of the coding and non-coding RNA (ncRNA) repertoire in strain N16961. To improve TSS detection, we developed a new technique to treat RNA extracted from cells grown in various conditions. This allowed for identification of 3078 TSSs with an average 5'UTR of 116 nucleotides, and peak distribution between 16 and 64 nucleotides; as well as 629 ncRNAs. Mitomycin C treatment induced transcription of 737 genes and 28 ncRNAs at least 2 fold, while it repressed 231 genes and 17 ncRNAs. Data analysis revealed that in addition to the core genes known to integrate the SOS regulon, several metabolic pathways were induced. This study allowed for expansion of the Vibrio SOS regulon, as twelve genes (ubiEJB, tatABC, smpA, cep, VC0091, VC1190, VC1369-1370) were found to be co-induced with their adjacent canonical SOS regulon gene(s), through transcriptional read-through. Characterization of UV and mitomycin C susceptibility for mutants of these newly identified SOS regulon genes and other highly induced genes and ncRNAs confirmed their role in DNA damage rescue and protection. CONCLUSIONS: We show that genotoxic stress induces a pervasive transcriptional response, affecting almost 20% of the V. cholerae genes. We also demonstrate that the SOS regulon is larger than previously known, and its syntenic organization is conserved among Vibrio species. Furthermore, this specific co-localization is found in other γ-proteobacteria for genes recN-smpA and rmuC-tatABC, suggesting SOS regulon conservation in this phylum. Finally, we comment on the limitations of widespread NGS approaches for identification of all RNA species in bacteria.
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Perfilación de la Expresión Génica , Regulón/genética , Respuesta SOS en Genética/genética , Vibrio cholerae/genética , Regiones no Traducidas 5'/genética , Mitomicina/farmacología , Fenotipo , Respuesta SOS en Genética/efectos de los fármacos , Sitio de Iniciación de la Transcripción/efectos de los fármacos , Vibrio cholerae/efectos de los fármacosRESUMEN
Genetic and molecular studies have indicated that an epigenetic imprint at mat1, the sexual locus of fission yeast, initiates mating type switching. The polar DNA replication of mat1 generates an imprint on the Watson strand. The process by which the imprint is formed and maintained through the cell cycle remains unclear. To understand better the mechanism of imprint formation and stability, we characterized the recruitment of early players of mating type switching at the mat1 region. We found that the switch activating protein 1 (Sap1) is preferentially recruited inside the mat1M allele on a sequence (SS13) that enhances the imprint. The lysine specific demethylases, Lsd1/2, that control the replication fork pause at MPS1 and the formation of the imprint are specifically drafted inside of mat1, regardless of the allele. The CENP-B homolog, Abp1, is highly enriched next to mat1 but it is not required in the process. Additionally, we established the computational signature of the imprint. Using this signature, we show that both sides of the imprinted molecule are bound by Lsd1/2 and Sap1, suggesting a nucleoprotein protective structure defined as imprintosome.
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The success of Staphylococcus aureus, as both a human and animal pathogen, stems from its ability to rapidly adapt to a wide spectrum of environmental conditions. Two-component systems (TCSs) play a crucial role in this process. Here, we describe a novel staphylococcal virulence factor, SpdC, an Abi-domain protein, involved in signal sensing and/or transduction. We have uncovered a functional link between the WalKR essential TCS and the SpdC Abi membrane protein. Expression of spdC is positively regulated by the WalKR system and, in turn, SpdC negatively controls WalKR regulon genes, effectively constituting a negative feedback loop. The WalKR system is mainly involved in controlling cell wall metabolism through regulation of autolysin production. We have shown that SpdC inhibits the WalKR-dependent synthesis of four peptidoglycan hydrolases, SceD, SsaA, LytM and AtlA, as well as impacting S. aureus resistance towards lysostaphin and cell wall antibiotics such as oxacillin and tunicamycin. We have also shown that SpdC is required for S. aureus biofilm formation and virulence in a murine septicemia model. Using protein-protein interactions in E. coli as well as subcellular localization in S. aureus, we showed that SpdC and the WalK kinase are both localized at the division septum and that the two proteins interact. In addition to WalK, our results indicate that SpdC also interacts with nine other S. aureus histidine kinases, suggesting that this membrane protein may act as a global regulator of TCS activity. Indeed, using RNA-Seq analysis, we showed that SpdC controls the expression of approximately one hundred genes in S. aureus, many of which belong to TCS regulons.
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Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Histidina Quinasa/metabolismo , Sepsis/microbiología , Infecciones Estafilocócicas/microbiología , Factores de Virulencia/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Femenino , Histidina Quinasa/genética , Ratones , Fosforilación , Regulón , Sepsis/metabolismo , Transducción de Señal , Infecciones Estafilocócicas/metabolismo , Staphylococcus aureus/patogenicidad , Virulencia , Factores de Virulencia/genéticaRESUMEN
BACKGROUND: An interferon signature is involved in the pathogenesis of primary Sjögren syndrome (pSS), but whether the signature is type 1 or type 2 remains controversial. Mouse models and genetic studies suggest the involvement of TH1 and type 2 interferon pathways. Likewise, polymorphisms of the IL-12A gene (IL12A), which encodes for IL-12p35, have been associated with pSS. The IL-12p35 subunit is shared by 2 heterodimers: IL-12 and IL-35. OBJECTIVE: We sought to confirm genetic association of the IL12A polymorphism and pSS and elucidate involvement of the IL-12/IL-35 balance in patients with pSS by using functional studies. METHODS: The genetic study involved 673 patients with pSS from 2 French pSS cohorts and 585 healthy French control subjects. Functional studies were performed on sorted monocytes, irrespective of whether they were stimulated. IL12A mRNA expression and IL-12 and IL-35 protein levels were assessed by using quantitative RT-PCR and ELISA and a multiplex kit for IL-35 and IL-12, respectively. RESULTS: We confirmed association of the IL12A rs485497 polymorphism and pSS and found an increased serum protein level of IL-12p70 in patients with pSS carrying the risk allele (P = .016). Serum levels of IL-12p70 were greater in patients than control subjects (P = .0001), especially in patients with more active disease (P = .05); conversely, IL-35 levels were decreased in patients (P = .0001), especially in patients with more active disease (P = .05). In blood cellular subsets both IL12p35 and EBV-induced gene protein 3 (EBI3) mRNAs were detected only in B cells, with a trend toward a lower level among patients with pSS. CONCLUSION: Our findings emphasize involvement of the IL-12/IL-35 balance in the pathogenesis of pSS. Serum IL-35 levels were associated with low disease activity, in contrast with serum IL-12p70 levels, which were associated with more active disease.
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Subunidad p35 de la Interleucina-12/genética , Subunidad p35 de la Interleucina-12/inmunología , Interleucinas/inmunología , Síndrome de Sjögren/inmunología , Anciano , Femenino , Genotipo , Humanos , Subunidad p35 de la Interleucina-12/sangre , Interleucinas/sangre , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Síndrome de Sjögren/sangre , Síndrome de Sjögren/genéticaRESUMEN
Bacteria of the genus Thiomonas are found ubiquitously in arsenic contaminated waters such as acid mine drainage (AMD), where they contribute to the precipitation and the natural bioremediation of arsenic. In these environments, these bacteria have developed a large range of resistance strategies among which the capacity to form particular biofilm structures. The biofilm formation is one of the most ubiquitous adaptive response observed in prokaryotes to various stresses, such as those induced in the presence of toxic compounds. This study focused on the process of biofilm formation in three Thiomonas strains (CB1, CB2 and CB3) isolated from the same AMD. The results obtained here show that these bacteria are all capable of forming biofilms, but the architecture and the kinetics of formation of these biofilms differ depending on whether arsenite is present in the environment and from one strain to another. Indeed, two strains favoured biofilm formation, whereas one favoured motility in the presence of arsenite. To identify the underlying mechanisms, the patterns of expression of some genes possibly involved in the process of biofilm formation were investigated in Thiomonas sp. CB2 in the presence and absence of arsenite, using a transcriptomic approach (RNA-seq). The findings obtained here shed interesting light on how the formation of biofilms, and the motility processes contribute to the adaptation of Thiomonas strains to extreme environments.
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Arsenitos/metabolismo , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Burkholderiales/efectos de los fármacos , Burkholderiales/fisiología , Contaminantes Ambientales/metabolismo , Locomoción/efectos de los fármacos , Burkholderiales/genética , Farmacorresistencia Bacteriana , Perfilación de la Expresión GénicaRESUMEN
The fungal cell wall is a rigid structure because of fibrillar and branched ß-(1,3)-glucan linked to chitin. Softening of the cell wall is an essential phenomenon during fungal morphogenesis, wherein rigid cell wall structures are cleaved by glycosylhydrolases. During the search for glycosylhydrolases acting on ß-(1,3)-glucan, we identified seven genes in the Aspergillus fumigatus genome coding for potential endo-ß-(1,3)-glucanase. ENG1 (previously characterized and named ENGL1, Mouyna et al., ), belongs to the Glycoside-Hydrolase 81 (GH81) family, while ENG2 to ENG7, to GH16 family. ENG1 and four GH16 genes (ENG2-5) were expressed in the resting conidia as well as during germination, suggesting an essential role during A. fumigatus morphogenesis. Here, we report the effect of sequential deletion of AfENG2-5 (GH16) followed by AfENG1 (GH81) deletion in the Δeng2,3,4,5 mutant. The Δeng1,2,3,4,5 mutant showed conidial defects, with linear chains of conidia unable to separate while the germination rate was not affected. These results show, for the first time in a filamentous fungus, that endo ß-(1,3)-glucanases are essential for proper conidial cell wall assembly and thus segregation of conidia during conidiation.
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Aspergillus fumigatus/enzimología , Pared Celular/enzimología , Proteínas Fúngicas/fisiología , Glicósido Hidrolasas/fisiología , Esporas Fúngicas/enzimología , Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus fumigatus/ultraestructura , Conformación de Carbohidratos , Pared Celular/ultraestructura , Glicosilación , Morfogénesis , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/ultraestructuraRESUMEN
The RIG-I-like receptors (RLRs) play a major role in sensing RNA virus infection to initiate and modulate antiviral immunity. They interact with particular viral RNAs, most of them being still unknown. To decipher the viral RNA signature on RLRs during viral infection, we tagged RLRs (RIG-I, MDA5, LGP2) and applied tagged protein affinity purification followed by next-generation sequencing (NGS) of associated RNA molecules. Two viruses with negative- and positive-sense RNA genome were used: measles (MV) and chikungunya (CHIKV). NGS analysis revealed that distinct regions of MV genome were specifically recognized by distinct RLRs: RIG-I recognized defective interfering genomes, whereas MDA5 and LGP2 specifically bound MV nucleoprotein-coding region. During CHIKV infection, RIG-I associated specifically to the 3' untranslated region of viral genome. This study provides the first comparative view of the viral RNA ligands for RIG-I, MDA5 and LGP2 in the presence of infection.
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Virus Chikungunya/inmunología , Proteína 58 DEAD Box/metabolismo , Helicasa Inducida por Interferón IFIH1/metabolismo , Virus del Sarampión/inmunología , ARN Helicasas/metabolismo , ARN Viral/metabolismo , Receptores Inmunológicos/metabolismo , Línea Celular , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Helicasa Inducida por Interferón IFIH1/aislamiento & purificación , ARN Helicasas/aislamiento & purificación , ARN Viral/genética , Receptores Inmunológicos/aislamiento & purificaciónRESUMEN
BACKGROUND AND AIMS: In cystic fibrosis (CF), Pseudomonas aeruginosa is not eradicated from the lower respiratory tract and is associated with epithelial inflammation that eventually causes tissue damage. To identify the molecular determinants of an effective response to P. aeruginosa infection, we performed a transcriptomic analysis of primary human bronchial epithelial cells from healthy donors (CTRL) 2, 4, and 6 h after induced P. aeruginosa infection. Compared to noninfected cells, infected cells showed changes in gene activity, which were most marked 6 h postinfection and usually consisted in upregulation. RESULTS: By comparing for each time point of infection, the transcriptomic response of epithelial cells from CF patients and healthy donors, we identified 851, 638, 667, and 980 differentially expressed genes 0, 2, 4, and 6 h postinfection, respectively. Gene selection followed by bioinformatic analysis showed that most of the differentially expressed genes, either up- or downregulated, were in the protein-binding and catalytic gene-ontology categories. Finally, we established that the protein products of the genes exhibiting the greatest differential upregulation (CSF2, CCL2, TNF, CSF3, MMP1, and MMP10) between CF patients and CTRL were produced in higher amounts by infected cells from CF patients versus CTRL. CONCLUSIONS: The differentially expressed genes in CF patients may constitute a signature for a detrimental inflammatory response and for an inefficient P. aeruginosa host-cell response.
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Bronquios/microbiología , Fibrosis Quística/microbiología , Células Epiteliales/microbiología , Regulación de la Expresión Génica , Infecciones por Pseudomonas/genética , Pseudomonas aeruginosa , Adulto , Bronquios/patología , Fibrosis Quística/genética , Fibrosis Quística/patología , Células Epiteliales/patología , Femenino , Humanos , Masculino , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología , Activación Transcripcional , Adulto JovenRESUMEN
Acid mine drainage (AMD) is a highly toxic environment for most living organisms due to the presence of many lethal elements including arsenic (As). Thiomonas (Tm.) bacteria are found ubiquitously in AMD and can withstand these extreme conditions, in part because they are able to oxidize arsenite. In order to further improve our knowledge concerning the adaptive capacities of these bacteria, we sequenced and assembled the genome of six isolates derived from the Carnoulès AMD, and compared them to the genomes of Tm. arsenitoxydans 3As (isolated from the same site) and Tm. intermedia K12 (isolated from a sewage pipe). A detailed analysis of the Tm. sp. CB2 genome revealed various rearrangements had occurred in comparison to what was observed in 3As and K12 and over 20 genomic islands (GEIs) were found in each of these three genomes. We performed a detailed comparison of the two arsenic-related islands found in CB2, carrying the genes required for arsenite oxidation and As resistance, with those found in K12, 3As, and five other Thiomonas strains also isolated from Carnoulès (CB1, CB3, CB6, ACO3 and ACO7). Our results suggest that these arsenic-related islands have evolved differentially in these closely related Thiomonas strains, leading to divergent capacities to survive in As rich environments.
Asunto(s)
Arsénico , Burkholderiaceae/genética , Genoma Bacteriano , Microbiología del Agua , Burkholderiaceae/aislamiento & purificaciónRESUMEN
The RpoS/σ(S) sigma subunit of RNA polymerase (RNAP) activates transcription of stationary phase genes in many Gram-negative bacteria and controls adaptive functions, including stress resistance, biofilm formation and virulence. In this study, we address an important but poorly understood aspect of σ(S)-dependent control, that of a repressor. Negative regulation by σ(S) has been proposed to result largely from competition between σ(S) and other σ factors for binding to a limited amount of core RNAP (E). To assess whether σ(S) binding to E alone results in significant downregulation of gene expression by other σ factors, we characterized an rpoS mutant of Salmonella enterica serovar Typhimurium producing a σ(S) protein proficient for Eσ(S) complex formation but deficient in promoter DNA binding. Genome expression profiling and physiological assays revealed that this mutant was defective for negative regulation, indicating that gene repression by σ(S) requires its binding to DNA. Although the mechanisms of repression by σ(S) are likely specific to individual genes and environmental conditions, the study of transcription downregulation of the succinate dehydrogenase operon suggests that σ competition at the promoter DNA level plays an important role in gene repression by Eσ(S).
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Factor sigma/metabolismo , Regiones Promotoras GenéticasRESUMEN
The RpoS/σS sigma subunit of RNA polymerase (RNAP) controls a global adaptive response that allows many Gram-negative bacteria to survive starvation and various stresses. σS also contributes to biofilm formation and virulence of the food-borne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). In this study, we used directional RNA-sequencing and complementary assays to explore the σS-dependent transcriptome of S. Typhimurium during late stationary phase in rich medium. This study confirms the large regulatory scope of σS and provides insights into the physiological functions of σS in Salmonella. Extensive regulation by σS of genes involved in metabolism and membrane composition, and down-regulation of the respiratory chain functions, were important features of the σS effects on gene transcription that might confer fitness advantages to bacterial cells and/or populations under starving conditions. As an example, we show that arginine catabolism confers a competitive fitness advantage in stationary phase. This study also provides a firm basis for future studies to address molecular mechanisms of indirect regulation of gene expression by σS. Importantly, the σS-controlled downstream network includes small RNAs that might endow σS with post-transcriptional regulatory functions. Of these, four (RyhB-1/RyhB-2, SdsR, SraL) were known to be controlled by σS and deletion of the sdsR locus had a competitive fitness cost in stationary phase. The σS-dependent control of seven additional sRNAs was confirmed in Northern experiments. These findings will inspire future studies to investigate molecular mechanisms and the physiological impact of post-transcriptional regulation by σS.