RESUMO
Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.
Assuntos
Infecções por Coronavirus/imunologia , Células Mieloides/imunologia , Mielopoese , Pneumonia Viral/imunologia , Adulto , Idoso , Antígenos CD11/genética , Antígenos CD11/metabolismo , COVID-19 , Células Cultivadas , Infecções por Coronavirus/sangue , Infecções por Coronavirus/patologia , Feminino , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Células Mieloides/citologia , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/patologia , Proteoma/genética , Proteoma/metabolismo , Proteômica , Análise de Célula ÚnicaRESUMO
RNA degradation is an essential process that allows bacteria to regulate gene expression and has emerged as an important mechanism for controlling virulence. However, the individual contributions of RNases in this process are mostly unknown. Here, we tested the influence of 11 potential RNases in the intestinal pathogen Yersinia pseudotuberculosis on the expression of its type III secretion system (T3SS) and associated effectors (Yops) that are encoded on the Yersinia virulence plasmid. We found that exoribonuclease PNPase and endoribonuclease RNase III inhibit T3SS and yop gene transcription by repressing the synthesis of LcrF, the master activator of Yop-T3SS. Loss of both RNases led to an increase in lcrF mRNA levels. Our work indicates that PNPase exerts its influence via YopD, which accelerates lcrF mRNA degradation. Loss of RNase III, on the other hand, results in the downregulation of the CsrB and CsrC RNAs, thereby increasing the availability of active CsrA, which has been shown previously to enhance lcrF mRNA translation and stability. This CsrA-promoted increase of lcrF mRNA translation could be supported by other factors promoting the protein translation efficiency (e.g. IF-3, RimM, RsmG) that were also found to be repressed by RNase III. Transcriptomic profiling further revealed that Ysc-T3SS-mediated Yop secretion leads to global reprogramming of the Yersinia transcriptome with a massive shift of the expression from chromosomal to virulence plasmid-encoded genes. A similar reprogramming was also observed in the RNase III-deficient mutant under non-secretion conditions. Overall, our work revealed a complex control system where RNases orchestrate the expression of the T3SS/Yop machinery on multiple levels to antagonize phagocytic uptake and elimination by innate immune cells.
RESUMO
Bacillus subtilis cells are well suited to study how bacteria sense and adapt to proteotoxic stress such as heat, since temperature fluctuations are a major challenge to soil-dwelling bacteria. Here, we show that the alarmones (p)ppGpp, well known second messengers of nutrient starvation, are also involved in the heat stress response as well as the development of thermo-resistance. Upon heat-shock, intracellular levels of (p)ppGpp rise in a rapid but transient manner. The heat-induced (p)ppGpp is primarily produced by the ribosome-associated alarmone synthetase Rel, while the small alarmone synthetases RelP and RelQ seem not to be involved. Furthermore, our study shows that the generated (p)ppGpp pulse primarily acts at the level of translation, and only specific genes are regulated at the transcriptional level. These include the down-regulation of some translation-related genes and the up-regulation of hpf, encoding the ribosome-protecting hibernation-promoting factor. In addition, the alarmones appear to interact with the activity of the stress transcription factor Spx during heat stress. Taken together, our study suggests that (p)ppGpp modulates the translational capacity at elevated temperatures and thereby allows B. subtilis cells to respond to proteotoxic stress, not only by raising the cellular repair capacity, but also by decreasing translation to concurrently reduce the protein load on the cellular protein quality control system.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Resposta ao Choque Térmico/genética , Ligases/genética , Regulação Bacteriana da Expressão Gênica/genéticaRESUMO
Conventional dendritic cells (cDCs) arise from committed precursor dendritic cells (pre-DCs) in the bone marrow that continuously seed the periphery. Pre-DCs and other upstream progenitors proliferate and mature in response to Fms-related receptor tyrosine kinase 3 ligand, which is considered the key cytokine for cDC development. However, other cytokines such as stem cell factor and colony-stimulating factor 1 (CSF1) were also shown to induce pre-DC maturation into DC-like cells. Yet, it is still only incompletely understood which cells contribute to cDC development once pre-DCs arrive in peripheral tissues. Here, we analysed the impact of lymph node (LN) fibroblastic stromal cells (FSCs) on the maturation of pre-DCs into cDC-like cells. We could demonstrate that ex vivo isolated LN FSCs co-cultured with pre-DCs induce precursor maturation into DC-like cells, which were capable of efficiently promoting the proliferation of naïve CD4+ T cells. Interestingly, FSCs isolated from distinct LNs induced DC-like cells with highly comparable transcriptomes, characterized by the expression of signature genes of both ex vivo isolated DCs and macrophages. Finally, by performing supplementation and receptor blocking studies, we could demonstrate that CSF1 is a driving factor for LN FSC-mediated pre-DC maturation into DC-like cells. In summary, we could identify CSF1 as a stromal cell-derived factor that has the potential to support the maturation of pre-DCs into cDC-like cells within secondary lymphoid organs.
Assuntos
Células Dendríticas , Fator Estimulador de Colônias de Macrófagos , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/metabolismo , Linfonodos , Fator Estimulador de Colônias de Macrófagos/metabolismo , Fator Estimulador de Colônias de Macrófagos/farmacologia , Células Estromais , Linfócitos TRESUMO
Methods for assembly, taxonomic profiling and binning are key to interpreting metagenome data, but a lack of consensus about benchmarking complicates performance assessment. The Critical Assessment of Metagenome Interpretation (CAMI) challenge has engaged the global developer community to benchmark their programs on highly complex and realistic data sets, generated from â¼700 newly sequenced microorganisms and â¼600 novel viruses and plasmids and representing common experimental setups. Assembly and genome binning programs performed well for species represented by individual genomes but were substantially affected by the presence of related strains. Taxonomic profiling and binning programs were proficient at high taxonomic ranks, with a notable performance decrease below family level. Parameter settings markedly affected performance, underscoring their importance for program reproducibility. The CAMI results highlight current challenges but also provide a roadmap for software selection to answer specific research questions.
Assuntos
Metagenômica , Software , Algoritmos , Benchmarking , Análise de Sequência de DNARESUMO
Gastrointestinal infections caused by enteric yersiniae can become persistent and complicated by relapsing enteritis and severe autoimmune disorders. To establish a persistent infection, the bacteria have to cope with hostile surroundings when they transmigrate through the intestinal epithelium and colonize underlying gut-associated lymphatic tissues. How the bacteria gain a foothold in the face of host immune responses is poorly understood. Here, we show that the CNFY toxin, which enhances translocation of the antiphagocytic Yop effectors, induces inflammatory responses. This results in extensive tissue destruction, alteration of the intestinal microbiota and bacterial clearance. Suppression of CNFY function, however, increases interferon-γ-mediated responses, comprising non-inflammatory antimicrobial activities and tolerogenesis. This process is accompanied by a preterm reprogramming of the pathogen's transcriptional response towards persistence, which gives the bacteria a fitness edge against host responses and facilitates establishment of a commensal-type life style.
Assuntos
Toxinas Bacterianas/genética , Deleção de Genes , Inflamação/genética , Fatores de Virulência/genética , Infecções por Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/genética , Animais , Ceco/microbiologia , Progressão da Doença , Feminino , Gastroenterite/genética , Gastroenterite/microbiologia , Gastroenteropatias/genética , Gastroenteropatias/microbiologia , Microbioma Gastrointestinal/fisiologia , Inflamação/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Organismos Geneticamente Modificados , Yersinia pseudotuberculosis/patogenicidade , Infecções por Yersinia pseudotuberculosis/patologiaRESUMO
Pathogenic bacteria need to rapidly adjust their virulence and fitness program to prevent eradication by the host. So far, underlying adaptation processes that drive pathogenesis have mostly been studied in vitro, neglecting the true complexity of host-induced stimuli acting on the invading pathogen. In this study, we developed an unbiased experimental approach that allows simultaneous monitoring of genome-wide infection-linked transcriptional alterations of the host and colonizing extracellular pathogens. Using this tool for Yersinia pseudotuberculosis-infected lymphatic tissues, we revealed numerous alterations of host transcripts associated with inflammatory and acute-phase responses, coagulative activities, and transition metal ion sequestration, highlighting that the immune response is dominated by infiltrating neutrophils and elicits a mixed TH17/TH1 response. In consequence, the pathogen's response is mainly directed to prevent phagocytic attacks. Yersinia up-regulates the gene and expression dose of the antiphagocytic type III secretion system (T3SS) and induces functions counteracting neutrophil-induced ion deprivation, radical stress, and nutritional restraints. Several conserved bacterial riboregulators were identified that impacted this response. The strongest influence on virulence was found for the loss of the carbon storage regulator (Csr) system, which is shown to be essential for the up-regulation of the T3SS on host cell contact. In summary, our established approach provides a powerful tool for the discovery of infection-specific stimuli, induced host and pathogen responses, and underlying regulatory processes.
Assuntos
Interações Hospedeiro-Patógeno/genética , Transcriptoma , Infecções por Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/genética , Animais , Feminino , Camundongos Endogâmicos BALB C , Nódulos Linfáticos Agregados/metabolismo , Nódulos Linfáticos Agregados/microbiologia , RNA Mensageiro/genética , Análise de Sequência de RNA , Fatores de Virulência/genética , Yersinia pseudotuberculosis/metabolismo , Yersinia pseudotuberculosis/fisiologia , Infecções por Yersinia pseudotuberculosis/imunologiaRESUMO
Intestinal regulatory T cells (Tregs) are fundamental in peripheral tolerance toward commensals and food-borne antigens. Accordingly, gut-draining mesenteric lymph nodes (mLNs) represent a site of efficient peripheral de novo Treg induction when compared to skin-draining peripheral LNs (pLNs), and we had recently shown that LN stromal cells substantially contribute to this process. Here, we aimed to unravel the underlying molecular mechanisms and generated immortalized fibroblastic reticular cell lines (iFRCs) from mLNs and pLNs, allowing unlimited investigation of this rare stromal cell subset. In line with our previous findings, mLN-iFRCs showed a higher Treg-inducing capacity when compared to pLN-iFRCs. RNA-seq analysis focusing on secreted molecules revealed a more tolerogenic phenotype of mLN- as compared to pLN-iFRCs. Remarkably, mLN-iFRCs produced substantial numbers of microvesicles (MVs) that carried elevated levels of TGF-ß when compared to pLN-iFRC-derived MVs, and these novel players of intercellular communication were shown to be responsible for the tolerogenic properties of mLN-iFRCs. Thus, stromal cells originating from mLNs contribute to peripheral tolerance by fostering de novo Treg induction using TGF-ß-carrying MVs. This finding provides novel insights into the subcellular/molecular mechanisms of de novo Treg induction and might serve as promising tool for future therapeutic applications to treat inflammatory disorders.
Assuntos
Vesículas Extracelulares/imunologia , Linfonodos/imunologia , Células Estromais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linhagem Celular , Vesículas Extracelulares/genética , Vesículas Extracelulares/ultraestrutura , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica/métodos , Mesentério/imunologia , Camundongos Endogâmicos BALB C , Camundongos Knockout , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Células Estromais/metabolismo , Células Estromais/ultraestrutura , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologiaRESUMO
One hallmark of pathogenic yersiniae is their ability to rapidly adjust their life-style and pathogenesis upon host entry. In order to capture the range, magnitude and complexity of the underlying gene control mechanisms we used comparative RNA-seq-based transcriptomic profiling of the enteric pathogen Y. pseudotuberculosis under environmental and infection-relevant conditions. We identified 1151 individual transcription start sites, multiple riboswitch-like RNA elements, and a global set of antisense RNAs and previously unrecognized trans-acting RNAs. Taking advantage of these data, we revealed a temperature-induced and growth phase-dependent reprogramming of a large set of catabolic/energy production genes and uncovered the existence of a thermo-regulated 'acetate switch', which appear to prime the bacteria for growth in the digestive tract. To elucidate the regulatory architecture linking nutritional status to virulence we also refined the CRP regulon. We identified a massive remodelling of the CRP-controlled network in response to temperature and discovered CRP as a transcriptional master regulator of numerous conserved and newly identified non-coding RNAs which participate in this process. This finding highlights a novel level of complexity of the regulatory network in which the concerted action of transcriptional regulators and multiple non-coding RNAs under control of CRP adjusts the control of Yersinia fitness and virulence to the requirements of their environmental and virulent life-styles.
Assuntos
Proteína Receptora de AMP Cíclico/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Regulon/genética , Yersinia pseudotuberculosis/genética , AMP Cíclico/genética , AMP Cíclico/metabolismo , Regulação Fúngica da Expressão Gênica , Interação Gene-Ambiente , RNA Antissenso/genética , RNA Antissenso/isolamento & purificação , Riboswitch/genética , Temperatura , Sítio de Iniciação de Transcrição , Yersinia pseudotuberculosis/crescimento & desenvolvimento , Yersinia pseudotuberculosis/patogenicidadeRESUMO
Clostridioides difficile is the major pathogen causing diarrhea following antibiotic treatment. It is considered to be a strictly anaerobic bacterium, however, previous studies have shown a certain and strain-dependent oxygen tolerance. In this study, the model strain C. difficile 630Δerm was shifted to micro-aerobiosis and was found to stay growing to the same extent as anaerobically growing cells with only few changes in the metabolite pattern. However, an extensive change in gene expression was determined by RNA-Seq. The most striking adaptation strategies involve a change in the reductive fermentation pathways of the amino acids proline, glycine and leucine. But also a far-reaching restructuring in the carbohydrate metabolism was detected with changes in the phosphotransferase system (PTS) facilitated uptake of sugars and a repression of enzymes of glycolysis and butyrate fermentation. Furthermore, a temporary induction in the synthesis of cofactor riboflavin was detected possibly due to an increased demand for flavin mononucleotid (FMN) and flavin adenine dinucleotide (FAD) in redox reactions. However, biosynthesis of the cofactors thiamin pyrophosphate and cobalamin were repressed deducing oxidation-prone enzymes and intermediates in these pathways. Micro-aerobically shocked cells were characterized by an increased demand for cysteine and a thiol redox proteomics approach revealed a dramatic increase in the oxidative state of cysteine in more than 800 peptides after 15â¯min of micro-aerobic shock. This provides not only a catalogue of oxidation-prone cysteine residues in the C. difficile proteome but also puts the amino acid cysteine into a key position in the oxidative stress response. Our study suggests that tolerance of C. difficile towards O2 is based on a complex and far-reaching adjustment of global gene expression which leads to only a slight change in phenotype.
Assuntos
Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/genética , Perfilação da Expressão Gênica , Estresse Oxidativo , Oxigênio/toxicidade , Aerobiose , Anaerobiose , Clostridioides difficile/crescimento & desenvolvimento , Genômica , Redes e Vias Metabólicas/genética , ProteômicaRESUMO
We recently found that Yersinia pseudotuberculosis can be used as a model of persistent bacterial infections. We performed in vivo RNA-seq of bacteria in small cecal tissue biopsies at early and persistent stages of infection to determine strategies associated with persistence. Comprehensive analysis of mixed RNA populations from infected tissues revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence when the pathogen resides within the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26oC, with for example, up-regulation of flagellar genes and invA. These findings are expected to have impact on future rationales to identify suitable bacterial targets for new antibiotics. Other genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, arcA, fnr, frdA, and wrbA play critical roles in persistence. Our findings suggest a model for the life cycle of this enteropathogen with reprogramming from a virulent to an adapted phenotype capable of persisting and spreading by fecal shedding.
Assuntos
Análise de Sequência de RNA/métodos , Virulência/genética , Infecções por Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/patogenicidade , Animais , Ceco/imunologia , Ceco/microbiologia , Ceco/patologia , Feminino , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Camundongos , Análise em Microsséries , Microbiota/imunologia , RNA Bacteriano/genética , Yersinia pseudotuberculosis/imunologia , Infecções por Yersinia pseudotuberculosis/imunologia , Infecções por Yersinia pseudotuberculosis/patologiaRESUMO
UNLABELLED: The twin-arginine translocation (Tat) system mediates the secretion of folded proteins that are identified via an N-terminal signal peptide in bacteria, plants, and archaea. Tat systems are associated with virulence in many bacterial pathogens, and our previous studies revealed that Tat-deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analyzed the global transcriptome of parental and ΔtatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the ΔtatC mutant were seen at 26°C during stationary-phase growth, and these included the altered expression of genes related to virulence, stress responses, and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes, including decreased YadA expression, impaired growth under iron-limited and high-copper conditions, as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than those in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. IMPORTANCE: In addition to its established role in mediating the secretion of housekeeping enzymes, the Tat system has been recognized as being involved in infection. In some clinically relevant bacteria, such as Pseudomonas spp., several key virulence determinants can readily be identified among the Tat substrates. In enteropathogens, such as Yersinia spp., there are no obvious virulence determinants among the Tat substrates. Tat mutants show no growth defect in vitro but are highly attenuated in in vivo This makes Tat an attractive target for the development of novel antimicrobials. Therefore, it is important to establish the causes of the attenuation. Here, we show that the attenuation is likely due to synergistic effects of different Tat-dependent phenotypes that each contributes to lowered in vivo fitness.
Assuntos
Proteínas de Bactérias/genética , Sistema de Translocação de Argininas Geminadas/metabolismo , Yersinia pseudotuberculosis/metabolismo , Proteínas de Bactérias/metabolismo , Cobre/metabolismo , Regulação Bacteriana da Expressão Gênica , Ferro/metabolismo , Fenótipo , Transporte Proteico , Transcriptoma , Sistema de Translocação de Argininas Geminadas/genética , Yersinia pseudotuberculosis/genéticaRESUMO
Gut-draining mesenteric and celiac lymph nodes (mLNs and celLNs) critically contribute to peripheral tolerance toward food and microbial antigens by supporting the de novo induction of regulatory T cells (Tregs). These tolerogenic properties of mLNs and celLNs are stably imprinted within stromal cells (SCs) by microbial signals and vitamin A (VA), respectively. Here, we report that a single, transient gastrointestinal infection in the neonatal, but not adult, period durably abrogates the efficient Treg-inducing capacity of celLNs by altering the subset composition and gene expression profile of celLNSCs. These cells carry information about the early-life pathogen encounter until adulthood and durably instruct migratory dendritic cells entering the celLN with reduced tolerogenic properties. Mechanistically, transiently reduced VA levels cause long-lasting celLN functional impairment, which can be rescued by early-life treatment with VA. Together, our data highlight the therapeutic potential of VA to prevent sequelae post gastrointestinal infections in infants.
Assuntos
Linfonodos , Linfócitos T Reguladores , Vitamina A , Animais , Linfonodos/imunologia , Linfonodos/patologia , Linfonodos/efeitos dos fármacos , Vitamina A/farmacologia , Vitamina A/uso terapêutico , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Camundongos , Animais Recém-Nascidos , Tolerância Imunológica/efeitos dos fármacos , Células Dendríticas/imunologia , Camundongos Endogâmicos C57BL , FemininoRESUMO
BACKGROUND: It is well known that the search for homologous RNAs is more effective if both sequence and structure information is incorporated into the search. However, current tools for searching with RNA sequence-structure patterns cannot fully handle mutations occurring on both these levels or are simply not fast enough for searching large sequence databases because of the high computational costs of the underlying sequence-structure alignment problem. RESULTS: We present new fast index-based and online algorithms for approximate matching of RNA sequence-structure patterns supporting a full set of edit operations on single bases and base pairs. Our methods efficiently compute semi-global alignments of structural RNA patterns and substrings of the target sequence whose costs satisfy a user-defined sequence-structure edit distance threshold. For this purpose, we introduce a new computing scheme to optimally reuse the entries of the required dynamic programming matrices for all substrings and combine it with a technique for avoiding the alignment computation of non-matching substrings. Our new index-based methods exploit suffix arrays preprocessed from the target database and achieve running times that are sublinear in the size of the searched sequences. To support the description of RNA molecules that fold into complex secondary structures with multiple ordered sequence-structure patterns, we use fast algorithms for the local or global chaining of approximate sequence-structure pattern matches. The chaining step removes spurious matches from the set of intermediate results, in particular of patterns with little specificity. In benchmark experiments on the Rfam database, our improved online algorithm is faster than the best previous method by up to factor 45. Our best new index-based algorithm achieves a speedup of factor 560. CONCLUSIONS: The presented methods achieve considerable speedups compared to the best previous method. This, together with the expected sublinear running time of the presented index-based algorithms, allows for the first time approximate matching of RNA sequence-structure patterns in large sequence databases. Beyond the algorithmic contributions, we provide with RaligNAtor a robust and well documented open-source software package implementing the algorithms presented in this manuscript. The RaligNAtor software is available at http://www.zbh.uni-hamburg.de/ralignator.
Assuntos
Algoritmos , Sequência de Bases , Análise de Sequência de RNA , Pareamento de Bases , Sequência de Bases/genética , Biologia Computacional/métodos , Simulação por Computador , Bases de Dados Factuais , RNA/química , RNA/genética , Alinhamento de Sequência , Análise de Sequência de RNA/métodos , SoftwareRESUMO
The current focus on renewable energy in global policy highlights the importance of methane production from biomass through anaerobic digestion (AD). To improve biomass digestion while ensuring overall process stability, microbiome-based management strategies become more important. In this study, metagenomes and metaproteomes were used for metagenomically assembled genome (MAG)-centric analyses to investigate a full-scale biogas plant consisting of three differentially operated digesters. Microbial communities were analyzed regarding their taxonomic composition, functional potential, as well as functions expressed on the proteome level. Different abundances of genes and enzymes related to the biogas process could be mostly attributed to different process parameters. Individual MAGs exhibiting different abundances in the digesters were studied in detail, and their roles in the hydrolysis, acidogenesis and acetogenesis steps of anaerobic digestion could be assigned. Methanoculleus thermohydrogenotrophicum was an active hydrogenotrophic methanogen in all three digesters, whereas Methanothermobacter wolfeii was more prevalent at higher process temperatures. Further analysis focused on MAGs, which were abundant in all digesters, indicating their potential to ensure biogas process stability. The most prevalent MAG belonged to the class Limnochordia; this MAG was ubiquitous in all three digesters and exhibited activity in numerous pathways related to different steps of AD.
RESUMO
A better understanding of the genetic regulation of the biosynthesis of microbial compounds could accelerate the discovery of new biologically active molecules and facilitate their production. To this end, we have investigated the time course of genome-wide transcription in the myxobacterium Sorangium sp. So ce836 in relation to its production of natural compounds. Time-resolved RNA sequencing revealed that core biosynthesis genes from 48 biosynthetic gene clusters (BGCs; 92% of all BGCs encoded in the genome) were actively transcribed at specific time points in a batch culture. The majority (80%) of polyketide synthase and non-ribosomal peptide synthetase genes displayed distinct peaks of transcription during exponential bacterial growth. Strikingly, these bursts in BGC transcriptional activity were associated with surges in the net production rates of known natural compounds, indicating that their biosynthesis was critically regulated at the transcriptional level. In contrast, BGC read counts from single time points had limited predictive value about biosynthetic activity, since transcription levels varied >100-fold among BGCs with detected natural products. Taken together, our time-course data provide unique insights into the dynamics of natural compound biosynthesis and its regulation in a wild-type myxobacterium, challenging the commonly cited notion of preferential BGC expression under nutrient-limited conditions. The close association observed between BGC transcription and compound production warrants additional efforts to develop genetic engineering tools for boosting compound yields from myxobacterial producer strains.
Assuntos
Myxococcales , Sorangium , Sorangium/genética , Policetídeo Sintases/genética , Família Multigênica , Myxococcales/genéticaRESUMO
BACKGROUND & AIMS: Thymic conventional dendritic cells (t-DCs) are crucial for the development of T cells. A substantial fraction of t-DCs originates extrathymically and migrates to the thymus. Here, these cells contribute to key processes of central tolerance like the clonal deletion of self-reactive thymocytes and the generation of regulatory T (Treg) cells. So far, it is only incompletely understood which impact the thymic microenvironment has on thymus-homing conventional DCs (cDCs), which phenotypic changes occur after the entry of peripheral cDCs into the thymus and which functional properties these modulated cells acquire. MATERIALS & METHODS: In the present study, we mimicked the thymus-homing of peripheral cDCs by introducing ex vivo isolated splenic cDCs (sp-DCs) into reaggregated thymic organ cultures (RTOCs). RESULTS: Already after two days of culture, the transcriptomic profile of sp-DCs was modulated and had acquired certain key signatures of t-DCs. The regulated genes included immunomodulatory cytokines and chemokines as well as costimulatory molecules. After four days of culture, sp-DCs appeared to have at least partially acquired the peculiar Treg cell-inducing capacity characteristic of t-DCs. DISCUSSION & CONCLUSION: Taken together, our findings indicate that peripheral cDCs possess a high degree of plasticity enabling them to quickly adapt to the thymus-specific microenvironment. We further provide indirect evidence that thymus-specific properties such as the efficient induction of Treg cells under homeostatic conditions can be partially transferred to thymus-homing peripheral cDC subsets.
Assuntos
Células Dendríticas , Linfócitos T Reguladores , FenótipoRESUMO
Epigenetic modifications such as DNA methylation play an essential role in imprinting specific transcriptional patterns in cells. We performed genome-wide DNA methylation profiling of murine lymph node-derived ILCs, which led to the identification of differentially methylated regions (DMRs) and the definition of epigenetic marker regions in ILCs. Marker regions were located in genes with a described function for ILCs, such as Tbx21, Gata3, or Il23r, but also in genes that have not been related to ILC biology. Methylation levels of the marker regions and expression of the associated genes were strongly correlated, indicating their functional relevance. Comparison with T helper cell methylomes revealed clear lineage differences, despite partial similarities in the methylation of specific ILC marker regions. IL-33-mediated challenge affected methylation of ILC2 epigenetic marker regions in the liver, while remaining relatively stable in the lung. In our study, we identified a set of epigenetic markers that can serve as a tool to study phenotypic and functional properties of ILCs.
Assuntos
Imunidade Inata , Linfócitos , Animais , Biomarcadores , Metilação de DNA/genética , Epigênese Genética , Estudo de Associação Genômica Ampla , CamundongosRESUMO
Gut-draining mesenteric lymph nodes (LN) provide the framework to shape intestinal adaptive immune responses. Based on the transcriptional signatures established by our previous work, the composition and immunomodulatory function of LN stromal cells (SC) vary according to location. Here, we describe the single-cell composition and development of the SC compartment within mesenteric LNs derived from postnatal to aged mice. We identify CD34+ SC and fibroblastic reticular stromal cell (FRC) progenitors as putative progenitors, both supplying the typical rapid postnatal mesenteric LN expansion. We further establish the location-specific chromatin accessibility and DNA methylation landscape of non-endothelial SCs and identify a microbiota-independent core epigenomic signature, showing characteristic differences between SCs from mesenteric and skin-draining peripheral LNs. The epigenomic landscape of SCs points to dynamic expression of Irf3 along the differentiation trajectories of FRCs. Accordingly, a mesenchymal stem cell line acquires a Cxcl9+ FRC molecular phenotype upon lentiviral overexpression of Irf3, and the relevance of Irf3 for SC biology is further underscored by the diminished proportion of Ccl19+ and Cxcl9+ FRCs in LNs of Irf3-/- mice. Together, our data constitute a comprehensive transcriptional and epigenomic map of mesenteric LNSC development in early life and dissect location-specific, microbiota-independent properties of non-endothelial SCs.
Assuntos
Linfonodos , Células Estromais , Camundongos , Animais , Camundongos Endogâmicos C57BL , Células Estromais/metabolismo , Linfonodos/patologia , Moléculas de Adesão Celular/metabolismo , Antígenos CD34/metabolismoRESUMO
BACKGROUND: The secondary structure of RNA molecules is intimately related to their function and often more conserved than the sequence. Hence, the important task of searching databases for RNAs requires to match sequence-structure patterns. Unfortunately, current tools for this task have, in the best case, a running time that is only linear in the size of sequence databases. Furthermore, established index data structures for fast sequence matching, like suffix trees or arrays, cannot benefit from the complementarity constraints introduced by the secondary structure of RNAs. RESULTS: We present a novel method and readily applicable software for time efficient matching of RNA sequence-structure patterns in sequence databases. Our approach is based on affix arrays, a recently introduced index data structure, preprocessed from the target database. Affix arrays support bidirectional pattern search, which is required for efficiently handling the structural constraints of the pattern. Structural patterns like stem-loops can be matched inside out, such that the loop region is matched first and then the pairing bases on the boundaries are matched consecutively. This allows to exploit base pairing information for search space reduction and leads to an expected running time that is sublinear in the size of the sequence database. The incorporation of a new chaining approach in the search of RNA sequence-structure patterns enables the description of molecules folding into complex secondary structures with multiple ordered patterns. The chaining approach removes spurious matches from the set of intermediate results, in particular of patterns with little specificity. In benchmark experiments on the Rfam database, our method runs up to two orders of magnitude faster than previous methods. CONCLUSIONS: The presented method's sublinear expected running time makes it well suited for RNA sequence-structure pattern matching in large sequence databases. RNA molecules containing several stem-loop substructures can be described by multiple sequence-structure patterns and their matches are efficiently handled by a novel chaining method. Beyond our algorithmic contributions, we provide with Structator a complete and robust open-source software solution for index-based search of RNA sequence-structure patterns. The Structator software is available at http://www.zbh.uni-hamburg.de/Structator.