RESUMO
Mucociliary clearance through coordinated ciliary beating is a major innate defense removing pathogens from the lower airways, but the pathogen sensing and downstream signaling mechanisms remain unclear. We identified virulence-associated formylated bacterial peptides that potently stimulated ciliary-driven transport in the mouse trachea. This innate response was independent of formyl peptide and taste receptors but depended on key taste transduction genes. Tracheal cholinergic chemosensory cells expressed these genes, and genetic ablation of these cells abrogated peptide-driven stimulation of mucociliary clearance. Trpm5-deficient mice were more susceptible to infection with a natural pathogen, and formylated bacterial peptides were detected in patients with chronic obstructive pulmonary disease. Optogenetics and peptide stimulation revealed that ciliary beating was driven by paracrine cholinergic signaling from chemosensory to ciliated cells operating through muscarinic M3 receptors independently of nerves. We provide a cellular and molecular framework that defines how tracheal chemosensory cells integrate chemosensation with innate defense.
Assuntos
Acetilcolina/imunologia , Proteínas de Bactérias/farmacologia , Cílios/imunologia , Depuração Mucociliar/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Canais de Cátion TRPM/imunologia , Traqueia/imunologia , Acetilcolina/metabolismo , Animais , Proteínas de Bactérias/imunologia , Transporte Biológico , Cílios/efeitos dos fármacos , Cílios/metabolismo , Feminino , Formiatos/metabolismo , Expressão Gênica , Humanos , Imunidade Inata , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Optogenética/métodos , Comunicação Parácrina/imunologia , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/patologia , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/imunologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Canais de Cátion TRPM/deficiência , Canais de Cátion TRPM/genética , Papilas Gustativas/imunologia , Papilas Gustativas/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/patologia , VirulênciaRESUMO
Organoids derived from mouse and human stem cells have recently emerged as a powerful tool to study organ development and disease. We here established a three-dimensional (3D) murine bronchioalveolar lung organoid (BALO) model that allows clonal expansion and self-organization of FACS-sorted bronchioalveolar stem cells (BASCs) upon co-culture with lung-resident mesenchymal cells. BALOs yield a highly branched 3D structure within 21 days of culture, mimicking the cellular composition of the bronchioalveolar compartment as defined by single-cell RNA sequencing and fluorescence as well as electron microscopic phenotyping. Additionally, BALOs support engraftment and maintenance of the cellular phenotype of injected tissue-resident macrophages. We also demonstrate that BALOs recapitulate lung developmental defects after knockdown of a critical regulatory gene, and permit modeling of viral infection. We conclude that the BALO model enables reconstruction of the epithelial-mesenchymal-myeloid unit of the distal lung, thereby opening numerous new avenues to study lung development, infection, and regenerative processes in vitro.
Assuntos
Pneumopatias/patologia , Pulmão/crescimento & desenvolvimento , Organoides/crescimento & desenvolvimento , Células-Tronco/fisiologia , Animais , Ataxina-1/genética , Ataxina-1/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Células Endoteliais/citologia , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Células Epiteliais/citologia , Fibroblastos , Humanos , Pulmão/citologia , Células-Tronco Mesenquimais , Camundongos , Morfogênese/genética , Morfogênese/fisiologia , Organogênese/fisiologia , Organoides/citologia , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/crescimento & desenvolvimento , RNA Mensageiro/metabolismo , Regeneração/genética , Regeneração/fisiologiaRESUMO
Bisphosphonate-related osteonecrosis of the jaw (BRONJ) represents a serious health condition, impacting the lives of many patients worldwide. The condition challenges clinical care due to its complex etiology and limited therapeutic options. A thorough understanding of the pathophysiological and patient-related factors that promote disease development is essential. Recently, the oral microbiome has been implicated as a potential driver and modulating factor of BRONJ by several studies. Modern genomic sequencing methods have provided a wealth of data on the microbial composition of BRONJ lesions; however, the role of individual species in the process of disease development remains elusive. A comprehensive PubMed search was conducted to identify relevant studies on the microbiome of BRONJ patients using the terms "microbiome", "osteonecrosis of the jaws", and "bisphosphonates". Studies focusing on symptoms, epidemiology, pathophysiology, risk factors, and treatment options were included. The principal risk factors for BRONJ are tooth extraction, surgical procedures, and the administration of high doses of bisphosphonates. Importantly, the oral microbiome plays a significant role in the progression of the disease. Several studies have identified alterations of microbial composition in BRONJ lesions. However, there is no consensus regarding bacterial species that are associated with BRONJ across studies. The bacterial genera typically found include Actinomyces, Fusobacterium, and Streptococcus. It is postulated that these microbes contribute to the pathogenesis of BRONJ by promoting inflammation and disrupting normal bone remodeling processes. Current therapeutic approaches are disease-stage-specific and the necessity for more effective treatment strategies remains. This review examines the potential causes of and therapeutic approaches to BRONJ, highlighting the link between microbial colonization and BRONJ development. Future research should seek to more thoroughly investigate the interactions between bisphosphonates, the oral microbiome, and the immune system in order to develop targeted therapies.
Assuntos
Osteonecrose da Arcada Osseodentária Associada a Difosfonatos , Difosfonatos , Microbiota , Humanos , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/etiologia , Osteonecrose da Arcada Osseodentária Associada a Difosfonatos/microbiologia , Microbiota/efeitos dos fármacos , Fatores de Risco , Difosfonatos/efeitos adversos , Difosfonatos/uso terapêutico , Boca/microbiologiaRESUMO
The conducting airways are lined by distinct cell types, comprising basal, secretory, ciliated, and rare cells, including ionocytes, solitary cholinergic chemosensory cells, and solitary and clustered (neuroepithelial bodies) neuroendocrine cells. Airway neuroendocrine cells are in clinical focus since they can give rise to small cell lung cancer. They have been implicated in diverse functions including mechanosensation, chemosensation, and regeneration, and were recently identified as regulators of type 2 immune responses via the release of the neuropeptide calcitonin gene-related peptide (CGRP). We here assessed the expression of the chemokine CXCL13 (B cell attracting chemokine) by these cells by RT-PCR, in silico analysis of publicly available sequencing data sets, immunohistochemistry, and immuno-electron microscopy. We identify a phenotype of neuroendocrine cells in the naïve mouse, producing the chemokine CXCL13 predominantly in solitary neuroendocrine cells of the tracheal epithelium (approx. 70% CXCL13+) and, to a lesser extent, in the solitary neuroendocrine cells and neuroepithelial bodies of the intrapulmonary bronchial epithelium (< 10% CXCL13+). In silico analysis of published sequencing data of murine tracheal epithelial cells was consistent with the results obtained by immunohistochemistry as it revealed that neuroendocrine cells are the major source of Cxcl13-mRNA, which was expressed by 68-79% of neuroendocrine cells. An unbiased scRNA-seq data analysis of overall gene expression did not yield subclusters of neuroendocrine cells. Our observation demonstrates phenotypic heterogeneity of airway neuroendocrine cells and points towards a putative immunoregulatory role of these cells in bronchial-associated lymphoid tissue formation and B cell homeostasis.
Assuntos
Quimiocina CXCL13 , Células Neuroendócrinas , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Colinérgicos , Células Epiteliais/metabolismo , Pulmão/metabolismo , Camundongos , Células Neuroendócrinas/metabolismo , RNA Mensageiro/genética , TraqueiaRESUMO
The intestinal microbiota is known to influence local immune homeostasis in the gut and to shape the developing immune system towards elimination of pathogens and tolerance towards self-antigens. Even though the lung was considered sterile for a long time, recent evidence using next-generation sequencing techniques confirmed that the lower airways possess their own local microbiota. Since then, there has been growing evidence that the local respiratory and intestinal microbiota play a role in acute and chronic pediatric lung diseases. The concept of the so-called gut-lung axis describing the mutual influence of local microbiota on distal immune mechanisms was established. The mechanisms by which the intestinal microbiota modulates the systemic immune response include the production of short-chain fatty acids (SCFA) and signaling through pattern recognition receptors (PRR) and segmented filamentous bacteria. Those factors influence the secretion of pro- and anti-inflammatory cytokines by immune cells and further modulate differentiation and recruitment of T cells to the lung. This article does not only aim at reviewing recent mechanistic evidence from animal studies regarding the gut-lung axis, but also summarizes current knowledge from observational studies and human trials investigating the role of the respiratory and intestinal microbiota and their modulation by pre-, pro-, and synbiotics in pediatric lung diseases.
Assuntos
Microbioma Gastrointestinal , Pneumopatias , Microbiota , Animais , Criança , Ácidos Graxos Voláteis , Microbioma Gastrointestinal/fisiologia , Humanos , PulmãoRESUMO
The human central nervous system (CNS) is separated from the blood by distinct cellular barriers, including the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CFS) barrier (BCSFB). Whereas at the center of the BBB are the endothelial cells of the brain capillaries, the BCSFB is formed by the epithelium of the choroid plexus. Invasion of cells of either the BBB or the BCSFB is a potential first step during CNS entry by the Gram-positive bacterium Listeria monocytogenes (Lm). Lm possesses several virulence factors mediating host cell entry, such as the internalin protein family-including internalin (InlA), which binds E-cadherin (Ecad) on the surface of target cells, and internalin B (InlB)-interacting with the host cell receptor tyrosine kinase Met. A further family member is internalin (InlF), which targets the intermediate filament protein vimentin. Whereas InlF has been shown to play a role during brain invasion at the BBB, its function during infection at the BCSFB is not known. We use human brain microvascular endothelial cells (HBMEC) and human choroid plexus epithelial papilloma (HIBCPP) cells to investigate the roles of InlF and vimentin during CNS invasion by Lm. Whereas HBMEC present intracellular and surface vimentin (besides Met), HIBCPP cells do not express vimentin (except Met and Ecad). Treatment with the surface vimentin modulator withaferin A (WitA) inhibited invasion of Lm into HBMEC, but not HIBCPP cells. Invasion of Lm into HBMEC and HIBCPP cells is, however, independent of InlF, since a deletion mutant of Lm lacking InlF did not display reduced invasion rates.
Assuntos
Listeria monocytogenes , Humanos , Barreira Hematoencefálica/metabolismo , Vimentina/metabolismo , Filamentos Intermediários/metabolismo , Células Endoteliais/metabolismo , Proteínas de Bactérias/metabolismoRESUMO
BACKGROUND: The technology of single cell RNA sequencing (scRNA-seq) has gained massively in popularity as it allows unprecedented insights into cellular heterogeneity as well as identification and characterization of (sub-)cellular populations. Furthermore, scRNA-seq is almost ubiquitously applicable in medical and biological research. However, these new opportunities are accompanied by additional challenges for researchers regarding data analysis, as advanced technical expertise is required in using bioinformatic software. RESULTS: Here we present WASP, a software for the processing of Drop-Seq-based scRNA-Seq data. Our software facilitates the initial processing of raw reads generated with the ddSEQ or 10x protocol and generates demultiplexed gene expression matrices including quality metrics. The processing pipeline is realized as a Snakemake workflow, while an R Shiny application is provided for interactive result visualization. WASP supports comprehensive analysis of gene expression matrices, including detection of differentially expressed genes, clustering of cellular populations and interactive graphical visualization of the results. The R Shiny application can be used with gene expression matrices generated by the WASP pipeline, as well as with externally provided data from other sources. CONCLUSIONS: With WASP we provide an intuitive and easy-to-use tool to process and explore scRNA-seq data. To the best of our knowledge, it is currently the only freely available software package that combines pre- and post-processing of ddSEQ- and 10x-based data. Due to its modular design, it is possible to use any gene expression matrix with WASP's post-processing R Shiny application. To simplify usage, WASP is provided as a Docker container. Alternatively, pre-processing can be accomplished via Conda, and a standalone version for Windows is available for post-processing, requiring only a web browser.
Assuntos
Análise de Célula Única , Software , Biologia Computacional , Perfilação da Expressão Gênica , RNA-Seq , Análise de Sequência de RNARESUMO
BACKGROUND AND AIMS: Intestinal adaptation in short bowel syndrome (SBS) includes morphologic processes and functional mechanisms. This study investigated whether digestive enzyme expression in the duodenum and colon is upregulated in SBS patients. METHOD: Sucrase-isomaltase (SI), lactase-phlorizin hydrolase (LPH), and neutral Aminopeptidase N (ApN) were analyzed in duodenal and colonic biopsies from nine SBS patients in a late stage of adaptation as well as healthy and disease controls by immunoelectron microscopy (IEM), Western blots, and enzyme activities. Furthermore, proliferation rates and intestinal microbiota were analyzed in the mucosal specimen. RESULTS: We found significantly increased amounts of SI, LPH, and ApN in colonocytes in most SBS patients with large variation and strongest effect for SI and ApN. Digestive enzyme expression was only partially elevated in duodenal enterocytes due to a low proliferation level measured by Ki-67 staining. Microbiome analysis revealed high amounts of Lactobacillus resp. low amounts of Proteobacteria in SBS patients with preservation of colon and ileocecal valve. Colonic expression was associated with a better clinical course in single cases. CONCLUSION: In SBS patients disaccharidases and peptidases can be upregulated in the colon. Stimulation of this colonic intestinalization process by drugs, nutrients, and pre- or probiotics might offer better therapeutic approaches.
Assuntos
Intestino Grosso/enzimologia , Síndrome do Intestino Curto/enzimologia , Aminopeptidases/metabolismo , Western Blotting , Dissacaridases/metabolismo , Feminino , Humanos , Lactase-Florizina Hidrolase/metabolismo , Lactobacillus/fisiologia , Masculino , Microscopia Imunoeletrônica , Peptídeo Hidrolases/metabolismo , Proteobactérias/fisiologia , Complexo Sacarase-Isomaltase/metabolismoRESUMO
Whole genome sequencing of bacteria has become daily routine in many fields. Advances in DNA sequencing technologies and continuously dropping costs have resulted in a tremendous increase in the amounts of available sequence data. However, comprehensive in-depth analysis of the resulting data remains an arduous and time-consuming task. In order to keep pace with these promising but challenging developments and to transform raw data into valuable information, standardized analyses and scalable software tools are needed. Here, we introduce ASA3P, a fully automatic, locally executable and scalable assembly, annotation and analysis pipeline for bacterial genomes. The pipeline automatically executes necessary data processing steps, i.e. quality clipping and assembly of raw sequencing reads, scaffolding of contigs and annotation of the resulting genome sequences. Furthermore, ASA3P conducts comprehensive genome characterizations and analyses, e.g. taxonomic classification, detection of antibiotic resistance genes and identification of virulence factors. All results are presented via an HTML5 user interface providing aggregated information, interactive visualizations and access to intermediate results in standard bioinformatics file formats. We distribute ASA3P in two versions: a locally executable Docker container for small-to-medium-scale projects and an OpenStack based cloud computing version able to automatically create and manage self-scaling compute clusters. Thus, automatic and standardized analysis of hundreds of bacterial genomes becomes feasible within hours. The software and further information is available at: asap.computational.bio.
Assuntos
Biologia Computacional/métodos , Análise de Sequência de DNA/métodos , Algoritmos , Bactérias/genética , Sequência de Bases/genética , Mapeamento Cromossômico/métodos , Computação em Nuvem , Genoma Bacteriano/genética , Análise de Sequência de DNA/estatística & dados numéricos , Software , Sequenciamento Completo do Genoma/métodosRESUMO
OBJECTIVES: We aimed at the high-resolution examination of the oral microbiome depending on oil pulling, compared it with saline pulling, and analyzed whether the method is capable of reducing the overall microbial burden of the oral cavity. MATERIALS AND METHODS: The study was a cohort study with three healthy subjects. Oil pulling samples, saline pulling samples, and saliva samples were microscoped and cultured under microaerophilic and anaerobic conditions; colony-forming units were counted; and cultivated bacteria were identified employing MALDI-TOF MS. The oral microbiomes (saliva) and the microbiota incorporated in oil and saline pulling samples were determined in toto by using 16S rDNA next-generation sequencing (NGS) and bioinformatics. RESULTS: Microscopy revealed that oral epithelial cells are ensheathed with distinct oil droplets during oil pulling. Oil pulling induced a higher production of saliva and the oil/saliva emulsion contained more bacteria than saline pulling samples. Oil pulling resulted in a significant and transient reduction of the overall microbial burden in comparison to saliva examined prior to and after pulling. Both oil and saline pulling samples mirrored the individual oral microbiomes in saliva. CONCLUSIONS: Within the limitations of this pilot study, it might be concluded that oil pulling is able to reduce the overall microbial burden of the oral cavity transiently and the microbiota in oil pulling samples are representative to the oral microbiome. CLINICAL RELEVANCE: Within the limitations of this pilot study, it might be concluded that oil pulling can be considered as an enlargement of standard oral hygiene techniques since it has the characteristic of an oral massage, enwrapping epithelial cells carrying bacteria in oil vesicles and reaching almost all unique habitats in oral cavity.
Assuntos
Microbiota , Estudos de Coortes , Voluntários Saudáveis , Humanos , Boca , Projetos Piloto , RNA Ribossômico 16S , Saliva , Óleo de GirassolRESUMO
Natural smectites have demonstrated efficacy in the treatment of diarrhea. The present study evaluated the prophylactic effect of a diosmectite (FI5pp) on the clinical course, colon damage, expression of tight junction (TJ) proteins and the composition of the gut microbiota in dextran sulfate sodium (DSS) colitis. Diosmectite was administered daily to Balb/c mice from day 1 to 7 by oral gavage, followed by induction of acute DSS-colitis from day 8 to 14 ("Control", n = 6; "DSS", n = 10; "FI5pp + DSS", n = 11). Mice were sacrificed on day 21. Clinical symptoms (body weight, stool consistency and occult blood) were checked daily after colitis induction. Colon tissue was collected for histological damage scoring and quantification of tight junction protein expression. Stool samples were collected for microbiome analysis. Our study revealed prophylactic diosmectite treatment attenuated the severity of DSS colitis, which was apparent by significantly reduced weight loss (p = 0.022 vs. DSS), disease activity index (p = 0.0025 vs. DSS) and histological damage score (p = 0.023 vs. DSS). No significant effects were obtained for the expression of TJ proteins (claudin-2 and claudin-3) after diosmectite treatment. Characterization of the microbial composition by 16S amplicon NGS showed that diosmectite treatment modified the DSS-associated dysbiosis. Thus, diosmectites are promising candidates for therapeutic approaches to target intestinal inflammation and to identify possible underlying mechanisms of diosmectites in further studies.
Assuntos
Bactérias/classificação , Colite/tratamento farmacológico , Sulfato de Dextrana/efeitos adversos , Microbiota/efeitos dos fármacos , Silicatos/administração & dosagem , Administração Oral , Animais , Bactérias/efeitos dos fármacos , Bactérias/genética , Bactérias/isolamento & purificação , Peso Corporal/efeitos dos fármacos , Colite/induzido quimicamente , Colite/metabolismo , Colite/microbiologia , DNA Bacteriano/genética , DNA Ribossômico/genética , Fezes/microbiologia , Masculino , Camundongos Endogâmicos BALB C , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Índice de Gravidade de Doença , Silicatos/farmacologia , Proteínas de Junções Íntimas/metabolismo , Resultado do TratamentoRESUMO
Enterococcus faecalis has to cope with major stress conditions during colonization. To understand the effects of stress encountered during infection, the present study assessed the transcriptomic response of the bacteria facing exposure to serum, urine, bile salts, acid pH, or oxidative stress. Compared to non-stressed culture, 30% of the E. faecalis genes were differentially expressed. The transcriptome analysis reveals common but also specific responses, depending on stresses encountered: thus, urine exposure has the most important impact, and the highest number of genes with modified expression is involved in transport and metabolism. The results also pinpoint many stress-related sRNA or intergenic regions not yet characterized. This study identified the general stress stimulon related to infection: when the commensal bacterium initiates its response to stress related to infection, it increases its ability to survive to rough conditions for colonization, rather than promoting expression of virulence factors, and becomes this opportunistic pathogen that thrives in hospital settings.
Assuntos
Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Estresse Fisiológico/fisiologia , Proteínas de Bactérias/genética , Enterococcus faecalis/genética , Perfilação da Expressão Gênica , Humanos , Estresse Oxidativo , Transcriptoma/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
We describe a strain of Lassa virus representing a putative new lineage that was isolated from a cluster of human infections with an epidemiologic link to Togo. This finding extends the known range of Lassa virus to Togo.
Assuntos
Febre Lassa/epidemiologia , Febre Lassa/virologia , Vírus Lassa/classificação , Animais , Chlorocebus aethiops , Genes Virais , História do Século XXI , Humanos , Febre Lassa/história , Filogenia , Togo/epidemiologia , Células VeroRESUMO
Staphylococcus aureus can cause wide range of infections from simple soft skin infections to severe endocarditis, bacteremia, osteomyelitis and implant associated bone infections (IABI). The focus of the present investigation was to study virulence properties of S. aureus isolates from acute and chronic IABI by means of their in vivo lethality, in vitro osteoblasts invasion, biofilm formation and subsequently whole genome comparison between high and low virulent strains. Application of insect infection model Galleria mellonella revealed high, intermediate and low virulence phenotypes of these clinical isolates, which showed good correlation with osteoblast invasion and biofilm formation assays. Comparative genomics of selected high (EDCC 5458) and low (EDCC 5464) virulent strains enabled the identification of molecular factors responsible for the development of acute and chronic IABI. Accordingly, the low virulent strain EDCC 5464 harbored point mutations resulting in frame shift mutations in agrC (histidine kinase in agr system), graS (histidine kinase in graSR, a two component system) and efeB (peroxidase in efeOBU operon, an iron acquisition system) genes. Additionally, we found a mobile element (present 11 copies in EDCC 5464) inserted at the end of ß-hemolysin (hlb) and sarU genes, which are involved in the pathogenesis and regulation of virulence gene expression in coordination with quorum sensing system. All these results are in good support with the low virulence behavior of EDCC 5464. From the previous literature, it is well known that agr defective S. aureus clinical strains are isolated from the chronic infections. Similarly, low virulent EDCC 5464 was isolated from chronic implant-associated bone infections infection whereas EDCC 5458 was obtained from acute implant-associated bone infections. Laboratory based in vitro and in vivo results and insights from comparative genomic analysis could be correlated with the clinical conclusion of IABIs and allows evidence-based treatment strategies based on the pathogenesis of the strain to cure life devastating implant-associated infections.
Assuntos
Osso e Ossos/microbiologia , Genoma Bacteriano/genética , Osteomielite/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Animais , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Osso e Ossos/patologia , Regulação Bacteriana da Expressão Gênica , Histidina Quinase/genética , Humanos , Sequências Repetitivas Dispersas/genética , Mariposas/microbiologia , Osteoblastos/microbiologia , Osteomielite/patologia , Peroxidase/genética , Proteínas Quinases/genética , Percepção de Quorum/genética , Staphylococcus aureus/isolamento & purificação , Virulência/genética , Sequenciamento Completo do GenomaRESUMO
Two Listeria-like isolates obtained from mangrove swamps in Goa, India were characterized using polyphasic combinations of phenotypic, chemotaxonomic and whole-genome sequence (WGS)-based approaches. The isolates presented as short, non-spore-forming, Gram-positive rods, that were non-motile, oxidase-negative, catalase-positive and exhibited α-haemolysis on 5â% sheep- and horse-blood agar plates. The 16S rRNA gene sequences exhibited 93.7-99.7â% nucleotide identity to other Listeria species and had less than 92â% nucleotide identity to species of closely related genera, indicating that the isolates are de facto members of the genus Listeria. Their overall fatty acid composition resembled that of other Listeria species, with quantitative differences in iso C15â:â0, anteiso C15â:â0, iso C16â:â0, C16â:â0, iso C17â:â0 and anteiso C17â:â0 fatty acid profiles. Phylogeny based on 406 core coding DNA sequences grouped these two isolates in a monophyletic clade within the genus Listeria. WGS-based average nucleotide identity and in silico DNA-DNA hybridization values were lower than the recommended cut-off values of 95 and 70â%, respectively, to the other Listeria species, indicating that they are founding members of a novel Listeria species. We suggest the name Listeriagoaensis sp. nov. be created and the type strain is ILCC801T (=KCTC 33909;=DSM 29886;=MCC 3285).
Assuntos
Listeria/classificação , Filogenia , Áreas Alagadas , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Índia , Listeria/genética , Listeria/isolamento & purificação , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Rhizophoraceae , Análise de Sequência de DNARESUMO
Each bacterium has to co-ordinate its growth with division to ensure genetic stability of the population. Consequently, cell division and growth are tightly regulated phenomena, albeit different bacteria utilise one of several alternative regulatory mechanisms to maintain control. Here we consider GpsB, which is linked to cell growth and division in Gram-positive bacteria. ΔgpsB mutants of the human pathogen Listeria monocytogenes show severe lysis, division and growth defects due to distortions of cell wall biosynthesis. Consistent with this premise, GpsB interacts both in vitro and in vivo with the major bi-functional penicillin-binding protein. We solved the crystal structure of GpsB and the interaction interfaces in both proteins are identified and validated. The inactivation of gpsB results in strongly attenuated virulence in animal experiments, comparable in degree to classical listerial virulence factor mutants. Therefore, GpsB is essential for in vitro and in vivo growth of a highly virulent food-borne pathogen, suggesting that GpsB could be a target for the future design of novel antibacterials.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Listeria monocytogenes/fisiologia , Proteínas de Ligação às Penicilinas/química , Proteínas de Ligação às Penicilinas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Divisão Celular/fisiologia , Parede Celular/metabolismo , Listeria monocytogenes/citologia , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Modelos Moleculares , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill-defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG-I, MDA5 and STING thereby triggering interferon ß production. Secreted Listeria nucleic acids also caused RIG-I-dependent IL-1ß-production and inflammasome activation. The signalling molecule CARD9 contributed to IL-1ß production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG-I provides a mechanistic explanation for efficient induction of immunity by live bacteria.
Assuntos
Citoplasma/metabolismo , RNA Helicases DEAD-box/fisiologia , DNA Bacteriano/imunologia , Imunidade Celular/imunologia , Inflamação/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , RNA Bacteriano/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Western Blotting , Proteínas Adaptadoras de Sinalização CARD , Células Cultivadas , Citoplasma/imunologia , Citoplasma/microbiologia , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Bacteriano/genética , Citometria de Fluxo , Imunofluorescência , Inflamação/microbiologia , Helicase IFIH1 Induzida por Interferon , Listeria monocytogenes/genética , Listeriose/genética , Listeriose/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Bacteriano/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
A total of 17 Enterobacter-like isolates were obtained from blood during a septicaemia outbreak in a neonatal unit, Tanzania, that could not be assigned based on phenotypic test to any existing Enterobacter species. Eight representative outbreak isolates were investigated in detail. Fermentation characteristics, biochemical assays and fatty acid profiles for taxonomic analysis were determined and supplemented with information derived from whole genome sequences. Phenotypic and morphological tests revealed that these isolates were Gram-stain-negative, rod-shaped, highly motile and facultatively anaerobic. The fatty acid profile was similar to those of the type strains for all recognized Enterobacter species, with quantitative differences in C17 : 0, C18 : 1ω7c and C17 : 0 cyclo fatty acids. Whole genome sequencing was used to identify taxonomically relevant characteristics, i.e. for 16S rRNA gene sequence analysis, multi-locus sequence analysis (MLSA), in silico DNA-DNA hybridization (isDDH) and average nucleotide identity (ANI). Draft genomes were approximately 4.9âMb in size with a G+C content of 56.0âmol%. The 16S rRNA gene sequence of these eight isolates showed >97 % similarity to all Enterobacter species, while MLSA clustered them closely with the type strains of Enterobacter xiangfangensis and Enterobacter hormaechei. These eight strains showed less than 70 % isDDH identity with the type strains of Enterobacter species. In addition, less than 95 % ANI to the type strains of Enterobacter species was observed. From these results, it is concluded that these isolates possess sufficient characteristics to differentiate them from all recognized Enterobacter species, and should therefore be considered as representing a novel species. The name Enterobacter bugandensis sp. nov. is proposed with EB-247T ( = DSM 29888T = NCCB 100573T) as the type strain.
RESUMO
Listeria monocytogenes is a firmicute bacterium causing serious infections in humans upon consumption of contaminated food. Most of its virulence factors are secretory proteins either released to the medium or attached to the bacterial surface. L. monocytogenes encodes at least six different protein secretion pathways. Although great efforts have been made in the past to predict secretory proteins and their secretion routes using bioinformatics, experimental evidence is lacking for most secretion systems. Therefore, we constructed mutants in the main housekeeping protein secretion systems, which are the Sec-dependent transport, the YidC membrane insertases SpoIIIJ and YqjG, as well as the twin-arginine pathway, and analyzed their secretion and virulence defects. Our results demonstrate that Sec-dependent secretion and membrane insertion of proteins via YidC proteins are essential for viability of L. monocytogenes. Depletion of SecA or YidC activity severely affected protein secretion, whereas loss of the Tat-pathway was without any effect on secretion, viability, and virulence. Two-dimensional gel electrophoresis combined with protein identification by mass spectrometry revealed that secretion of many virulence factors and of enzymes synthesizing and degrading the cell wall depends on the SecA route. This finding was confirmed by SecA inhibition experiments using sodium azide. Analysis of secretion of substrates typically dependent on the accessory SecA2 ATPase in wild type and azide resistant mutants of L. monocytogenes revealed for the first time that SecA2-dependent protein secretion also requires the ATPase activity of the house-keeping SecA protein.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Listeria monocytogenes/patogenicidade , Proteínas de Membrana Transportadoras/genética , Adenosina Trifosfatases/metabolismo , Animais , Arginina/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Células HeLa , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , Camundongos , Proteômica , Canais de Translocação SEC , Proteínas SecA , Fatores de Virulência/genéticaRESUMO
BACKGROUND: Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type. RESULTS: We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro). CONCLUSION: Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.