Intestinal Anti-inflammatory Effects of Outer Membrane Vesicles from Escherichia coli Nissle 1917 in DSS-Experimental Colitis in Mice

Escherichia coli Nissle 1917 (EcN) is a probiotic strain with proven efficacy in inducing and maintaining remission of ulcerative colitis. However, the microbial factors that mediate these beneficial effects are not fully known. Gram-negative bacteria release outer membrane vesicles (OMVs) as a direct pathway for delivering selected bacterial proteins and active compounds to the host. In fact, vesicles released by gut microbiota are emerging as key players in signaling processes in the intestinal mucosa. In the present study, the dextran sodium sulfate (DSS)-induced colitis mouse model was used to investigate the potential of EcN OMVs to ameliorate mucosal injury and inflammation in the gut. The experimental protocol involved pre-treatment with OMVs for 10 days before DSS intake, and a 5-day recovery period. Oral administration of purified EcN OMVs (5 μg/day) significantly reduced DSS-induced weight loss and ameliorated clinical symptoms and histological scores. OMVs treatment counteracted altered expression of cytokines and markers of intestinal barrier function. This study shows for the first time that EcN OMVs can mediate the anti-inflammatory and barrier protection effects previously reported for this probiotic in experimental colitis. Remarkably, translation of probiotics to human healthcare requires knowledge of the molecular mechanisms involved in probiotic–host interactions. Thus, OMVs, as a non-replicative bacterial form, could be explored as a new probiotic-derived therapeutic approach, with even lower risk of adverse events than probiotic administration.his work was funded by the “Ministerio de Economía y Competitividad”, Spain (Grants AGL2012-34985, AGL2016-79113-R (AEI/FEDER, UE) and AGL2015-67995-C3-3-R, all co-financed with European Commission ERDF funds), by the Generalitat de Catalunya, AGAUR (Grant 2014SGR1017), and by the Junta de Andalucía (Grant CTS-164). The CIBEREHD is funded by the Instituto de Salud Carlos III. M-JF acknowledges her FPI fellowship from the Spanish Ministry of Economy and Competitiveness

Glyceraldehyde-3-phosphate dehydrogenase as a moonlighting protein in bacteria

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/63704Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a housekeeping protein that is present in virtually all organisms, where it performs metabolic functions essential for survival. GAPDH plays an essential role in the process of energy production, and is also involved in numerous biological processes. GAPDH belongs to a subset of proteins called moonlighting proteins, in which different functions are associated with a single polypeptide chain. The multifunctionality of GAPDH has been described in pathogenic and probiotic microorganisms, in mammals and in plants. In this review, we summarize the moonlighting role of GAPDH in bacteria

Dual role of LldR in regulation of the lldPRD operon, involved in l-Lactate metabolism in Escherichia coli.

The lldPRD operon of Escherichia coli, involved in L-lactate metabolism, is induced by growth in this compound. We experimentally identified that this system is transcribed from a single promoter with an initiation site located 110 nucleotides upstream of the ATG start codon. On the basis of computational data, it had been proposed that LldR and its homologue PdhR act as regulators of the lldPRD operon. Nevertheless, no experimental data on the function of these regulators have been reported so far. Here we show that induction of an lldP-lacZ fusion by L-lactate is lost in an lldR mutant, indicating the role of LldR in this induction. Expression analysis of this construct in a pdhR mutant ruled out the participation of PdhR in the control of lldPRD. Gel shift experiments showed that LldR binds to two operator sites, O1 (positions 105 to 89) and O2 (positions 22 to 38), with O1 being filled at a lower concentration of LldR. L-Lactate induced a conformational change in LldR that did not modify its DNA binding activity. Mutations in O1 and O2 enhanced the basal transcriptional level. However, only mutations in O1 abolished induction by L-lactate. Mutants with a change in helical phasing between O1 and O2 behaved like O2 mutants. These results were consistent with the hypothesis that LldR has a dual role, acting as a repressor or an activator of lldPRD. We propose that in the absence of L-lactate, LldR binds to both O1 and O2, probably leading to DNA looping and the repression of transcription. Binding of L-lactate to LldR promotes a conformational change that may disrupt the DNA loop, allowing the formation of the transcription open complex

Cell-to-Cell Communication by Host-Released Extracellular Vesicles in the Gut: Implications in Health and Disease

Communication between cells is crucial to preserve body homeostasis and health. Tightly controlled intercellular dialog is particularly relevant in the gut, where cells of the intestinal mucosa are constantly exposed to millions of microbes that have great impact on intestinal homeostasis by controlling barrier and immune functions. Recent knowledge involves extracellular vesicles (EVs) as mediators of such communication by transferring messenger bioactive molecules including proteins, lipids, and miRNAs between cells and tissues. The specific functions of EVs principally depend on the internal cargo, which upon delivery to target cells trigger signal events that modulate cellular functions. The vesicular cargo is greatly influenced by genetic, pathological, and environmental factors. This finding provides the basis for investigating potential clinical applications of EVs as therapeutic targets or diagnostic biomarkers. Here, we review current knowledge on the biogenesis and cargo composition of EVs in general terms. We then focus the attention to EVs released by cells of the intestinal mucosa and their impact on intestinal homeostasis in health and disease. We specifically highlight their role on epithelial barrier integrity, wound healing of epithelial cells, immunity, and microbiota shaping. Microbiota-derived EVs are not reviewed here

Outer membrane vesicles and soluble factors released by probiotic Escherichia coli Nissle 1917 and commensal ECOR63 enhance barrier function by regulating expression of tight junction proteins in intestinal epithelial cells

The gastrointestinal epithelial layer forms a physical and biochemical barrier that maintains the segregation between host and intestinal microbiota. The integrity of this barrier is critical in maintaining homeostasis in the body and its dysfunction is linked to a variety of illnesses, especially inflammatory bowel disease. Gut microbes, and particularly probiotic bacteria, modulate the barrier integrity by reducing gut permeability and reinforcing tight junctions. Probiotic Escherichia coli Nissle 1917 (EcN) is a good colonizer of the human gut with proven therapeutic efficacy in the remission of ulcerative colitis in humans. EcN positively modulates the intestinal epithelial barrier through upregulation and redistribution of the tight junction proteins ZO-1, ZO-2 and claudin-14. Upregulation of claudin-14 has been attributed to the secreted protein TcpC. Whether regulation of ZO-1 and ZO-2 is mediated by EcN secreted factors remains unknown. The aim of this study was to explore whether outer membrane vesicles (OMVs) released by EcN strengthen the epithelial barrier. This study includes other E. coli strains of human intestinal origin that contain the tcpC gene, such as ECOR63. Cell-free supernatants collected from the wild-type strains and from the derived tcpC mutants were fractionated into isolated OMVs and soluble secreted factors. The impact of these extracellular fractions on the epithelial barrier was evaluated by measuring transepithelial resistance and expression of several tight junction proteins in T-84 and Caco-2 polarized monolayers. Our results show that the strengthening activity of EcN and ECOR63 does not exclusively depend on TcpC. Both OMVs and soluble factors secreted by these strains promote upregulation of ZO-1 and claudin-14, and down-regulation of claudin-2. The OMVs-mediated effects are TcpC-independent. Soluble secreted TcpC contributes to the upregulation of ZO-1 and claudin-14, but this protein has no effect on the transcriptional regulation of claudin-2. Thus, in addition to OMVs and TcpC, other active factors released by these microbiota strains contribute to the reinforcement of the epithelial barrier. Keywords: probiotics, gut microbes, Escherichia coli, phylogenetic group B2, membrane vesicles, tight junctions, intestinal barrier, Tcp

Outer membrane vesicles from probiotic and commensal Escherichia coli activate NOD1-mediated immune responses in intestinal epithelial cells

Gut microbiota plays a critical role in maintaining human intestinal homeostasis and host health. Bacterial extracellular vesicles are key players in bacteria-host communication, as they allow delivery of effector molecules into the host cells. Outer membrane vesicles (OMVs) released by Gram-negative bacteria carry many ligands of pattern recognition receptors that are key components of innate immunity. NOD1 and NOD2 cytosolic receptors specifically recognize peptidoglycans present within the bacterial cell wall. These intracellular immune receptors are essential in host defense against bacterial infections and in the regulation of inflammatory responses. Recent contributions show that NODs are also fundamental to maintain intestinal homeostasis and microbiota balance. Peptidoglycan from non-invasive pathogens is delivered to cytosolic NODs through OMVs, which are internalized via endocytosis. Whether this pathway could be used by microbiota to activate NOD receptors remains unexplored. Here, we report that OMVs isolated from the probiotic Escherichia coli Nissle 1917 and the commensal ECOR12 activate NOD1 signaling pathways in intestinal epithelial cells. NOD1 silencing and RIP2 inhibition significantly abolished OMV-mediated activation of NF-κB and subsequent IL-6 and IL-8 expression. Confocal fluorescence microscopy analysis confirmed that endocytosed OMVs colocalize with NOD1, trigger the formation of NOD1 aggregates, and promote NOD1 association with early endosomes. This study shows for the first time the activation of NOD1-signaling pathways by extracellular vesicles released by gut microbiota. Keywords: gut microbiota, Escherichia coli Nissle 1917, NF-κB activation, bacterial extracellular vesicles, NOD

The UlaG protein family defines novel structural and functional motifs grafted on an ancient RNase fold

Background: Bacterial populations are highly successful at colonizing new habitats and adapting to changing environmental conditions, partly due to their capacity to evolve novel virulence and metabolic pathways in response to stress conditions and to shuffle them by horizontal gene transfer (HGT). A common theme in the evolution of new functions consists of gene duplication followed by functional divergence. UlaG, a unique manganese-dependent metallo-b-lactamase (MBL) enzyme involved in L-ascorbate metabolism by commensal and symbiotic enterobacteria, provides a model for the study of the emergence of new catalytic activities from the modification of an ancient fold. Furthermore, UlaG is the founding member of the so-called UlaG-like (UlaGL) protein family, a recently established and poorly characterized family comprising divalent (and perhaps trivalent)metal-binding MBLs that catalyze transformations on phosphorylated sugars and nucleotides. Results: Here we combined protein structure-guided and sequence-only molecular phylogenetic analyses to dissect the molecular evolution of UlaG and to study its phylogenomic distribution, its relatedness with present-day UlaGL protein sequences and functional conservation. Phylogenetic analyses indicate that UlaGL sequences are present in Bacteria and Archaea, with bona fide orthologs found mainly in mammalian and plant-associated Gramnegative and Gram-positive bacteria. The incongruence between the UlaGL tree and known species trees indicates exchange by HGT and suggests that the UlaGL-encoding genes provided a growth advantage under changing conditions. Our search for more distantly related protein sequences aided by structural homology has uncovered that UlaGL sequences have a common evolutionary origin with present-day RNA processing and metabolizing MBL enzymes widespread in Bacteria, Archaea, and Eukarya. This observation suggests an ancient origin for the UlaGL family within the broader trunk of the MBL superfamily by duplication, neofunctionalization and fixation. Conclusions: Our results suggest that the forerunner of UlaG was present as an RNA metabolizing enzyme in the last common ancestor, and that the modern descendants of that ancestral gene have a wide phylogenetic distribution and functional roles. We propose that the UlaGL family evolved new metabolic roles among bacterial and possibly archeal phyla in the setting of a close association with metazoans, such as in the mammalian gastrointestinal tract or in animal and plant pathogens, as well as in environmental settings. Accordingly, the major evolutionary forces shaping the UlaGL family include vertical inheritance and lineage-specific duplication and acquisition of novel metabolic functions, followed by HGT and numerous lineage-specific gene loss events

Membrane vesicles released by a hypervesiculating Escherichia coli Nissle 1917 tolR mutant are highly heterogeneous and show reduced capacity for epithelial cell interaction and entry.

Membrane vesicles (MVs) produced by Gram-negative bacteria are being explored for novel clinical applications due to their ability to deliver active molecules to distant host cells, where they can exert immunomodulatory properties. MVs released by the probiotic Escherichia coli Nissle 1917 (EcN) are good candidates for testing such applications. However, a drawback for such studies is the low level of MV isolation from in vitro culture supernatants, which may be overcome by the use of mutants in cell envelope proteins that yield a hypervesiculation phenotype. Here, we confirm that a tolR mutation in EcN increases MV production, as determined by protein, LPS and fluorescent lipid measurements. Transmission electron microscopy (TEM) of negatively stained MVs did not reveal significant differences with wild type EcN MVs. Conversely, TEM observation after high-pressure freezing followed by freeze substitution of bacterial samples, together with cryo-TEM observation of plunge-frozen hydrated isolated MVs showed considerable structural heterogeneity in the EcN tolR samples. In addition to common one-bilayer vesicles (OMVs) and the recently described double-bilayer vesicles (O-IMVs), other types of MVs were observed. Time-course experiments of MV uptake in Caco-2 cells using rhodamine- and DiO-labelled MVs evidenced that EcN tolR MVs displayed reduced internalization levels compared to the wild-type MVs. The low number of intracellular MVs was due to a lower cell binding capacity of the tolR-derived MVs, rather than a different entry pathway or mechanism. These findings indicate that heterogeneity of MVs from tolR mutants may have a major impact on vesicle functionality, and point to the need for conducting a detailed structural analysis when MVs from hypervesiculating mutants are to be used for biotechnological applications

An overview on the modulation of the intestinal barrier and immune response by membrane vesicles secreted by the probiotic Escherichia coli Nissle 1917

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/128014Probiotic Escherichia coli Nissle 1917 (EcN) is a good colonizer of the human gut and its efficacy in the inflammatory process undergone in ulcerative colitis has been demonstrated. The probiotic action is mainly through the modulation of intestinal epithelial tight junctions and immune system. Here we review the role of outer membrane vesicles (OMVs) released by this probiotic strain on the modulation of intestinal homeostasis. EcN OMVs enter into host epithelial cells via clathrin-mediated endocytosis and are sorted to lysosomes via endocytic compartments. In cellular models of intestinal barrier, EcN OMVs stimulate the underlying immune system through the intestinal epithelium, triggering immune and defense responses. Thus, the use of probiotic derived OMVs could be a safe probiotic-derived strategy targeting intestinal inflammatory processes

Membrane vesicles from the probiotic Nissle 1917 and gut resident Escherichia coli strains distinctly modulate human dendritic cells and subsequent T cell responses

Extracellular membrane vesicles (MVs) released by gut microbiota are key players in the communication with the host. The aim of this study was to evaluate the immunomodulatory properties of MVs from the probiotic E. coli Nissle 1917 (EcN) in terms of DC-derived adaptive immune responses and to compare the effects with those elicited by commensal E. coli. The effects of MVs were analysed in monocyte-derived DCs by measuring cytokine expression and the ability of activated-DCs to differentiate CD4+ T cells towards specific effector subsets. EcN MVs derived intricate Th1/Th2/Th17/Th22/Treg responses consistent with the beneficial effects of this probiotic. Th2/Th17/Th22 responses were common to commensal E. coli-derived vesicles but specific differences were observed for Th1 and Treg responses. Since MVs activate DCs in a strain-specific manner, probiotic-derived MVs could be explored as a safe (bacteria-free) strategy to develop new functional food ingredients targeting gut microbiota balance or intestinal inflammation