Extracellular vesicles and soluble factors secreted by Escherichia coli Nissle 1917 and ECOR63 protect against enteropathogenic E. coli-induced intestinal epithelial barrier dysfunction.

BACKGROUND: Enteric pathogens have developed mechanisms to disrupt tight junctions and increase gut permeability. Many studies have analysed the ability of live probiotics to protect intestinal epithelial cells against tight junction damage caused by bacterial pathogens. Escherichia coli Nissle 1917 (EcN) is among the probiotics that positively modulates the intestinal epithelial barrier by regulating expression and distribution of tight junction proteins. We previously reported that regulation of ZO-1, claudin-14 and claudin-2 is mediated by EcN secreted factors, either free-released or associated with outer membrane vesicles (OMVs). Factors secreted by commensal ECOR63 elicited comparable effects in intact epithelial T-84 and Caco-2 cell monolayers. RESULTS: Here we analyse the ability of OMVs and soluble secreted factors to protect epithelial barrier function in polarized T-84 and Caco-2 cells infected with enteropathogenic Escherichia coli (EPEC). Transepithelial electrical resistance, paracellular permeability, mRNA levels and subcellular distribution of tight junction proteins were monitored in the absence or presence of EcN and ECOR63 extracellular fractions. EPEC downregulated expression of ZO-1 ZO-2, occludin and claudin-14 and altered the subcellular localization of ZO-1, occludin and F-actin cytoskeleton. OMVs and soluble factors secreted by EcN and ECOR63 counteracted EPEC-altered transepithelial resistance and paracellular permeability, preserved occludin and claudin-14 mRNA levels, retained ZO-1 and occludin at tight junctions in the cell boundaries and ameliorated F-actin disorganization. Redistribution of ZO-1 was not accompanied by changes at mRNA level. CONCLUSION: This study provides new insights on the role of microbiota secreted factors on the modulation of intestinal tight junctions, expanding their barrier-protective effects against pathogen-induced disruption.

Alcohol consumption in college students from the pharmacy faculty.

Alcohol consumption is highly prevalent in university students. Early detection in future health professionals is important: their consumption might not only influence their own health but may determine how they deal with the implementation of preventive strategies in the future. The aim of this paper is to detect the prevalence of risky alcohol consumption in first- and last-degree year students and to compare their drinking patterns.Risky drinking in pharmacy students (n=434) was assessed and measured with the AUDIT questionnaire (Alcohol Use Disorders Identification Test). A comparative analysis between college students from the first and fifth years of the degree in pharmacy, and that of a group of professors was carried to see differences in their alcohol intake patterns.Risky drinking was detected in 31.3% of students. The highest prevalence of risky drinkers, and the total score of the AUDIT test was found in students in their first academic year. Students in the first academic level taking morning classes had a two-fold risk of risky drinking (OR=1.9 (IC 95%1.1-3.1)) compared with students in the fifth level. The frequency of alcohol consumption increases with the academic level, whereas the number of alcohol beverages per drinking occasion falls.Risky drinking is high during the first year of university. As alcohol consumption might decrease with age, it is important to design preventive strategies that will strengthen this tendency.

El consumo de alcohol es muy prevalente entre los estudiantes universitarios. La detección precoz en futuros profesionales sanitarios es muy importante  puesto que  puede incidir no solo en su salud, sino también en su enfoque en futuras acciones preventivas como profesionales de la salud.Detectar la prevalencia del consumo de riesgo de alcohol en estudiantes de farmacia y en el profesorado y comparar el patrón de consumo entre estos grupos, y según el curso académico.Se realiza un cribado del consumo de alcohol mediante el cuestionario AUDIT (Alcohol Use Disorders Identification Test) a estudiantes universitarios de farmacia (n=434) en el marco de un proyecto de innovación docente. Se realiza un análisis comparativo entre los estudiantes de primero y quinto curso, y el profesorado. El 31,3% de los estudiantes fueron identificados como bebedores de riesgo. La mayor prevalencia de consumidores de riesgo y las mayores puntuaciones totales se observaron en los alumnos de primer curso. Los estudiantes de primero de turno de mañana presentaron un riesgo de 1,9 (IC 95%1,1-3,1) comparado con los de quinto. La frecuencia de consumo de alcohol se incrementa con el curso académico, mientras que el número de consumiciones por día de consumo se reduce. Durante el primer año en la Facultad los estudiantes presentan una elevada prevalencia de consumo de riesgo. Puesto que con la edad se observa una tendencia decreciente en dichos consumos, es importante diseñar intervenciones preventivas que la favorezcan.

The secreted autotransporter toxin (Sat) does not act as a virulence factor in the probiotic Escherichia coli strain Nissle 1917.

BACKGROUND: Escherichia coli Nissle 1917 (EcN) is a probiotic used in the treatment of intestinal diseases. Although it is considered safe, EcN is closely related to the uropathogenic E. coli strain CFT073 and contains many of its predicted virulence elements. Thus, it is relevant to assess whether virulence-associated genes are functional in EcN. One of these genes encodes the secreted autotransporter toxin (Sat), a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs) that are secreted following the type V autotransporter pathway. Sat is highly prevalent in certain E. coli pathogenic groups responsible for urinary and intestinal infections. In these pathogens Sat promotes cytotoxic effects in several lines of undifferentiated epithelial cells, but not in differentiated Caco-2 cells. RESULTS: Here we provide evidence that sat is expressed by EcN during the colonization of mouse intestine. The EcN protein is secreted as an active serine protease, with its 107 kDa-passenger domain released into the medium as a soluble protein. Expression of recombinant EcN Sat protein in strain HB101 increases paracellular permeability to mannitol in polarized Caco-2 monolayers. This effect, also reported for the Sat protein of diffusely adherent E. coli, is not observed when this protein is expressed in the EcN background. In addition, we show that EcN supernatants confer protection against Sat-mediated effects on paracellular permeability, thus indicating that other secreted EcN factors are able to prevent barrier disruption caused by pathogen-related factors. Sat is not required for intestinal colonization, but the EcNsat::cat mutant outcompetes wild-type EcN in the streptomycin-treated mouse model. Analysis of the presence of sat in 29 strains of the ECOR collection isolated from stools of healthy humans shows 34.8 % positives, with high prevalence of strains of the phylogenetic groups D and B2, related with extra-intestinal infections. CONCLUSIONS: Sat does not act as a virulence factor in EcN. The role of Sat in intestinal pathogenesis relies on other genetic determinants responsible for the bacterial pathotype.

Influence of antioxidant compounds, total sugars and genetic background on the chilling injury susceptibility of a non-melting peach (Prunus persica (L.) Batsch) progeny.

BACKGROUND: To identify genotypes with good organoleptic properties, antioxidant-rich content and low susceptibility to chilling injury (CI), fruits from 130 peach cultivars were studied over three consecutive years. Pomological traits, l-ascorbic acid, flavonoids, total phenolics, relative antioxidant capacity (RAC) and sugars were determined. Major symptoms of CI developed at 5 °C, such as leatheriness, flesh browning, bleeding and loss of flavor, were evaluated. RESULTS: The population exhibited wide phenotypic variation in agronomic and biochemical traits. Six genotypes with high total phenolics, RAC, flavonoids and total sugars were selected. The progeny also showed variability for all evaluated CI symptoms, and 16 genotypes showed considerably lower susceptibility to CI. After 2 weeks of cold storage, leatheriness and bleeding were the main CI symptoms observed, whereas flesh browning was predominant after 4 weeks. CONCLUSION: It was possible to find varieties with high phenolic concentration and relatively low or intermediate CI susceptibility (22, 33, 68, 80, 81, 96 and 120). However, the correlations observed between CI and phenolic contents highlight their potential influence on susceptibility to internal browning. This relationship should be considered in the current breeding programs to select cultivars with high bioactive compound contents, health-enhancing properties and good postharvest performance.

Proteomic analysis of outer membrane vesicles from the probiotic strain Escherichia coli Nissle 1917.

Escherichia coli Nissle 1917 (EcN) is a probiotic used for the treatment of intestinal disorders. EcN improves gastrointestinal homeostasis and microbiota balance; however, little is known about how this probiotic delivers effector molecules to the host. Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria and have a relevant role in bacteria-host interactions. Using 1D SDS-PAGE and highly sensitive LC-MS/MS analysis we identified in this study 192 EcN vesicular proteins with high confidence in three independent biological replicates. Of these proteins, 18 were encoded by strain-linked genes and 57 were common to pathogen-derived OMVs. These proteins may contribute to the ability of this probiotic to colonize the human gut as they fulfil functions related to adhesion, immune modulation or bacterial survival in host niches. This study describes the first global OMV proteome of a probiotic strain and provides evidence that probiotic-derived OMVs contain proteins that can target these vesicles to the host and mediate their beneficial effects on intestinal function. All MS data have been deposited in the ProteomeXchange with identifier PXD000367 (http://proteomecentral.proteomexchange.org/dataset/PXD000367).

The Impact of Rootstock on "Big Top" Nectarine Postharvest Concerning Chilling Injury, Biochemical and Molecular Parameters.

Peaches and nectarines have a short shelf life even when harvested at appropriate physiological maturity. Market life is increased by storage at low temperatures. However, chilling injury symptoms can appear, causing physiological disorders and limiting shipping potential. The rootstock effect on the post-harvest quality has hardly been explored. Thus, the principal aim of this work was to study the influence of seven different Prunus rootstocks on the "Big Top" nectarine cv, considering harvest and post-harvest quality parameters and their correlation with chilling injury disorders. Basic fruit quality traits, individual sugars and organic acids analyzed by HPLC and other biochemical compounds such as relative antioxidant capacity, total phenolics content, flavonoids, anthocyanins, vitamin C and related enzyme activities (PAL, POD, PPO) were considered. In addition, correlations with possible candidate genes for chilling injury (CI) tolerance were searched by qPCR. Although a low susceptibility to CI symptoms has been found in "Big Top", rootstocks "PADAC 9902-01", "PADAC 99-05" and "ReplantPAC" exhibited lower CI symptoms. A statistically significant influence of the evaluated rootstocks was found concerning the parameters of this study. Phenols and anthocyanins seem to be important parameters to be considered in the prevention of chilling injury disorders. Moreover, PAL1, PPO4, PG2 and LDOX genes relative expressions were positively associated with chilling injury susceptibility. This study opens new perspectives for understanding peach fruit adaptation and response to cold storage temperatures during the post-harvest period.

Population Structure and Association Mapping for Agronomical and Biochemical Traits of a Large Spanish Apple Germplasm.

A basic knowledge of linkage disequilibrium and population structure is necessary in order to determine the genetic control and identify significant associations with agronomical and phytochemical compounds in apple (Malus × domestica Borkh). In this study, 186 apple accessions (Pop1), representing both Spanish native accessions (94) and non-Spanish cultivars (92) from the EEAD-CSIC apple core collection, were assessed using 23 SSRs markers. Four populations were considered: Pop1, Pop2, Pop3, and Pop4. The initial Pop1 was divided into 150 diploid (Pop2) and 36 triploid accessions (Pop3), while for the inter-chromosomal linkage disequilibrium and the association mapping analysis, 118 phenotype diploid accessions were considered Pop4. Thus, the average number of alleles per locus and observed heterozygosity for the overall sample set (Pop1) were 15.65 and 0.75, respectively. The population structure analysis identified two subpopulations in the diploid accessions (Pop2 and Pop4) and four in the triploids (Pop3). Regarding the Pop4, the population structure with K = 2 subpopulations segregation was in agreement with the UPGMA cluster analysis according to the genetic pairwise distances. Moreover, the accessions seemed to be segregated by their origin (Spanish/non-Spanish) in the clustering analysis. One of the two subpopulations encountered was quite-exclusively formed by non-Spanish accessions (30 out of 33). Furthermore, agronomical and basic fruit quality parameters, antioxidant traits, individual sugars, and organic acids were assessed for the association mapping analysis. A high level of biodiversity was exhibited in the phenotypic characterization of Pop4, and a total of 126 significant associations were found between the 23 SSR markers and the 21 phenotypic traits evaluated. This study also identified many new marker-locus trait associations for the first time, such as in the antioxidant traits or in sugars and organic acids, which may be useful for predictions and for a better understanding of the apple genome.

Transcriptional regulation of the gene cluster encoding allantoinase and guanine deaminase in Klebsiella pneumoniae.

Purines can be used as the sole source of nitrogen by several strains of K. pneumoniae under aerobic conditions. The genes responsible for the assimilation of purine nitrogens are distributed in three separated clusters in the K. pneumoniae genome. Here, we characterize the cluster encompassing genes KPN_01787 to KPN_01791, which is involved in the conversion of allantoin into allantoate and in the deamination of guanine to xanthine. These genes are organized in three transcriptional units, hpxSAB, hpxC, and guaD. Gene hpxS encodes a regulatory protein of the GntR family that mediates regulation of this system by growth on allantoin. Proteins encoded by hpxB and guaD display allantoinase and guanine deaminase activity, respectively. In this cluster, hpxSAB is the most tightly regulated unit. This operon was activated by growth on allantoin as a nitrogen source; however, addition of allantoin to nitrogen excess cultures did not result in hpxSAB induction. Neither guaD nor hpxC was induced by allantoin. Expression of guaD is mainly regulated by nitrogen availability through the action of NtrC. Full induction of hpxSAB by allantoin requires both HpxS and NAC. HpxS may have a dual role, acting as a repressor in the absence of allantoin and as an activator in its presence. HpxS binds to tandem sites, S1 and S2, overlapping the -10 and -35 sequences of the hpxSAB promoter, respectively. The NAC binding site is located between S1 and S2 and partially overlaps S2. In the presence of allantoin, interplay between NAC and HpxS is proposed.

Biochemical Characterization and Differential Expression of PAL Genes Associated With "Translocated" Peach/Plum Graft-Incompatibility.

Grafting is an ancient plant propagation technique widely used in horticultural crops, particularly in fruit trees. However, the involvement of two different species in grafting may lead to lack of affinity and severe disorders between the graft components, known as graft-incompatibility. This complex agronomic trait is traditionally classified into two categories: "localized" (weak graft unions with breaks in cambial and vascular continuity at the graft interface and absence of visual symptoms in scion leaves and shoots) and "translocated" (degeneration of the sieve tubes and phloem companion cells at the graft interface causing translocation problems in neighboring tissues, and reddening/yellowing of scion leaves). Over the decades, more attention has been given to the different mechanisms underlying the "localized" type of graft-incompatibility; whereas the phenylpropanoid-derived compounds and the differential gene expression associated with the "translocated" graft-incompatibility remain unstudied. Therefore, the aim of this study was to shed light on the biochemical and molecular mechanisms involved in the typical "translocated" graft-incompatibility of peach/plum graft-combinations. In this study, the "Summergrand" (SG) nectarine cultivar was budded on two plum rootstocks: "Adara" and "Damas GF 1869". "Translocated" symptoms of incompatibility were shown and biochemically characterized in the case of "SG/Damas GF 1869" graft-combination, 3 years after grafting. Non-structural carbohydrates (soluble sugars and starch), phenolic compounds and antioxidant activity, were significantly enhanced in the incompatible graft-combination scion. Similarly, the enzymatic activities of the antioxidant enzyme peroxidase, the phenylalanine ammonia-lyase (PAL) and polyphenol oxidase involved in the phenylpropanoid pathway were significantly affected by the incompatible rootstock "Damas GF 1869", inducing higher activities in the scion than those induced by the compatible rootstock "Adara". In addition, a positive and strong correlation was obtained between total phenol content, antioxidant capacity and the expression of the key genes involved in the phenylpropanoid pathway, PAL1 and PAL2. Regarding the "SG/Adara" graft-combination, there were neither external symptoms of "translocated" incompatibility nor significant differences in the biochemical and molecular parameters between scion and rootstock, proving it to be a compatible combination. The differential expression of PAL genes together with the biochemical factors cited above could be good markers for the "translocated" peach/plum graft-incompatibility.

Transcriptomic microRNA Profiling of Dendritic Cells in Response to Gut Microbiota-Secreted Vesicles.

The interconnection between nutrients, metabolites and microbes is a key factor governing the healthy/pathological status of an individual. Thus, microbiota-based research is essential in order to better understand human health and nutrition. Gut bacteria release membrane vesicles (MVs) as an intercellular communication mechanism that allows the direct delivery of factors that prime the host's innate immune system. We have previously shown that MVs from intestinal E. coli activate dendritic cells (DCs) in a strain-specific manner. To gain insights into the regulatory mechanisms involved, here, we have used an RNA deep sequencing approach to identify differentially expressed miRNAs (microRNAs) in DCs which are challenged by the MVs of the probiotic Nissle 1917 (EcN) or the commensal ECOR12. MicroRNAs are post-transcriptional regulatory mediators that permit the fine tuning of signaling pathways. This approach allowed the identification of a common set of miRNAs which are modulated by MVs from both strains and miRNAs which are differentially expressed in response to EcN or ECOR12 MVs. Based on the differential expression of the target genes and subsequent validation experiments, we correlated some of the selected miRNAs with the reported cytokine profile and specific T cell responses. As far as we know, this is the first study to analyze the regulation of miRNAs in DCs by MVs released by gut microbiota.

Increased production of the ether-lipid platelet-activating factor in intestinal epithelial cells infected by Salmonella enteritidis.

When exposed to enteric pathogens intestinal epithelial cells produce several cytokines and other proinflammatory mediators. To date there is no evidence that the ether-lipid platelet-activating factor (PAF) is one of these mediators. Our results revealed a significant increase in PAF production by human colonic tissue 4 h after infection by enterohemorrhagic Escherichia coli (EHEC) or Salmonella enteritidis. PAF is produced in the gut by cells of the immune system in response to bacterial infection. To determine whether the epithelial cells of colonic mucosa might also modulate PAF levels, we carried out PAF quantification and analysis of the enzymes involved in PAF synthesis in 5-day-old (undifferentiated) or 28-day-old (differentiated) Caco-2 cell cultures. Infection of undifferentiated Caco-2 cells with either bacterium had no effect on PAF levels, whereas in differentiated cells, infection by S. enteritidis increased PAF levels. Following infection by S. enteritidis, there were no changes in the activity of dithiothreitol-insensitive choline phosphotransferase. However, the enzymes of the remodeling pathway cytosolic phospholipase A(2), which catalyzes the formation of the PAF precursor lysoPAF, and lysoPAF acetyltransferase, are activated in the infected epithelial cells. This response is Ca(2+)-dependent.

NAD+-dependent post-translational modification of Escherichia coli glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional housekeeping protein reported to be a target of several covalent modifications in many organisms. In a previous study, enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains were shown to secrete GAPDH and the protein to bind human plasminogen and fibrinogen. Here we report that GAPDH of these pathogens is ADP-ribosylated either in the cytoplasm or in the extracellular medium. GAPDH catalyzes its own modification, which involves Cys-149 at the active site. ADP-ribosylation of extracellular GAPDH may play an important role in the host-pathogen interaction, as also proposed in other pathogens.

Role of YiaX2 in L-ascorbate transport in Klebsiella pneumoniae 13882.

The yiaK-S operon is required for aerobic growth on L-ascorbate in several Enterobacteriaceae. Here we present evidence that the yiaX2 gene belonging to the yiaK-S operon of Klebsiella pneumoniae 13882, which encodes a protein similar to the putative transporters classified as the major facilitator superfamily, is involved in the uptake of L-ascorbate. Concentration kinetic analysis yielded an apparent K(m) of YiaX2 for L-ascorbate of 161.38 +/-8.28 micromol x L(-1) and a Vmax of 3.81 +/- 0.60 nmol x mg(-1) x min(-1). This carrier uses the energy from electrochemical gradients, since it was inhibited by carbonyl cyanide m-chlorophenylhydrazone, a hydrophobic proton conductor that dissipates proton motive force.

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 Delta 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.

The yiaKLX1X2PQRS and ulaABCDEFG gene systems are required for the aerobic utilization of L-ascorbate in Klebsiella pneumoniae strain 13882 with L-ascorbate-6-phosphate as the inducer.

The capacity to both ferment and oxidize L-ascorbate has been widely documented for a number of enteric bacteria. Here we present evidence that all the strains of Klebsiella pneumoniae tested in this study ferment L-ascorbate using the ula regulon-encoded proteins. Under aerobic conditions, several phenotypes were observed for the strains. Our results showed that the yiaK-S system is required for this aerobic metabolic process. Gel shift experiments performed with UlaR and YiaJ and probes corresponding to the specific promoters indicated that L-ascorbate-6-phosphate is the effector molecule recognized by both regulators, since binding of the repressors to their recognition sites was impaired by the presence of this compound. We demonstrated that in K. pneumoniae cells L-ascorbate-6-phosphate is formed only by the action of the UlaABC phosphotransferase system. This finding explains why strains that lack the ula genetic system and therefore are unable to form the inducer intracellularly cannot efficiently use this vitamin as a carbon source under either anaerobic or aerobic conditions. Thus, efficient aerobic metabolism of L-ascorbate in K. pneumoniae is dependent on the presence of both the yiaK-S and ula systems. The expression of the yiaK-S operon, but not the expression of the ula regulon, is controlled by oxygen availability. Both systems are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and by IHF.

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.

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.

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.

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.