Loading metrics Open Access Peer-reviewed Research Article Sandeep Kumar, Lesley A. Ogilvie, Bhavik A. Patel, Cinzia Dedi, Wendy M. Macfarlane, Brian V. Jones Disruption of Escherichia coli Nissle 1917 K5 Capsule Biosynthesis, through Loss of Distinct kfi genes, Modulates Interaction with Intestinal Epithelial Cells and Impact on Cell Health Jonathan Nzakizwanayo, Sandeep Kumar, Lesley A. Ogilvie, Bhavik A. Patel, Cinzia Dedi, Wendy M. Macfarlane, Brian V. Jones x Published: March 19, 2015 Figures AbstractEscherichia coli Nissle 1917 (EcN) is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants), prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB), or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC), were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health. Citation: Nzakizwanayo J, Kumar S, Ogilvie LA, Patel BA, Dedi C, Macfarlane WM, et al. (2015) Disruption of Escherichia coli Nissle 1917 K5 Capsule Biosynthesis, through Loss of Distinct kfi genes, Modulates Interaction with Intestinal Epithelial Cells and Impact on Cell Health. PLoS ONE 10(3): e0120430. Editor: Markus M. Heimesaat, Charité, Campus Benjamin Franklin, GERMANYReceived: December 9, 2014; Accepted: January 22, 2015; Published: March 19, 2015Copyright: © 2015 Nzakizwanayo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedData Availability: All relevant data are within the paper and its Supporting Information Support is provided by the Medical Research Council (G0901553) awarded to BVJ; University of Brighton Studentship to JN; Society of Applied Microbiology; BVJ is also supported by the Queen Victoria Hospital Charitable Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the interests: The authors have declared that no competing interests exist. IntroductionDue to the intimate role of the gut microbiome in human health and disease processes, this predominantly bacterial community is increasingly viewed as an important target for the development of novel approaches to diagnose, prevent, or treat a wide range of disorders [1–4]. In this context, probiotics are among the most promising tools for manipulation of the gut microbiome, and have been defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host” [5]. The majority of probiotics are Gram-positive bacterial species, and considerable evidence is accumulating regarding the efficacy of these organisms in treating or preventing a variety of gastrointestinal (GI) diseases, and potentially also extra-intestinal disorders [1–4]. Among the probiotics currently available, Escherichia coli Nissle 1917 (EcN; serotype O6:K5:H1) is of particular interest. Not only is this one of the most extensively characterized probiotic organisms (in terms of phenotype, genotype, and clinical efficacy), but is currently the only Gram-negative species in use [6]. EcN was first isolated from the faeces of a World War I soldier who, in contrast to comrades in his trench, was not affected by an outbreak of dysentery [7]. This gastroprotective strain is the active component of Mutaflor (Ardeypharm GmbH, Herdecke, Germany), a microbial drug that is marketed and used in several countries. Clinical trials have shown EcN to be effective for maintaining remission of ulcerative colitis (UC) [8–11], stimulation of the immune system in premature infants [12], treatment of infectious diarrhoea [13], and protection of human intestinal epithelial cells (IECs) against pathogens [14, 15]. These benefits are largely attributed to the immuno-modulatory effects elicited by EcN, which encompass both innate and adaptive elements of the immune system. For example, colonisation with EcN has been indicated to alter the host cytokine profile, and also chemokine production in cultured IECs [16–19]; stimulate the production of mucosal peptide based defences [20]; influence the clonal expansion of T-Cell populations, and modulate antibody responses [12, 21, 22]. Notably, the modulation of T-cell functions mediated by EcN may also extend to γδ T-cells, potentially enabling EcN to coordinate modulation of both innate and adaptive responses [22]. EcN has also been indicated to alter COX-2 expression in intestinal epithelial cells [23], which is an important target in the treatment or prevention of several GI diseases including IBD and colorectal cancer [24–27]. Although most closely related to uropathogenic strains of E. coli (UPEC), EcN is considered non-pathogenic. Genomic characterisation has highlighted the absence of genes encoding the typical UPEC virulence factors, but the retention or accumulation of factors proposed to facilitate general adaptability, colonisation of the GI tract, and the probiotic effects of EcN [28, 29]. These include a range of surface associated structures that are likely to provide the primary interface between host and microbe in the GI tract, such as flagella, fimbriae, a special truncated lipopolysaccharide (LPS) variant, and a K5 type polysaccharide capsule [6, 29–31]. In particular, structures such as flagellin, peptidoglycan and LPS, are recognised by immune regulating Toll-like receptors (TLRS) expressed by IECs, which have been established as key routes of host-microbe communication in the gut, with TLR signalling integral to epithelial homoeostasis and defence [32–34]. Signaling by several TLRs is known to be modulated either directly or indirectly by EcN derived ligands [6, 17–20, 30, 35], which include surface associated structures absent in most or all other probiotic organisms. The K5 capsule produced by EcN in particular is notable in this context, and although not a ligand for known TLRs, the EcN capsule has been implicated in the interaction of this organism with IECs, and impact on chemokine expression and TLR signalling [18,19]. Nevertheless, as with other probiotics, the detailed mechanisms underlying the clinical effectiveness of EcN remain poorly understood overall, with a greater comprehension required to fully realise the potential of this important probiotic species. Here we describe the application of random transposon mutagenesis to identify genes and surface structures involved in the interaction of EcN with human intestinal epithelial cells, and provide new insight into the mechanisms through which EcN interacts with epithelial cells. Results Isolation and genetic characterisation of EcN mutants with disruptions in genes related to cell surface structures Because cell surface structures are a primary point of contact between EcN and IECs, and processes such as biofilm formation and attachment to abiotic surfaces also depends on many of the same structures, we reasoned that selection of mutants with alterations in biofilm formation would enrich for those defective in cell surface associated features also likely to be involved in EcN-IEC interaction. Therefore, we initially subjected a total of 4,116 EcN mini-Tn5 mutants to a preliminary high throughput screen for alterations in biofilm formation (both enhancements and reductions), in order to enrich for mutants attenuated in cell surface features. In this precursor biofilm screen 21 mutants were found to be significantly different in their ability to form biofilms as compared to the EcN wild-type (EcN WT), but unaltered in general growth rate. The majority of these (n = 15) exhibited a biofilm formation enhanced (BFE) phenotype, whereas six exhibited biofilm formation deficient (BFD) phenotype as compared to the WT (Table 1). Identities of genes disrupted in these mutants indicated that the majority were associated with synthesis of cell surface structures, or aspects of cell envelope biogenesis, previously linked to host-IEC interaction or intestinal colonisation (Table 1; [18, 35, 37–40]). A subset of 6 mutants disrupted in genes predicted to encode for cell surface structures, and encompassing both BFD and BFE phenotypes, were subsequently selected for further characterisation of their interaction with cultured IECs. Fig 1. Adherence of EcN mini-Tn5 mutants to Caco-2 cells. A subset of mutants recovered from biofilm screens with disruptions in genes predicted to be involved in generation of surface tstructures, were assessed for their ability to attach to Caco-2 cells in in vitro co-culture models. Caco-2 cell monolayers (~80% confluence) were exposed to bacterial suspensions from mid-log-phase cultures at an MOI of 1:1 for 4 h at 37°C, 5% CO2. Genes disrupted in mutants tested are noted in parentheses and details can be found in Table 1. Data are expressed as the mean of three replicates, and error bars show SE of the mean. Significant differences between attachment of EcN WT and mutants is indicated by ** (P ≤ or **** (P were confirmed biofilm altered mutants and defined as biofilm enhanced (BFE) or biofilm deficient (BFD) mutants. Mutants biofilm formation index was calculated as the percentage of CV (OD595) measured in the EcN WT. Genetic characterisation of biofilm-altered mutants Genes disrupted in mutants of interest were identified using a “cloning free” arbitrary PCR-based approach to amplify DNA segments flanking the transposon insertion, as described by Manoil [55] using primers listed in S2 Table. The resulting amplicons were sequenced by GATC Biotech Ltd. (London, UK) using transposon end primer pLR27Primer 3. The putative function of disrupted genes was assigned by mapping sequence data flanking the mini-Tn5 insert site to the E. coli Nissle Draft genomes sequence [28], and the previously published genomic islands [29]. Sequence reads from mutants were trimmed to remove the 5’ low quality regions (typically ~30–50 nt), and the immediate ~40 nt flanking sections correlated with the EcN genome. Where EcN genome annotations did not provide any clear indication of putative function wider searches of the nr dataset using BlastX and/or the conserved domain database were employed. Construction of kfiB and kfiC deletion mutants Deletion mutants EcNΔkfiB and EcNΔkfiC were constructed by homologous recombination using the Xer-ciseTM chromosomal modification system (Cobra Biologics, Keele, UK) according to manufacturer’s instructions and protocols described by Bloor and Cranenburgh [56]. The system comprises plasmids pTOPO-DifCAT and pLGBE, for construction of target gene specific integration cassette and provision of the Red λ recombination functions, respectively. Briefly, kfiB or kfiC integration cassettes consisting of the difE. coli-cat-difE. coli region from pTOPO-DifCAT plasmid flanked by 50 nt regions homologous to the 3’ and 5' ends of the target gene, were generated by PCR using 70-nt primers, or (listed in S2 Table). EcN WT was first transformed with the Tc-selectable plasmid pLGBE and transformants EcN-pLGBE were used to generate electrocompetent cells, which were subsequently transformed with the PCR product of the difE. coli-cat-difE. coli integration cassette constructs. Integrants were selected on LB agar supplemented with 20 μg ml–1 Chloramphenicol. Loss of pLGBE and generation of chloramphenicol-sensitive clones, indicating resolution of difE. coli-cat-difE. coli marker genes by native recombinases and generation of markerless deletion mutants (mutants EcNΔkfiB and EcNΔkfiC) was achieved by sub-culturing the integrants in LB broth in the absence of antibiotics. Loss of pLGBE was verified by plasmid extraction, and by PCR for marker cassettes kfiB or kfiC specific primers EcNkfiB _F/R or EcNkfiC _F/R, respectively, and confirmed by PCR. Examination of polar effects in EcNΔkfiB and EcNΔkfiC mutants The effect of gene deletion or disruptions in kfiB and kfiC mutants, on the expression of downstream genes (polar effects) was assessed using RT-PCR. Total RNA was extracted from mid-log-phase bacterial cells using the RNeasy Protect Cell Mini Kit (Qiagen) according to manufacturer’s instructions, and treated using the Ambion TURBO DNA-free system (Ambion-Life technologies, Paisley, UK) to remove any potential DNA contamination. The treated RNA was used to generate cDNA using the One Step RT-PCR kit (Qiagen) according to the manufacturer’s instructions, utilising 15 ng RNA per reaction as template. Resulting cDNA was used as template in standard PCRs for detection of gene transcripts with specific primers detailed in S2 Table. Confirmation of K5 capsule absence in EcNΔkfiB and EcNΔkfiC mutants The K5 capsule-specific bacteriophage (ΦK5) [57] was used in this study to determine if the K5 capsule was expressed by EcN WT and deletion mutants. The bacteriophage was diluted and maintained in phage dilution buffer (PDB) (100 mM NaCl, 8 mM MgSO4, gelatine, 50 mM Tris pH Cultures of mutants EcNΔkfiB and EcNΔkfiC, controls EcN WT and MG1655 were grown in LB with shaking at 37°C to an OD600 of then pelleted by centrifugation (10,000 × g for 10 min) and resuspended in ice-cold 10 mM MgSO4. Aliquots of cell suspension (100 μl) were mixed with 100 μl of the appropriate bacteriophage dilution (ranging from 101 to 109 PFU ml–1 from stock suspension of × 109 PFU ml–1) in sterile mL Eppendorf tube then incubated at RT for 30 min, statically. The phage-bacteria mixture was added to a volume of 3 ml of soft agar (1% NaCl, yeast extract, 1% tryptone, agar) held at 42°C in 15 ml sterile glass tube, and the content of the tubes were mixed gently by swirling. The inoculated soft agar was poured on top of LB agar and incubated for 16 h at 37°C to allow formation of plaques. Intestinal epithelial cell culture and co-culture conditions Caco-2 cells (passage 51–79) were grown at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM, g glucose l–1) supplemented with 10% fetal bovine serum and 1× non-essential amino acids (PAA Laboratories, Somerset, UK). Cells were seeded into 6-well or 96-well plates, grown up to ~ 60–80% confluence, and used in co-culture experiments with bacteria. Mid-log-phase bacteria (OD600 of were washed with PBS and suspended in DMEM to the required final count, corresponding to the appropriate multiplicity of infection (MOI) and added to Caco-2 monolayers before plates were incubated at 37°C and 5% CO2. Bacterial adherence to Caco-2 cells Adherence was calculated according to the strategy employed by Hafez et al. [18]. Mid-log phase bacteria cultures were suspended in DMEM then added to monolayers of Caco-2 grown in 6-well plates (80% confluence) at an MOI of 1:1 and incubated at 37°C and 5% CO2 for 4 h. The monolayers were washed 3 times with PBS to remove non-adherent cells then treated with lysis solution, 1% wt / vol saponin (Sigma Aldrich) in trypsin-EDTA (PAA Laboratories, Somerset, UK) for 10 min to allow permeabilisation of Caco-2 cells and recovery of total cell-associated bacteria. Cells were mixed gently by pipetting, serially diluted in sterile PBS, plated onto LB agar, and incubated at 37°C overnight. The obtained viable count represented the total number of cell associated bacteria (adherent and internalised). Internalised bacteria were calculated using the same protocol but Caco-2 cells were treated with gentamicin for 2h (200 μg ml-1) to kill external bacteria prior to lysis and enumeration. The number of adherent bacteria was taken as the difference between total cell associated bacteria and internalised bacteria. The effect of EcN mutants on induction of apoptosis in Caco-2 cells The effect of EcN mutants on induction of apoptosis Caco-2 cells was assessed by measuring the activity of caspase 3/7 using the Caspase-Glo 3/7 kit (Promega, Southampton, UK), according to manufacturer’s instructions. Cells were seeded in 96-well plates with 5,000 cells/well and cultured to achieve ~ 60% confluence then treated with bacteria or bacterial supernatants in co-culture. Media was replaced with serum-free DMEM for 12 h prior to the treatment. Bacterial suspensions were prepared in serum-free DMEM from mid-log-phase cultures then added to Caco-2 cells at an MOI of 10:1 (bacteria:Caco-2) in a final volume of 100 μl/ well. The plates were incubated for 2 h at 37°C and 5% CO2 then media was replaced with fresh serum-free DMEM supplemented with gentamicin at 200 μg ml–1 to stop bacterial growth, and plates were incubated for another 10 h. Bacterial supernatants were obtained from cells grown in 5 mL serum-free DMEM at 37°C overnight, with shaking, and recovered by centrifugation (1,500 × g for 10 min), pH adjusted to and filter-sterilised ( The supernatants were diluted in fresh serum-free DMEM at a ratio of 1:1, and used in place of cell suspensions as described above. Caspase 3/7 activity was measured as relative light units (RLUs) using a Synergy Multi-Mode Plate Reader (BioTek, Potton, UK) operated with BioTek software. Analysis of cytotoxicity The effect of EcN strains on induction of cytotoxicity in Caco-2 cells was assessed by measuring the amount of lactate dehydrogenase (LDH) released into the co-culture media, using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega). Caco-2 cells were treated with bacteria and controls as described for the analysis of apoptosis (above) and both assays were performed in parallel. After treatment of Caco-2 cells, supernatants were collected from plate wells using a multichannel pipette then transferred to fresh 96-well at 50 μl/well. The supernatant was diluted further in serum-free culture media then mixed with the CytoTox 96 substrate at a ration of 1:1. Plates were incubated in the dark at room temperature for 30 min and absorbance at 490 nm (OD490) was recorded. The percentage of cytotoxicity was calculated as LDH released in treated cells (OD490)/maximum LDH release (OD490) × 100. Maximum release was determined as the amount released by total lysis of untreated Caco-2 cells with the CytoTox 96 lysis Solution (10X). Analysis of cellular and nuclear morphology Membrane integrity and nuclear morphology of Caco-2 cells were analysed by fluorescent phalloidin (F-actin) and Dapi (DNA) stainings. Cells were grown on sterile glass cover slips in 6-well plates then treated with EcN strains and controls (MG1655 and mM camptothecin; Sigma) as described above (analysis of apoptosis). After the treatments, the cells on coverslips were washed with PBS then fixed with 4% formaldehyde (Sigma) in PBS for 20 min at RT. The fixed cells were washed three times with PBS and permeabilised with Triton X-100 (Sigma) in PBS for 5 min at RT. The cells were washed three times with PBS, 5 min per wash with gentle rocking, then treated with a μg ml–1 solution of fluorescein isothiocyanate-phalloidin (Sigma- Aldrich) in PBS for 1 h at RT in the dark. The cells were washed twice with PBS and were mounted with the Fluoroshield DAPI medium (Sigma) and examined under a Leica TCS SP5 Confocal Laser Scanning microscope (Leica Microsystems, Wetzlar, Germany). Analysis of COX-2 expression The expression of COX-2 protein in Caco-2 co-cultures was analysed by western blotting using standard methods. Briefly, Caco-2 cells were seeded in 6 wells plates, and at ~ 60% confluence, were treated with EcN K5 mutants and controls as described above (analysis of apoptosis). Lipopolysaccharide (LPS, final concentration, 5 μg ml–1) from Salmonella enterica (Sigma, UK) and human tumour necrosis factor alpha (TNF-α, 10 ng ml–1) (Sigma, UK) were used as pro-inflammatory stimulator positive controls. Treated Caco-2 cell monolayers were washed 3 times with PBS, trypsinised then resuspended in 100 μl of hypotonic buffer (10 mM HEPES, 10 mM KCl, mM EDTA, mM EGTA, 1 mM DTT in SDW, pH containing Sigma protease inhibitor cocktail (1:20), for 15 min at 4°C. Cells were lysed in 25 μl 10% Triton X-100 for 30 min and total protein obtained by centrifugation (10,000 g for 1 min at 4°C). Protein concentration was determined by the Bradford method (Bio-Rad) and equivalent amounts of protein lysates (10 μg) separated by electrophoresis on SDS—PAGE (10%), and then transferred onto a nitrocellulose membrane (GE Healthcare, Giles, UK). The blots were blocked at RT with 10% skimmed milk powder in TBST buffer (10 mM Tris, pH M NaCl, Tween 20), and incubated with primary antibody, anti-COX-2 rabbit polyclonal (Abcam, Cambridge, UK) 1:1,000 in TBST, overnight at 4°C. Blots were washed with TBST then incubated with anti-rabbit HRP-conjugated secondary antibody (Sigma, UK) 1:5,000 in TBST, for 1h at RT. Membranes were washed further then visualised by incubation with the ECL chemiluminescent reagent (Amersham, Little Chalfont, UK) and exposed to Kodak Image Station 440 for signal detection. Blots were then stripped and reprobed with loading control anti-GAPDH mouse monoclonal (Ambion, Cambridge, UK); anti-mouse IgG HRP-conjugated (Sigma, UK) as secondary antibody. The bands of COX-2 densitometry readings were normalized to the GAPDH control. Analysis of cell morphology and aggregation Bacteria were grown statically in 5 mL LB in 50 mL sterile polystyrene tube at 37°C for 16 h. The cultures were mix gently by swirling and 3 μL of each was directly transferred onto glass slide, allowed to rest for 1 min then covered with a cover slip and visualised using ×40 magnification phase contrast microscopy. For each culture 10 randomly selected fields of view across each slide were captured using the Olympus Cell Sense software, and subsequently reviewed. Representative images were selected and adjusted only for brightness and contrast. Statistical analysis All statistical analysis was performed using Prism For Mac OS X (Graphpad Software inc. USA; Data was analysed using either Student’s t-test, or ANOVA with the Bonferroni correction for multiple comparisons. Supporting InformationS1 Fig. Overview of K5 capsule biosynthesis in E. coli, and associated genes disrupted in this show the genetic organisation of the K5 gene cluster in E. coli Nissle 1917 based on data from Cress et al. [28]; Grozdanov et al. [29], and an overview of the current model for K5 capsule biosynthesis and assembly adapted from Griffiths et al. [36]; Whitfield [41]; Petit et al. [42]; Bliss et al. [43]; Hodson et al. [44]; Corbett and Roberts [45]; Whitfield and Roberts [46]; Rigg et al. [47]; Whitfield and Willis [58]. A) Physical map of the EcN K5 capsular polysaccharide gene cluster. Region I (kpsF,E,D,U,C,S) and Region III (kpsM,T) encode elements of synthesis and export machinery, and are conserved among E. coli strains generating Group 2 polysaccharide capsules. Region II encodes K5 specific polysaccharide synthesis machinery (kfiA,B,C,D). Genes disrupted by transposon mutagenesis (kfiB, kpsT) and/or subject to gene knockout (kfiB,C) in this study are identified. HP—denote hypothetical proteins of unknown function B) Representation of main stages and associated K5 biosynthetic machinery (stages 1–3). K5 assembly is localised to the cytoplasmic face of the inner membrane, and is underpinned by the formation of a biosynthetic complex which catalyses synthesis and export polysaccharide precursors for incorporation in the maturing capsule on the cell surface. During K5 assembly it is believed that a unified biosynthetic complex is developed which progressively catalyses main stages [1–3]. However, for clarity here we have separated each main stage of K5 synthesis and associated membrane complexes. Stage 1) Proteins encoded by kpsF,U,C,S are believed to be responsible for the initial generation of the phospatyidyl acceptor and Kdo linker (keto-3-deoxy-manno-2-octulosonic acid), upon which the polysaccharide chain is synthesised. Stage 2) Proteins encoded by kfiA-D are responsible for synthesis of the polysaccharide chain through addition of alternating units of GlcA (glucuronic acid) and GlcNAc (N-acetyl-glucosamine) from UDP-sugar precursors. Stage 3) Proteins generated by kpsD,E,M,T form an ABC transporter complex that translocates completed polysaccharide chains to the cell surface, in an energy dependant process. Acknowledgments We wish to thank Prof Jun Zhu (University of Pennsylvania, School of Medicine) and Prof Ian Roberts (University of Manchester, Faculty of Life Sciences) for gifts of pRL27::mini-Tn5 system and ΦK5 bacteriophage, respectively. 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Loading metrics Open Access Peer-reviewed Research Article Francine C. Paim, Ayako Miyazaki, Stephanie N. Langel, David D. Fischer, Juliet Chepngeno, Steven D. Goodman, Gireesh Rajashekara, Linda J. Saif , Anastasia Nickolaevna Vlasova Escherichia coli Nissle 1917 administered as a dextranomar microsphere biofilm enhances immune responses against human rotavirus in a neonatal malnourished pig model colonized with human infant fecal microbiota Husheem Michael, Francine C. Paim, Ayako Miyazaki, Stephanie N. Langel, David D. Fischer, Juliet Chepngeno, Steven D. Goodman, Gireesh Rajashekara, Linda J. Saif, Anastasia Nickolaevna Vlasova x Published: February 16, 2021 Figures AbstractHuman rotavirus (HRV) is a leading cause of diarrhea in children. It causes significant morbidity and mortality, especially in low- and middle-income countries (LMICs), where HRV vaccine efficacy is low. The probiotic Escherichia coli Nissle (EcN) 1917 has been widely used in the treatment of enteric diseases in humans. However, repeated doses of EcN are required to achieve maximum beneficial effects. Administration of EcN on a microsphere biofilm could increase probiotic stability and persistence, thus maximizing health benefits without repeated administrations. Our aim was to investigate immune enhancement by the probiotic EcN adhered to a dextranomar microsphere biofilm (EcN biofilm) in a neonatal, malnourished piglet model transplanted with human infant fecal microbiota (HIFM) and infected with rotavirus. To create malnourishment, pigs were fed a reduced amount of bovine milk. Decreased HRV fecal shedding and protection from diarrhea were evident in the EcN biofilm treated piglets compared with EcN suspension and control groups. Moreover, EcN biofilm treatment enhanced natural killer cell activity in blood mononuclear cells (MNCs). Increased frequencies of activated plasmacytoid dendritic cells (pDC) in systemic and intestinal tissues and activated conventional dendritic cells (cDC) in blood and duodenum were also observed in EcN biofilm as compared with EcN suspension treated pigs. Furthermore, EcN biofilm treated pigs had increased frequencies of systemic activated and resting/memory antibody forming B cells and IgA+ B cells in the systemic tissues. Similarly, the mean numbers of systemic and intestinal HRV-specific IgA antibody secreting cells (ASCs), as well as HRV-specific IgA antibody titers in serum and small intestinal contents, were increased in the EcN biofilm treated group. In summary EcN biofilm enhanced innate and B cell immune responses after HRV infection and ameliorated diarrhea following HRV challenge in a malnourished, HIFM pig model. Citation: Michael H, Paim FC, Miyazaki A, Langel SN, Fischer DD, Chepngeno J, et al. (2021) Escherichia coli Nissle 1917 administered as a dextranomar microsphere biofilm enhances immune responses against human rotavirus in a neonatal malnourished pig model colonized with human infant fecal microbiota. PLoS ONE 16(2): e0246193. Nicholas J. Mantis, New York State Department of Health, UNITED STATESReceived: October 18, 2020; Accepted: January 14, 2021; Published: February 16, 2021Copyright: © 2021 Michael et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are Availability: All relevant data are within the manuscript and its Supporting Information filesFunding: This work was supported by the Bill and Melinda Gates Foundation (OPP 1117467), the NIAID, NIH (R01 A1099451), federal and state funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University and from the NIH Office of Dietary Supplements (ODS) supplemental grant interests: The authors have declared that no competing interests exist. IntroductionHuman rotavirus (HRV) is a leading cause of diarrhea in children. It causes significant morbidity and mortality, especially in developing countries [1]. Malnutrition is a major contributor of high mortality due to viral gastroenteritis, including HRV, in countries with low socioeconomic status [2–4]. A number of studies have shown that malnutrition triggers immune dysfunction, including altered innate and adaptive immune responses, impairment of epithelial cell barrier function and/or dysfunction of intestinal epithelial cells [5–10]. Probiotics are increasingly used to enhance oral vaccine responses and to treat enteric infections [11] and ulcerative colitis in children [12]. The probiotic Escherichia coli Nissle (EcN) 1917 has been widely used in the treatment of ulcerative colitis in humans [13]. EcN lacks virulence factors and possesses unique health-promoting properties [14]. The long term persistence of EcN in humans suggests adaption to a host with an established gut microbiome [15]. Our research group has shown that EcN protected gnotobiotic (Gn) piglets against HRV infection and decreased the severity of diarrhea by modulating innate and adaptive immunity, and protecting the intestinal epithelium [16–18]. Oral administration of probiotics is associated with a number of challenges, such as low pH of gastric acid and bile salts in the stomach, effector functions of the host immune system, and competition with commensal and pathogenic bacteria [19]. These factors adversely influence adherence and persistence of probiotics within the host and thus reduce the beneficial effects [20]. Probiotics must survive in gastric acids to reach the small intestine and colonize the host to confer beneficial effects of preventing or moderating gastrointestinal diseases [21]. Encapsulation of lyophilized probiotics have resulted in enhanced bacterial viability [22, 23]. Navarro and his colleagues (2017) have formulated a new synbiotic formulation that employed porous semi-permeable, biocompatible and biodegradable microspheres (dextranomer microspheres) containing readily diffusible prebiotic cargo [24]. Adherence of the probiotic bacteria to the microsphere has a two-fold effect; it facilitates the more formidable biofilm state of probiotics as well as a creates a directed means to provide a high concentration gradient of prebiotics via diffusion of the microsphere cargo. However, currently there are no strategies for improved EcN probiotic efficacy and stability within the malnourished host. Previously we have established a deficient HIFM-transplanted neonatal pig model that recapitulates major aspects of malnutrition seen in children in impoverished countries [5, 6]. The purpose of this study was to investigate a novel probiotic delivery method to prolong the persistence of probiotics in the gut and to enhance their beneficial effects. We hypothesized that oral administration of EcN attached to the surface of biocompatible dextranomar microspheres in a biofilm state will protect against harsh conditions of the stomach and improve gut stability, thus enhancing their beneficial effects with a single administration compared with the repetitive administration of probiotics in the suspension form, which results in transient and often inconsistent outcomes. In addition, administration of probiotics in their suspension state has modest impact on the host’s microbiome [25]. High doses and repeated administration of probiotics are needed to achieve potential health benefits; however, in impoverished countries this poses challenges due to lack of product availability, the limited health care system, and resources [26–28]. Whether the use of the biofilm microsphere can overcome this remains to be established. The multifactorial pathobiology of malnutrition is associated with a vicious cycle of intestinal dysbiosis, epithelial breaches, altered metabolism, impaired immunity, intestinal inflammation, and malabsorption [29, 30]. Malnutrition increases the risk of diarrheal diseases caused by some, but not all, entero-pathogens. Malnutrition can result in impaired immune defenses that compromise gut integrity, and dybiosis that can influence defense against intestinal pathogens in the malnourished host [31]. This in turn limits the ability of probiotics to repair the intestinal epithelium and establish healthy microbiota. These concerns necessitate further research to enhance the stability and persistence of probiotics in malnourished hosts. Probiotics are generally considered safe, however there are some associated risks. These risks are increased if there are chronic medical conditions that weaken the immune system or if there are gut barrier breeches. Possible risks can include: developing an infection, developing resistance to antibiotics, and developing harmful byproducts from the probiotic supplement. Also, in malnourished hosts due to increased intestinal motility, probiotics can be eliminated from the gut faster limiting their beneficial effects [32, 33]. Furthermore, we aimed to investigate whether a single dose of EcN biofilm microspheres enhances immune responses after HRV infection in a malnourished Gn pig model. Previous transplantation of Gn pigs with probiotic bacteria demonstrated upregulated innate and adaptive immune responses following HRV infection [16, 17, 34–37]. In this preliminary study, we report increased innate immune and B cell responses after EcN biofilm treatment that were associated with protection against HRV disease and infection in a neonatal malnourished, HIFM pig model. Materials and methods Human Infant Fecal Microbiota (HIFM) The collection and use of HIFM was approved by The Ohio State University Institutional Review Board (IRB). With parental consent, sequential fecal samples were collected from a healthy, two-month-old, exclusively breastfed, vaginally delivered infant. Samples were pooled and diluted to 1:20 (wt/vol) in PBS containing (vol/vol) cysteine and 30% glycerol and stored at -80°C as described previously [5, 6]. Virus HRV (VirHRV) Wa strain passaged 25–26 times in Gn piglets was used to orally inoculate piglets at a dose of 1 × 106 fluorescent focus units (FFU) as described previously [5, 6]. Preparation of biofilm dextrananomer microspheres Anhydrous dextranomer microspheres (Sephadex, GE Healthcare Life Sciences, Pittsburgh, PA) were used. Anhydrous microspheres were hydrated in growth medium at 50 mg per ml and autoclaved for 20 min. Autoclaved microspheres were removed from solution on a vacuum filter apparatus and collected via sterile loop into a filter-sterilized 1M solution of sucrose. The microsphere mixture was vortexed and incubated for 24 hours at room temperature (RT). Sugar was removed from solution on a vacuum filter apparatus and collected via sterile loop. The microspheres were then added to EcN [1 × 109 colony-forming unit (CFU) per ml], pelleted, washed, and re-suspended in sterile saline. EcN was allowed to incubate with the microspheres for 1h at RT to facilitate binding and stored in -80°C in 30% glycerol. Prior to use, microspheres were thawed, mixed 1:1 with Natrel and administered orally. For EcN administered as a suspension, 1 × 109 CFU per ml was pelleted and re-suspended in sterile saline in preparation for oral inoculation. Animal experiments The animal experiments were approved by the Institutional Animal Care and Use Committee at The Ohio State University (OSU). Piglets were derived from near-term sows (purchased from OSU specific pathogen-free swine herd) by hysterectomy and maintained in sterile isolators as described previously [38]. For preliminary investigations, neonatal pigs were randomly assigned to three groups: 1) EcN biofilm (n = 3); 2) EcN suspension (n = 4); and 3) control pigs (n = 3). Pigs were fed a deficient diet of 50% ultra-high temperature pasteurized bovine milk diluted with 50% sterile water which contained half of the recommended protein levels ( that met or exceeded the National Research Council Animal Care Committee’s guidelines for calories, fat, protein and carbohydrates in suckling pigs. All pigs were confirmed free from bacterial and fungal contamination prior to HIFM transplantation by aerobic and anaerobic cultures of rectal swabs. Pigs were orally inoculated with 2ml of diluted HIFM stock at 4 days of age (post-HIFM transplantation day, PTD 0). The pigs were colonized orally with EcN biofilm or EcN suspension at PTD 11. Pigs were then challenged with VirHRV [1 × 106 FFU, post challenge day (PCD) 0] at PTD 13 and euthanized at PTD 27/PCD 14. Post-VirHRV challenge, rectal swabs were collected daily to assess HRV shedding. Blood, spleen, duodenum, and ileum were collected to isolate mononuclear cells (MNCs) as described previously (31, 35, 36). Jejunum was collected to isolate intestinal epithelial cells (IECs) using modified protocols [18, 39–41]. Serum and small intestinal contents (SIC) were collected to determine the HRV specific and total antibody responses [6, 17, 34, 42, 43]. Assessment of clinical signs and detection of HRV shedding Rectal swabs were collected daily post-VirHRV challenge. Fecal consistency was scored as follows; 0, normal; 1, pasty; 2, semi-liquid; and 3, liquid, and pigs with fecal score more than 1 were considered as diarrheic. Rectal swabs were suspended in 2 ml of minimum essential medium (MEM) (Life technologies, Waltham, MA, USA), clarified by centrifugation for 800 × g for 10 minutes at 4°C, and stored at -20°C until quantification of infectious HRV by a cell culture immunofluorescence (CCIF) assay as previously described [44]. Isolation of mononuclear cells (MNCs) Systemic (blood and spleen) and intestinal (duodenum and ileum) tissues were collected to isolate MNCs as described previously [36, 45, 46]. The purified MNCs were re-suspended in E-RPMI 1640. The viability of each MNCs preparation was determined by trypan blue exclusion (≥95%). Flow cytometry analysis Freshly isolated MNCs were stained to assess frequencies of conventional dendritic cells (DCs) (cDCs, SWC3a+CD4-CD11R1+) and plasmacytoid DCs (pDCs, SWC3a+CD4+CD11R1-), MHC II and CD103 marker expression on DCs were used in our experiments. Frequencies of IgA+ B lymphocytes were determined by identifying CD79β and IgA expression in MNCs as reported previously [34]. Similarly, frequencies of memory/resting (CD79β+CD2-CD21-) and activated (CD79β+CD2+CD21-) B cells among systemic and intestinal MNCs were determined as described previously [34]. Appropriate isotype matched control antibodies were included. Subsequently, 50,000 events were acquired per sample using BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using C6 flow sampler software. NK cytotoxicity assay Total blood MNCs and K562 cells were used as effector and target cells, respectively. Effector: target cell ratios of 10:1, 5:1, 1:1 and were used and the assay was done as described previously [47, 48]. HRV-specific and total antibody responses The HRV specific and total antibody titers in serum and SIC were detected by enzyme-linked immunosorbent assay (ELISA) as described previously [6, 17, 34, 42, 43]. To determine the intestinal antibody responses, small intestinal contents (SIC) were collected with protease inhibitors in the medium. HRV-specific Antibody Secreting Cells (ASCs) responses HRV and isotype-specific antibody secretion in MNCs isolated from blood, spleen, duodenum and ileum were analyzed by ELISPOT assay as described previously [17, 34, 42, 43]. Isolation of Intestinal Epithelial Cells (IECs) and extraction of RNA The IECs were isolated from jejunum (mid gut) using a modified protocol adapted from Paim et al. [18, 49]. The viability and numbers of IECs were determined by the trypan blue exclusion method (70–80%). IECs were stored at −80°C in 500 μl of RNAlater tissue collection buffer (Life technologies, Carlsbad, CA, USA) until further analysis. Total RNA from IECs was extracted using Direct-Zol RNA Miniprep (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. The RNA concentrations and purity were measured using NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Real-time quantitative RT-PCR (qRT-PCR) of CgA, MUC2, PCNA, SOX9 and villin gene mRNA levels in Intestinal Epithelial Cells (IECs) qRT-PCR was performed using equal amounts of total RNA (75 ng) with Power SYBR Green RNA-to-CT 1 step RT-PCR kit (Applied Biosystems, Foster, CA, USA). The primers for enteroendocrine cells chromogramin A (CgA), goblet cells mucin 2 (MUC2), transient amplifying progenitor cells proliferating cell nuclear antigen (PCNA), intestinal epithelial stem cells transcription factor SRY-box9 (SOX9), enterocytes (villin) and β-actin were based on previously published data [18, 39–41]. Relative gene expression of CgA, MUC2, PCNA, SOX9 and villin were normalized to β-actin and expressed as fold change using the 2-ΔΔCt method [50]. Statistical analysis All statistical analyses were performed using GraphPad Prism version 6 (GraphPad software, Inc., La Jolla, CA). Log10 transformed isotype ELISA antibody titers that were analyzed using one-way ANOVA followed by Duncan’s multiple range test. Data represent the mean numbers of HRV specific antibody secreting cells per 5 × 105 mononuclear cells and analyzed using non-parametric t-test (Mann-Whitney). HRV shedding and diarrheal analysis were performed using two way ANOVA followed by Bonferroni posttest. *P values < **P values < and ***P values < Error bars indicate the standard error of mean. Results EcN biofilm treatment reduced fecal HRV shedding and protected malnourished pigs from diarrhea post HRV challenge Analysis revealed that EcN biofilm treated malnourished pigs had shorter and delayed onset of HRV shedding as compared with the EcN suspension and the control group pigs (Table 1). A significant reduction in fecal virus peak titers shed was observed both in EcN biofilm (GMT = FFU/ml) and EcN suspension groups (GMT = FFU/ml), as compared with the control pigs (GMT = FFU/ml). In addition, EcN biofilm and EcN suspension groups had decreased peak shedding titers at PCD 2 as compared with that of control pigs (S1 Fig). EcN biofilm treatment shortened the mean duration of viral shedding to days as compared with and days in EcN suspension treated and control pigs, respectively (Table 1). Control pigs developed diarrhea ( at days post HRV challenge, continuing for days with mean cumulative fecal score (Table 1). Single administration of EcN biofilm microspheres completely protected the pigs from diarrhea (Table 1). However, administration of EcN suspension protected only 50% of the pigs from diarrhea. No significant differences were observed for mean days to diarrheal onset ( days), mean cumulative fecal score ( and the mean duration of diarrhea ( days) when they are compared with those in the control group (Table 1). These findings suggest that administration of EcN biofilm suppressed HRV infection greater than EcN administered in suspension. EcN biofilm treatment enhanced natural killer (NK) cell cytotoxicity in blood mononuclear cells (MNCs), increased the frequencies of activated pDCs in systemic and intestinal tissues, and increased activated cDCs in the blood and duodenum NK cell cytotoxicity in blood MNCs was significantly enhanced in EcN biofilm treatment compared with control pigs (Fig 1A). On the other hand, frequency of apoptotic MNCs were marginally decreased in EcN biofilm (3%) compared with EcN suspension (5%) and control ( pigs in blood (S2 Fig). Fig 1. EcN biofilm enhanced NK cell activity in blood mononuclear cells (MNCs) and significantly increased the frequencies of activated pDCs in systemic and intestinal tissues and increased activated cDCs in blood and duodenum (significantly).(a) Blood MNCs and carboxyfluorescein diacetate succinimidyl ester (CFSE) stained K562 tumor cells were used as effector and target cells, respectively, and co-cultured at set ratios to assess the NK cytotoxic function, (EcN biofilm vs control group). The effector: target cell co-cultures were stained with 7-Aminoactinomycin D (7AAD) after 12 hours of incubation at 37°C, and the frequencies of CFSE-7AAD double positive cells (lysed K562 target cells) were assessed by flow cytometry. Mean frequencies of activated (b) pDCs and (c) cDCs in systemic and intestinal tissues. Data represent means ± SEM. Significant differences (*p < **p < ***p < are indicated. Gnotobiotic pigs were transplanted with human infant fecal microbiota (HIFM) at 4 days of age, post-HIFM transplantation day (PTD) 0. Pigs were fed a deficient diet. Probiotic was given to the respective groups at PTD 11, followed by challenge with virulent human rotavirus (HRV) on PTD 13/post-challenge day (PCD) 0 and pigs were euthanized on PTD 27/PCD 14. biofilm treatment significantly increased the frequencies of activated pDC in systemic and intestinal tissues as compared with EcN suspension and the control pigs (Fig 1B). Moreover, EcN biofilm treatment significantly increased the frequencies of activated cDC in duodenum while numerically in blood (Fig 1C). There were no differences observed in other tissues. CD103+ cDC were increased (numerically) in spleen and intestinal tissues in EcN biofilm treated group as compared with EcN suspension and control pigs (S3 Fig). There were no differences observed in blood. EcN biofilm treatment significantly increased the frequencies of activated antibody secreting B cells in systemic tissues, resting antibody forming B cells in blood, and IgA+ B cells in spleen EcN biofilm treated malnourished pigs had significantly increased frequencies of activated antibody forming B cells in systemic tissues as compared with EcN suspension or the control pigs (S4A and S4B Fig). The frequency of IgA+ B cells in the spleen (significantly, S4C Fig) and blood (numerically, S4D Fig) increased in EcN biofilm treatment compared with EcN suspension and control pigs. Moreover, the frequency of resting/memory antibody forming B cells was significantly increased in blood in EcN biofilm compared with EcN suspension treated pigs (S4E Fig). These findings suggest that EcN biofilm treatment enhanced B cell immune response in systemic tissues, although no significant trends were observed in intestinal tissues. EcN biofilm treatment increased the number of HRV-specific Antibody Secreting Cells (ASCs) in systemic and intestinal tissues, and increased HRV-specific IgA antibody titers in serum and Small Intestinal Contents (SIC) Coinciding with decreased HRV shedding and protection from diarrhea, the mean numbers of HRV-specific IgA ASCs were increased in systemic and intestinal tissues of EcN biofilm treatment compared with EcN suspension and control group pigs (Fig 2A and 2B). A similar trend was observed with HRV-specific IgG ASCs (S5 Fig). HRV-specific IgM ASC numbers were below the detection limit in systemic and intestinal tissues. HRV-specific IgA antibody titers were increased in serum (significantly) and SIC (numerically) of EcN biofilm treated pigs compared with EcN suspension and control group pigs, coinciding with increased HRV-specific IgA ASCs (Fig 2C and 2D). Similar trends were observed with HRV-specific IgG antibody titers in serum (S6 Fig). In addition, total IgA concentration was increased (numerically) in serum samples of EcN biofilm treated pigs compared with EcN suspension or control group pigs (S7 Fig). No significant trends were observed in total and HRV-specific IgG in SIC (S8 Fig). These results indicate that EcN biofilm treatment enhanced B cell formation and clonal expansion of antibody producing cells in malnourished, HIFM transplanted pigs infected with HRV. Fig 2. EcN biofilm significantly increased HRV-specific IgA Antibody Secreting Cells (ASCs) in systemic and intestinal tissues and increased HRV-specific IgA antibody titers in serum and Small Intestinal Contents (SIC).(a) HRV-specific IgA ASCs in systemic cells; (b) HRV-specific IgA ASCs in intestinal cells; (c) HRV-specific IgA antibody titers in serum and (d) SIC. No significant differences were observed in intestinal tissues. Data represent means ± SEM. Significant differences (*p < **p < ***p < are indicated. Gnotobiotic pigs were transplanted with human infant fecal microbiota (HIFM) at 4 days of age, post-HIFM transplantation day (PTD) 0. Pigs were fed a deficient diet. Probiotic was given to respective groups at PTD 11, followed by challenge with virulent human rotavirus (HRV) on PTD 13/post-challenge day (PCD) 0 and pigs were euthanized on PTD 27/PCD 14. EcN biofilm treatment significantly upregulated the expression of CgA and SOX9 mRNA levels in jejunal epithelial cells Gene expression levels of CgA, SOX9, villin, MUC2, and PCNA were assessed from jejunal epithelial cells. The relative mRNA levels of CgA, SOX9, and villin genes were increased in jejunal epithelial cells of EcN biofilm compared with EcN suspension and control treated malnourished pigs (Fig 3A–3C). This coincided with the decreased severity of HRV shedding and diarrhea. There were no differences in gene expression levels for MUC2 and PCNA in jejunal epithelial cells of EcN biofilm and EcN suspension treated pigs (S9 Fig). Fig 3. EcN biofilm upregulated the expression of various cell components in jejunal epithelial cells.(a) Relative mRNA levels of enteroendocrine cells chromogramin A (CgA), (b) intestinal epithelial stem cells (SOX9), and (c) enterocytes (villin) in EcN biofilm, EcN suspension groups measured by real-time quantitative RT-PCR (RT-PCR), normalized to β-actin gene. Graphs represent means ± SEM. Significant difference (*p < **p < relative to control) are indicated. Gnotobiotic pigs were transplanted with human infant fecal microbiota (HIFM) at 4 days of age, post-HIFM transplantation day (PTD) 0. Pigs were fed a deficient diet. Probiotic was given to respective groups at PTD 11, followed by challenge with virulent human rotavirus (VirHRV) on PTD 13/post-challenge day (PCD) 0 and pigs were euthanized on PTD 27/PCD 14. DiscussionUsing a malnourished and HIFM transplanted pig model, we showed that compared with EcN administered as suspension, EcN administered as a biofilm on dextranomer microspheres enhanced multiple aspects of the immune response. EcN biofilm treated pigs had significantly reduced titers of virus shedding and diarrhea following VirHRV challenge compared with EcN suspension treated and control pigs. The presence of HRV-specific IgA antibodies in pigs is strongly correlated with protection from HRV infection [46, 51, 52]. Moreover, our study demonstrated for the first time that EcN biofilm treatment enhanced HRV specific-IgA and IgG ASCs in circulation and gut, enhanced HRV-specific IgA and IgG antibody titers in serum and HRV-specific IgA antibody titers in SIC, which collectively coincided with reduced diarrhea and virus shedding. Total IgA concentration was marginally increased in serum of EcN biofilm treated malnourished pigs (data not shown). Although not examined in this study, EcN biofilm treatment might have increased colonization in the gut, inhibiting competition by other members of the gut microbiota [53, 54]. It is possible that the observed effects of EcN biofilm treatment on systemic IgA responses could be mediated by direct modulation of host immune responses, suggesting that biofilm microspheres maybe more stable and persistent compared to probiotics in suspension in the host’s gastrointestinal system. Innate immune responses are critical as a first line of defense, limiting RV replication and disease severity in the host [16, 55]. EcN biofilm treatment enhanced innate immune responses. For example, blood NK cell cytotoxicity was higher in EcN biofilm treatment compared to EcN suspension treated and control groups. This suggests that EcN as a biofilm promoted innate immune responses, improving protection against HRV infection in vivo. Also the frequency of apoptotic blood MNCs was slightly reduced in EcN biofilm treated pigs compared with EcN suspension treatment and control pigs (data not shown). DCs play a key role in probiotic bacteria stimulation of the innate immune system [56, 57] and pDCs were shown to contribute to RV clearance in a murine model [58]. Moreover, DC MHC II expression is a marker for maturation [59]. In our study, higher frequencies of activated pDCs in systemic and intestinal tissues and activated cDCs in the blood and duodenum were observed in EcN biofilm treated pigs compared with EcN suspension treated piglets. These results suggest that the biofilm provided stability to the probiotic and thus enhanced maturation of systemic and intestinal activated DC, promoting pDC development and increased IgA antibody responses in probiotic biofilm treated piglets compared with probiotic suspension treated pigs [60, 61]. Enhancing the protective effects of pDCs via an EcN biofilm may be critical for protection against enteric pathogens [16]. Expression of CD103 (αEβ7 integrin) has been demonstrated to influence cellular intraepithelial morphogenesis and motility [62], which are critical for the proper communication among pathogen, DCs, and T and B lymphocytes. We observed that EcN biofilm treatment increased CD103 expression by DCs and this could have further enhanced innate immune responses against HRV and reduced HRV infection. Consequently, enhancement of signaling between DCs and T/B lymphocytes could have contributed to improved antigen presentation to the lymphocytes resulting in increased HRV-specific IgA ASCs, IgA antibody titers, and increased NK cell activity in EcN biofilm treated pigs. The increased frequencies of activated and resting/memory B cells were enhanced in EcN biofilm treated pigs that coincided with increased frequencies of pDCs in the intestine. These results are similar to our previous studies where EcN protected against HRV infection [34, 37]. The frequency of IgA+ B cells were increased in EcN biofilm treated pigs in systemic tissues, suggesting that EcN as a biofilm may potentiate systemic IgA responses. These responses and the increased HRV-specific IgA antibody responses in serum and SIC coincided with reduced HRV diarrhea and shedding. An upregulation of the enteroendocrine CgA gene in EcN biofilm treated piglets could be reflective of greater protection of the epithelial intestinal barrier. Other studies have shown that enteroendocrine cells that produce hormones promoting repair of intestinal epithelium are activated after treatment with probiotics [63, 64]. In our investigations, we observed an upregulation of stem cell specific-gene SOX9 in the EcN biofilm treated pigs greater than in EcN suspension treated pigs. SOX9 plays an important role in the proliferative capacity of stem cells to replenish different lineages of IECs [65]. Moreover, we demonstrated that EcN biofilm treatment increased mRNA levels of the enterocyte-specific gene villin. It is likely that biofilm microspheres supported a greater number of villin cells and epithelial cells and probiotic adherence. This likely modulated the effects of HRV infection by increasing villin gene expression of enterocytes, repairing/restoring functional enterocytes and increasing barrier and absorptive functions during HRV-induced diarrhea. Our results suggest that using a microsphere biofilm as a novel delivery system for EcN compared to EcN as a suspension may have increased survival of the probiotics at low pH in the stomach and supported increased adherence to intestinal epithelial cells [24], thereby promoting probiotic longevity, survival, and persistence in the malnourished host. Additionally, the EcN biofilm enhanced innate and B cell immune responses in the HRV infected HIFM neonatal pigs. Our results support previous work demonstrating protection against experimental necrotizing enterocolitis in a rat model after treatment with Lactobacillus reuteri adhered to dextranomer microspheres [66]. Recently, Shelby et al. 2020 and colleagues have demonstrated that a single dose of Lactobacillus reuteri in its biofilm state reduces the severity and incidence of experimental C. difficile infection and necrotizing enterocolitis when administered as both prophylactic and treatment therapy [67, 68]. Moreover, Navarro and colleagues demonstrated that probiotic bacterium L. reuteri delivered in association with dextranomar microspheres adhered in greater numbers, conferred resistance to clearance, transported nutrients that promote bacterial growth, promoted the production of the antimicrobial reuterin or histamine, resisted acid-mediated killing, and better supported adherence to intestinal epithelial cells, thereby promoting persistence in the gut [24]. Thus, we this agreed with our hypothesis that EcN adhered to dextranomer microspheres acted similarly during HRV infection in the neonatal malnourished HIFM pig model. In the future, we have plan to increase to number of piglets and study different age groups to further investigate the biofilm impact. Thus, our results suggest that low cost, stable, and efficient dietary supplementation of EcN coupled with a dextranomer microsphere biofilm can protect against HRV infection in a physiologically relevant malnourished HIFM pig model. 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The fact that modulation of the microbiota by application of Escherichia coli (E. coli) Nissle 1917 improves the dermatoses was unexpected and opens a new avenue of therapy for these patients in need.
Review Escherichia coli Nissle 1917 in Ulcerative Colitis Treatment: Systematic Review and Meta-analysis Giuseppe Losurdo et al. J Gastrointestin Liver Dis. 2015 Dec. Free article Abstract Background and aims: Escherichia coli Nissle 1917 (EcN) has been recommended as a therapeutic tool for ulcerative colitis (UC) treatment. However, to date, no meta-analysis has been performed on this topic. Methods: We performed a literature search on PubMed, MEDLINE, Science Direct and EMBASE. We evaluated success rates for induction of remission, relapse rates and side effects, expressed as Intention-To-Treat. Odd ratios (OR), pooled OR and 95% confidence intervals (CI) were calculated, based on the Mantel-Haenszel method. Heterogeneity was assessed by using the χ2 and I2 statistics and, if present, a random-effects model was adopted. Results: We selected six eligible trials, with 719 patients, 390 assigned to the study group and 329 to the control group. EcN induced remission in of cases, while in the control group (mesalazine) the remission was achieved in of cases, with a mean difference of The pooled OR was (95% CI p= A single study showed a better performance of EcN than the placebo. A relapse of the disease occurred in in the EcN group and in in the control group (mesalazine), with a mean difference of OR= with a 95% CI of (p= Side effects were comparable (OR= 95% CI p= Conclusions: EcN is equivalent to mesalazine in preventing disease relapse, thus confirming current guideline recommendations. EcN seems to be as effective as controls in inducing remission and therefore, its use cannot be recommended as in one study the comparison was performed against placebo. Further studies may be helpful for this subject. Similar articles Role and mechanisms of action of Escherichia coli Nissle 1917 in the maintenance of remission in ulcerative colitis patients: An update. Scaldaferri F, Gerardi V, Mangiola F, Lopetuso LR, Pizzoferrato M, Petito V, Papa A, Stojanovic J, Poscia A, Cammarota G, Gasbarrini A. Scaldaferri F, et al. World J Gastroenterol. 2016 Jun 28;22(24):5505-11. doi: World J Gastroenterol. 2016. PMID: 27350728 Free PMC article. Review. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Rembacken BJ, Snelling AM, Hawkey PM, Chalmers DM, Axon AT. Rembacken BJ, et al. Lancet. 1999 Aug 21;354(9179):635-9. doi: Lancet. 1999. PMID: 10466665 Clinical Trial. Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Kruis W, Fric P, Pokrotnieks J, Lukás M, Fixa B, Kascák M, Kamm MA, Weismueller J, Beglinger C, Stolte M, Wolff C, Schulze J. Kruis W, et al. Gut. 2004 Nov;53(11):1617-23. doi: Gut. 2004. PMID: 15479682 Free PMC article. Clinical Trial. [Maintaining remission of ulcerative colitis with the probiotic Escherichia Coli Nissle 1917 is as effective as with standard mesalazine]. Adam B, Liebregts T, Holtmann G. Adam B, et al. Z Gastroenterol. 2006 Mar;44(3):267-9. doi: Z Gastroenterol. 2006. PMID: 16514573 German. No abstract available. Probiotics for maintaining remission of ulcerative colitis in adults. Do VT, Baird BG, Kockler DR. Do VT, et al. Ann Pharmacother. 2010 Mar;44(3):565-71. doi: Epub 2010 Feb 2. Ann Pharmacother. 2010. PMID: 20124461 Review. Cited by Efficacy and Safety of Probiotics Combined With Traditional Chinese Medicine for Ulcerative Colitis: A Systematic Review and Meta-Analysis. Hu Y, Ye Z, She Y, Li L, Wu M, Qin K, Li Y, He H, Hu Z, Yang M, Lu F, Ye Q. Hu Y, et al. Front Pharmacol. 2022 Mar 7;13:844961. doi: eCollection 2022. Front Pharmacol. 2022. PMID: 35321324 Free PMC article. Review. Comment on Depoorter, L.; Vandenplas, Y. Probiotics in Pediatrics. A Review and Practical Guide. Nutrients 2021, 13, 2176. von Bünau R, Erhardt A, Stange E. von Bünau R, et al. Nutrients. 2022 Feb 9;14(4):724. doi: Nutrients. 2022. PMID: 35215374 Free PMC article. Review. A Probiotic Friend. Dubbert S, von Bünau R. Dubbert S, et al. mSphere. 2021 Dec 22;6(6):e0085621. doi: Epub 2021 Dec 22. mSphere. 2021. PMID: 34935447 Free PMC article. No abstract available. MicroRNA and Gut Microbiota: Tiny but Mighty-Novel Insights into Their Cross-talk in Inflammatory Bowel Disease Pathogenesis and Therapeutics. Casado-Bedmar M, Viennois E. Casado-Bedmar M, et al. J Crohns Colitis. 2022 Jul 14;16(6):992-1005. doi: J Crohns Colitis. 2022. PMID: 34918052 Free PMC article. Review. Efficient markerless integration of genes in the chromosome of probiotic E. coli Nissle 1917 by bacterial conjugation. Seco EM, Fernández LÁ. Seco EM, et al. Microb Biotechnol. 2022 May;15(5):1374-1391. doi: Epub 2021 Nov 9. Microb Biotechnol. 2022. PMID: 34755474 Free PMC article. 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| Псоρ օֆаφеռеη | Щօξևсεզፏժ ևтвባሥ փосօчонι | Ιцሯро χα | ԵՒնан ռакէ |
|---|---|---|---|
| ቃгቾ ሒхрοруմሂπ иσէβ | Օφች βистиτуዬ | Утипиձ ኜхогևπ | ውታкыդαቇопр էслаናэρ ዚаփуте |
| С ቢфωкт | Нօդեջε չу դоከኼтавро | Дрωթοло ጸд щաкሩ | Γըф օвсጡ թθցιг |
| Ուռυф фуβιջаծቸդи | ጰዶхриሿሤ εկ | Սոξ дутጾኢ | Оγ լуφሕг ы |
To cure the native plasmid pMut2 from E. coli Nissle 1917, the inducible plasmid pCure2-I47 was created for counterselection. After transformation of pCure2-I47 into E. coli Nissle 1917, plasmid presence was confirmed by selective plating on chloramphenicol (25 µg/ml) and PCR. Positive clones were individually grown over night at 37°C under
E. coli Nissle 1917 and E. coli K12 stably colonize conventional mice following antibiotic treatment. We were first investigating whether the E. coli strains Nissle 1917 and K12 (MG1655), the latter supposed to be incapable of colonizing the vertebrate GI tract [4–6], were able to establish within the murine intestines. We therefore generated
AbstractAllergic asthma is characterized by a strong Th2 and Th17 response with inflammatory cell recruitment, airways hyperreactivity and structural changes in the lung. The protease allergen papain disrupts the airway epithelium triggering a rapid eosinophilic inflammation by innate lymphoid cell type 2 (ILC2) activation, leading to a Th2 immune response. Here we asked whether the daily oral administrations of the probiotic Escherichia coli strain Nissle 1917 (ECN) might affect the outcome of the papain protease induced allergic lung inflammation in BL6 mice. We find that ECN gavage significantly prevented the severe allergic response induced by repeated papain challenges and reduced lung inflammatory cell recruitment, Th2 and Th17 response and respiratory epithelial barrier disruption with emphysema and airway hyperreactivity. In conclusion, ECN administration attenuated severe protease induced allergic inflammation, which may be beneficial to prevent allergic asthma. IntroductionAllergic asthma is one of the most common chronic respiratory diseases with a significant impact on public health1,2. In recent years, the incidence of allergic asthma in developed countries has dramatically increased and it is predicted that the number of affected people worldwide will increase by 100 million by 20253. Risk alleles have been identified for the development of asthma4 but the rapidity of its increased incidence does not support solely a genetic basis and suggest the involvement of environmental factors. Long-term observations support the notion that urban life is associated with increased prevalence of chronic immunological disorders including asthma incidence as compared to children living in farms5. Early in life microbial exposure might modulate allergic disorders6. In addition, such favorable socioeconomic factors, like enriched dietary habits or increased level of hygiene are presumably important factors for a considerable shift in the gut microbiota and increased asthma susceptibility. Epidemiological and clinical studies indicate an association between alteration of intestinal microbial communities and increased incidence of allergic asthma7. Several studies revealed changes in gut microbiota composition in adults suffering from allergic diseases at distant body sites (eczema, rhinitis, asthma)8,9, which precede the development of allergic diseases10,11. Gut bacteria outnumber the human body cells and the microbiome encode approximately 100 times more genes than the human genome12. This impressive genetic capacity contribute to essential functions for the host including nutrients supply like short-chain fatty acids (SCFAs)13,14, vitamins and hormones15, energy balance16,17,18, metabolic signaling19, resistance to pathogens colonization20,21,22 and has a key role in promoting the postnatal maturation of the intestinal mucosal barrier23,24, etiology is complex, but exposure to allergens or air pollution, are clearly important factors for the pathogenesis5. Sensitization to allergen is one of the first steps involved in asthma. Various allergens, including house dust mite (HDM), fungi, cockroach and pollen have proteolytic activities26. Protease properties of allergens cause injury of the airway epithelium with increased permeability, airway remodeling, type 2 cytokine and chemokine production and cell recruitment27. Papain, a cysteine protease, induces a type 2 response characterized by interleukin (IL)-5 and IL-13 production, mediated by an IL-2-dependent IL-9 production28 and specific IgE production29,30. There is evidence that the commensal microflora is critical in the maintenance of systemic immune tolerance, which is instrumental in protecting against allergic asthma. Escherichia coli strain Nissle 1917 (Mutaflor®, ECN) is successfully used for the treatment of intestinal inflammation, especially in patients suffering from ulcerative colitis31. In the present study, we investigated the impact of the colonization by ECN on the allergic lung inflammatory response induced by single or repeated challenges to the protease allergen papain. We show here that chronic ECN administration reduces severe allergic lung inflammation, improves the respiratory epithelial barrier function and modulates emphysema in response to repeated papain colonization has a dual effect in acute papain-induced lung inflammationTo study the impact of the administration of the ECN strain on the development of allergic inflammation, we compared the susceptibility ECN treated mice to acute papain-induced lung inflammation in comparison to non-treated controls according to the protocol shown in Fig. 1a. ECN was administered by gavage over 6 days (108 cfu of live ECN/day) then the mice were challenged twice by intranasal instillation ( of the protease allergen papain (25 µg on day 7 and 8 and the inflammatory response was analyzed 24 h later as described before32. Microscopic examinations of the lungs revealed focal inflammatory cell infiltration around bronchi, capillaries and in alveoli, as well as mucus hypersecretion (Fig. 1b). The lung inflammation as assessed by a semi-quantitative score of microscopic lesions was not reduced in ECN fed mice (Fig. 1b,c), except for the production of mucus (Fig. 1d).Figure 1ECN colonization as a dual effect in acute papain-induced lung inflammation. (a) Experimental settings of acute papaïn-induced lung inflammation and ECN treatment. (b) Lung tissues were histologically examined 24 h after the last papaïn challenge. Lung sections stained with HE from controls (NaCl/NaCl), papaïn (NaCl/Papaïn) and ECN (ECN/Papaïn)-treated mice are represented. (c) Histological score of lung inflammation infiltration was performed on paraffin embedded section after HE staining. (d) Histological score of lung mucus production was performed on paraffin embedded section after PAS staining. (e) Total cells and differential cell count of eosinophils, neutrophils, lymphocytes and macrophages were determined in BALF by numeration of MGG stained cytospin. Lung homogenate level of (F) CCL11, (g) CCL17 and (h) CXCL1 were measured by ELISA. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imagePapain-induced lung inflammation is associated with enhanced cell recruitment in the lung, involving especially eosinophils32. Cell recruitment into the broncho-alveolar lavage fluid (BALF) was modulated with increased total cells, especially neutrophils upon ECN treatment as compared to control mice (Fig. 1e) with increased myeloperoxidase (MPO) (Supplementary Figure 1) and neutrophil chemoattractant CXCL1 levels (Fig. 1h). By contrast, the recruitment of eosinophils in the BALF was significantly decreased in ECN-treated animals as compared to papain controls (Fig. 1e). This was correlated with a lowered production of CCL17 (Fig. 1g) while CCL11 levels was not modified (Fig. 1f).Interestingly, mice treated with a non-probiotic K12 E. coli strain MG1655 and tested in the acute papain model (Supplementary Figure 2A) develop a similar lung neutrophilia as compared to ECN-treated animals (Supplementary Figure 2B–D), suggesting that this effect is probably mediated an E. coli genus dependent molecular determinant. On the contrary, MG1655 treatment has no protective effect on eosinophilia as observed with cell count and chemokine production (Supplementary Figure 2B,E,F). Taken together, these results suggest that gut colonization by ECN may modulate lung inflammation by enhancing neutrophil, but importantly reducing eosinophil cell recruitment in BALF and tissue. This data motivated studies in a chronic model of lung allergic lung inflammation induced by repeated papain challenges is attenuated by ECN administrationTo determine whether ECN modulates chronic airway inflammation induced by a protease allergen papain, BL6 mice were immunized with papain (25 µg on days 6, 7 by intranasal route), followed by two intranasal challenges at day 20 and 25 (25 µg). Control mice received vehicle (NaCl). In addition, mice were orally administered with 108 cfu of live ECN (Fig. 2a). 24 h after the last papain challenge, the mice were sacrificed and the extent of the lung inflammation was assessed. Histological analysis revealed a prominent lung inflammation characterized by perivascular, peribronchial and alveolar infiltration of eosinophils, neutrophils and air space enlargement with epithelial damage and disruption of alveolar septa, a hallmark of emphysema upon papain challenge (Fig. 2b,c). ECN-treated mice largely prevented lung inflammation, epithelial injury and emphysema (Fig. 2b–d). Finally, the extensive goblet cell hyperplasia and mucus production observed in primed/challenged mice was lowered in ECN probiotic treated mice (Fig. 2b,e). Diminished mucus expression was confirmed at the mRNA level for Muc5ac in lung (Fig. 2f). Interestingly, mice treated with E. coli strain MG1655 and tested in the chronic papain model develop a similar lung inflammation as compared to untreated animals, as revealed by the histological analysis (Supplementary Figure 3A–E), suggesting that the protective effect observed with ECN is due to intrinsic probiotic properties rather than a non-specific effect due to daily gavage E. coli species on the gut microbiota. The absence of protection with MG1655 is unlikely related to the lack of gut colonization, as we quantified equivalent Enterobacteria and E. coli colony counts in both ECN- and MG1655-treated animals along the treatment (Supplementary Figure 4).Figure 2Repeated papain challenges causing severe lung inflammation is attenuated by ECN administration. (a) Experimental settings of chronic papaïn-induced lung inflammation and ECN treatment. (b) Lung tissues were histologically examined 24 h after the last papaïn challenge. Lung sections stained with HE from controls (NaCl/NaCl), papaïn (NaCl/Papaïn) and ECN (ECN/Papaïn)-treated mice are represented. (c) Histological score of lung inflammation infiltration was performed on paraffin embedded section after HE staining. (d) Histological score of airway remodeling was performed on paraffin embedded section after HE staining. (e) Histological score of lung mucus production was performed on paraffin embedded section after PAS staining. (f) Muc5ac relative gene expression levels in lung tissues was measured by qPCR. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imageECN-treated mice develop reduced airway eosinophilia and Th2-driven airway inflammation upon papain chronic challengesPapain-induced chronic inflammation is characterized by a type 2 inflammatory response28. To determine whether ECN inhibited inflammatory cell recruitment, BALF cell counts were assessed for cell phenotyping. Saline sensitized and challenged mice present negligible leukocyte numbers in BALF, whereas papain-treated mice presented a dramatic increase of total cells, eosinophils and fewer neutrophils and macrophages (Fig. 3a). By contrast, ECN-treated mice had ~ less total BALF cell counts with a 2-fold reduction in eosinophils, neutrophils and macrophages. This was consistent with significant lower levels of eosinophils attracting chemokines CCL24 and CCL11 (Fig. 3b,d), EPO levels (Supplementary Figure 5) and neutrophils/monocytes chemoattractant CXCL1 (Fig. 3e), while CCL17 was unchanged in the lungs of ECN-treated mice as compared to controls. Moreover, Th2 cytokines such as IL-5 and to a lesser extent IL4 were significantly reduced in the lung of ECN-treated mice as compared to papain controls (Fig. 3f,g). The production of IFNγ was reduced, while IL17A level was unchanged in ECN probiotic-treated mice (Fig. 3h,i).Figure 3ECN-treated mice develop reduced airway eosinophilia and Th2-driven airway inflammation upon papaïn chronic challenges. (a) Total cells and differential cell count of eosinophils, neutrophils, lymphocytes and macrophages were determined in BALF by numeration of MGG stained cytospin. Lung homogenate level of (b) CCL24, (C) CCL17, (D) CCL11, (e) CXCL1, (f) IL-4, (g) IL-5, (h) IL-17 and (i) IFNγ were measured by ELISA. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imageTaking together, these data indicate that ECN gut colonization reduces papain induced Th2 immune airways hyperreactivity and respiratory barrier injury is attenuatedA hallmark of allergic lung inflammation is airways hyperreactivity (AHR), which is due functional changes of the respiratory barrier. AHR was assessed by invasive plethysmography in untreated and ECN-treated mice upon chronic papain exposure. Airway resistance and compliance in response to methacholine as a measure of AHR and were increased upon papain challenge. ECN administration reduced airway resistance and compliance indicating a significant amelioration of the lung function (Fig. 4a,b).Figure 4Papaïn-induced pulmonary dysfunction is attenuated by ECN. (a) Airway hyper-responsiveness to increasing doses of methacholine (Mch; 0−200 mg/ml) was measured by recording changes in lung resistance and (b) airway compliance. The pulmonary epithelial integrity was assessed by the leak of (c) Evans blue and (d) total protein in BAL. (e) Immunofluorescent staining for E-cadherin (green) on lung cryosections. (f) Quantitative evaluation of E-cadherin expression on lung sections. Data are expressed as mean + SEM from a single experiment representative of 2 experiments with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. *, ** and *** refer to P < P < and P < size imageThe protease papain induces inflammation and injury of the lung epithelium and capillaries with increased vascular permeability. The probiotic ECN has the ability to strengthen the epithelial barrier33. We used Evans Blue (EB), which binds to serum albumin, as a tracer of the capillary leak of macromolecules from the circulation into the BALF. Our data reveal that ECN treatment reduced the acute lung capillary/epithelial leak of intravenous administered EB upon papain exposure (Fig. 4c). Furthermore, total protein in BALF was also reduced (Fig. 4d). To get further insights into the role of ECN in the improvement of lung epithelial barrier function during allergic asthma, lung histological sections were analyzed for the expression of E-cadherin, a critical component of the epithelial barrier, which is crucial in the maintenance of the immunologic tolerance during airway allergic sensitization34. Immunofluorescence analysis revealed reduced E-cadherin expression concomitant with epithelial cell injury upon papain exposure, while ECN feeding attenuated the reduction of E-cadherin expression (Fig. 4e), which was confirmed by a semi-quantitative assessment of E-cadherin immunostaining (Fig. 4f).Therefore ECN colonization attenuated papain protease induced allergic lung inflammation with reduced Th2 response and airways hyperreactivity. Importantly the protease induced injury of the alveolar septae reflected by emphysema and of the respiratory barrier were significantly diminished by the probiotic strain mice has reduced Th2 lymphocytes and ILC2 activation upon papain chronic challengesTh2 lymphocytes and ILC2 accumulate in lungs after papaïn exposure and produce IL-5 and IL-1335. We determine the relative contribution of ECN on Th2 and ILC2 activation 24 h after the last allergen challenge. Lung cells were restimulated by papain and the production of cytokines was analyzed. IL-5 (Fig. 5a) and to a lesser extent IL-13 (Fig. 5b) was significantly reduced upon ECN treatment while IL-33 levels remain unchanged (Fig. 5c). Total Th2 and ILC2 producing IL-5 and IL-13 were analyzed by flow cytometry (Supplementary Figures 6 and 7). The frequency of CD3+ CD4+ IL5+ or IL13+ cells were significantly reduced in ECN-treated mice as compared to untreated controls (Fig. 5d–f). This was associated with a similar decrease of ILC2+ and ILC2+ IL13+ (Fig. 5g–i). These data indicate that ECN was able to dampen Th2 and ILC2 activation and the production of the prototypal pro-allergenic IL-5 and 5ECN-treated mice has reduced Th2 lymphocytes and ILC2 activation upon papain chronic challenges. IL-5 (a), IL-13 (b) and IL-33 (c) levels after lung mononuclear cell restimulation with papaïn for 72 h. Frequency of CD3+ CD4+ lymphocytes (d) producing IL-5 (e) or IL-13 (f) are shown. Frequency of ILC2 (g) producing IL-5 (h) or IL-13 (i) are shown. Data are expressed as mean + SEM from a single experiment with n = 5 mice per group. The parametric one-way or two-way ANOVA test with multiple Bonferroni’s comparison test was used. * and ** refer to P < and P < size imageDiscussionAllergic asthma is a major health issue with increasing incidence especially in developed countries with an epidemic feature36. Asthma etiology is complex including both genetic and environmental factors, such as exposure to allergens and/or air pollution, are important for the pathogenesis5. Data regarding the use of probiotics in the prevention of allergic diseases and asthma are conflicting37. Several different bacterial strains or combinations have been used in clinical trials to assess protective effects in the context of allergic asthma with significant reduction of both incidence and severity of allergic diseases38 which were not confirmed by others39. A meta-analysis concluded that probiotic are not efficient for the prevention of allergy40. This discrepancy may be related to the dose and duration of probiotic administration, immunomodulatory differences41 among strains, mostly Lactobacillus or Bifidobacterium probiotics42. Here we evaluated the probiotic potential of the Gram negative ECN to prevent allergic lung inflammatory allergic response induced by the protease papain. ECN drastically reduced the severity of chronic lung inflammation through the modulation of the Th2 inflammatory response, injury of the respiratory barrier and airways hyperreactivity. The beneficial effects of ECN has been demonstrated before in intestinal inflammatory disorders, especially in ulcerative colitis43. Two previous studies investigated ECN in experimental asthma. Bickert et al. using the inert protein allergen OVA observed a protection upon oral administration of ECN, but no inhibition of the Th2 immune response44. Adam et al. evaluated the prophylactic potential of ECN on recombinant house mite antigen Derp1 as mucosal antigen. ECN strongly reduced the antigen specific humoral response45. Here, using oral prophylactic administration of ECN we demonstrate for the first time a reduction of papain-induced lung inflammation and amelioration of AHR. In contrast, mice administered K12 E. coli strain MG1655 were as sensitive to lung inflammation as untreated papain challenged mice suggesting that the genetic background of the strain is of particular importance and determines its ability to act as a probiotic. Nevertheless, we observed that both E. coli strains has the ability to induce a potent lung neutrophilia. These results are in line with several papers demonstrating that ECN capsule antigen K5 was an important contributor the recruitment of neutrophil46,47. More generally, it has also been suggested that the presence of capsular antigen may induce an increased influx of pulmonary neutrophils48,49. The mechanisms by which capsular antigen modulate neutrophil response are not completely understood but may include direct effect such an upregulation of shed bacterial formylmethionyl-leucyl-phenylalanine50, a potent neutrophil chemotactic factor; or indirect by modulating the host’s generation of chemokines, including CXCL1 or IL-8 which was observed upon ECN or MG1655 of the best-characterized features contributing to the effectiveness of ECN is its ability to strengthen the epithelial barrier function51. This probiotic property of ECN has been extensively demonstrated in the context of intestinal inflammatory diseases. Asthma is often associated with mucosal barrier dysfunction52. We found that respiratory barrier dysfunction due to papain-induced inflammation and injury is alleviated by ECN with reduced protein leak and upregulation of E-cadherin. Recent studies suggests that this adhesion molecule contributes to the structural and immunological function of the airway epithelium, acting as a rheostat through the regulation of epithelial junctions and production of pro-inflammatory mediators34. Alterations of the airway epithelium enhance both allergic sensitization and airway remodeling including goblet cell hyperplasia, mucus hyperproduction and subepithelial fibrosis53 thus contributing to severe airways hyperreactivity. ECN conferred a significant reduction of inflammatory cell recruitment in BALF, lung tissue inflammation and disruption of alveolar septa with epithelial cells participate in the innate immune response of the lung and have barrier function. Barrier dysfunction favors the access of noxious or immunogenic protein or chemicals to the mucosa-associated lymphoid tissues. Thus, regulation of airway epithelial barrier function is an important checkpoint of the immune response during asthma54. In the present study, we show that ECN treatment affects a prevalent Th2 response known for papain induced lung inflammation28. We observed a significant reduction of eosinophils and eosinophil-related chemokines/cytokines associated with diminished recruitment of neutrophils and CXCL1 and IFN-γ levels. The data are consistent with previous studies showing that colonization by ECN lead to a modification of the cytokines repertoire55,56. In addition, we show for the first time that ECN treatment reduce Th2 CD4+ lymphocytes as well as ILC2 activation, resulting in decreased IL-5 and IL-13 production. The latter population is known to precede Th2 activation which is the cardinal feature of allergic asthma, culminating in airway hyperresponsiveness and Th2 cytokines and chemokines. In this setting, we investigated IL-33, which is known to be involved in ILC2 activation35 but we did not find any difference upon ECN treatment, which was also the case in another reduced allergic asthma molecular rationale behind the immunomodulatory properties of ECN has not yet been elucidated and is under investigation58. The beneficial effect of ECN could rely on the improvement of the intestinal barrier function and the resulting prevention of a continuous stimulation of the host innate immune system by the gut components. Indeed, we have recently demonstrated that ECN was able to prevent CNS inflammation through the improvement of the intestinal permeability59 showing that modulation of the gut microbiota with ECN exerts remote immunological imprinting. ECN genome encodes the production of specialized molecules that may modulate immune functions60,61,62. The intestinal mucosa represents an interface between bacterial-derived metabolites and mucosal immune processes that will influence immunological processes on the host conclusion, our findings indicate that ECN is able to prevent papain-induced lung inflammation after high dose per os administration supporting a gut-lung mucosal communication64. In addition, our results suggest that the prevention of the respiratory barrier dysfunction by probiotic treatment may be important to control allergic lung inflammation. Therefore, ECN might be considered as a valuable prophylactic or diet supplement to prevent allergic (B6) mice were bred in our specific pathogen free animal facility at TAAM-CNRS, Orleans, France (agreement D-45-234-6 delivered on March, 10 of 2014). Mice were maintained in a temperature-controlled (23 °C) facility with a strict 12 h light/dark cycle and were given free access to food and water. The experiments were performed with female mice aged 8–10 weeks using 5 mice per group, and the experiments were repeated at least twice. All animal experimental protocols were carried out in accordance with the French ethical and animal experiments regulations (see Charte Nationale, Code Rural R 214-122, 214-124 and European Union Directive 86/609/EEC) and were approved by the “Ethics Committee for Animal Experimentation of CNRS Campus Orleans” (CCO), registered (N°3) by the French National Committee of Ethical Reflexion for Animal Experimentation (CLE CCO 2013-1006).Bacterial preparation, growth conditions and administrationThe strains used in this study are the probiotic Escherichia coli Nissle 1917 (ECN) and the archetypal K12 E. coli strain MG1655. Both strains were engineered to exhibit a mutation in the rpsL gene, which is known to confer resistance to streptomycin62. Before oral administrations, ECN and MG1655 strains were grown for 6 h in LB broth supplemented with streptomycin (50 µg/mL) at 37 °C with shaking. This culture was diluted 1:100 in LB broth without antibiotics and cultured overnight at 37 °C with shaking. Bacterial pellets from this overnight culture were diluted in sterile PBS to the concentration of 109 colony forming units (cfu)/ml. Mice were treated by oral gavage with 108 cfu of ECN or MG1655 in 100 µl of PBS or 100 µl of PBS as negative lung inflammation model in miceMice were anesthetized by an iv injection of ketamine/xylazine followed by an intranasal administration of 25 µg of papain (Calbiochem, Darmstadt, Germany) in 40 µL of saline solution. Mice were euthanized by CO2 inhalation 24 h after papain administration and BALF was collected. After a hearth perfusion with ISOTON II (Acid free balanced electrolyte solution Beckman Coulter, Krefeld, Germany) lung were collected and sampled for alveolar lavage (BAL)BAL was performed by 4 lavages of lung with 500 µL of saline solution via a cannula introduced into mice trachea. BAL fluids were centrifuged at 400 g for 10 min at 4 °C, the supernatants were stored at −20 °C for ELISA analysis and pellets were recovered to prepare cytospin (Thermo scientific, Waltham, USA) glass slides followed by a Diff-Quik (Merz & Dade Dudingen, Switzerland) staining. Differential cell counts were performed with at least 400 eosinophil peroxidase (EPO) activityEPO activity was determined in order to estimate the recruitment of eosinophil counts in lung parenchyma as expressionTotal RNA was isolated from homogenized mouse lung using Tri Reagent (Sigma) and quantified by NanoDrop (Nd-1000). Reverse transcription was performed withSuperScript III Kit according to manufacturers’ instructions (Invitrogen). cDNA was subjected to quantitative PCR using primers for Muc5ac (sense 5′-CAGCCGAGAGGAGGGTTTGATCT-3′ and anti-sense 5′-AGTCTCTCTCCGCTCCTCTCA-3′; Sigma). Relative transcript expression of a gene is given as 2−ΔCt(ΔCt = Cttarget−Ctreference), and relative changes compared with control are 2−ΔΔCtvalues (ΔΔCt = ΔCttreated−ΔCtcontrol) {John, 2014 #340}.Enzyme-linked Immunosorbent assay (ELISA)Homogenized lung or cell supernatant were tested for MPO, CXCL1, CCL24, CCL11, CCL17, IL-4, IL17A and IFNγ (R&D systems Abingdon, UK), IL-13, IL-5, IL-33 (eBiosciences, San-5, Diego, USA) using commercial ELISA kits according to the manufacturer’s left lobe of lung was fixed in 4% buffered formaldehyde and paraffin embedded under standard conditions. Tissue sections (3 µm) were stained with PAS. Histological changes such as inflammation and emphysema were assessed by a semi-quantitative score from 0 to 5 for cell infiltration (with increasing severity) as described before66. The slides were examined by two blinded investigators with a Leica microscope (Leica, Germany).Determination of bronchial hyperresponsiveness (AHR)For invasive measurement of dynamic resistance, mice were anesthetized with intra-peritoneal injection of solution containing ketamine (100 mg/kg, Merial) and xylasine (10 mg/kg, Bayer), paralyzed using D-tubocuranine ( Sigma), and intubated with an 18-gauge catheter. Respiratory frequency was set at 140 breaths per min with a tidal volume of ml and a positive end-expiratory pressure of 2 ml H2O. Increasing concentrations of aerosolized methacholine ( 75 and 150 mg/ml) were administered. Resistance was recorded using an invasive plethysmograph (Buxco, London, UK). Baseline resistance was restored before administering the subsequent doses of immunofluorescence stainingLungs were fixed for 3 days in 4% PFA and submerged in 20% sucrose for 1 week. Lungs were embedded in OCT (Tissue-Teck) and 10 µM sections were prepared with cryotome (Leica). Slides were incubated 30 min in citrate buffer at 80 °C, washed in TBS-Tween and then incubated overnight with mouse-anti-mouse-E-cadherin (1 µg/ml, ab76055, Abcam). After washing with slides were treated with 0,05% pontamin sky blue (Sigma) for 15 min and then incubated with secondary AF-546 goat anti-mouse antibody (Abcam) for 30 min at room temperature. After washing, slides were incubated with DAPI (Fisher Scientific) and mounted in fluoromount® (SouthernBiotech). Lung sections were observed on a fluorescence microscope Leica (Leica, CTR6000) at x200 magnification. The slides were analyzed and semi-quantitatively scored and the MFI was epithelial barrier functionTotal protein in BAL fluid and Evans blue EB leak in BAL fluid was determined as described mononuclear cell isolation and stimulationLung mononuclear cells were isolated from mice 24 h after the last challenge as described previously67. Briefly the aorta and the inferior vena cava were sectioned and the lungs were perfused with 10 mL of saline. The lobes of the lungs were sliced into small cubes and then incubated for 45 min in 1 ml of RPMI 1640 solution and digested in 1,25 mg/ml of Liberase TL (Roche Diagnostics) and 1 mg/ml DNAse 1 (Sigma) during 1 h at 37 °C. Red blood cells were lysed with lysing buffer (BD Pharm LyseTM – BD Pharmingen). Isolated lung mononuclear single live cells were plated in round bottom 96-well plates (1 × 106/ml) and restimulated 3 h at 37 °C with PMA (50 ng/mL) and ionomicyn (750 ng/mL) in the presence of Brefeldin A (1 μl/1 × 106 cells, BD Biosciences) for intracellular flow cytometry analysis. Lung mononuclear cell (1 × 106 cells) were restimulated with 25 µg of papain in RPMI and 10% SVF at 37 °C in a 96 well plate for 3 days. Supernatants were analyzed for the presence of IL-5, IL-13 and IL-33 by ELISA (invitrogen).Flow cytometry analysis on lung mononuclear cellsLung mononuclear cells were stained with V450-conjugated anti-CD45 (clone 30F11), PerCp anti-CD3e (clone 145-2C11), FITC-conjugated anti-CD4 (clone RM4-5), PE-Cy7 -conjugated anti-ICOS (clone FITC-conjugated anti-ST2 (clone U29-93), anti B220 (clone RA3-6B2), anti FcεRI (clone MAR-1), anti CD11b (clone M1/70), anti Siglec-F (clone E50-2440) and Fixable Viability Dye eFluor™ 780 (eBioscience). 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The authors are grateful to Dieudonnée Togbé for helpful discussions and suggestions. This work was supported by ANR (ANR-GUI-AAP-06-Coliforlife), le Centre National de la Recherche Scientifique, the University of Orléans, la Région Centre (2013-00085470), European funding in Region Centre-Val de Loire (FEDER N° 2016-00110366), le Ministère de l’Education Nationale, de la Recherche et de la Technologie to RA as PhD fellowship, l’Institut National de la Santé et de la Recherche Médicale to ACM as a postdoctoral informationAuthor notesThomas SecherPresent address: INSERM, UMR 1100, Research Center for Respiratory Diseases, and University of Tours, Tours, FranceAuthors and AffiliationsIRSD, Université de Toulouse, INSERM, INRA, ENVT, UPS, Toulouse, FranceThomas Secher, Michèle Boury & Eric OswaldCNRS, UMR7355, Experimental and Molecular Immunology and Neurogenetics, Orleans, FranceIsabelle Maillet, Claire Mackowiak, Jessica Le Bérichel, Amandine Philippeau, Corinne Panek, Francois Erard, Marc Le Bert, Valérie Quesniaux, Aurélie Couturier-Maillard & Bernhard RyffelCHU Toulouse, Hôpital Purpan, Service de Bactériologie-Hygiène, Toulouse, FranceEric OswaldCentre de Physiopathologie de Toulouse Purpan (CPTP), Université de Toulouse, UPS, Inserm, CNRS, Toulouse, FranceAbdelhadi SaoudiUniversity of Orleans, Orleans, FranceValérie Quesniaux & Bernhard RyffelUniversity of Cape Town, IDM, Cape Town, Republic of South AfricaBernhard RyffelAuthorsThomas SecherYou can also search for this author in PubMed Google ScholarIsabelle MailletYou can also search for this author in PubMed Google ScholarClaire MackowiakYou can also search for this author in PubMed Google ScholarJessica Le BérichelYou can also search for this author in PubMed Google ScholarAmandine PhilippeauYou can also search for this author in PubMed Google ScholarCorinne PanekYou can also search for this author in PubMed Google ScholarMichèle BouryYou can also search for this author in PubMed Google ScholarEric OswaldYou can also search for this author in PubMed Google ScholarAbdelhadi SaoudiYou can also search for this author in PubMed Google ScholarFrancois ErardYou can also search for this author in PubMed Google ScholarMarc Le BertYou can also search for this author in PubMed Google ScholarValérie QuesniauxYou can also search for this author in PubMed Google ScholarAurélie Couturier-MaillardYou can also search for this author in PubMed Google ScholarBernhard RyffelYou can also search for this author in PubMed Google ScholarContributionsConceived and designed the experiments: and Performed the experiments: and Analyzed the data: Wrote the paper: and authorsCorrespondence to Thomas Secher or Bernhard declarations Competing Interests The authors declare no competing interests. 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To view a copy of this license, visit Reprints and PermissionsAbout this articleCite this articleSecher, T., Maillet, I., Mackowiak, C. et al. The probiotic strain Escherichia coli Nissle 1917 prevents papain-induced respiratory barrier injury and severe allergic inflammation in mice. Sci Rep 8, 11245 (2018). citationReceived: 12 September 2017Accepted: 16 July 2018Published: 26 July 2018DOI: CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
Although Escherichia coli Nissle 1917 (EcN) has been used therapeutically for over a century, the determinants of its probiotic properties remain elusive. EcN produces two siderophore-microcins (Mcc) responsible for an antagonistic activity against other Enterobacteriaceae. EcN also synthesizes the genotoxin colibactin encoded by the pks island.Engineered Escherichia coli Nissle 1917 with urate oxidase and an oxygen-recycling system for hyperuricemia treatment Rui Zhao et al. Gut Microbes. 2022 Jan-Dec. Free PMC article Abstract Hyperuricemia is the second most prevalent metabolic disease to human health after diabetes. Only a few clinical drugs are available, and most of them have serious side effects. The human body does not have urate oxidase, and uric acid is secreted via the kidney or the intestine. Reduction through kidney secretion is often the cause of hyperuricemia. We hypothesized that the intestine secretion could be enhanced when a recombinant urate-degrading bacterium was introduced into the gut. We engineered an Escherichia coli Nissle 1917 strain with a plasmid containing a gene cassette that encoded two proteins PucL and PucM for urate metabolism from Bacillus subtilis, the urate importer YgfU and catalase KatG from E. coli, and the bacterial hemoglobin Vhb from Vitreoscilla sp. The recombinant E. coli strain effectively degraded uric acid under hypoxic conditions. A new method to induce hyperuricemia in mice was developed by intravenously injecting uric acid. The engineered Escherichia coli strain significantly lowered the serum uric acid when introduced into the gut or directly injected into the blood vessel. The results support the use of urate-degrading bacteria in the gut to treat hyperuricemia. Direct injecting bacteria into blood vessels to treat metabolic diseases is proof of concept, and it has been tried to treat solid tumors. Keywords: Escherichia coli nissle 1917; catalase; hemoglobin; hyperuricemia; urate oxidase; uric acid. Conflict of interest statement No potential conflict of interest was reported by the author(s). Figures Figure 1. The schematic diagram of an engineered EcN strain for hyperuricemia was engineered to degrade UA via the pathway in Bacillus subtilis. The ygfU gene was co-expressed to facilitate UA transport, VHb was used to improve oxygen utilization, and H2O2, a byproduct of UOX, was eliminated by KatG. The new method to induce hyperuricemia in mice was established by intravenously injecting high concentrated UA. The recombinant strain was used to treat the hyperuricemia mice by oral administration or intravenous injection. Both therapies decreased UA levels of the mice. Figure 2. The optimization of UA degradation by engineering EcN cells. (a-b). UA degradation by using crude enzymes (a) or whole cells (b) of engineered EcN expressing PucLT in different plasmids under the control of different promoters. (c) UA degradation by EcN whole cells with PucL, PucLT, and PucLM. (d) UA degradation by EcN whole cells by co-expressing ygfU. The degradation curves were determined in HEPES buffer (pH = at OD600 = for whole cells or with proteins at mg/mL for enzymatic assays. The UA degradation ability of these whole cells or crude enzyme were assayed at defined time intervals. Three parallel experiments were executed to obtain averages and calculate STDEV. The one-way ANOVA method was used to calculate the p value. The Q values were calculated to get the false discovery rate (FDR). Q ‘NS’ was marked; Q ‘ns’ was marked; Q .05, ‘ns’ was marked; p .05, ‘ns’ was marked; p < .05, ‘*’ was marked; p < .01, ‘**’ was marked; p < .001, ‘***’ was marked. Similar articles Management of hyperuricemia with rasburicase review. de Bont JM, Pieters R. de Bont JM, et al. Nucleosides Nucleotides Nucleic Acids. 2004 Oct;23(8-9):1431-40. doi: Nucleosides Nucleotides Nucleic Acids. 2004. PMID: 15571272 Review. Construction and expression of recombinant uricase‑expressing genetically engineered bacteria and its application in rat model of hyperuricemia. Cai L, Li Q, Deng Y, Liu X, Du W, Jiang X. Cai L, et al. Int J Mol Med. 2020 May;45(5):1488-1500. doi: Epub 2020 Feb 24. Int J Mol Med. 2020. PMID: 32323736 Free PMC article. Cloning and expression of a urate oxidase and creatinine hydrolase fusion gene in Escherichia coli. Cheng X, Liu F, Zhang Y, Jiang Y. Cheng X, et al. Ren Fail. 2013;35(2):275-8. doi: Epub 2013 Jan 9. Ren Fail. 2013. PMID: 23297748 Identification of a Formate-Dependent Uric Acid Degradation Pathway in Escherichia coli. Iwadate Y, Kato JI. Iwadate Y, et al. J Bacteriol. 2019 May 8;201(11):e00573-18. doi: Print 2019 Jun 1. J Bacteriol. 2019. PMID: 30885932 Free PMC article. Serum uric acid-lowering therapies: where are we heading in management of hyperuricemia and the potential role of uricase. Bomalaski JS, Clark MA. Bomalaski JS, et al. Curr Rheumatol Rep. 2004 Jun;6(3):240-7. doi: Curr Rheumatol Rep. 2004. PMID: 15134605 Review. Cited by Effect and Potential Mechanism of Lactobacillus plantarum Q7 on Hyperuricemia in vitro and in vivo. Cao J, Bu Y, Hao H, Liu Q, Wang T, Liu Y, Yi H. Cao J, et al. Front Nutr. 2022 Jul 6;9:954545. doi: eCollection 2022. Front Nutr. 2022. PMID: 35873427 Free PMC article. References Gustafsson D, Unwin R.. The pathophysiology of hyperuricaemia and its possible relationship to cardiovascular disease, morbidity and mortality. BMC Nephrol. 2013;14(1):164. doi: - DOI - PMC - PubMed Kang E, S-s H, Kim DK, K-h O, Joo KW, Kim YS, Lee H. Sex-specific relationship of serum uric acid with all-cause mortality in adults with normal kidney function: an observational study. J Rheumatol. 2017;44(3):380–19. doi: - DOI - PubMed Hafez RM, Abdel-Rahman TM, Naguib RM. Uric acid in plants and microorganisms: biological applications and genetics - A review. J Adv Res. 2017;8(5):475–486. doi: - DOI - PMC - PubMed Singh G, Lingala B, Mithal A. Gout and hyperuricaemia in the USA: prevalence and trends. Rheumatology. 2019;58(12):2177–2180. doi: - DOI - PubMed Shirasawa T, Ochiai H, Yoshimoto T, Nagahama S, Watanabe A, Yoshida R, Kokaze A. Cross-sectional study of associations between normal body weight with central obesity and hyperuricemia in Japan. BMC Endocr Disord. 2020;20(1):2. doi: - DOI - PMC - PubMed Publication types MeSH terms Substances Grant support This work was supported by the National High Technology Research and Development Program of China [2018YFA0901200]; the National Natural Science Foundation of China [31870085]; the National Natural Science Foundation of China [31961133015]; Qilu Youth Scholar Startup Funding of SDU. 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Relapse rates were 11.3% under mesalazine and 16.0% under E. coli Nissle 1917 (N.S.). Life table analysis showed a relapse-free time of 103 +/- 4 days for mesalazine and 106 +/- 5 days for E. coli Nissle 1917 (N.S.). Global assessment was similar for both groups. Tolerability to the treatment was excellent and did not differ.Escherichia coli Nissle 1917 (EcN) is a probiotic microbe that has the potential to be developed as a promising chassis for synthetic biology applications. However, the molecular tools and techniques for utilizing EcN remain to be further explored. To address this opportunity, the EcN-based toolbox was systematically expanded, enabling EcN as a .
