We recently reported the use of the probiotic E. coli strain Nissle 1917 (EcN) to sufficiently produce heme containing proteins, as it encodes the outer membrane heme receptor, ChuA, which allows for natural uptake of heme. 大肠杆菌Nissle 1917(EcN)(血清型为 06：K5：H1)是一株被德国医生Alfred Nissle在一 战期间发现并分离得到的无致病性的革兰氏阴性 益生菌 。 EcN的益生功能与其对肠道微生物的 调控和肠道内细胞因子引起免疫反应存在关 联。 The aim of this study was to improve our understanding of the E. coli Nissle 1917-host interaction by analyzing the gene expression pattern initiated by this probiotic in human intestinal epithelial cells. Methods: Gene expression profiles of Caco-2 cells treated with E. coli Nissle 1917 were analyzed with microarrays. A second human intestinal Effects of Escherichia coli Nissle 1917 on the Porcine Gut Microbiota, Intestinal Epithelium and Immune System in Early Life. The pre-weaning period offers a ‘window of opportunity’ to modulate the porcine gut microbiota and immune system through dietary interventions such as EcN supplementation, and EcN induced T cell proliferation and Bacterial cancer therapy was developed using probiotic Escherichia coli Nissle 1917 (EcN) for medical intervention of colorectal cancer. EcN was armed with HlyE, a small cytotoxic protein, under Consumption of fructose leads to metabolic syndrome, but it is also known to increase iron absorption. Present study investigates the effect of genetically modified Escherichia coli Nissle 1917 (EcN) synbiotic along with fructose on non-heme iron absorption. Charles foster rats weighing 150-200 g were fed with iron-deficient diet for 2 months. . 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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Ultrastructure of Proteus mirabilis swarmer cell rafts and role of swarming in catheter-associated urinary tract infection. Infect Immun. 2004; 72: 3941–3950. View Article Google Scholar Review Escherichiacoli Nissle 1917 as a Novel Microrobot for Tumor-Targeted Imaging and Therapy Qingyao Liu et al. Pharmaceutics. 2021. Free PMC article Abstract Highly efficient drug delivery systems with excellent tumor selectivity and minimal toxicity to normal tissues remain challenging for tumor treatment. Although great effort has been made to prolong the blood circulation and improve the delivery efficiency to tumor sites, nanomedicines are rarely approved for clinical application. Bacteria have the inherent properties of homing to solid tumors, presenting themselves as promising drug delivery systems. Escherichia coli Nissle 1917 (EcN) is a commonly used probiotic in clinical practice. Its facultative anaerobic property drives it to selectively colonize in the hypoxic area of the tumor for survival and reproduction. EcN can be engineered as a bacteria-based microrobot for molecular imaging, drug delivery, and gene delivery. This review summarizes the progress in EcN-mediated tumor imaging and therapy and discusses the prospects and challenges for its clinical application. EcN provides a new idea as a delivery vehicle and will be a powerful weapon against cancer. Keywords: E. coli Nissle 1917; bacteria-mediated tumor imaging; bacteria-mediated tumor therapy; microrobot; tumor colonization. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Figures Figure 1 EcN-mediated tumor imaging and therapy. (A) Schematic illustration of the ability of preferential tumor colonization in hypoxic regions. EcN can be designed to load drugs or integrate nanoparticles and express exogenous genes; (B) Schematic diagram of the strategies of various imaging modalities and treatment patterns for EcN, EcN minicell, and EcN ghost. Figure 2 (A) [18F]-FDS PET imaging in CT26-bearing mice treated with E. coli. (A) PET imaging was performed at day 0, 1, 3, 5 after intravenous injection of E. coli. The radioactivity uptake of the tumor was significantly higher at day 1, 3, 5 than in pre-treatment. (B) Positive correlation between SUVmax and the number of viable bacteria. (C) Schematic illustration of the synthesis of [18F]-FDS from [18F]-FDG. Reproduced with permission from Jung-Joon Min, Theranostics; published by Ivyspring International Publisher, 2020. Figure 3 (A) Schematic illustration of the synthesis of MTdox@EcN; (B) Schematic illustration of the mechanism of MTDOX@EcN as a biorocket for drug delivery in tumor; (C) Typical SEM and (D) CLSM images of MTDOX@EcN. (E) Tumor inhibition and (F) survival rates of MTdox@EcN treatment in tumor-bearing mice. Reproduced with permission from Xiaohong Li, Chemical Engineering Journal; published by Elsevier, 2020. Figure 4 (A) Schematic illustration of the engineering EcN strain named SYNB1891; (B) Tumor inhibition and (C) survival rates of SYNB1891 treatment in B16F10 tumor-bearing mice. Reproduced with permission from Jose M. Lora, Nature Communications; published by Springer Nature, 2020. ** p = (blue stars), **** p < (pink stars), ** p = (pink stars), ** p = (black stars), *** p = (black stars). Figure 5 The future application of nano-bacteria hybrid system. Similar articles Expressing cytotoxic compounds in Escherichia coli Nissle 1917 for tumor-targeting therapy. Li R, Helbig L, Fu J, Bian X, Herrmann J, Baumann M, Stewart AF, Müller R, Li A, Zips D, Zhang Y. Li R, et al. Res Microbiol. 2019 Mar;170(2):74-79. doi: Epub 2018 Nov 14. Res Microbiol. 2019. PMID: 30447257 Intestinal probiotics E. coli Nissle 1917 as a targeted vehicle for delivery of p53 and Tum-5 to solid tumors for cancer therapy. He L, Yang H, Tang J, Liu Z, Chen Y, Lu B, He H, Tang S, Sun Y, Liu F, Ding X, Zhang Y, Hu S, Xia L. He L, et al. J Biol Eng. 2019 Jun 28;13:58. doi: eCollection 2019. J Biol Eng. 2019. PMID: 31297149 Free PMC article. High density fermentation of probiotic E. coli Nissle 1917 towards heparosan production, characterization, and modification. Datta P, Fu L, Brodfuerer P, Dordick JS, Linhardt RJ. Datta P, et al. Appl Microbiol Biotechnol. 2021 Feb;105(3):1051-1062. doi: Epub 2021 Jan 22. Appl Microbiol Biotechnol. 2021. PMID: 33481068 Genetic engineering of probiotic Escherichia coli Nissle 1917 for clinical application. Ou B, Yang Y, Tham WL, Chen L, Guo J, Zhu G. Ou B, et al. Appl Microbiol Biotechnol. 2016 Oct;100(20):8693-9. doi: Epub 2016 Sep 17. Appl Microbiol Biotechnol. 2016. PMID: 27640192 Review. [Escherichia coli Nissle 1917 as safe vehicles for intestinal immune targeted therapy--a review]. Xia P, Zhu J, Zhu G. Xia P, et al. Wei Sheng Wu Xue Bao. 2013 Jun 4;53(6):538-44. Wei Sheng Wu Xue Bao. 2013. PMID: 24028055 Review. Chinese. Cited by Encoding with a fluorescence-activating and absorption-shifting tag generates living bacterial probes for mammalian microbiota imaging. Cao Z, Wang L, Liu R, Lin S, Wu F, Liu J. Cao Z, et al. Mater Today Bio. 2022 Jun 6;15:100311. doi: eCollection 2022 Jun. Mater Today Bio. 2022. PMID: 35711290 Free PMC article. Native and Engineered Probiotics: Promising Agents against Related Systemic and Intestinal Diseases. Shen H, Zhao Z, Zhao Z, Chen Y, Zhang L. Shen H, et al. Int J Mol Sci. 2022 Jan 6;23(2):594. doi: Int J Mol Sci. 2022. PMID: 35054790 Free PMC article. Review. References Wilhelm S., Tavares Dai Q., Ohta S., Audet J., Dvorak Chan Analysis of nanoparticle delivery to tumours. Nat. Rev. Mater. 2016;1:16014. doi: - DOI Heldin Rubin K., Pietras K., Östman A. High interstitial fluid pressure—an obstacle in cancer therapy. Nat. Rev. Cancer. 2004;4:806–813. doi: - DOI - PubMed Khawar Kim Kuh Improving drug delivery to solid tumors: Priming the tumor microenvironment. J. Control. Release. 2015;201:78–89. doi: - DOI - PubMed Malmgren Flanigan Localization of the vegetative form of Clostridium tetani in mouse tumors following intravenous spore administration. Cancer Res. 1955;15:473–478. - PubMed Starnes Coley’s toxins in perspective. Nature. 1992;357:11–12. doi: - DOI - PubMed Publication types LinkOut - more resources Full Text Sources Europe PubMed Central Multidisciplinary Digital Publishing Institute (MDPI) PubMed Central 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. LinkOut - more resources Full Text Sources Europe PubMed Central PubMed Central Taylor & Francis Medical MedlinePlus Health Information Escherichia coli (E. coli) to pospolita bakteria występująca w mikroflorze jelita grubego u ludzi i zwierząt stałocieplnych. W większości to nieszkodliwe bakterie, niektóre jednak powodować mogą poważne zatrucia pokarmowe, zapalenia żołądka, czy jelit. Jest jednak jeden wyjątkowy szczep, który stosuje się do zapobiegania i leczenia wszelkich dolegliwości trawiennych – Escherichia coli Nissle 1917. Bakterie te zostały odkryte ponad 100 lat temu, przez fryburskiego higienistę, prof. dr Alfreda Nissle, który założył we Freiburgu w 1938 r. prywatny instytut badań bakteriologicznych, którym kierował aż do śmierci w 1965 r. Podczas I wojny światowej, w 1917 roku, w pewnej grupie żołnierzy, w szpitalu wojskowym nieopodal Freiburga, wybuchła czerwonka. Tylko jeden żołnierz pozostał zdrowy, nie wykazując żadnych objawów choroby jelit. Widząc to, prof. Nissle przebadał jego kał pod kątem zawartości bakterii jelitowych i wyizolował szczep E. coli, który następnie użył do leczenia pozostałych żołnierzy. Od tego czasu, szczep ten zaczęto nazwać E. coli Nissle 1917, i stosować go w leczeniu różnych zaburzeń żołądkowo-jelitowych. Na Uniwersytecie we Freiburgu, studenci prof. Nissle, podczas zajęć praktycznych z mikrobiologii, mieszali własne próbki kału z czystymi hodowlami patogennych szczepów Salmonelli. Zazwyczaj obserwowali szybki rozrost Salmonelli, wypierających tym samym, inne bakterie jelitowe. Były jednak i takie przypadki, w których rozrost był nieznaczny, a nawet wcale niezauważalny. W ten sposób powstała hipoteza, że mikroflora niektórych próbek kału zawiera takie szczepy, które hamują rozwój mikroorganizmów patogennych. Później podejrzenia te zostały potwierdzone w laboratorium, w trakcie badań hodowli mieszanin szczepów Salmonella z różnymi izolatami E. coli, uzyskanymi z próbek kału zdrowych ludzi. Okazało się, że patogenne szczepy E. coli posiadają dodatkowe geny, tzw. „geny zjadliwości”, które czynią je chorobotwórczymi. Escherichia coli Nissle 1917 natomiast, wyróżnia się na tle innych bakterii ze swojej rodziny, tym, że na drodze ewolucji, poprzez poziomy transfer genów z innych bakterii jelitowych, nabyła dodatkowe elementy genetyczne, nazywane „Wyspami Genomowymi”. To one są odpowiedzialne m. in. za zdolność hamowania rozwoju różnego rodzaju enteropatogenów. Tę szczególną właściwość, prof. Nissle nazwał „aktywnością antagonistyczną”. Niepatogenny szczep bakterii Escherichia coli wykazuje wiele korzystnych właściwości, pełni istotne funkcje w ludzkim organizmie. Odpowiedzialny jest za rozkład produktów spożywczych, bierze udział w produkcji witamin z grupy B i K, poprawia wchłanianie żelaza. Jest bakterią tlenową, więc po przez zużycie tlenu obecnego w jelitach przyczynia się do wytworzenia pozytywnego środowiska dla anaerobów – bakterii beztlenowych. Wspomaga proces zasiedlania innych bakterii probiotycznych jednocześnie usuwając patogeny z mikroflory jelit. Szczep E-coli Nissle 1917 posiada właściwości probiotyczne oraz adhezyjne – przyczepia się do ścian jelitowych uszczelniając je i wpływając aprobująco na wchłanianie organizmu. Szczep Escherichia coli Nissle 1917 sprzyja tworzeniu substancji przeciwzapalnych i autogennych antybiotyków oraz wpływa pozytywnie na system immunologiczny. Niepatogenna E-coli sprawdza się w leczeniu wrzodziejącego zapalenia jelita grubego, zespołu jelita drażliwego, w walce z alergiami pokarmowymi, a także wykazuje korzystne działanie w profilaktyce raka jelita grubego. Niedobór tej bakterii w organizmie przynieść może przykre skutki w postaci częstego występowania nawracających infekcji moczowo-płciowych, czy oddechowych, a to wszystko za sprawą obniżonej odporności śluzówek. Niestety, wraz z pojawieniem się antybiotyków, zgasło zainteresowanie mikroflorą jelitową i terapeutycznym zastosowaniem żywych bakterii. Dopiero niedawno, medyczne osiągnięcia i rozwój mikrobiologii, spowodowały, że wcześniejsze doświadczenia mogły zostać dokładnie potwierdzone, a leczenie probiotykami znalazło się na powrót w centrum zainteresowania lekarzy i naukowców. Obecnie jest to prawdopodobnie najintensywniej badany szczep bakteryjny. The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens Artur Altenhoefer et al. FEMS Immunol Med Microbiol. 2004. Free article Abstract The probiotic Escherichia coli strain Nissle 1917 (Mutaflor) of serotype O6:K5:H1 was reported to protect gnotobiotic piglets from infection with Salmonella enterica serovar Typhimurium. An important virulence property of Salmonella is invasion of host epithelial cells. Therefore, we tested for interference of E. coli strain Nissle 1917 with Salmonella invasion of INT407 cells. Simultaneous administration of E. coli strain Nissle 1917 and Salmonella resulted in up to 70% reduction of Salmonella invasion efficiency. Furthermore, invasion of Yersinia enterocolitica, Shigella flexneri, Legionella pneumophila and even of Listeria monocytogenes were inhibited by the probiotic E. coli strain Nissle 1917 without affecting the viability of the invasive bacteria. The observed inhibition of invasion was not due to the production of microcins by the Nissle 1917 strain because its isogenic microcin-negative mutant SK22D was as effective as the parent strain. Reduced invasion rates were also achieved if strain Nissle 1917 was separated from the invasive bacteria as well as from the INT407 monolayer by a membrane non-permeable for bacteria. We conclude E. coli Nissle 1917 to interfere with bacterial invasion of INT407 cells via a secreted component and not relying on direct physical contact with either the invasive bacteria or the epithelial cells. Similar articles Detection and distribution of probiotic Escherichia coli Nissle 1917 clones in swine herds in Germany. Kleta S, Steinrück H, Breves G, Duncker S, Laturnus C, Wieler LH, Schierack P. Kleta S, et al. J Appl Microbiol. 2006 Dec;101(6):1357-66. doi: J Appl Microbiol. 2006. PMID: 17105567 E. coli Nissle 1917 Affects Salmonella adhesion to porcine intestinal epithelial cells. Schierack P, Kleta S, Tedin K, Babila JT, Oswald S, Oelschlaeger TA, Hiemann R, Paetzold S, Wieler LH. Schierack P, et al. PLoS One. 2011 Feb 17;6(2):e14712. doi: PLoS One. 2011. PMID: 21379575 Free PMC article. Nonpathogenic Escherichia coli strain Nissle 1917 inhibits signal transduction in intestinal epithelial cells. Kamada N, Maeda K, Inoue N, Hisamatsu T, Okamoto S, Hong KS, Yamada T, Watanabe N, Tsuchimoto K, Ogata H, Hibi T. Kamada N, et al. Infect Immun. 2008 Jan;76(1):214-20. doi: Epub 2007 Oct 29. Infect Immun. 2008. PMID: 17967864 Free PMC article. Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli Nissle 1917 in live mice. Stritzker J, Weibel S, Hill PJ, Oelschlaeger TA, Goebel W, Szalay AA. Stritzker J, et al. Int J Med Microbiol. 2007 Jun;297(3):151-62. doi: Epub 2007 Apr 19. Int J Med Microbiol. 2007. PMID: 17448724 Effect of probiotic strains on interleukin 8 production by HT29/19A cells. Lammers KM, Helwig U, Swennen E, Rizzello F, Venturi A, Caramelli E, Kamm MA, Brigidi P, Gionchetti P, Campieri M. Lammers KM, et al. Am J Gastroenterol. 2002 May;97(5):1182-6. doi: Am J Gastroenterol. 2002. PMID: 12014725 Cited by The potential utility of fecal (or intestinal) microbiota transplantation in controlling infectious diseases. Ghani R, Mullish BH, Roberts LA, Davies FJ, Marchesi JR. Ghani R, et al. Gut Microbes. 2022 Jan-Dec;14(1):2038856. doi: Gut Microbes. 2022. PMID: 35230889 Free PMC article. Review. The microbial ecology of Escherichia coli in the vertebrate gut. Foster-Nyarko E, Pallen MJ. Foster-Nyarko E, et al. FEMS Microbiol Rev. 2022 May 6;46(3):fuac008. doi: FEMS Microbiol Rev. 2022. PMID: 35134909 Free PMC article. Review. Quantifying cumulative phenotypic and genomic evidence for procedural generation of metabolic network reconstructions. Moutinho TJ Jr, Neubert BC, Jenior ML, Papin JA. Moutinho TJ Jr, et al. PLoS Comput Biol. 2022 Feb 7;18(2):e1009341. doi: eCollection 2022 Feb. PLoS Comput Biol. 2022. PMID: 35130271 Free PMC article. 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. Escherichia coli Nissle 1917 secondary metabolism: aryl polyene biosynthesis and phosphopantetheinyl transferase crosstalk. Jones CV, Jarboe BG, Majer HM, Ma AT, Beld J. Jones CV, et al. Appl Microbiol Biotechnol. 2021 Oct;105(20):7785-7799. doi: Epub 2021 Sep 21. Appl Microbiol Biotechnol. 2021. 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escherichia coli nissle 1917