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.
ABSTRACT: Escherichia 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 (EcN?kfiB), or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcN?kfiC), were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcN?kfiC and the EcN wild-type, EcN?kfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcN?kfiC 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.
Project description:Rapidly growing antibiotic resistance among gastrointestinal pathogens, and the ability of antibiotics to induce the virulence of these pathogens makes it increasingly difficult to rely on antibiotics to treat gastrointestinal infections. The probiotic Escherichia coli strain Nissle 1917 (EcN) is the active component of the pharmaceutical preparation Mutaflor® and has been successfully used in the treatment of gastrointestinal disorders. Gut bacteriophages are dominant players in maintaining the microbial homeostasis in the gut, however, their interaction with incoming probiotic bacteria remains to be at conception. The presence of bacteriophages in the gut makes it inevitable for any probiotic bacteria to be phage resistant, in order to survive and successfully colonize the gut. This study addresses the phage resistance of EcN, specifically against lytic T4 phage infection. From various experiments we could show that (i) EcN is resistant toward T4 phage infection, (ii) EcN's K5 polysaccharide capsule plays a crucial role in T4 phage resistance and (iii) EcN's lipopolysaccharide (LPS) inactivates T4 phages and notably, treatment with the antibiotic polymyxin B which neutralizes the LPS destroyed the phage inactivation ability of isolated LPS from EcN. Combination of these identified properties in EcN was not found in other tested commensal E. coli strains. Our results further indicated that N-acetylglucosamine at the distal end of O6 antigen in EcN's LPS could be the interacting partner with T4 phages. From our findings, we have reported for the first time, the role of EcN's K5 capsule and LPS in its defense against T4 phages. In addition, by inactivating the T4 phages, EcN also protects E. coli K-12 strains from phage infection in tri-culture experiments. Our research highlights phage resistance as an additional safety feature of EcN, a clinically successful probiotic E. coli strain.
Project description:Rapidly growing antibiotic resistance among gastrointestinal pathogens, and the ability of antibiotics to induce the virulence of these pathogens makes it increasingly difficult to rely on antibiotics to treat gastrointestinal infections. The probiotic E. coli strain Nissle 1917 (EcN) is the active component of the pharmaceutical preparation Mutaflor® and has been successfully used in the treatment of gastrointestinal disorders. Gut bacteriophages are dominant players in maintaining the microbial homeostasis in the gut, however, their interaction with incoming probiotic bacteria remains to be at conception. The presence of bacteriophages in the gut makes it inevitable for any probiotic bacteria to be phage resistant, in order to survive and successfully colonize the gut. This study addresses the phage resistance of EcN, specifically against lytic T4 phage infection. From various experiments we could show that i) EcN is resistant towards T4 phage infection, ii) EcN’s K5 polysaccharide capsule plays a crucial role in T4 phage resistance and iii) EcN’s lipopolysaccharide (LPS) inactivates T4 phages and notably, treatment with the antibiotic polymyxin B which neutralizes the LPS destroyed the phage inactivation ability of isolated LPS from EcN. Our results further indicate that N-acetylglucosamine at the distal end of O6 antigen in EcN’s LPS could be the interacting partner with T4 phages. From our findings, we have reported for the first time, the role of EcN’s K5 capsule and LPS in its defense against T4 phages. In addition, by inactivating the T4 phages, EcN also protects E. coli K-12 strains from phage infection in tri-culture experiments. The combination of the identified properties is not found in other tested commensal E. coli strains. Furthermore, our research highlights phage resistance as an additional safety feature of EcN, a clinically successful probiotic E. coli strain. Overall design: EcN culture (1e8 CFUs/ml) and 100 µl of a phage extract (~1e8 pfus/ml) were added to final volume of 1 ml LB medium in a 24 well plate. The plates were incubated in a static manner for 2 h of time at 37 °C.
Project description:Two Klebsiella bacteriophages K5-2 and K5-4, which are able to infect and grow on either capsular types K30/K69 and K5 or K8 and K5 of Klebsiella strains, were isolated and characterized. Each phage contained two open reading frames (ORFs), which encoded two putative capsule depolymerases, respectively. The first ORF encoded tail fiber proteins, which have K30/K69 depolymerase and K8 depolymerase activities. The second ORF encoded hypothetical proteins, which are almost identical in amino acid sequences, and have K5 depolymerase activity. Alcian blue staining of enzyme-treated capsular polysaccharides (CPS) showed that purified depolymerases can cleave purified Klebsiella CPS in vitro and liberate monosaccharaides. Capsule K5 deletion mutants were not lysed by either phage, suggesting that the capsule was essential for phage infection. Bacterial killing was observed when incubated Klebsiella strains with phages but not with purified depolymerases. Treatment with the K5-4 phage significantly increased the survival of mice infected with a K. pneumoniae K5 strain. In conclusion, two dual host-specific Klebsiella phages and their tailspikes exhibit capsule depolymerase activity were characterized. Each phage and phage-encoded depolymerase has specificity for capsular type K30/K69, K8 or K5, and could be used for the typing and treatment of K. pneumoniae infection.
Project description:Membrane vesicles (MVs) produced by Gram-negative bacteria are being explored for novel clinical applications due to their ability to deliver active molecules to distant host cells, where they can exert immunomodulatory properties. MVs released by the probiotic Escherichia coli Nissle 1917 (EcN) are good candidates for testing such applications. However, a drawback for such studies is the low level of MV isolation from in vitro culture supernatants, which may be overcome by the use of mutants in cell envelope proteins that yield a hypervesiculation phenotype. Here, we confirm that a tolR mutation in EcN increases MV production, as determined by protein, LPS and fluorescent lipid measurements. Transmission electron microscopy (TEM) of negatively stained MVs did not reveal significant differences with wild type EcN MVs. Conversely, TEM observation after high-pressure freezing followed by freeze substitution of bacterial samples, together with cryo-TEM observation of plunge-frozen hydrated isolated MVs showed considerable structural heterogeneity in the EcN tolR samples. In addition to common one-bilayer vesicles (OMVs) and the recently described double-bilayer vesicles (O-IMVs), other types of MVs were observed. Time-course experiments of MV uptake in Caco-2 cells using rhodamine- and DiO-labelled MVs evidenced that EcN tolR MVs displayed reduced internalization levels compared to the wild-type MVs. The low number of intracellular MVs was due to a lower cell binding capacity of the tolR-derived MVs, rather than a different entry pathway or mechanism. These findings indicate that heterogeneity of MVs from tolR mutants may have a major impact on vesicle functionality, and point to the need for conducting a detailed structural analysis when MVs from hypervesiculating mutants are to be used for biotechnological applications.
Project description:Respiratory syncytial virus (RSV) exploits cell surface heparan sulfate proteoglycans (HSPGs) as attachment receptors. The interaction between RSV and HSPGs thus presents an attractive target for the development of novel inhibitors of RSV infection. In this study, selective chemical modification of the Escherichia coli K5 capsular polysaccharide was used to generate a collection of sulfated K5 derivatives with a backbone structure that mimics the heparin/heparan sulfate biosynthetic precursor. The screening of a series of N-sulfated (K5-NS), O-sulfated (K5-OS), and N,O-sulfated (K5-N,OS) derivatives with different degrees of sulfation revealed the highly sulfated K5 derivatives K5-N,OS(H) and K5-OS(H) to be inhibitors of RSV. Their 50% inhibitory concentrations were between 1.07 nM and 3.81 nM in two different cell lines, and no evidence of cytotoxicity was observed. Inhibition of RSV infection was maintained in binding and attachment assays but not in preattachment assays. Moreover, antiviral activity was also evident when the K5 derivatives were added postinfection, both in cell-to-cell spread and viral yield reduction assays. Finally, both K5-N,OS(H) and K5-OS(H) prevented RSV infection in human-derived tracheal/bronchial epithelial cells cultured to form a pseudostratified, highly differentiated model of the epithelial tissue of the human respiratory tract. Together, these features put K5-N,OS(H) and K5-OS(H) forward as attractive candidates for further development as RSV inhibitors.
Project description:Lactobacillus paracasei K5 is a lactic acid bacteria (LAB) strain that has been isolated from dairy products. Previous studies have established its probiotic potential in a series of in vitro tests, including molecular characterization, safety profiling, and tolerability of the gastrointestinal tract conditions. To characterize its beneficial actions on the host, we have shown previously that L. paracasei K5 adheres to Caco-2 cells and exerts anti-proliferative effects through the induction of apoptosis. In the present study, we focused on the immunomodulatory potential of this strain. We employed the dorsal-air-pouch mouse model of inflammation and recorded an eight-fold increase in the recruitment of immune cells in mice treated with the probiotic strain, compared to the control group. Analysis of the exudates revealed significant changes in the expression of pro-inflammatory mediators on site. Treatment of Caco-2 cells with L. paracasei K5 induced significant upregulation of cytokines interleukin-1? (IL-1?), ?L-1?, IL-6, tumor necrosis factor-alpha (TNF-?), the chemokine C-X-C motif ligand 2 (CXCL2), and the inflammation markers soluble intercellular adhesion molecule (sICAM) and metallopeptidase inhibitor-1 (TIMP-1). Transient induction of the Toll-like receptors (TLRs) 2, 4, 6, and 9 expression levels was recorded by real-time PCR analysis. These results highlight the immunomodulatory potential of this strain and further support its probiotic character.
Project description:Colonization of the human small intestine by Vibrio cholerae is an essential step in pathogenesis that requires the type IV toxin-coregulated pilus (TCP). To date, three functions of TCP have been characterized: it serves as the CTX? receptor, secretes the colonization factor TcpF, and functions in microcolony formation by mediating bacterium-bacterium interactions. Although type IV pili in other pathogenic bacteria have been characterized as playing a major role in attachment to epithelial cells, there are very few studies to suggest that TCP acts as an attachment factor. Taking this into consideration, we investigated the function of TCP in attachment to Caco-2 cells and found that mutants lacking TCP were defective in attachment compared to the wild type. Overexpression of ToxT, the activator of TCP, significantly increased attachment of wild-type V. cholerae to Caco-2 cells. Using field-emission scanning electron microscopy (FESEM), we also observed TCP-mediated attachment to the small intestines of infected infant mice by using antibodies specific to TCP and V. cholerae. Remarkably, we also visualized matrices comprised of TCP appearing to engulf V. cholerae during infection, and we demonstrated that these matrices protected the bacteria from a component of bile, disclosing a possible new role of this pilus in protection of the bacterial cells from antimicrobial agents. This study provides new insights into TCP's function in V. cholerae colonization of the small intestine, describing additional roles in mediating attachment and protection of V. cholerae bacterial cells.
Project description:K5 lyase A (KflA) is a tailspike protein from the K5A phage that catalyzes the degradation of the capsule polysaccharide of K5 strains of Escherichia coli. The K5 E. coli capsule polysaccharide, also known as heparosan, is composed of the disaccharide repeating unit of [-4)-GlcA-?(1,4)-GlcNAc-?(1-] and therefore identical to the biological precursor of heparin and heparan sulfate (HS). KflA could supplement the heparin lyases for heparin and HS analysis. The first part of this study aimed to clarify ambiguity resulting from the revision of the KflA amino acid sequence in 2010 from that published in 2000. We found that only the expression of the updated sequence gave a soluble active enzyme, which produced heparosan degradation products similar to those of previous studies. Next, we examined the specificity of KflA toward heparosan oligosaccharides of varying sizes, all containing a single N-sulfated glucosamine (GlcNS) residue. The presence of GlcNS in an octasaccharide and a nonasaccharide chain directed cleavage by KflA to a single position at the reducing end of the substrate. However, an N-sulfated decasaccharide exhibited extensive cleavage at the nonreducing end of the chain, illustrating a distinct change in the cleavage pattern of KflA toward substrates of differing sizes. Because KflA is able to cleave a substrate containing isolated GlcNS residues, this enzyme could be used for the analysis of low-sulfate content HS domains.
Project description:The Escherichia coli K5 capsular polysaccharide [-4)-betaGlcA-(1, 4)-alphaGlcNAc-(1-] is a receptor for the capsule-specific bacteriophage K5A. Associated with the structure of bacteriophage K5A is a polysaccharide lyase which degrades the K5 capsule to expose the underlying bacterial cell surface. The bacteriophage K5A lyase gene (kflA) was cloned and sequenced. The kflA gene encodes a polypeptide with a predicted molecular mass of 66.9 kDa and which exhibits amino acid homology with ElmA, a K5 polysaccharide lyase encoded on the chromosome of E. coli SEBR 3282. There was only limited nucleotide homology between the kflA and elmA genes, suggesting that these two genes are distinct and either have been derived from separate progenitors or have diverged from a common progenitor for a considerable length of time. Southern blot analysis revealed that kflA was not present on the chromosome of the E. coli strains examined. In contrast, elmA was present in a subset of E. coli strains. Homology was observed between DNA flanking the kflA gene of bacteriophage K5A and DNA flanking a small open reading frame (ORF(L)) located 5' of the endosialidase gene of the E. coli K1 capsule-specific bacteriophage K1E. The DNA homology between these noncoding sequences indicated that bacteriophages K5A and K1E were related. The deduced polypeptide sequence of ORF(L) in bacteriophage K1E exhibited homology to the N terminus of KflA from bacteriophage K5A, suggesting that ORF(L) is a truncated remnant of KflA. The presence of this truncated kflA gene implies that bacteriophage K1E has evolved from bacteriophage K5A by acquisition of the endosialidase gene and subsequent loss of functional kflA. A (His)(6)-KflA fusion protein was overexpressed in E. coli and purified to homogeneity with a yield of 4.8 mg per liter of bacterial culture. The recombinant enzyme was active over a broad pH range and NaCl concentration and was capable of degrading K5 polysaccharide into a low-molecular-weight product.
Project description:The short-chain fatty acid butyrate plays critical roles in human gut health, affecting immunomodulation, cell differentiation, and apoptosis, while also serving as the preferred carbon source for colon cells. In this work, we have engineered a model probiotic organism, Escherichia coli Nissle 1917 (EcN, serotype O6:K5:H1), to produce butyrate from genomic loci up to approximately 1 g/L (11 mM). Then, for real-time monitoring of butyrate production in cultures, we developed a high-throughput biosensor that responds to intracellular butyrate concentrations, with green fluorescent protein as the reporter. This work provides a foundation for studies of butyrate for therapeutic applications.