Project description:Gut commensal bacterial strains were cultured with or without added sugars (glucose, sucrose and kestose). The base culture medium without sugar added were modified based on the Yeast Casitone Fatty Acids (YCFA) broth. Samples were analyzed for proteomics using LC-MS/MS.
Project description:Escherichia coli is an important human pathogen, among others a cause of severe diarrhea diseases and urinary tract infections. The ability to distinguish different pathogenic E. coli subspecies is crucial for correct treatment of the infection. Characterization and quantification of clinical isolates proteomes can provide details of the organisms’ metabolism and specific virulence factors. We performed a systematic quantitative proteomic analysis on a representative selection of 16 pathogenic and 2 commensal E. coli strains, together with 5 pathogenic Shigella strains. The analysis yielded a dataset of more than 4 thousand proteins, with an average of 2 thousand proteins per strain and 980 proteins common to all strains. Statistical comparison of label-free quantitative levels of 750 proteins, which were quantified in all strains, revealed that levels of a majority of the shared proteins vary substantially among specific strains. Theses quantitative protein profiles clearly distinguished E. coli strains from Shigella and largely separated commensal E. coli strains from intestinal and extraintestinal E. coli isolates.
Project description:Pathogenic biofilms have been associated with persistent infections due to high resistance to antimicrobial agents while commensal biofilms often fortify host immune system. Hence, controlling biofilm formation of both pathogenic bacteria and commensal bacteria is important in bacteria-related diseases. We investigated the effect of plant flavonoids on biofilm formation of both enterohemorrhagic Escherichia coli O157:H7 and three commensal E. coli K-12 strains. Phloretin abundant in apples markedly reduced E. coli O157:H7 biofilm formation without affecting the growth of planktonic cells while phloretin did not harm commensal E. coli K-12 biofilms. Also, phloretin reduced E. coli O157:H7 attachment to human colon epithelial cells. Global transcriptome analyses revealed that phloretin repressed toxin genes (hlyE and stx2), autoinducer-2 importer genes (lsrACDBF), a curli gene (csgA), and a dozens of prophage genes in E. coli O157:H7 cells. Electron microscopy confirmed that phroretin reduced the curli production in E. coli O157:H7. In addition, phloretin suppressed TNF-α-induced inflammatory response in vitro using human colonic epithelial cells. Moreover, in the trinitrobenzene sulfonic acid (TNBS)-induced rat colitis model, phloretin significantly ameliorated colon inflammation and body weight loss. Taken together, our results suggest that phloretin may act as an inhibitor of E. coli O157:H7 biofilm formation as well as anti-inflammatory agent on inflammatory bowel diseases while leaving beneficial commensal E. coli biofilm intact.
Project description:The goal was to determine the chemotherapy-induced, mammalian cell death-dependent transcriptional response in a human commensal strain of E. coli. Murine intestinal epithelial cells were used to induce chemotherapy-driven cell death, and a human commensal strain of E. coli was used as the 'recipient' bacteria.
Project description:Honey has been widely used against bacterial infection for centuries. Previous studies suggested that honeys in high concentrations inhibited bacterial growth due to the presence of anti-microbial compounds, such as methylglyoxal, hydrogen peroxide, and peptides. In this study, we found that three honeys (acacia, clover, and polyfloral) in a low concentration as below as 0.5% (v/v) significantly suppress virulence and biofilm formation in enterohemorrhagic E. coli O157:H7 affecting the growth of planktonic cells while these honeys do not harm commensal E. coli K-12 biofilm formation. Transcriptome analyses show that honeys (0.5%) markedly repress quorum sensing genes (e.g., AI-2 import and indole biosynthesis), virulence genes (e.g., LEE genes), and curli genes (csgBAC). We found that glucose and fructose in honeys are key compounds to reduce the biofilm formation of E. coli O157:H7 via suppressing curli production, but not that of E. coli K-12. Additionally, we observed the temperature-dependent response of honeys and glucose on commensal E. coli K-12 biofilm formation; honey and glucose increase E. coli K-12 biofilm formation at 37°C, while they decrease E. coli K-12 biofilm formation at 26°C. These results suggest that honey can be a practical tool for reducing virulence and colonization of the pathogenic E. coli O157:H7, while honeys do not harm commensal E. coli community in the human.
Project description:Pathogenic biofilms have been associated with persistent infections due to high resistance to antimicrobial agents while commensal biofilms often fortify host immune system. Hence, controlling biofilm formation of both pathogenic bacteria and commensal bacteria is important in bacteria-related diseases. We investigated the effect of plant flavonoids on biofilm formation of both enterohemorrhagic Escherichia coli O157:H7 and three commensal E. coli K-12 strains. Phloretin abundant in apples markedly reduced E. coli O157:H7 biofilm formation without affecting the growth of planktonic cells while phloretin did not harm commensal E. coli K-12 biofilms. Also, phloretin reduced E. coli O157:H7 attachment to human colon epithelial cells. Global transcriptome analyses revealed that phloretin repressed toxin genes (hlyE and stx2), autoinducer-2 importer genes (lsrACDBF), a curli gene (csgA), and a dozens of prophage genes in E. coli O157:H7 cells. Electron microscopy confirmed that phroretin reduced the curli production in E. coli O157:H7. In addition, phloretin suppressed TNF-α-induced inflammatory response in vitro using human colonic epithelial cells. Moreover, in the trinitrobenzene sulfonic acid (TNBS)-induced rat colitis model, phloretin significantly ameliorated colon inflammation and body weight loss. Taken together, our results suggest that phloretin may act as an inhibitor of E. coli O157:H7 biofilm formation as well as anti-inflammatory agent on inflammatory bowel diseases while leaving beneficial commensal E. coli biofilm intact. For the microarray experiments, E. coli O157:H7 EDL933 was inoculated in 25 ml of LB in 250 ml flasks with overnight cultures that were diluted at 1:100. Cells were shaken at 100 rpm and 37°C for 7 hrs. Cells were immediately chilled with dry ice and 95% ethanol (to prevent RNA degradation) for 30 sec before centrifugation in 50 ml centrifuge tubes at 13,000 g for 2 min; cell pellets were frozen immediately with dry ice and stored -80°C. RNA was isolated using Qiagen RNeasy mini Kit (Valencia, CA, USA). To eliminate DNA contamination, Qiagen RNase-free DNase I was used to digest DNA. RNA quality was assessed by Agilent 2100 bioanalyser using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and quantity was determined by ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., DE, USA).
Project description:Escherichia coli spans a genetic continuum from enteric strains to several phylogenetically distinct, atypical lineages that are rare in humans, but more common in extra-intestinal environments. To investigate the link between gene regulation, phylogeny and diversification in this species, we analyzed global gene expression profiles of four strains representing distinct evolutionary lineages, including a well-studied laboratory strain, a typical commensal (enteric) strain and two environmental strains. RNA-Seq was employed to compare the whole transcriptomes of strains grown under batch, chemostat and starvation conditions. Highly differentially expressed genes showed a significantly lower nucleotide sequence identity compared with other genes, indicating that gene regulation and coding sequence conservation are directly connected. Overall, distances between the strains based on gene expression profiles were largely dependent on the culture condition and did not reflect phylogenetic relatedness. Expression differences of commonly shared genes (all four strains) and E. coli core genes were consistently smaller between strains characterized by more similar primary habitats. For instance, environmental strains exhibited increased expression of stress defense genes under carbon-limited growth and entered a more pronounced survival-like phenotype during starvation compared with other strains, which stayed more alert for substrate scavenging and catabolism during no-growth conditions. Since those environmental strains show similar genetic distance to each other and to the other two strains, these findings cannot be simply attributed to genetic relatedness but suggest physiological adaptations. Our study provides new insights into ecologically relevant gene-expression and underscores the role of (differential) gene regulation for the diversification of the model bacterial species. Four E.coli strains, laboratory strain K12 (MG1655), a commensal model strain (IAI1), a soil-isolated strain (TW11588-Clade IV), and a freshwater-isolated strain (TW09308âClade V) were used. Each strain was grown on a minimal growth medium (Ihssen and Egli, 2004) in three treatment modes: chemostat, batch, and starvation. Cells from batch culture were collected when reaching steady-state. For starvation, the medium flow was stopped during steady-state and bacteria were collected after 4âh.
Project description:Strain specific growth of C. jejuni on fucose has been linked to a plasticity region of the chromosome (PR2) and confers a competitive advantage during intestinal colonization. Growth on fucose induces gene expression of PR2 genes, but the regulatory mechanism of the structural genes involved with fucose utilization is unknown. Additionally, the mechanism of fucose dissimilation by C. jejuni is not known since no fucose catabolism homologs are found in the C. jejuni genome. Transcriptional profiles of C. jejuni grown with and without fucose may provide insight in to the genes that are necessary for fucose utilization. The design utilized an available two color microarray slide for the entire transcriptome of Campylobacter jejuni wild type strain NCTC 11168. Each sample represents one competitive hybridization: sham-treated NCTC 11168 v.s. 25mM fucose treated NCTC 11168. There were four biological replicates of each sample with a dye swap introduced in alternating replicates. Samples were independently grown, treated and harvested.
Project description:Metabolomics data from E. Coli Nissle (wild type, WT) and 3 E. Coli Nissle knockout (KO) strains grown in M-9 media and enriched M-9 media.
Project description:Transcriptome profiling of E. coli MG1655, E. coli EDL933, E. coli ECOR26, and E. coli Nissle 1917 grown on mouse cecal mucus as carbon source The carbon sources that support growth of pathogenic E. coli O157:H7 in the mammalian intestine have not previously been investigated. In vivo, pathogenic E. coli EDL933 primarily grows as dispersed single cells within the mucus layer that overlies the mouse cecal epithelium. We therefore compared the pathogen and commensal E. coli MG1655 for their mode of metabolism in vitro on a mixture of the sugars known to be present in cecal mucus and found that the two strains used the thirteen sugars in a similar order and co-metabolized as many as nine sugars at a time. We conducted a systematic mutational analysis of E. coli EDL933 and E. coli MG1655 with lesions in the pathways used for catabolism of thirteen mucus-derived sugars and five other compounds for which the corresponding gene system was induced in the transcriptome of cells grown on cecal mucus. Each of 18 catabolic mutants in both genetic backgrounds was fed to streptomycin-treated mice together with the respective wildtype parent strain and their colonization was monitored in fecal plate counts. None of the mutations corresponding to the five compounds not found in mucosal polysaccharides resulted in colonization defects. Based on the mutations that caused colonization defects, we determined that both E. coli EDL933 and E. coli MG1655 used arabinose, fucose, and N-acetylglucosamine in the intestine. In addition, E. coli EDL933 used galactose, hexuronates, mannose and ribose, whereas E. coli MG1655 used gluconate and N-acetylneuraminic acid. The colonization defects of six catabolic lesions were found to be additive in E. coli EDL933, but not E. coli MG1655. The data indicate that pathogenic E. coli EDL933 uses sugars that are not used by commensal E. coli MG1655 to colonize the mouse intestine. The results suggest a strategy whereby invading pathogens gain advantage by simultaneously consuming several sugars that may be available because they are not consumed by the commensal intestinal microbiota. Keywords: growth condition: carbon source was mouse cecal mucus