Project description:Unlike many other types of diarrheagenic bacteria that act primarily in the small intestine, O157:H7 expresses virulence primarily in the large intestine. In this study, microarray analysis is employed to examine the transcriptional response of O157:H7 to bile treatment, to gain insight into how bile affects virulence and whether bile might be temporally defending the small intestine against virulence by these bacteria. Keywords: Expression profiling of two different growth conditions Two groups of three replicates were used: E.coli O157:H7 grown in Luria broth with or without 0.8% bile salts
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:Unlike many other types of diarrheagenic bacteria that act primarily in the small intestine, O157:H7 expresses virulence primarily in the large intestine. In this study, microarray analysis is employed to examine the transcriptional response of O157:H7 to bile treatment, to gain insight into how bile affects virulence and whether bile might be temporally defending the small intestine against virulence by these bacteria. Keywords: Expression profiling of two different growth conditions
Project description:There is increasing evidence to support a role for sigma factor 54 (RpoN) in the regulation of stress resistance factors and protein secretion systems important to bacterial transmission and pathogenesis. In enterohemorrhagic E. coli O157:H7, acid resistance and type III secretion are essential determinants of gastric passage and colonization. This study thus described the transcriptome of an rpoN null strain of E. coli O157:H7 (EcJR-8) to determine the influence of RpoN on virulence and stress resistance gene regulation, and further explored its contribution to glutamate-dependent acid resistance (GDAR). Inactivation of rpoN resulted in the growth phase-dependent, differential expression of 104 genes. This included type III secretion structural and regulatory genes encoded on the locus of enterocyte effacement (LEE), as well as GDAR genes gadA, gadBC and gadE. Upregulation of gad transcript levels in EcJR-8 during logarithmic growth correlated with increased GDAR and survival in a model stomach. Acid susceptibility was reconstituted in EcJR-8 complemented in trans with wild-type rpoN. Acid resistance in EcJR-8 was dependent on exogenous glutamate, gadE and rpoS, but was independent of hns. Results also suggest that GDAR may be controlled by RpoN at multiple regulatory levels. This study supports the hypothesis that RpoN is an important regulator of virulence and stress resistance factors in E. coli O157:H7, and is the first to examine the mechanism by which it represses GDAR.
Project description:The effect of pooled immunoglobulins (IgG) on E. coli O157:H7 colonization and the course of disease in an EHEC mouse model was investigated showing an improved survival and decreased intestinal and renal pathology. Treatment was given after inoculation thereby corresponding to the clinical setting. In vitro studies identified E. coli serine protease EspP as the E. coli O157:H7 protein that IgG bound to, via the Fc fragment, in both murine and human IgG preparations, and blocked its enzymatic activity. EspP is a virulence factor previously shown to promote colonic cell injury and the uptake of Shiga toxin by intestinal cells. The results suggest that IgG in commercial preparations binds to EspP protecting the host from E. coli O157:H7 infection and could potentially be beneficial in patients.
Project description:Pathogenic biofilms have been associated with persistent infections due to their high resistance to antimicrobial agents. To identify non-toxic biofilm inhibitors for enterohemorrhagic Escherichia coli O157:H7, indole-3-acetaldehyde was used and reduced E. coli O157:H7 biofilm formation. Global transcriptome analyses revealed that indole-3-acetaldehyde most repressed two curli operons, csgBAC and csgDEFG, and induced tryptophanase (tnaAB) in E. coli O157:H7 biofilm cells. Electron microscopy showed that indole-3-acetaldehyde reduced curli production in E. coli O157:H7. Together, this study shows that Actinomycetales are an important resource of biofilm inhibitors as well as antibiotics.
Project description:Enterohemorrhagic Escherichia coli (EHEC), including serotype O157:H7, cause severe food-borne illness. On route to the human colon, they encounter and resist, numerous anti-microbial ingestion stresses. We hypothesize that these stresses cue EHEC to alter virulence properties. This study investigated the impact of bile salts on virulence properties and examined the genetic basis of the phenotypes. Established assays were used to examine adhesion to human epithelial cells, motility, verotoxin (VT) production and antimicrobial resistance with/without bile salt stress. Bacteria treated for 90 minute in DMEM plus 0.15% (w/v) bile salt mix demonstrated significantly enhanced adhesion to epithelial cells and resistance to several antibiotics but did not increase motility or VT production. To determine the genetic basis of these phenotypes a microarray experiment was conducted. EHEC strain 86-24, in mid-log phase of growth, were grown in DMEM pH 7.4 (control), or DMEM plus bile salt mix (0.15% w/v), for 90 minutes, statically at 37˚C, 5% CO2 prior to harvesting RNA for the microarray study. Four biological replicates were produced for each treatment. Microarray and gene expression analysis (semi-quantitative RT-PCR and beta-galactosidase reporter assays) of bile salt-treated EHEC revealed significant up-regulation of genes for lipid A modification, fimbriae, an efflux pump, and a two-component regulatory system relative to the bacteria grown in DMEM alone. This work points to several mechanisms that EHEC employs to resist the stresses of the human small intestine, notably efflux, antimicrobial resistance, and outer membrane alterations. Bile salts enhanced the virulence-related properties of increased adhesion and resistance to antimicrobials but not VT production or motility. This research contributes to our understanding of how EHEC senses and responds to host environmental signals and the mechanisms this pathogen uses to successfully colonize and infect the human host.
Project description:There is increasing evidence to support a role for sigma factor 54 (RpoN) in the regulation of stress resistance factors and protein secretion systems important to bacterial transmission and pathogenesis. In enterohemorrhagic E. coli O157:H7, acid resistance and type III secretion are essential determinants of gastric passage and colonization. This study thus described the transcriptome of an rpoN null strain of E. coli O157:H7 (EcJR-8) to determine the influence of RpoN on virulence and stress resistance gene regulation, and further explored its contribution to glutamate-dependent acid resistance (GDAR). Inactivation of rpoN resulted in the growth phase-dependent, differential expression of 104 genes. This included type III secretion structural and regulatory genes encoded on the locus of enterocyte effacement (LEE), as well as GDAR genes gadA, gadBC and gadE. Upregulation of gad transcript levels in EcJR-8 during logarithmic growth correlated with increased GDAR and survival in a model stomach. Acid susceptibility was reconstituted in EcJR-8 complemented in trans with wild-type rpoN. Acid resistance in EcJR-8 was dependent on exogenous glutamate, gadE and rpoS, but was independent of hns. Results also suggest that GDAR may be controlled by RpoN at multiple regulatory levels. This study supports the hypothesis that RpoN is an important regulator of virulence and stress resistance factors in E. coli O157:H7, and is the first to examine the mechanism by which it represses GDAR. Hybridizations measured transcriptional differences between an rpoN null and wild-type (WT) strain of E. coli O157:H7 Sakai at logarithmic and transition phase. Image files (TIFF) of hybridized microarray slides were generated using an Axon 4000B scanner (Molecular Devices), and analyzed using GenePix Pro software (Molecular Devices, ver. 6.0). The resulting microarray intensity data was log2-transformed, and normalized using the LOWESS algorithm in MAANOVA ver. 0.98-8 (R ver. 2.2.1).
Project description:The human intestinal microbiota associated with rats produces in vivo a soluble(s) factor(s) that down-regulates the expression of genes encoding for the Shiga toxin II in E. coli O157:H7. The Shiga toxin II is one of the major virulence factors of E. coli enterohemorragic leading to the deadly hemolitic and uremic syndrome. Investigation of the effect of the human intestinal microbiota on the whole transcriptome of EHEC O157:H7 is of major importance to increase our understanding of the pathogen transcriptomic adaptation in response to the human microbiota. We analysed by microarray hybridization the gene expression pattern of EHEC O157:H7 grown in the caecal content of germ-free rats or rats associated with the human microbiota of a healthy human subject. By doing so, we increased our understanding of the regulatory activities of the human gut microbiota on E. coli O157:H7
Project description:Secreted proteins constitute a major part of virulence factors that are responsible for pathogenesis caused by gram negative bacteria. Enterohemorrhagic Escherichia coli (E. coli), EHEC O157:H7 is the major pathogen often causing outbreaks. There is growing evidence that non-O157:H7 E. coli strains may also be involved in the recent outbreaks. However, there is no systematic study describing differential secreted proteins from non-O157:H7 E. coli strains. Here, we have applied isobaric tag-based TMT labeling combined with high-resolution Fourier transform mass spectrometry to study the differential secretome analysis of major non-O157:H7 E. coli strains, O103, O111, O121, O145, O26 and O45, which is known as diarrhea inducing non-O175:H7 ATCC “big six” serogroup E. coli strains. We identified 1,240 proteins quantitatively identified, 565 proteins were found to be secreted as predicted by PSORTb and SecretomeP. We identified 310 proteins containing signal peptide and 255 proteins as secreted. We identified 20 strain specific proteins with in big-six group and was confirmed by proteogenomics approach. Further we enriched and have shown relative expression of type III secretion system. To our knowledge, this study is the first comparative proteomic study on secretome of E. coli big six serogroup and the several of these strain specific secreted proteins can be further studied to develop potential markers for identification and strain level differentiation. Moreover, the results of this study can be utilized in several applications, including food safety, diagnostics of E. coli outbreaks, and biodefense.