bis(3--5-)-cyclic-diguanylic-acid and Escherichia-coli-Infections

Topics: Bacterial Proteins; Biofilms; Biological Variation, Population; Cyclic GMP; Escherichia coli; Escherichia coli Infections; Extracellular Matrix; Fluorescent Antibody Technique; Gene Expression Regulation, Bacterial; Models, Biological; Mutation; Phenotype; Signal Transduction

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Topics: Animals; ATP-Binding Cassette Transporters; Cyclic GMP; Escherichia coli Infections; Escherichia coli Proteins; Fimbriae, Bacterial; Gene Expression Regulation, Bacterial; Humans; Mice; Multigene Family; Mutation; Operon; Phosphates; Phosphorus-Oxygen Lyases; Recombinases; Regulon; Transcription Factors; Urinary Bladder; Urinary Tract Infections; Uropathogenic Escherichia coli; Virulence

In 2011, nearly 4,000 people in Germany were infected by Shiga toxin (Stx)-producing Escherichia coli O104:H4 with > 22% of patients developing haemolytic uraemic syndrome (HUS). Genome sequencing showed the outbreak strain to be related to enteroaggregative E. coli (EAEC), suggesting its high virulence results from EAEC-typical strong adherence and biofilm formation combined to Stx production. Here, we report that the outbreak strain contains a novel diguanylate cyclase (DgcX)--producing the biofilm-promoting second messenger c-di-GMP--that shows higher expression than any other known E. coli diguanylate cyclase. Unlike closely related E. coli, the outbreak strain expresses the c-di-GMP-controlled biofilm regulator CsgD and amyloid curli fibres at 37°C, but is cellulose-negative. Moreover, it constantly generates derivatives with further increased and deregulated production of CsgD and curli. Since curli fibres are strongly proinflammatory, with cellulose counteracting this effect, high c-di-GMP and curli production by the outbreak O104:H4 strain may enhance not only adherence but may also contribute to inflammation, thereby facilitating entry of Stx into the bloodstream and to the kidneys where Stx causes HUS.

Topics: Biofilms; Cyclic GMP; Disease Outbreaks; Escherichia coli Infections; Escherichia coli Proteins; Female; Germany; Hemolytic-Uremic Syndrome; Humans; Middle Aged; Phosphorus-Oxygen Lyases; Shiga Toxin; Shiga-Toxigenic Escherichia coli

Escherichia coli O157:H7 is an important foodborne pathogen that causes serious illness in humans at low infectious doses. The main source of infections is beef or greens contaminated with E. coli O157:H7 shed by cattle. Here we investigated the role of c-di-GMP-dependent signal transduction in cattle gut colonization of E. coli O157:H7. To manipulate intracellular c-di-GMP levels, we introduced into E. coli O157:H7 a c-di-GMP specific phosphodiesterase (PDE). Liquid chromatography tandem mass spectrometry analysis confirmed that in E. coli O157:H7, over-expression of PDE decreased c-di-GMP level. Consistent with the altered c-di-GMP level, PDE overexpression resulted in decreased biofilm formation in E. coli O157:H7. Furthermore, this diminished c-di-GMP levels reduced adhesion of E. coli O157:H7 to both cultured HT-29 cells and cattle colon explants. Consistently, mRNA levels of genes involved in adhesion were down-regulated including genes encoding E. coli common pili, long polar fimbriae 1, hemorrhagic coli pilus, as well as intimin and tir. We further observed decreased curli fimbriae synthesis in the strain with decreased c-di-GMP levels, which was supported by the reduction in the transcription of curli large subunit gene csgA and the curli expression regulator gene csgD. Genes for enterocyte effacement encoded regulator (Ler) and type III secretion system effectors, EspA and EspB, were also down-regulated. Collectively, data indicated that c-di-GMP signaling positively regulates E. coli O157:H7 intestinal epithelial cell and tissue colonization and expression of associated adhesion factors.

Topics: Animals; Cattle; Cyclic GMP; Enterocytes; Epithelial Cells; Escherichia coli Infections; Escherichia coli O157; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; HT29 Cells; Humans; Intestines; Signal Transduction; Time Factors

During urinary tract infections (UTIs), uropathogenic Escherichia coli must maintain a delicate balance between sessility and motility to achieve successful infection of both the bladder and kidneys. Previous studies showed that cyclic dimeric GMP (c-di-GMP) levels aid in the control of the transition between motile and nonmotile states in E. coli. The yfiRNB locus in E. coli CFT073 contains genes for YfiN, a diguanylate cyclase, and its activity regulators, YfiR and YfiB. Deletion of yfiR yielded a mutant that was attenuated in both the bladder and the kidneys when tested in competition with the wild-type strain in the murine model of UTI. A double yfiRN mutant was not attenuated in the mouse model, suggesting that unregulated YfiN activity and likely increased cytoplasmic c-di-GMP levels cause a survival defect. Curli fimbriae and cellulose production were increased in the yfiR mutant. Expression of yhjH, a gene encoding a proven phosphodiesterase, in CFT073 ΔyfiR suppressed the overproduction of curli fimbriae and cellulose and further verified that deletion of yfiR results in c-di-GMP accumulation. Additional deletion of csgD and bcsA, genes necessary for curli fimbriae and cellulose production, respectively, returned colonization levels of the yfiR deletion mutant to wild-type levels. Peroxide sensitivity assays and iron acquisition assays displayed no significant differences between the yfiR mutant and the wild-type strain. These results indicate that dysregulation of c-di-GMP production results in pleiotropic effects that disable E. coli in the urinary tract and implicate the c-di-GMP regulatory system as an important factor in the persistence of uropathogenic E. coli in vivo.

Topics: Animals; Bacterial Proteins; Cellulose; Cyclic GMP; Cytoplasm; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Female; Fimbriae, Bacterial; Gene Deletion; Hydrogen Peroxide; Iron; Mice; Phosphorus-Oxygen Lyases; Urinary Tract; Urinary Tract Infections; Urine; Uropathogenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of morbidity and mortality due to diarrheal illness in developing countries. There is currently no effective vaccine against these important pathogens. Because genes modulated by pathogen-host interactions potentially encode putative vaccine targets, we investigated changes in gene expression and surface morphology of ETEC upon interaction with intestinal epithelial cells in vitro. Pan-genome microarrays, quantitative reverse transcriptase PCR (qRT-PCR), and transcriptional reporter fusions of selected promoters were used to study changes in ETEC transcriptomes. Flow cytometry, immunofluorescence microscopy, and scanning electron microscopy were used to investigate alterations in surface antigen expression and morphology following pathogen-host interactions. Following host cell contact, genes for motility, adhesion, toxin production, immunodominant peptides, and key regulatory molecules, including cyclic AMP (cAMP) receptor protein (CRP) and c-di-GMP, were substantially modulated. These changes were accompanied by visible changes in both ETEC architecture and the expression of surface antigens, including a novel highly conserved adhesin molecule, EaeH. The studies reported here suggest that pathogen-host interactions are finely orchestrated by ETEC and are characterized by coordinated responses involving the sequential deployment of multiple virulence molecules. Elucidation of the molecular details of these interactions could highlight novel strategies for development of vaccines for these important pathogens.

Topics: Adhesins, Bacterial; Antigens, Surface; Caco-2 Cells; Cell Line, Tumor; Cyclic GMP; DNA-Binding Proteins; Enterotoxigenic Escherichia coli; Epithelial Cells; Escherichia coli Infections; Escherichia coli Proteins; Gene Expression; Host-Pathogen Interactions; Humans; Intestinal Mucosa; Intestines; Promoter Regions, Genetic; Receptors, Cyclic AMP; Transcription, Genetic; Transcriptome; Virulence