To analyze whether Escherichia coli strains that cause urinary tract infections (UPEC) share virulence characteristics with the diarrheagenic E. coli

Enteroaggregative Escherichia coli (EAEC) causes watery diarrhoea that is often mildly inflammatory. Previous studies have reported that the flagellin of EAEC induces IL-8 from intestinal epithelial cells (IECs) in culture. To characterize more fully the inflammatory response to EAEC, we infected IECs with EAEC prototype strain 042 and assessed cellular responses by macroarray and reverse transcriptase polymerase chain reaction (RT-PCR). Genes upregulated in 042-infected non-polarized T84 cells included IL-8, IL-6, TNF-alpha, GRO-alpha, GRO-gamma, ICAM-1, GM-CSF and IL-1alpha. RT-PCR analyses performed with cDNA from T84 and HT-29 cells infected with an aflagellar mutant (042fliC) suggested that these responses were primarily mediated by flagellin. To better reproduce the conditions of the infection for this non-invasive pathogen, we assessed the responses of polarized IECs to strain 042 infection. As expected, 042 induced IL-8 production from both polarized T84 and HT-29 cells. However, significant IL-8 secretion was induced in polarized T84 cells infected with 042fliC, suggesting that a factor other than flagellin contributes to inflammation in this model. This non-flagellar IL-8 response required expression of the aggregative adherence fimbria (AAF) adhesin, and was related to the presence of the minor fimbria-associated protein AafB. Our data suggest that multiple factors contribute to EAEC-induced inflammation, and further characterization of the nature of EAEC proinflammatory factors will greatly advance our understanding of this emerging pathogen.

EnteroaggregativeEscherichia coli(EAEC) is defined by a characteristic “stacked-brick” aggregative adherence (AA) pattern to cultured cells. In well-studied EAEC prototype strains (called typical EAEC strains), the AA phenotype requires aggregative adherence fimbriae (AAFs). However, previous studies suggest that known AAF alleles are not found in all EAEC strains. To define mechanisms contributing to adherence in an atypical strain, we studied EAEC strain C1096. AnE. coliK12 derivative carrying two plasmids, designated pSERB1 and pSERB2, from C1096 adhered to cell lines and exhibited an AA pattern. Nucleotide sequence analysis of pSERB1 indicated that it is related to plasmids of the IncI1 incompatibility group. These plasmids encode genes involved in pilus-mediated conjugal transfer, as well aspilL-V, which encodes a second pilus of the type IV family. Insertional inactivation of the gene predicted to encode the major type IV pilin subunit (pilS) reduced conjugal transfer of the plasmid by 4 orders of magnitude. Adherence of the mutant strain to polystyrene and to HT29 cells was reduced by approximately 21% and 75%, respectively. In a continuous-flow microfermentor, thepilSinactivation reduced mature biofilm formation on a glass slide by approximately 50%. In addition, the simultaneous presence of both pSERB1 and pSERB2 plasmids promotedpilS-independent biofilm formation. We conclude that the IncI1 plasmid of EAEC C1096 encodes a type IV pilus that contributes to plasmid conjugation, epithelial cell adherence, and adherence to abiotic surfaces. We also observe that AA can be mediated by factors distinct from AAF adhesins.

Escherichia coli produces polysaccharide capsules that, based on their mechanisms of synthesis and assembly, have been classified into four groups. The group 4 capsule (G4C) polysaccharide is frequently identical to that of the cognate lipopolysaccharide O side chain and has, therefore, also been termed the O-antigen capsule. The genes involved in the assembly of the group 1, 2, and 3 capsules have been described, but those required for G4C assembly remained obscure. We found that enteropathogenic E. coli (EPEC) produces G4C, and we identified an operon containing seven genes, ymcD , ymcC , ymcB , ymcA , yccZ , etp , and etk , which are required for formation of the capsule. The encoded proteins appear to constitute a polysaccharide secretion system. The G4C operon is absent from the genomes of enteroaggregative E. coli and uropathogenic E. coli. E. coli K-12 contains the G4C operon but does not express it, because of the presence of IS 1 at its promoter region. In contrast, EPEC, enterohemorrhagic E. coli , and Shigella species possess an intact G4C operon.

The type III secretion system (TTSS) is a macromolecular structure that spans the cell wall of Gram-negative bacterial pathogens, enabling delivery of virulence effector proteins directly to the membranes and cytosol of host eukaryotic cells. TTSS consists of a conserved needle complex (NC) that is composed of sets of inner and outer membranes rings connected by a periplasmic rod. Enteropathogenic Escherichia coli (EPEC) is an extracellular diarrhoeagenic pathogen that uses TTSS to induce actin polymerization and colonizes the intestinal epithelium. In EPEC, EscJ is predicted to be targeted to the periplasm, in a sec-dependent manner, and to bridge the TTSS membrane-associated rings. In this study we determined the global fold of EscJ using Nuclear Magnetic Resonance spectroscopy. We show that EscJ comprises two subdomains (D1 – amino acid residues 1–55 in the mature protein, and D2 – amino acid residues 90–170), each comprising a three-stranded β-sheet flanked by two α-helices. A flexible region (residues 60–85) couples the structured regions D1 and D2. Periplasmic overexpression of EscJ_D1 and EscJ_D2 in a single escJ mutant bacterium failed to restore protein secretion activity, suggesting that the flexible linker is essential for the rod function. In contrast, periplasmic overexpression of EscJ_D1 and EscJ_D2 in the same wild-type bacterium had a dominant-negative phenotype suggesting defective assembly of the TTSS and protein translocation.