No abstract

A second region containing five genes homologous to the long polar fimbrial operon of Salmonella enterica serovar Typhimurium is located in the chromosome of enterohemorrhagic Escherichia coli (EHEC) O157:H7. A non-fimbriated E. coli K-12 strain carrying the cloned EHEC lpf (lpf2) genes expressed thin fibrillae-like structures on its surface and displayed reduced adherence to tissue culture cells. Neither mutation in the lpfA2 gene in either the parent or lpfA1 mutant strains showed an effect in adherence or in the formation of A/E lesions on HeLa cells. lpfA2 isogenic mutant strains adhere to Caco-2 cells almost as well as the wild-type at 5 h, but they were deficient in adherence at early time points. A collection of diarrheagenic E. coli strains were investigated for the presence of lpfA1 and lpfA2 and results showed that these genes are present in specific serogroups which are phylogenetically related. Our results suggest that LP fimbriae 2 may contribute to the early stages of EHEC adhesion and that genes encoding the major LP fimbrial subunits are present in a small group of EHEC and EPEC serotypes.

Outer membrane protein A (OmpA) is located in the membrane of Escherichia coli and other gram-negative bacteria and plays a multifunctional role in bacterial physiology and pathogenesis. In enterohemorrhagic E. coli (EHEC), especially serotype O157:H7, OmpA interacts with cultured human intestinal cells and likely acts as an important component to stimulate the immune response during infection. To test this hypothesis, we analyzed the effect of EHEC OmpA on cytokine production by dendritic cells (DCs) and on DC migration across polarized intestinal epithelial cells. OmpA induced murine DCs to secrete interleukin-1 (IL-1), IL-10, and IL-12 in a dose-dependent manner, and this effect was independent of Toll-like receptor 4. Although DCs displayed differential responses to EHEC OmpA and OmpA-specific antibodies enhanced DC cytokine secretion, we cannot discard that other EHEC surface elements were likely to be involved.

The adherence of pathogenic Escherichia coli strains to intestinal epithelium is essential for initiation of infection. The cad operon encodes the lysine decarboxylase (LDC) system responsible for metabolizing lysine, and this operon has been proposed as an antivirulence mechanism in enteroinvasive E. coli and Shigella flexneri and as a factor mediating E. coli O157:H7 adherence. We sought to determine whether the LDC activity was present in a phylogenetically characterized collection of diarrheagenic E. coli (DEC) strains and to establish whether its expression was associated with their adherence to tissue culture cells. LDC activity was found in most of the pathogenic E. coli strains tested and was absent from Shiga toxin-producing E. coli (STEC) O111 strains (DEC pathotype 8). Analysis of the cad region in these O111 strains indicates that the operon has been rearranged and some of the genes are either missing or disrupted. A similar rearrangement was found in an E. coli O111:H8 strain recently isolated from an outbreak in Texas. Complementation of the LDC-negative strains with the cad operon in trans restored the LDC activity and resulted in a reduction in adherence to tissue culture cells. Initial analysis of the protein profiles on the surface of the O111 strains indicates that the LDC activity has an effect on the expression of the adhesin intimin. Cadaverine had a slight effect on LDC-negative strain adhesion but none on intimin expression. Our data suggest that this pathoadaptive mutation is an important mechanism to control functions potentially implicated in the pathogenesis of these organisms.

SummaryAtypical enteropathogenic Escherichia coli (aEPEC) strains are frequently implicated in infant diarrhoea in developing countries. Not much is known about the adherence properties of aEPEC; however, it has been shown that these strains can adhere to tissuecultured cells. A chromosomal region designated the locus for diffuse adherence (LDA) confers aEPEC strain 22 the ability to adhere to culture cells. LDA is an afimbrial adhesin that contains a major subunit, LdaG, whose expression is induced on MacConkey agar at 37°C. We hypothesized that the bile salts found in this culture media induce the expression of LdaG. Strain 22 and the LdaG mutant were grown in Luria-Bertani (LB) media in the presence or absence of bile salts and heat-extracted surface-expressed proteins were separated by SDS-PAGE to determine whether expression of the 25 kDa LdaG protein was induced. Western blot analysis with anti-LdaG confirmed that bile salts enhance LdaG expression at 37°C. Adhesion assays on HeLa cells revealed that adhesion in a diffuse pattern of strain 22 increased in the presence of bile salts. We also confirmed that expression of the localized adherence pattern observed in the ldaG mutant required the presence of a large cryptic plasmid found in strain 22 and that this phenotype was not induced by bile salts. At the transcriptional level, the ldaG-lacZ promoter fusion displayed maximum beta-galactosidase activity when the parent strain was grown in LB supplemented with bile salts. Fluorescence Activated Cell Sorting analysis, immunogold labelling electron microscopy and immunofluorescence using antiLdaG sera confirmed that LDA is a bile saltsinducible surface-expressed afimbrial adhesin. Finally, LdaG expression was induced in presence of individual bile salts but not by other detergents. We concluded that bile salts increase expression of LDA, conferring a diffuse adherence pattern and having an impact on the adhesion properties of this aEPEC strain.