Quorum sensing is a cell-to-cell signaling mechanism in which bacteria respond to hormone-like molecules called autoinducers (AIs). The AI-3 quorum-sensing system is also involved in interkingdom signaling with the eukaryotic hormones epinephrine͞ norepinephrine. This signaling activates transcription of virulence genes in enterohemorrhagic Escherichia coli O157:H7. However, this signaling system has never been shown to be involved in virulence in vivo, and the bacterial receptor for these signals had not been identified. Here, we show that the QseC sensor kinase is a bacterial receptor for the host epinephrine͞norepinephrine and the AI-3 produced by the gastrointestinal microbial flora. We also found that an ␣-adrenergic antagonist can specifically block the QseC response to these signals. Furthermore, we demonstrated that a qseC mutant is attenuated for virulence in a rabbit animal model, underscoring the importance of this signaling system in virulence in vivo. Finally, an in silico search found that the periplasmic sensing domain of QseC is conserved among several bacterial species. Thus, QseC is a bacterial adrenergic receptor that activates virulence genes in response to interkingdom cross-signaling. We anticipate that these studies will be a starting point in understanding bacterial-host hormone signaling at the biochemical level. Given the role that this system plays in bacterial virulence, further characterization of this unique signaling mechanism may be important for developing novel classes of antimicrobials.AI-3 ͉ enterohemorrhagic Escherichia coli ͉ epinephrine ͉ quorum sensing ͉ two-component systems

Shiga toxin 1 and 2 production is a cardinal virulence trait of enterohemorrhagic Escherichia coli infection that causes a spectrum of intestinal and systemic pathology. However, intestinal sites of enterohemorrhagic E. coli colonization during the human infection and how the Shiga toxins are taken up and cross the globotriaosylceramide (Gb3) receptor-negative intestinal epithelial cells remain largely uncharacterized. We used samples of human intestinal tissue from patients with E. coli O157:H7 infection to detect the intestinal sites of bacterial colonization and characterize the distribution of Shiga toxins. We further used a model of largely Gb3-negative T84 intestinal epithelial monolayers treated with B-subunit of Shiga toxin 1 to determine the mechanisms of non-receptor-mediated toxin uptake. We now report that E. coli O157:H7 were found at the apical surface of epithelial cells only in the ileocecal valve area and that both toxins were present in large amounts inside surface and crypt epithelial cells in all tested intestinal samples. Our in vitro data suggest that macropinocytosis mediated through Src activation significantly increases toxin endocytosis by intestinal epithelial cells and also stimulates toxin transcellular transcytosis. We conclude that Shiga toxin is taken up by human intestinal epithelial cells during E. coli O157:H7 infection regardless of the presence of bacterial colonies. Macropinocytosis might be responsible for toxin uptake by Gb3-free intestinal epithelial cells and transcytosis. These observations provide new insights into the understanding of Shiga toxin contribution to enterohemorrhagic E. coli-related intestinal and systemic diseases.

The pathogenicity island termed the locus of enterocyte effacement (LEE) is found in diverse attaching and effacing pathogens associated with diarrhea in humans and other animal species. To explore the relation of variation in LEE sequences to host specificity and genetic lineage, we determined the nucleotide sequence of the LEE region from a rabbit diarrheagenic Escherichia coli strain RDEC-1 (O15:H؊) and compared it with those from human enteropathogenic E. coli (EPEC, O127:H6) and enterohemorrhagic E. coli (EHEC, O157:H7) strains. Differing from EPEC and EHEC LEEs, the RDEC-1 LEE is not inserted at selC and is flanked by an IS2 element and the lifA toxin gene. The RDEC-1 LEE contains a core region of 40 open reading frames, all of which are shared with the LEE of EPEC and EHEC. orf3 and the ERIC (enteric repetitive intergenic consensus) sequence present in the LEEs of EHEC and EPEC are absent from the RDEC-1 LEE. The predicted promoters of LEE1, LEE2, LEE3, tir, and LEE4 operons are highly conserved among the LEEs, although the upstream regions varied considerably for tir and the crucial LEE1 promoter, suggesting differences in regulation. Among the shared genes, high homology (>95% identity) between the RDEC-1 and the EPEC and EHEC LEEs at the predicted amino acid level was observed for the components of the type III secretion apparatus, the Ces chaperones, and the Ler regulator. In contrast, more divergence (66 to 88% identity) was observed in genes encoding proteins involved in host interaction, such as intimin (Eae) and the secreted proteins (Tir and Esps). A comparison of the highly variable genes from RDEC-1 with those from a number of attaching and effacing pathogens infecting different species and of different evolutionary lineages was performed. Although RDEC-1 diverges from some human-infecting EPEC and EHEC, most of the variation observed appeared to be due to evolutionary lineage rather than host specificity. Therefore, much of the observed hypervariability in genes involved in pathogenesis may not represent specific adaptation to different host species.Attaching and effacing Escherichia coli (AEEC) induce a unique intestinal histopathological phenotype termed attaching and effacing (A/E), which is characterized by intimate bacterial attachment to the epithelial cells with effacement of microvilli and rearrangement of the host cell cytoskeleton to form a pedestal-like structure that cups individual bacterial cells (35). The A/E phenotype is encoded by the locus of enterocyte effacement (LEE) pathogenicity island (PAI) (32). The LEE in human enteropathogenic E. coli (EPEC) strain E2348/69 is a 36-kb cluster of genes containing five major polycistronic operons: LEE1, LEE2,

Nonenterotoxigenic porcine Escherichia coli strains belonging to the serogroup O45 have been associated with postweaning diarrhea in swine and adhere to intestinal epithelial cells in a characteristic attaching and effacing (A/E) pattern. O45 porcine enteropathogenic E. coli (PEPEC) strain 86-1390 induces typical A/E lesions in a pig ileal explant model. Using TnphoA transposon insertion mutagenesis on strain 86-1390, we found a mutant that did not induce A/E lesions. The insertion was identified in a gene designated paa (porcine A/E-associated gene). Sequence analysis of paa revealed an open reading frame of 753 bp encoding a 27.6-kDa protein which displayed 100, 51.8, and 49% homology with Paa of enterohemorrhagic E. coli O157:H7 strains (EDL933 and Sakai), PEB3 of Campylobacter jejuni, and AcfC of Vibrio cholerae, respectively. Chromosomal localization studies indicated that the region containing paa was inserted between the yciD and yciE genes at about 28.3 min of the E. coli K-12 chromosome. The presence of paa and eae sequences in the porcine O45 strains is highly correlated with the A/E phenotype. However, the observation that three eae-positive but paa-negative PEPEC O45 strains were A/E negative provides further evidence for the importance of the paa gene in the A/E activity of O45 strains. As well, the complementation of the paa mutant restored the A/E activity of the 86-1390 strain, showing the involvement of Paa in PEPEC pathogenicity. These observations suggest that Paa contributes to the early stages of A/E E. coli virulence.Attaching and effacing (A/E) Escherichia coli (AEEC) induces distinctive histopathological lesions on the intestinal mucosa, known as the A/E lesions. These lesions are characteristic of enteric pathogens such as enteropathogenic E. coli (EPEC), responsible for severe childhood diarrhea in developing countries (14, 38), enterohemorrhagic E. coli (EHEC), causing hemorrhagic colitis and hemolytic-uremic syndrome, a diarrheagenic E. coli strain of rabbits (RDEC-1), strains of Hafnia alvei isolated from children with diarrhea, and Citrobacter rodentium, causing transmissible colonic hyperplasia in mice (4,16,53). A/E lesions have also been associated with diarrhea in different animal species such as rabbits, calves, dogs, cats, lambs, pigs, and tamarins (8,9,22,32,37,55).A/E lesions result from intimate bacterial adherence to the apical surfaces of enterocytes and activation of several chromosomal gene products that interact with components of the host cell, leading to host cell protein phosphorylation, effacement of target brush borders, and disruption of the underlying actin cytoskeleton (11, 38). The genes are clustered in a chromosomal pathogenicity island called the locus of enterocyte effacement (LEE). Its location and size vary in different strains. In EPEC strain E2348/69 and EHEC O157:H7 strains, the LEE is inserted in the selC locus at about 82 min on the E. coli K-12 chromosome, but its size varies from 35 kb for EPEC to 43 kb for EHEC. In strains of serotype O26:H-, the...

Enteropathogenic Escherichia coli (EPEC) infection triggers the release of ATP from host intestinal cells, and the ATP is broken down to ADP, AMP, and adenosine in the lumen of the intestine. Ecto-5-nucleotidase (CD73) is the main enzyme responsible for the conversion of 5-AMP to adenosine, which triggers fluid secretion from host intestinal cells and also has growth-promoting effects on EPEC bacteria. In a recent study, we examined the role of the host enzyme CD73 in EPEC infection by testing the effect of ecto-5-nucleotidase inhibitors. Zinc was a less potent inhibitor of ecto-5-nucleotidase in vitro than the nucleotide analog ␣,␤-methylene-ADP, but in vivo, zinc was much more efficacious in preventing EPEC-induced fluid secretion in rabbit ileal loops than ␣,␤-methylene-ADP. This discrepancy between the in vitro and in vivo potencies of the two inhibitors prompted us to search for potential targets of zinc other than ecto-5-nucleotidase. Zinc, at concentrations that produced little or no inhibition of EPEC growth, caused a decrease in the expression of EPEC protein virulence factors, such as bundle-forming pilus (BFP), EPEC secreted protein A, and other EPEC secreted proteins, and reduced EPEC adherence to cells in tissue culture. The effects of zinc were not mimicked by other transition metals, such as manganese, iron, copper, or nickel, and the effects were not reversed by an excess of iron. Quantitative real-time PCR showed that zinc reduced the abundance of the RNAs encoded by the bfp gene, by the plasmid-encoded regulator (per) gene, by the locus for the enterocyte effacement (LEE)-encoded regulator (ler) gene, and by several of the esp genes. In vivo, zinc reduced EPEC-induced fluid secretion into ligated rabbit ileal loops, decreased the adherence of EPEC to rabbit ileum, and reduced histopathological damage such as villus blunting. Some of the beneficial effects of zinc on EPEC infection appear to be due to the action of the metal on EPEC bacteria as well as on the host.Enteropathogenic Escherichia coli (EPEC) infection is a common cause of watery diarrhea in children in developing countries. The mechanism by which EPEC triggers watery diarrhea is obscure, since unlike several other types of diarrheaproducing E. coli strains, EPEC produces no toxins. We recently theorized that the release of adenine nucleotides from the host intestinal cells, followed by the breakdown to adenosine, could trigger watery diarrhea by the activation of adenosine receptors in the intestine (8).Our interest in zinc was prompted by studies of ecto-5Ј-nucleotidase, a key extracellular enzyme which catalyzes the hydrolysis of extracellular 5Ј-AMP to adenosine. Zinc and the nucleotide analog ␣,␤-methylene-ADP are the classical inhibitors of this enzyme that were tested in vitro and in vivo in a recent study by one of our laboratories (9). Although ␣,␤-methylene-ADP is about eightfold more potent than zinc acetate in the inhibition of ecto-5Ј-nucleotidase, in vivo, zinc acetate was superior to ␣,␤-methylene-ADP in its ability to...