Role of host xanthine oxidase in infection due to enteropathogenic and Shiga-toxigenic Escherichia coli.

Xanthine oxidase (XO), also known as xanthine oxidoreductase, has long been considered an important host defense molecule in the intestine and in breastfed infants. Here, we present evidence that XO is released from and active in intestinal tissues and fluids in response to infection with enteropathogenic Escherichia coli (EPEC) and Shiga-toxigenic E. coli (STEC), also known as enterohemorrhagic E. coli (EHEC). XO is released into intestinal fluids in EPEC and STEC infection in a rabbit animal model. XO activity results in the generation of surprisingly high concentrations of uric acid in both cultured cell and animal models of infection. Hydrogen peroxide (H(2)O(2)) generated by XO activity triggered a chloride secretory response in intestinal cell monolayers within minutes but decreased transepithelial electrical resistance at 6 to 22 h. H(2)O(2) generated by XO activity was effective at killing laboratory strains of E. coli, commensal microbiotas, and anaerobes, but wild-type EPEC and STEC strains were 100 to 1,000 times more resistant to killing or growth inhibition by this pathway. Instead of killing pathogenic bacteria, physiologic concentrations of XO increased virulence by inducing the production of Shiga toxins from STEC strains. In vivo, exogenous XO plus the substrate hypoxanthine did not protect and instead worsened the outcome of STEC infection in the rabbit ligated intestinal loop model of infection. XO released during EPEC and STEC infection may serve as a virulence-inducing signal to the pathogen and not solely as a protective host defense.

Effect of zinc in enteropathogenic Escherichia coli infection.

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 alpha,beta-methylene-ADP, but in vivo, zinc was much more efficacious in preventing EPEC-induced fluid secretion in rabbit ileal loops than alpha,beta-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.

Ecto-5'-nucleotidase and intestinal ion secretion by enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) triggers a large release of adenosine triphosphate (ATP) from host intestinal cells and the extracellular ATP is broken down to adenosine diphosphate (ADP), AMP, and adenosine. Adenosine is a potent secretagogue in the small and large intestine. We suspected that ecto-5'-nucleotidase (CD73, an intestinal enzyme) was a critical enzyme involved in the conversion of AMP to adenosine and in the pathogenesis of EPEC diarrhea. We developed a nonradioactive method for measuring ecto-5'-nucleotidase in cultured T84 cell monolayers based on the detection of phosphate release from 5'-AMP. EPEC infection triggered a release of ecto-5'-nucleotidase from the cell surface into the supernatant medium. EPEC-induced 5'-nucleotidase release was not correlated with host cell death but instead with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Ecto-5'-nucleotidase was susceptible to inhibition by zinc acetate and by alpha,beta-methylene-adenosine diphosphate (alpha,beta-methylene-ADP). In the Ussing chamber, these inhibitors could reverse the chloride secretory responses triggered by 5'-AMP. In addition, alpha,beta-methylene-ADP and zinc blocked the ability of 5'-AMP to stimulate EPEC growth under nutrient-limited conditions in vitro. Ecto-5'-nucleotidase appears to be the major enzyme responsible for generation of adenosine from adenine nucleotides in the T84 cell line, and inhibitors of ecto-5'-nucleotidase, such as alpha,beta-methylene-ADP and zinc, might be useful for treatment of the watery diarrhea produced by EPEC infection.

Mutual enhancement of virulence by enterotoxigenic and enteropathogenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) and enteropathogenic E. coli (EPEC) are common causes of diarrhea in children in developing countries. Dual infections with both pathogens have been noted fairly frequently in studies of diarrhea around the world. In previous laboratory work, we noted that cholera toxin and forskolin markedly potentiated EPEC-induced ATP release from the host cell, and this potentiated release was found to be mediated by the cystic fibrosis transmembrane conductance regulator. In this study, we examined whether the ETEC heat-labile toxin (LT) or the heat-stable toxin (STa, also known as ST) potentiated EPEC-induced ATP release. We found that crude ETEC culture filtrates, as well as purified ETEC toxins, did potentiate EPEC-induced ATP release in cultured T84 cells. Coinfection of T84 cells with live ETEC plus EPEC bacteria also resulted in enhanced ATP release compared to EPEC alone. In Ussing chamber studies of chloride secretion, adenine nucleotides released from the host by EPEC also significantly enhanced the chloride secretory responses that were triggered by crude ETEC filtrates, purified STa, and the peptide hormone guanylin. In addition, adenosine and LT had additive or synergistic effects in inducing vacuole formation in T84 cells. Therefore, ETEC toxins and EPEC-induced damage to the host cell both enhance the virulence of the other type of E. coli. Our in vitro data demonstrate a molecular basis for a microbial interaction, which could result in increased severity of disease in vivo in individuals who are coinfected with ETEC and EPEC.

Two pathways for ATP release from host cells in enteropathogenic Escherichia coli infection.

We previously reported that enteropathogenic Escherichia coli (EPEC) infection triggered a large release of ATP from the host cell that was correlated with and dependent on EPEC-induced killing of the host cell. We noted, however, that under some circumstances, EPEC-induced ATP release exceeded that which could be accounted for on the basis of host cell killing. For example, EPEC-induced ATP release was potentiated by noncytotoxic agents that elevate host cell cAMP, such as forskolin and cholera toxin, and by exposure to hypotonic medium. These findings and the performance of the EPEC espF mutant led us to hypothesize that the CFTR plays a role in EPEC-induced ATP release that is independent of cell death. We report the results of experiments using specific, cell-permeable CFTR activators and inhibitors, as well as transfection of the CFTR into non-CFTR-expressing cell lines, which incriminate the CFTR as a second pathway for ATP release from host cells. Increased ATP release via CFTR is not accompanied by an increase in EPEC adherence to transfected cells. The CFTR-dependent ATP release pathway becomes activated endogenously later in EPEC infection, and this activation is mediated, at least in part, by generation of extracellular adenosine from the breakdown of released ATP.