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 ,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-5nucleotidase, a key extracellular enzyme which catalyzes the hydrolysis of extracellular 5-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-5-nucleotidase, in vivo, zinc acetate was superior to ,-methylene-ADP in its ability to block EPEC infection-induced fluid accumulation in rabbit ileal loops. Zinc treatment also decreased EPEC adherence to intestinal villi, an effect not explained by the known roles of ecto-5-nucleotidase. We initiated the present study to try to determine if zinc had molecular targets other than ecto-5