Broad and efficient control of major foodborne pathogenic strains of Escherichia coli by mixtures of plant-produced colicins.

Enterohemorrhagic Escherichia coli (EHEC) is one of the leading causes of bacterial enteric infections worldwide, causing ∼100,000 illnesses, 3,000 hospitalizations, and 90 deaths annually in the United States alone. These illnesses have been linked to consumption of contaminated animal products and vegetables. Currently, other than thermal inactivation, there are no effective methods to eliminate pathogenic bacteria in food. Colicins are nonantibiotic antimicrobial proteins, produced by E. coli strains that kill or inhibit the growth of other E. coli strains. Several colicins are highly effective against key EHEC strains. Here we demonstrate very high levels of colicin expression (up to 3 g/kg of fresh biomass) in tobacco and edible plants (spinach and leafy beets) at costs that will allow commercialization. Among the colicins examined, plant-expressed colicin M had the broadest antimicrobial activity against EHEC and complemented the potency of other colicins. A mixture of colicin M and colicin E7 showed very high activity against all major EHEC strains, as defined by the US Department of Agriculture/Food and Drug Administration. Treatments with low (less than 10 mg colicins per L) concentrations reduced the pathogenic bacterial load in broth culture by 2 to over 6 logs depending on the strain. In experiments using meats spiked with E. coli O157:H7, colicins efficiently reduced the population of the pathogen by at least 2 logs. Plant-produced colicins could be effectively used for the broad control of pathogenic E. coli in both plant- and animal-based food products and, in the United States, colicins could be approved using the generally recognized as safe (GRAS) regulatory approval pathway.

Isolation of an isoenzyme of human glutaminyl cyclase: retention in the Golgi complex suggests involvement in the protein maturation machinery.

Mammalian glutaminyl cyclase isoenzymes (isoQCs) were identified. The analysis of the primary structure of human isoQC (h-isoQC) revealed conservation of the zinc-binding motif of the human QC (hQC). In contrast to hQC, h-isoQC carries an N-terminal signal anchor. The cDNAs of human and murine isoQCs were isolated and h-isoQC, lacking the N-terminal signal anchor and the short cytosolic tail, was expressed as a fusion protein in Escherichia coli. h-isoQC exhibits 10fold lower activity compared to hQC. Similar to hQC, h-isoQC was competitively inhibited by imidazoles and cysteamines. Inactivation by metal chelators suggests a conserved metal-dependent catalytic mechanism of both isoenzymes. A comparison of the expression pattern of m-isoQC and murine QC revealed ubiquitous expression of both enzymes. However, murine QC transcript formation was higher in neuronal tissue, whereas the amount of m-isoQC transcripts did not vary significantly between different organs. h-isoQC was exclusively localized within the Golgi complex, obviously retained by the N-terminus. Similar resident enzymes of the Golgi complex are the glycosyltransferases. Golgi apparatus retention implies a "housekeeping" protein maturation machinery conducting glycosylation and pyroglutamyl formation. For these enzymes, apparently similar strategies evolved to retain the proteins in the Golgi complex.