Engineering plants to produce protein to kill E.coli

Colicins are non-antibiotic antimicrobial proteins, produced by E. coli strains that kill or inhibit the growth of others.

Several colicins are effective against key Enterohemorrhagic Escherichia coli (EHEC) strains with potential reduction in bacterial load of up to 5 logs, said the study.

Yuri Gleba, CEO of Nomad Bioscience and colleagues, looked to see if edible plants could be engineered to produce colicins and whether the proteins might prevent EHEC contamination in food.

Current interventions involve heating or organic acids, which can change taste and quality of products.

Antimicrobial activity against E.coli​

Among those evaluated, colicin M was found to possess the broadest antimicrobial activity against pathogenic E. coli strains.

Mixtures of colicin M and other colicins, and of colicin M and colicin E7, showed very high activity against all seven pathogenic serotypes defined by the US Department of Agriculture (USDA)/Food and Drug Administration (FDA) as major foodborne pathogens.

These mixtures were also effective in controlling the emerging pathogenic serotype O104:H4.

Treatments with colicin mixtures at low levels (nanomolar concentrations, or less than 10mg total colicins per kg of treated food product) reduce bacterial load of different pathogenic strains by 2 to >6 logs, found the study.

So mixtures of colicins, applied at low concentrations to bacterial cultures, could reduce growth of E. coli.

The team selected 12 colicin proteins representing all four activity groups and various receptor specificities, including colicins E2, E3, E6, E7, D, N, K, 5, U, B, Ia, and M.

Edible plants tested​

They tested the transient expression of recombinant colicin M in crops including leafy beets, spinach, chicory, and lettuce using agroinfiltration with needleless syringe.

Colicin-containing plant extracts were tested in radial diffusion assays via the spot-on-lawn method, and antimicrobial activity was evaluated semi-quantitatively based on the highest dilution of colicin-containing plant extracts causing a clearing effect.

Colicin M demonstrated high antimicrobial activity against five and moderate against two of the eight pathogenic strains tested.

For more precise quantitative evaluation of antimicrobial activity against EHEC strains, colicin-containing plant extracts were then tested in broth culture.

Colicin M used singly in concentrations of 1, 3.75, and 7.5 mg/L reduced bacterial populations by 2.5–5 cfu logs. Applied together, colicins M and E7 demonstrated synergistic effect for certain EHEC strains.

“These results suggest that using selective mixtures of two or more colicins with complementary or synergistic activities may allow for significant reductions in the amount of total colicin protein applied to food products​,” said the researchers.

Researchers said because compositions of plant-produced colicins are identical to those of native colicins produced by colicinogenic strains in environments, including the gastrointestinal (GI) tract of humans, colicins could use the GRAS (generally recognized as safe) regulatory approval pathway.

“To our knowledge we were first to suggest that plant-based production should be scalable and can yield colicins or colicin mixtures at price points low enough to make them attractive for industrial adoption as a novel food safety strategy.” ​

Source: Proceedings of the National Academy of Sciences

Online ahead of print, DOI: 10.1073/pnas.1513311112​

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

Authors: Steve Schulz, Anett Stephan, Simone Hahn, Luisa Bortesi, Franziska Jarczowski, Ulrike Bettmann, Anne-Katrin Paschke, Daniel Tusé, Chad H. Stahl, Anatoli Giritch and Yuri Gleba