New Approach to Defeating Gram-Negative Bugs

Ronald Woodard's team set out looking for a way to kill a stubborn type of bacteria and they succeeded---but not in the way he expected.

"We didn't get there the way we thought we'd get there, but in the end, we were right," said Woodard, chair of medicinal chemistry at the University of Michigan College of Pharmacy.

Woodard is senior author of an article describing way he and his research team genetically modified Escherichia coli bacteria, known as a Gram-negative bug, to weaken its defenses. That article appears in the recently released inaugural issue of the American Chemical Society's journal ACS Chemical Biology.

Some of the better-known Gram negatives are salmonella, gonorrhea, cholera and meningicoccal meningitis, along with the bacteria that caused the black plague.

Woodard and his collaborators worked on E. coli in part because it is one of the more common Gram-negative bacteria, and it is considered by researchers the gold standard of Gram-negative bacteria.

After their genetic modifications, E. coli was killed with just a fraction of the antibiotic dose typically needed. It was 512 times more susceptible to Rifampin, 256 times more vulnerable to Novobiocin, and eight times more susceptible to Bacitracin, suggesting doses could be dramatically cut and still be effective, Woodard said. Antibiotics typically only effective against Gram-positive bacteria could work against Gram-negative bacteria if a compound can be designed to mimic this genetic modification, Woodard said.

Also, E. coli can typically withstand the bile salts found in the human digestive tract, but by weakening it, Woodard's team found E. coli would die in the presence of normal levels of bile salts to which the bacteria would be exposed in the human gut.

Besides differing in how they respond to Gram's coloring test, Gram-positive and Gram-negative bacteria look different. Gram-positive cells are smooth on the outside, while Gram-negative cells have sugars and carbohydrates on the outside in structures that look like hairs.

That exterior protection is part of what makes Gram-negative bacteria harder to kill antibiotics, Woodard said.

Woodard's team set out to genetically modify the cells to eliminate the key sugar to which the hair anchors on the outside of the cell.

"Unfortunately, the bug didn't die," Woodard said. The researchers found that a "backup" gene from a different pathway also could form the anchor, so they knocked out that gene, as well. Initially the cell with both genomic knockouts did not survive without special nutritional supplements. Later, they were surprised to see that with different growth conditions, the cell began to grow again but without the hair-like structure.

The cells survived---but they looked a lot like Gram-positive cells, without all the sugars on the outside.

"We, as well as the entire scientific community, always thought Gram-negative cells could not survive without this external structure. This shows that is not true," Woodard said. Though they didn't die, they were weakened, and that made the cells an easy target for antibiotics.

Because Woodard suspected he might be flying in the face of conventional wisdom on bacteria, he solicited second opinions from the Borstel Research Center in Germany, which does a good deal of work on Gram-negative bacteria. Scientists there were initially skeptical, he said, but eventually, Uwe Mamat and Buko Lindner from Borstel signed on to the project and became co-authors of the current paper.

Other members of the team were U-M medicinal chemistry doctoral students Timothy Meredith and Parag Aggarwal. Meredith, lead author of the publication, has since joined Harvard Medical School as a researcher.

Aggarwal, Mamat and Woodard continue to work on the approach, encouraged by the potential of developing a safer way to treat patients. They hope their research leads to combination therapies, which include compounds that could duplicate the effect caused by the genetic mutation of bacteria together with low-dose antibiotics.

"Bugs are very smart," Woodard said. "It's not a matter of if a bug will become antibiotic resistant, but when. We have to work hard to get ahead of them."

Woodard's research is funded in part by a $2 million, five-year grant from the National Institutes of Health.

Source: University of Michigan