Sep. 18, 2001 DURHAM, N.C. -- Duke biochemists have identified a key chemical reaction by which some important virulent bacteria alter their outer coat to make it antibiotic-resistant. The scientists say that their finding could lead to drugs to block such protective alteration, preventing bacteria from developing resistance.
Antibiotic resistance is a major public health threat worldwide, with commonly used antibiotics proving less and less effective. According to the World Health Organization, the rapid rise of antibiotic resistance threatens to profoundly undermine the dramatic breakthroughs made in medical science over the past 50 years see http://www.who.int/emc/amr.html.
Duke University Medical Center biochemist Christian Raetz and his colleagues report their discovery in three papers scheduled for publication in the Journal of Biological Chemistry, and now posted on its Web site http://www.jbc.org/. Raetz is the George Barth Geller Professor and chairman of biochemistry. Lead author on the papers is postdoctoral fellow M. Stephen Trent, with co-authors Anthony Ribeiro, Shanhua Lin and Robert Cotter. The research was supported by the National Institutes of Health.
Raetz, Trent and their colleagues report discovery of a new enzyme that attaches an unusual sugar molecule, called aminoarabinose, to the key lipid molecule (known as lipid A) that constitutes much of the outer coat of gram negative bacteria -- including disease-causing E. coli, Salmonella and Pseudomonas. Attachment of aminoarabinose to lipid A serves to reduce the net negative charge on the bacterial coat. This coat alteration reduces the ability of positively charged antibiotics like polymyxin or certain peptides, to attach to the outer coat -- a key step in the antibiotic's process of piercing the bacterial coat to kill it.
According to Raetz, positively charged peptides are components of many antibiotics, including those employed by the body's innate immune system, as a first-strike weapon against bacteria, and by bacteria themselves in their continual war against one another for survival.
"It was suspected that aminoarabinose was involved in the resistance, but how it is made, and the enzyme that attaches it were not known," Raetz said. "So, we have discovered the enzyme that attaches this modifying group to lipid A, as well as a novel precursor molecule (named undecaprenyl phosphate-aminoarabinose) that donates this aminoarabinose to lipid A.
"Although much is known about the varied mechanisms of drug-resistance, we have found a new one that uses novel natural products and novel enzymes that have not been described before in the biochemical literature," he said. According to Raetz, the discovery not only offers new fundamental insight into bacterial drug resistance, but a potential approach to overcoming it.
"It might be possible to redesign peptide antibiotics to work even against bacteria with aminoarabinose attached to their lipid A," he said. "Also, one could imagine devising inhibitors of our aminoarabinose transferase enzyme that would render polymyxin resistant mutants sensitive again."
In their experiments, the Duke biochemists studied the mechanism behind resistance to the antibiotic polymyxin, which is normally used as a topical ointment against skin infections. Thus, for example, said Raetz, adding a transferase inhibitor to a polymyxin ointment might make the polymyxin more effective.
The scientists searched for the transferase enzyme by analyzing a polymyxin-resistant strain of Salmonella. Previous studies by other scientists had found that such resistance is associated with activation of several genes of unknown function in a particular region of the bacterial genome. Using genetic and biochemical analyses, the scientists were able to pinpoint within that region the specific gene coding for a protein that is responsible for transferring aminoarabinose to lipid A, which they called arnT. The scientists also identified a highly similar version of the arnT gene in resistant strains of E. coli.
The scientists also identified the lipid donor of the aminoarabinose molecule, by comparing minor lipids in strains of the gut bacterium E. coli that were both polymyxin sensitive and resistant. This identification of the donor further elucidated the pathway by which the bacteria "armor" themselves against antibiotic peptides. In future studies, the scientists will trace the full metabolic pathway, said Raetz, which could yield additional enzyme targets for inhibitory drugs.
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