The mechanism of action for traditional antibiotic compounds has been well characterized over the past several decades. However, how bacteria can survive the drug onslaught and “rebuild” has not been fully understood. Uncovering these enigmatic mechanisms could hold the key to elucidating the sources of antibiotic resistance. Now, investigators at the University of Notre Dame have illuminated how a single enzyme helps bacteria rebound from damage inflicted by antibiotics not strong enough to immediately eliminate the microbes. Findings from the new study were published recently in PNAS through an article entitled “Exolytic and endolytic turnover of peptidoglycan by lytic transglycosylase Slt of Pseudomonas aeruginosa.”
This new study focuses on an enzyme in the gram-negative bacterium Pseudomonas aeruginosa, a pathogen that causes pneumonia and sepsis. The enzyme, called lytic transglycosylase Slt, rapidly attempts repair of the organism's cell wall, which allows the bacterium to survive and infection to proceed unabated.
“It's a survival strategy,” noted senior study investigator Shahriar Mobashery, Ph.D., professor of bioorganic chemistry and biochemistry at Notre Dame. “The cell wall is the structural entity that encases the entire bacterium, and its health is critical for the survival of the bacteria. If you have a drug that inflicts damage to the cell wall, the bacterium cannot cope with it, and it dies.”
P. aeruginosa is often called a “nightmare bacteria” and has been highlighted in a recent report from the Centers for Disease Control and Prevention. The report stated that lab tests had found “unusual resistance more than 200 times in 2017 in 'nightmare bacteria' alone.”
Structurally, P. aeruginosa has cell walls made of long, adjacent units that are cross-linked together. In the presence of a beta-lactam antibiotic, the cross-links are not formed. However, long chains of uncross-linked polymers remain, which signal that the cell wall is damaged. That is where Slt comes in. The enzyme recognizes the damage and chops down the long chains of uncross-linked polymers, and the organism rebuilds the cell wall.
“It's sort of like if you're driving home and get into a fender bender, and by the time you get home, your car is already repaired,” Dr. Mobashery added.
Scientists have known about both families of enzymes for some time. Dr. Mobashery's and his team synthesized pieces of the cell wall and studied them with Slt to determine how the enzyme degradation process works. Moreover, the scientists sent purified Slt and cell-wall samples to collaborators at the Spanish National Research Council to determine its structure.
“We documented in this report that Slt turns over the peptidoglycan by both exolytic and endolytic reactions, which cause glycosidic bond scission from a terminus or in the middle of the peptidoglycan, respectively,” the authors wrote. “These reactions were characterized with complex synthetic peptidoglycan fragments that ranged in size from tetrasaccharides to octasaccharides.”
The authors continued: “the X-ray structure of the wild-type apo Slt revealed it to be a doughnut-shaped protein. In a series of six additional X-ray crystal structures, we provide insights with authentic substrates into how Slt is enabled for catalysis for both the endolytic and exolytic reactions. The substrate for the exolytic reaction binds Slt in a canonical arrangement and reveals how both the glycan chain and the peptide stems are recognized by the Slt.”
Dr. Mobashery has studied antibiotic resistance for 30 years. He said penicillin-binding proteins had been studied since the 1960s and lytic transglycosylases since the 1990s—but the issue of how they come together is new. Because of antibiotic resistance, this bacterium has become one of the most difficult bacterial pathogens to treat.
