Protein May Serve as Potential Model for Antibacterial Mechanism

Scientists at the U.S. Department of Energy’s Brookhaven National Laboratory and their collaborators have discovered a protein that’s deadly to bacteria. The newly discovered protein could serve as a model to help scientists unravel details of those drugs’ lethal effects on bacteria—and potentially point the way to future antibiotics.

The new findings are described in the journal PLOS ONE, in a paper titled, “A polypeptide model for toxic aberrant proteins induced by aminoglycoside antibiotics.”

“Aminoglycoside antibiotics interfere with the selection of cognate tRNAs during translation, resulting in the synthesis of aberrant proteins that are the ultimate cause of cell death,” the researchers wrote. “However, the toxic potential of aberrant proteins and how they avoid degradation by the cell’s protein quality control (QC) machinery are not understood. Here we report that levels of the heat shock (HS) transcription factor σ32 increased sharply following exposure of Escherichia coli to the aminoglycoside kanamycin (Kan), suggesting that at least some of the aberrant proteins synthesized in these cells were recognized as substrates by DnaK, a molecular chaperone that regulates the HS response, the major protein QC pathway in bacteria.”

“Identifying new targets in bacteria and alternative strategies to control bacterial growth is going to become increasingly important,” said Brookhaven biologist Paul Freimuth, PhD, who led the research. Bacteria have been developing resistance to many commonly used drugs, and many scientists and doctors have been concerned about the potential for large-scale outbreaks triggered by these antibiotic-resistant bacteria, he explained.

“What we’ve discovered is a long way from becoming a drug, but the first step is to understand the mechanism,” Freimuth said. “We’ve identified a single protein that mimics the effect of a complex mixture of aberrant proteins made when bacteria are treated with aminoglycosides. That gives us a way to study the mechanism that kills the bacterial cells. Then maybe a new family of inhibitors could be developed to do the same thing.”

The group discovered that the toxic factor wasn’t a plant protein at all. It was a strand of amino acids, the building blocks of proteins, that made no sense.

This nonsense strand had been churned out by mistake when the bacteria’s ribosomes (the cells’ protein-making machinery) translated the letters that make up the genetic code “out of phase.” Instead of reading the code in chunks of three letters that code for a particular amino acid, the ribosome read only the second two letters of one chunk plus the first letter of the next triplet. That resulted in putting the wrong amino acids in place.

“It would be like reading a sentence starting at the middle of each word and joining it to the first half of the next word to produce a string of gibberish,” Freimuth said.

The protein reminded Freimuth of a class of antibiotics called aminoglycosides. These antibiotics force ribosomes to make similar “phasing” mistakes and other sorts of errors when building proteins. The result: all the bacteria’s ribosomes make gibberish proteins.

“If a bacterial cell has 50,000 ribosomes, each one churning out a different aberrant protein, does the toxic effect result from one specific aberrant protein or from a combination of many? This question emerged decades ago and had never been resolved,” Freimuth said.

The new research shows that just a single aberrant protein can be sufficient for the toxic effect.

Freimuth and his team found that the aberrant plant protein activated the initial step in protein quality control, but that later stages of the process directly required for degradation of aberrant proteins were blocked. They also discovered that the difference between cell life and death was dependent on the rate at which the aberrant protein was produced.

“When cells contained many copies of the gene coding for the aberrant plant protein, the quality control machinery detected the protein but was unable to fully degrade it,” Freimuth said. “When we reduced the number of gene copies, however, the quality control machinery was able to eliminate the toxic protein and the cells survived.”

The same thing happens, he noted, in cells treated with sublethal doses of aminoglycoside antibiotics. “The quality control response was strongly activated, but the cells still were able to continue to grow,” he said.

“The good news is that now we have a single protein, with a known amino acid sequence, that we can use as a model to explore that mechanism,” Freimuth said.

“A next step would be to determine structures of our protein in complex with membrane channels, to investigate how the protein might inhibit normal channel function,” Freimuth said.