New model for antibacterial mechanism

Date:
April 29, 2022
Source:
DOE/Brookhaven National Laboratory
Summary:
Biologists have discovered an aberrant protein that's deadly
to bacteria.

This erroneously built protein mimics the action of aminoglycosides,
a class of antibiotics. 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.



FULL STORY ========================================================================== Biologists at the U.S. Department of Energy's Brookhaven National
Laboratory and their collaborators have discovered an aberrant protein
that's deadly to bacteria. In a paper just published in the journalPLOS
ONE, the scientists describe how this erroneously built protein mimics the action of aminoglycosides, a class of antibiotics. 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.


========================================================================== "Identifying new targets in bacteria and alternative strategies to
control bacterial growth is going to become increasingly important,"
said Brookhaven biologist Paul Freimuth, 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." Following an interesting branch The Brookhaven scientists, who normally
focus on energy-related research, weren't thinking about human health
when they began this project. They were using E. coli bacteria to study
genes involved in building plant cell walls.

That research could help scientists learn how to convert plant matter
(biomass) into biofuels more efficiently.

But when they turned on expression of one particular plant gene, enabling
the bacteria to make the protein, the cells stopped growing immediately.



========================================================================== "This protein had an acutely toxic effect on the cells. All the cells
died within minutes of turning on expression of this gene," Freimuth said.

Understanding the basis for this rapid inhibition of cell growth made
an ideal research project for summer interns working in Freimuth's lab.

"Interns could run experiments and see the effects within a single day,"
he said. And maybe they could help figure out why a plant protein would
cause such dramatic damage.

Misread code, unfolded proteins "That's when it really started to get interesting," Freimuth said.



==========================================================================
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 gibberish 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.

That wouldn't be too farfetched. Nonsense strands of amino acids can't
fold up properly to become fully functional. Although misfolded proteins
get produced in all cells by chance errors, they usually are detected
and eliminated completely by "quality control" machinery in healthy
cells. Breakdown of quality control systems could make aberrant proteins accumulate, causing disease.

Messed-up quality control The next step was to find out if the aberrant
plant protein could activate the bacterial cells' quality control system
-- or somehow block that system from working.

Freimuth and his team found that the aberrant plant protein indeed
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.

Model for mechanism These experiments indicated that the single aberrant
plant protein killed cells by the same mechanism as the complex mixture
of aberrant proteins induced by aminoglycoside antibiotics. But the
precise mechanism of cell death is still a mystery.

"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.

Scientists know that cells treated with the antibiotics become leaky,
allowing things like salts to seep in at toxic levels. One hypothesis is
that the misfolded proteins might form new channels in cellular membranes,
or alternatively jam open the gates of existing channels, allowing
diffusion of salts and other toxic substances across the cell membrane.

"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.

That would help advance understanding of how the aberrant proteins induced
by aminoglycoside antibiotics kill bacterial cells -- and could inform
the design of new drugs to trigger the same or similar effects.

This work was supported by a Laboratory Directed Research and Development
award from Brookhaven Lab and in part by the DOE Office of Science,
Office of Workforce Development for Teachers and Scientists (WDTS) under
the Visiting Faculty Program (VFP). Additional funding from the National Science Foundation (NSF) supported students participating in internships
under NSF's Science, Technology, Engineering, and Mathematics Talent
Expansion Program (STEP) and the Louis Stokes Alliances for Minority Participation (LSAMP) program.


========================================================================== Story Source: Materials provided by
DOE/Brookhaven_National_Laboratory. Note: Content may be edited for
style and length.


========================================================================== Journal Reference:
1. Mangala Tawde, Abdelaziz Bior, Michael Feiss, Feiyue Teng,
Paul Freimuth.

A polypeptide model for toxic aberrant proteins induced by
aminoglycoside antibiotics. PLOS ONE, 2022; 17 (4): e0258794 DOI:
10.1371/ journal.pone.0258794 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2022/04/220429144857.htm

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