NEW
YORK, Aug. 30, 2023 /PRNewswire/ -- Modern
medicine depends on antibiotics to treat infections by disabling
targets inside bacterial cells. Once inside these cells,
antibiotics bind to certain sites on specific enzyme targets to
stop bacterial growth. Randomly occurring changes (mutations) in
the genes for these targets occur naturally, in some cases making
the target harder for the antibiotic to attach to, and that
bacterial version resistant to treatment.
For this reason, the more antibiotics have been used over time,
the greater the chances that bacterial populations will evolve to
have mutants resistant to existing antibiotics, and the more urgent
the call for new approaches to keep the treatments from becoming
obsolete. Researchers have for decades studied resistant mutants in
hopes that related mechanisms would guide the design of new
treatments to overcome resistance. The effort has been limited,
however, because naturally occurring resistant mutants represent a
small fraction of the mutations that could possibly occur (the
complete mutational space), with most drug binding-site mutations
to date having been overlooked.
To address this challenge, a new study led by researchers at NYU
Grossman School of Medicine applied a technology called MAGE
(Multiplex Automated Genome Engineering) to generate the full
inventory of mutations in the bacterial species Escherichia
coli where the antibiotic rifampicin attaches to and disables
an essential bacterial enzyme known as RNA polymerase (RNAP). The
study authors created 760 unique RNAP mutants by replacing each of
the 38 amino acid building blocks that make up the rifampicin
binding site on E. coli with each of the twenty amino acid
options present in nature. Growth of this mutant pool was then
tested under different conditions, including treatment with
rifampicin.
Published online August 30 in the
journal Nature, the study found two mutants, L521Y and
T525D, that are hyper-sensitive to rifampicin. Not only does the
antibiotic prevent these mutants from growing, it nearly
obliterates the mutant bacterial populations. This is a remarkable
finding, say the authors, because rifampicin normally does not kill
E. coli, or many other bacterial pathogens, but only stops
their growth.
"This work provides a map of antibiotic-bacterial RNAP
interactions that will be of value to chemists working to build on
the study effects by changing, not bacterial binding site residues,
but instead the structure of rifampicin and other antibiotics so
that they bind tighter for increased potency," says study co-senior
investigator Evgeny Nudler, PhD, The
Julie Wilson Anderson Professor of Biochemistry, in the Department
of Biochemistry and Molecular Pharmacology, at NYU Langone Health.
"Our findings also suggest ways of improving rifampicin's ability
to bind to proteobacteria, actinobacter and firmicutes, bacterial
groups that include natural RNAP mutations that render them
vulnerable to rifampicin."
How Rifampicin Kills Bacteria
E. coli stores genetic instructions in DNA chains, but
then converts them into a related genetic material in RNA, with
RNAP building the RNA chains that guide the building of proteins
out of amino acids. The mutants created in the new study revealed
that rifampicin kills bacteria by stalling RNAP, and so causing
collisions between it and cellular machinery that operates in the
same molecular space to duplicate DNA as cells divide and multiply.
This in turn causes lethal breaks in both strands of bacterial
DNA.
In other insights from the study, some of the E. coli
RNAP binding site mutations were found to greatly increase the
speed with which RNAP builds RNA, and so the speed that it uses up
raw materials, including nucleotide building blocks like
pyrimidines. The work has significant implications, say the
researchers, for the understanding of the mechanism of action used
by nucleotide analogues like the anti-cancer drug 5FU.
Understanding how nucleotide depletion sensitizes cells to
nucleotide supplies may help in the design of new combination
therapies, they say.
"These techniques could be applied to map the binding sites of
other drug types, and especially to those vulnerable to
resistance," says co-senior study investigator, Aviram Rasouly, PhD, a research scientist at NYU
Langone.
Funding support for the study was provided through National
Institute of Health grants T32 AI007180 and R01GM126891 and the
Blavatnik Family Foundation. The study was led by MD-PhD student
Kevin Yang. Other NYU Langone
researchers involved in this study were Maria Cameranesi, Criseyda Martinez, Manjunath Gowder, Yosef
Shamovsky, Vitaliy Epshtein,
Khaled Alzoubi, Zhitai Hao, and
Ilya Shamovsky. Evgeny Nudler is also an investigator with the
Howard Hughes Medical Institute.
Contact:
Gregory Williams
gregory.williams@nyulangone.org
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SOURCE NYU Grossman School of Medicine and NYU Langone
Health
