Antibiotics are used in modern medicine to treat infections by impeding targets inside bacterial cells. Antibiotics attach to precise locations on enzyme targets once they are within these cells, preventing the development of bacteria. These targets' genes naturally experience mutations, which can sometimes make the target more difficult for an antibiotic to latch onto and the resulting bacterial variant resistant to treatment.
The likelihood that bacterial populations could develop mutants resistant to current antibiotics increases with the amount of antibiotics administered over time, making the need for novel techniques to prevent the treatments from becoming ineffective even more essential. For decades, scientists have researched resistant mutants in the hopes that similar processes could guide the development of novel therapies to overcome resistance.
Despite this need, efforts have been limited as only a small portion of the possible mutations can be attributed to naturally occurring resistant mutants, with most drug binding-site mutations to date having been overlooked.
The bacterial species Escherichia coli, which the antibiotic rifampicin renders inactive by attaching to an essential bacterial enzyme known as RNA polymerase (RNAP), presents a challenge that has recently been addressed by a new study led by researchers at NYU Grossman School of Medicine.
By substituting each of the 20 amino acid alternatives found in nature for one of the 38 amino acid components that make up the rifampicin binding site on E. coli, the study authors were able to produce 760 distinct RNAP mutants. The development of this mutant pool was then examined under various circumstances, including rifampicin therapy.
The study discovered two mutants, L521Y and T525D, which are extremely sensitive to rifampicin. It was published online on August 30th, 2023 in the journal Nature. The antibiotic not only stops the growth of these mutants, but it also almost completely wipes off the mutant bacterial populations.
The scientists call this a significant discovery because rifampicin typically only inhibits the growth of bacterial pathogens like E. coli and many others, not killing them.
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.”
Evgeny A. Nudler, Ph.D., Julie Wilson Anderson Professor of Biochemistry, Department of Biochemistry and Molecular Pharmacology, NYU Langone Health
Nudler added, “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
As RNAP constructs the RNA chains that direct the synthesis of proteins from amino acids, E. coli changes the genetic instructions it saves in DNA chains into comparable genetic material. Rifampicin kills bacteria by delaying RNAP, which results in collisions between it and cellular machinery that duplicates DNA when cells divide and grow.
This was discovered by the mutants made in the latest study. This results in fatal breaks in the DNA of the bacterial cells on both strands.
Some of the E. coli RNAP binding site alterations were shown to significantly speed up the rate at which RNAP manufactures RNA and, therefore, the rate at which it depletes resources, including nucleotide building blocks like pyrimidines.
According to the researchers, the study has important ramifications for the understanding of the mechanism of action of nucleotide analogs like the anti-cancer medication 5FU. According to them, developing new combination treatments could profit from an understanding of how nucleotide depletion makes cells more sensitive to nucleotide supply.
These techniques could be applied to map the binding sites of other drug types, and especially to those vulnerable to resistance.
Aviram Rasouly, Ph.D., Study Co-Senior Investigator and Research Scientist, NYU Langone Health
Yang, K. B., et al. (2023). High-resolution landscape of an antibiotic binding site. Nature. doi.org/10.1038/s41586-023-06495-6