LONDON - A novel antisense technique is capable of inhibiting bacterial genes, including those responsible for resistance to antibiotics, researchers in Denmark have shown.

The method could lead to new ways of overcoming resistance to existing antibiotics. It also will help scientists trying to design new antimicrobials find which genes are crucial to bacterial survival.

The antisense agent used is called peptide nucleic acid (PNA). This is a DNA mimic composed of bases attached to a peptide backbone. PNA can hybridize with complementary DNA, RNA or PNA by establishing Watson and Crick base pairs and forming a helix. The peptide backbone makes the molecule very stable, as well as resistant to breakdown by cellular enzymes.

Liam Good and Peter Nielsen at the Center for Biomolecular Recognition in the Panum Institute of the University of Copenhagen, in Denmark, have demonstrated PNA targeted at the RNA for certain bacterial enzymes can specifically inhibit translation. They showed PNA designed to knock out the beta-lactamase gene of Escherichia coli was able to sensitize cells to the antibiotic ampicillin, to which they previously were resistant.

Nielsen, professor of biochemistry, told BioWorld International, “If this technique can be further developed, then this finding is of major importance. In principle, the technology can be used for any gene. If you want to determine what function a given gene has, it allows you to knock it out and see the phenotypic effect.“

Good and Nielsen report their findings in a paper in the April issue of Nature Biotechnology titled “Antisense inhibition of gene expression in bacteria by PNA targeted to mRNA.“

They first designed PNAs that targeted the start codon regions of the E. coli genes for beta-galactosidase and beta-lactamase. In an in vitro system, they showed the PNAs specifically inhibited the production of these enzymes. A control PNA with a different sequence of bases did not have this effect. Further tests in vitro also showed the inhibition observed was taking place at the level of translation rather than transcription.

Good and Nielsen wanted to be sure the inhibition was specific and caused by the PNA binding to the target site. So they introduced base substitutions (which caused only silent mutations) in the PNA target site within the beta-lactamase gene. When six of these base substitutions were present, the antisense PNA failed to inhibit translation of the gene, even at high concentrations.

When they then changed the PNA so its base sequence matched the mutated base sequence of the target site of the beta-lactamase gene, this was able to inhibit transcription of the gene - although it was unable to inhibit the wild-type gene.

These results confirm, the authors wrote, “that the observed inhibition involves a true antisense mechanism through PNA base pairing to the target site.“

The two researchers went on to show that if they added the two types of PNA to the culture medium of E. coli, the antisense agent could inhibit both genes. Again, they were able to demonstrate this inhibition took place by base pairing of the PNAs to the start codon regions of the genes.

Compounds Target Drug Resistance, Pathogenesis

The next experiment involved adding the anti-beta-lactamase PNA to cultures of E. coli carrying the beta-lactamase gene, which confers resistance to ampicillin. The following day, Good and Nielsen found that, in cultures containing the PNA, the number of bacteria capable of forming new colonies after dilution of the culture and plating out was reduced by up to seven orders of magnitude.

In their paper, Good and Nielsen concluded, “Our results showing that antisense PNAs can alter a bacterial growth phenotype suggest possibilities for developing novel antibiotics by designing PNAs to limit the expression of genes required for pathogenesis or drug resistance.“

This possibility seems particularly important, they added, in view of the increasing resistance to available antibiotics displayed by many pathogenic bacteria, although improved uptake of PNAs by the target organisms would be required.

Commenting on the paper in the same issue of Nature Biotechnology, David Ecker and Susan Freier, of Isis Pharmaceuticals Inc., in Carlsbad, Calif., wrote that it might be possible to improve the limited uptake of PNAs by bacterial cells by chemically modifying the molecules.

They added, “Chemical modification and formulation of oligonucleotides have resulted in successful delivery of oligonucleotides. Similar success should be possible for delivery of PNAs to bacteria.“

Nielsen said this is next on his agenda: “Now that we know that we have something that - when it gets into bacterial cells - is very potent, we will try other formulations and chemical modifications to see whether we can increase the uptake.“ *

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