If you’ve ever been to the doctor and diagnosed with a bacterial infection, you were probably prescribed an antibiotic. From their discovery in the early 1900s, antibiotics were defined as inhibiting the growth of or killing bacteria via a chemical substance (Waksman, 1947).
The most prevalent antibiotics are B-lactam derivatives which target penicillin binding proteins, inhibiting peptidoglycan synthesis and resulting in cell lysis.
Antibiotic resistance’s prevalence came about in the 1930s, beginning with cases of antibiotic resistant syphilis in Western Europe and increased toward the end of the decade (Beckh and Vulgar 1939). Since then, it has become one of the world’s leading public health issues (WHO). This has been due to the incorporation of antibiotics into farming as well as the sanitation conditions in developed countries (NIH). Specifically, the overuse of antibiotics allows the non resistant bacteria to be killed, and the resistant survive and pass on the gene to the next generations. There have been many efforts to efficiently combat this antibiotic resistance: intracellular antibiotic delivery, lipopeptide antibiotic tripopeptin C, and B-lactamase inhibitors (Laws et al, 2019). The specific mechanism of antibiotic resistance occurs via B-lactamase, which hydrolyzes the B-lactam ring by cleaving the amide.
Recently, antisense therapy has been used as a method of combating antibiotic resistance when these have proved to show poor efficacy (Chan et al., 2006). This constitutes the use of oligonucleotides, which are short sequences of synthetic DNA. They are known as oligonucleotides (ONs) or antisense oligonucleotides (ASOs). They target certain mRNA sequences by binding them via a complementary sequence of a gene.
They can either work within the nucleus preventing 5’ cap formation and telomere addition, or in the cytosol by binding to the Shine-Dalgarno sequence and blocking the ribosome from transcribing the gene. However, the largest issue occurs when the oligonucleotide can not enter into the cell to reach the mRNA due to its large phosphate backbone not being able to cross the membrane. This led to the authors’ decision to attempt lipid conjugation of the molecule (Kauss et al, 2020).
In solution, oligonucleotides remain aqueous and therefore cannot pass through the membrane. Lipid oligonucleotides will form micelles, or an aggregate of the molecule where the hydrophobic parts will surround the outside with the hydrophilic parts facing the inside. This in turn allows the molecule to pass through the hydrophobic core of the lipid bilayer. As demonstrated by Karaki et al, lipid conjugated oligonucleotides enter the cell by endocytosis, and at a much greater rate than non conjugated molecules (2017). They were able to decrease tumor proteins that led to prostate cancer cell characteristics.
Cephalosporins are one of the major classes of antibiotics, which can be targeted by B-lactamases. Using lipid oligonucleotides to target the B-lactamase mRNA is proposed to restore antibiotic sensitivity by blocking its transcription and enzyme production.
The authors used a sensitive and resistant laboratory strain as well as another resistant clinical strain to demonstrate their findings. First, they tested the viability of cells in the presence of oligonucleotides and lipid oligonucleotides respectively. The sensitive strain’s reaction to cephalosporins was unaffected by either oligonucleotides. However, both the laboratory and clinical resistant strains had a decreased minimum inhibitory concentration (MIC) with the lipid oligonucleotides, meaning that a lower concentration of antibiotic was able to inhibit the cells. Their control was a scrambled sequence of nucleotides that would not target specific mRNA, and that did not have any effect on the MIC.
They experimented with structure in a few different ways in order to engineer the best possible molecule. They tagged the lipid onto the 5’ and 3’ ends and found that the 3’ end increased the MIC slightly. This led them to use the 5’ version as it had the greatest efficacy of decreasing MIC. Additionally, they altered the number of base pairs and found that the 19-25 bp range produced the lowest MIC.
Then, they sought visual proof that the lipid oligonucleotide entered the cell. To visualize intra bacterial localization they tagged LON with Cyanine 5 fluorescent dye and analyzed by fluorescent microscopy.
In order to track the B-lactamase activity they used a cephalosporin called nitrocefin that changes color from yellow to red when hydrolyzed. They measured the absorption at 492 nm with varied oligonucleotide concentrations. They found that 5 uM lipid oligonucleotide hydrolyzed the least antibiotic, displaying that it was the most effective concentration. However, the figure does not show if more than 5 uM was tested, so we do not know if there was a more effective concentration or if increasing the concentration did not decrease MIC after 5 uM.
RT-PCR and western blotting on the transcripts and protein products was performed to test expression. Similar levels were present when the nonconjugated versus the lipid oligonucleotide were used, indicating that the lipid conjugated molecule did not affect the production of B-lactamase compared to untreated control. A specific mechanism was not investigated by the authors, but it is probable the oligonucleotide produces some off-target pathway based on the decreased MIC but lack of change in transcript concentration.
Ultimately the authors were able to demonstrate that lipid oligonucleotides provided increased efficacy of antibiotics by decreasing B-lactamase activity in hydrolyzing B-lactam rings. They designed their molecule through trial and error and deduced that lowering the minimum inhibitory concentration is LON concentration-dependent. This was the first time this was performed in a gram negative species, the lipid micelle able to enter the cell by endocytosis through the peptidoglycan and cell wall layers. This technique of lipid conjugation could lead to discoveries in drug delivery across cell membranes as well as further advancements in combating antibiotic resistance if the mechanism is investigated.
Original article: Kauss, T., Arpin, C., Bientz, L. et al. Lipid oligonucleotides as a new strategy for tackling the antibiotic resistance. Sci Rep10, 1054 (2020). https://doi.org/10.1038/s41598-020-58047-x
Waksman, Selman A. “What Is an Antibiotic or an Antibiotic Substance?” Mycologia, vol. 39, no. 5, 1947, pp. 565–569. JSTOR, www.jstor.org/stable/3755196. Accessed 5 Feb. 2020.
World Health Organization Antibiotic Resistance. https://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance
Causes of Antimicrobial (Drug) Resistance. https://www.niaid.nih.gov/research/antimicrobial-resistance-causes
Mark Laws, Ali Shaaban, Khondaker Miraz Rahman, Antibiotic resistance breakers: current approaches and future directions, FEMS Microbiology Reviews, Volume 43, Issue 5, September 2019, Pages 490–516, https://doi.org/10.1093/femsre/fuz014
Chan, J. H. P., Lim, S. & Wong, W. S. F. Antisense oligonucleotides: from design to therapeutic application. Clin. Exp. Pharmacol. Physiol. 33, 533–540 (2006).
Karaki, S., Benizri, S., Mejías, R., Baylot, V., Branger, N., Nguyen, T., Vialet, B., Oumzil, K., Barthélémy, P., and Rocchi, P. (2017) Lipid-oligonucleotide conjugates improve cellular uptake and efficiency of TCTP-antisense in castration-resistant prostate cancer. Journal of Controlled Release 258, 1–9.
Beckh W, Kulchar GV. Treatment-Resistant Syphilis: An Evaluation Of The Causative Factors In Eighteen Cases. Arch Derm Syphilol. 1939;40(1):1–12. doi:10.1001/archderm.1939.01490010004001