Lipid Biotechnology: the War on Antibiotic Resistance

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).

Waksman, Selman A. “What Is an Antibiotic or an Antibiotic Substance?” Mycologia, vol. 39, no. 5, 1947, pp. 565–569. JSTOR, Accessed 5 Feb. 2020. 

World Health Organization Antibiotic Resistance.

Causes of Antimicrobial (Drug) Resistance.

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,

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

4 Replies to “Lipid Biotechnology: the War on Antibiotic Resistance”

  1. I am curious as to how this therapy may be utilized in gram-positive bacteria as they are not as privileged to exogenous substance uptake due to their thick peptidoglycan layer. You mentioned that it is unknown if greater than 5 µM lipid conjugate had a greater effect, however it is unlikely that this is the case as they would have certainly reported such data if it were the case. You also mention the possibility of an off-target effect due to decreased MIC despite the same concentrations of beta-lactamase mRNA. It is possible that the antisense RNA was simply binding the mRNA and not targeting it for degradation, simply blocking translation. This is somewhat rarer as these types of modifications are typically destabilizing. I am curious if they specifically designed this antisense RNA with any RISC recruiting motifs, that could be exactly what they need to boost the efficacy of such a therapy.

  2. Hi Victoria!

    This was a terrific post! You do an excellent job of describing the science in this paper as well as the authors’ motivations for doing what they did at each step along the way. This method of “shuttling” therapeutics, as it is often called, seems to be a common motif in the medicinal chemistry and molecular pharmacology literature (especially for diseases for which treatment is largely precluded due to the Blood-Brain-Barrier, such as Alzheimer’s or HIV viral reservoirs). I think this is another terrific example of how simple application of fundamental biochemical principles is used to effectively inform drug design. I loved the authors use of oligonucleotides (a la RNAi) to perturb translation of the enzyme implicated in clinical resistance. One really cool feature of a therapy like this is that given the limited size of the human genome, a LON that incorporates an oligo with a sequence of sufficient length (and thus variation) should in principle have VERY LIMITED (if not virtually zero) potential for off target interactions with any other mRNAs in the cytosol. Compared to the prospect of delivering a traditional enzyme-targeted inhibitor, this seems like it ought to improve the specificity (and therefore the safety) of a drug like this.

    In response to your speculation about why the authors’ spectroscopic assay to measure the relative amounts of antibiotic breakdown did not keep on going beyond 5 uM (or at least did not present data for it), my thought was this: In this preclinical context, it was sufficient for the authors to demonstrate the dose-dependent behavior of the LON they were delivering. Certainly, further study involving infected mammalian cell lines would be necessary to determine whether it can enable circumvention of antibiotic resistance for bacteria that have affected animal cells. I’m curious, in your background research did you come across anything that indicated previous attempts by either this group or others to use ASOs in humans or any other animals, or even other strains of bacteria? I know the authors’ precedent for their work here was the enhanced uptake of lipid-conjugated ASOs in prostate cancer cell lines, but the difficulty of effectively delivering this drug, getting it taken into one cell (the host), and then getting it uptaken into yet another cell (the pathogen) seems like an entirely different problem in and of itself.

    – Eli

  3. The topic of controlling antibiotic resistance is particularly interesting and undoubtedly increase the efficacy of antibiotics across the board. I am now interested in whether or not we might end up seeing some mutant strains of bacteria develop with a resistance to certain lipid oligonucleotides like the very resistance it is trying combat for antibiotics. Chances of this occurring may actually be effectively nonexistent as specific misuse of lipid oligonucleotide on its own probably won’t select for lipid oligonucleotide resistant bacteria as it doesn’t actually harm the bacteria as antibiotics do and that misuse of the compound may be the only real way resistant strains of bacteria can really appear. I suppose in that sense my question is more one of evolutionary biology than anything else.

  4. Hi Vic!! You did a great job of explaining the goals and experiments of the paper. I received a very good understanding of the paper by reading your summary. The large interest in the antibiotic shuttling across membranes is so important because there has been a great increase in overall antibiotic-resistant bacteria. I wonder if the use of lipid oligonucleotides has been tested against something more persistent like MRSA, since it is such a widely resistant Gram-positive bacteria. Although MRSA is resistant to beta-lactam antibiotics, I wonder if the shuttling of the antibiotic with the use of lipid oligonucleotides has a slight chance of success. Perhaps this paper can serve as a stepping stone into more research on that.

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