Dr Luke Clifton, Instrument Scientist at ISIS Neutron and Muon Source, explains the molecular mechanism behind antibiotics.
Since the initial large-scale production and widespread use of antibiotics in the 1940s there has been a continual race between the development of antibiotics and bacterial resistance to them1,2. Alexander Fleming, the discoverer of penicillin, warned, as early as 1945, against the overuse of antibiotics, suggesting it would select for resistant bacterial species3 – in fact, penicillin-resistant bacteria were identified before penicillin was approved for use1,4. However, this did not become a clinical problem until the 1950s, at which point it was solved by the development of new antibiotics2.
Bacterial resistance has since returned as a major issue. Since the late 1990s there have been relatively few new commercial antibiotics developed1.
Until recently many large pharmaceutical companies had stopped antibiotic research as the returns on investment were too low and regulatory requirements too stringent3. Concurrently, due to the overuse of antibiotics in agriculture and misuse in medicine, antibiotic resistant bacterial species have essentially been selected for both inside the hospital environment and beyond2. Anti-microbial resistance (AMR) has now been recognised by the World Health Organisation as one of the biggest threats to global health, food security and development5.
Bacteria are single celled organisms which are separated from their external environment by their biological membrane, a thin bilayer of lipids with embedded and bound proteins, and in the case of Gram-positive bacteria an additional thick layer of peptidoglycan, a polymer mesh, above the membrane.
The two main classes of bacteria are separated based on their differing cell surface architecture. Gram positive bacteria are named as the Gram stain binds to its thick peptidoglycan cell surface coating, whereas Gram negative bacteria only have a thin peptidoglycan between its two membranes so does not stain.
The surface architecture of Gram-negative bacteria is unique, with two membranes separating the bacteria from its external environment, the outer most of which is asymmetric in terms of its lipid composition. The make-up of the bacterial membranes is quite different to animal, plant, and fungal cells.
Since the 1940s a large variety of antibiotics have come into widespread use, which achieve their effects through a range of methods. Certain antimicrobial agents work by targeting specific processes within bacteria to halt their growth and ultimately eliminate them. For example, some drugs like penicillin, carbapenem, and vancomycin disrupt the synthesis of bacterial cell walls. Others, like tetracycline and aminoglycosides, inhibit the function of ribosomes, the cellular machinery responsible for protein production. Additionally, drugs like quinolones and rifampin disrupt the synthesis of nucleic acids, which are essential for bacterial DNA replication. By understanding how these different antimicrobial agents act on bacteria, scientists can develop more effective treatments to combat infections6.
The critical importance of membrane disruption
Antibiotics like daptomycin and colistin have another way of dealing with bacteria. These directly target the bacterial membranes, the protective outer layer of the bacteria.
Colistin works specifically against Gram-negative bacteria by interacting with lipopolysaccharides in their outer membrane7, causing the membrane to break down like a detergent. On the other hand, daptomycin works against Gram-positive bacteria by forming tiny pores in their membranes, leading to cell leakage and death8.
Crucially, disrupting bacterial membranes is a more fundamental way to fight infections compared to targeting specific biochemical pathways. This means bacteria may have a harder time developing resistance to these types of antibiotics, making them appealing for new treatments.
However, there is a downside. Some of these antibiotics, like colistin, can also harm our own cells in high doses, making them a last-resort option when no other antibiotics work. Researchers are still working on finding ways to use these antibiotics safely while taking advantage of their powerful antimicrobial properties.
To develop innovative antibiotics, understanding how antibiotics function at the molecular level as well as their interaction with mammalian membranes provides key information on how drugs interact with both the membranes of bacteria, and how they might affect our own cells’ membranes.
Harnessing neutrons to investigate membranes and antibiotics
Neutron beams and biological membranes may seem an odd coupling. Neutrons are sub-atomic particles without charge, theorised by Earnest Rutherford (when he proposed the structure of the atom) in the early 20th century and discovered by James Chadwick in 1932. Non-nucleus bound neutrons are produced during nuclear fission and their use as a probe of atomic and molecular level structure was essentially a by-product of nuclear energy and weapon research. By the 1970s dedicated facilities were being built just for neutron scattering studies which offered a range of different neutron scattering based analytical techniques.
Neutron scattering is a useful tool for examining antimicrobial/membrane interactions where the structure of a model pathogen membrane can be examined prior to and after an antibiotic interaction and the changes brought about by the drug can be deciphered.
Due to the unique ability to remove the contribution of individual structural components within the neutron scattering data through the collection of data sets in differing hydrogen isotope conditions. This allows a picture of the relative distribution of the antibiotic and the membrane to be determined, which allows for a structural understanding of its binding to, and disruption of, the pathogen membrane.
Neutron reflectometry is used to probe the structure across surfaces. In recent years a growing use of this technique has been to structurally examine membrane biochemistry events with molecular level precision. The caveat for this use is that the membranes studied are models, that is, recreations of real biological membranes.
However, several accurate bacterial surface models have been developed9 which allow for biological accuracy in these precision structural studies. This is particularly the case for the Gram-negative envelope where surface models of the asymmetric outer membranes have been fabricated that are accurate representations of the real bacterial membranes being modelled, using extracted bacterial lipopolysaccharides and the membrane being fully asymmetric in terms of its lipid distribution9,10.
Using a combination of neutron reflectometry measurements and the accurate models of the Gram-negative outer membrane, the dependence of Colistin activity on the dynamics of the lipopolysaccharides in the Gram negative bacterial outer membrane was uncovered11,12. This showed that the drug was able to bind to the lipopolysaccharides and disrupt the outer membrane.
Detailed mechanistic information on the membrane disrupting activity of a series of rationally designed anti-microbial peptides was also deciphered using the accurate outer membrane models and neutron reflectometry measurements13. In both cases the structural information provided a detailed view of not only how the antimicrobial bound to the membrane but also the nature of the disruptive effects on the membrane. This precision mechanistic structural information is not possible to view in the live cell or by other analytical techniques.
Understanding antimicrobial function through neutron scattering studies is still in its infancy although a growing group of researchers are using these techniques to benchmark membrane perturbing antimicrobial function. New developments in neutron scattering facilities such as the European spallation source currently under construction in Sweden or the LMX and SANDALS-II beamlines which are part of the Endeavour upgrade program at the ISIS neutron and muon source in the UK will enable new capabilities in the study of antimicrobial activity and function to provide further insight into future therapeutics at the molecular level.
DDW Volume 24 – Issue 4, Fall 2023
References
- Hede K. Nature 2014;509:4-5.
- Ventola CL. The antibiotic resistance crisis: part 1: causes and threats. PT 2015;40:277-283.
- B. Spellberg, J. G. Bartlett and D. N. Gilbert, N. Engl. J. Med., 2013, 368, 299–302.
- Finland M. N Engl J Med 1995;253:909-922.
- WHO, World Health Organisation: Antibiotic Resistance, https://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance
- Tenover FC. Am J Med 2006;119:S3-10; discussion S62-70.
- El-Sayed Ahmed MAEG. Emerg Microbes Infect 2020;9:868-885.
- Steenbergen JN. Antimicrob Chemother 2005;55:283-288.
- Clifton LA. Chemie Int Ed 2015;54:11952-11955.
- Clifton LA. J R Soc Interface 2013;10:20130810.
- Paracini N. Proc Natl Acad Sci 2018;115:E7587-E7594.
- Lakey JH. Biophys Rev 2022;3:021307.
- Gong H. Appl Mater Interfaces 2021;13:16062-16074.
About the author:
Dr Luke Clifton, PhD in Biophysical Chemistry, works as an Instrument Scientist in the neutron reflectometry group at the ISIS Neutron and Muon Source at Rutherford Appleton Laboratory in the UK. Dr Clifton has developed novel bio-membrane sample systems which have been used to answer applied questions of membrane biology. His work with the neutron scattering community includes studies on antimicrobial resistance, cancer, food security and bio-membrane homeostasis.
