Antimicrobial peptides – ancient weapons and new drugs Open Access

Antimicrobial resistance is a major health challenge globally, for both humans and farm and companion animals. One way to tackle this is the discovery of new drug candidates and antimicrobial peptides offer several leads to new drugs. Antimicrobial peptides (AMPs) are a diverse group of evolutionary conserved molecules found across in all three domains of life: the bacteria, the archaea and the eukaryotes. Cationic AMPs typically consist of 10–60 amino acids, although some are shorter or longer. Many of those peptides attack because of their positive charge, bacterial membranes. However, other AMPs also have intra-cellular targets, such as those peptides rich in the amino acid proline. AMPs contain hydrophobic residues, fold into unique structures and are classified into different groups based on structural features. Here, we look at two aspects: first, the fact that AMPs are always expressed as cocktails, as opposed to medical applications where most often a single drug is applied. From this observation, we then review how combinations of AMPs function and could be used.

Like other forms of life, insects produce a variety of AMPs as first line of their innate immune system. The antimicrobial activity of insects AMPs was first reported in the giant silk moths, leading to discovery of Cecropins in 1981, which laid the foundation for studying AMPs in multi-cellular organisms. To date, over 400 insect AMPs have been identified, highlighting their importance in insect immunity. For example, mealworms, Tenebrio molitor, have 14 known AMPs classified into six families: Attacins, Tenecins, Coleoptericins, Defensins, Cecropins and Thaumatin-like proteins (Figure 1). Fruit flies, Drosophila melanogaster, possess 25 identified AMPs, categorised into several families, including Attacins, Cecropins, Drosomycins and unclassified AMPs. Honeybees, Apis mellifera, have six identified AMPs, while bumblebees, Bombus spp. possess four.

Studies show that insects do not depend on one single AMP to fight against infections; instead, they express cocktails of AMPs that can synergistically work together. This antimicrobial strategy is not accidental, but the result of millions of years of evolution and refining their immune system and the persistence of AMP cocktails across insect species is a testament to their success. AMPs in insects exhibit synergy through different mechanisms. Some disrupt bacterial membranes, making it easier for others to enter and kill the cell. Others interfere with bacterial metabolism or enhance immune recognition. In vivo experiments in mealworms and fruit flies, where multiple AMP genes were knock downed, have shown higher pathogen growth and mortality, highlighting their collective importance. In vitro studies in honeybees and bumblebees have also demonstrated little activity of AMPs on their own but dramatic increases in antimicrobial effects in combinations, with certain AMP pairs being more effective together than individually, constituting a nice example of synergy. Synergies are cases of the whole being greater than the sum of the individual parts; two or more AMPs yielding a greater effect than would be expected by using each drug alone. Despite their potency in vitro, AMPs do not always act similarly in vivo. Host immunological environment and the presence of microbiota in different tissues introduce additional complexities.

AMPs are both ancient and ubiquitous in innate immune systems, and the evolutionary imperative has been for insects, as discussed above, to retain AMPs for millions of years and to produce them in cocktails (Figure 2). This, conversely, implies an evolutionary disadvantage to infecting bacteria as, otherwise, the bacteria would evolve resistance and kill their insect hosts. Studying AMPs in their natural contexts such as insects, therefore, provides a window into learning how combinatorial approaches that also avoid resistance evolution are moulded by natural selection. This can then be used to inform combination drug treatments in human and veterinary medicines.

Compared to conventional antibiotics, AMPs kill bacteria at faster rates and cause resistance at slower rates. The speed at which bacteria are killed by an antimicrobial (pharmacodynamics) is, of course, important in clearing infecting bacteria, as well as in determining whether or not antimicrobial resistance arises; the longer bacteria are exposed to an agent without dying, the more likely resistance is to arise to that agent. In addition to their faster killing rates, many AMPs kill in seconds as opposed to several antibiotics that require hours; when applied alone, it has been demonstrated that the pharmacodynamics of AMPs in combinations is even faster than that of those same AMPs alone, further reducing the likelihood of resistance evolution and also reducing effective concentrations. AMP combinations are better both at killing bacteria and at preventing bacteria from developing resistance.

Taking combinations and synergisms of individual AMPs a step further and expanding them is the production and use of random peptide mixtures, peptides of defined sizes and compositions but which are assembled randomly to produce an AMP cocktail. These AMP cocktails can contain millions (or even more) of different, individual peptides. One such peptide cocktail, known as FK20, is a potent antimicrobial against bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus, with a fast pharmacodynamic rate. In addition, when those bacteria were exposed to FK20 over extended periods, they did not develop resistance to the effects of the peptide mixture in a manner consistently seen for conventional antibiotics and even other AMPs. Intriguingly, in the case of P. aeruginosa post-exposure to FK20, mutations arose that are often found in isolates, which do have resistance to other AMPs; yet, the bacterium remained susceptible to FK20, in spite of these normally protective mutations.

Another set of studies that investigated a random peptide mixture, called glatiramer acetate (GA) and being used as a drug for treating multiple sclerosis, showed synergy between the peptide mixture and the antibiotic drug tobramycin. Despite having only moderate antibacterial activity itself, GA was able to reduce the concentrations of tobramycin that need to kill clinical P. aeruginosa isolates from people with respiratory infections. Results indicate this to be case of potentiation as even though GA only has minor antimicrobial activity on its own, it has the ability to breach bacterial membranes, allowing tobramycin access to the interior of the cell, where its targets are. In keeping with the FK20 results, no connection was seen between GA exposure and common AMP resistance mechanisms. GA did not stimulate the expression of AMP resistance genes by P. aeruginosa in the manner that other AMPs do, so the bacteria was unable to mount a strong defence of itself in the face of GA.

The cases of both FK20 and GA show the potential of random peptide cocktails as direct antimicrobials and potentiators of antibiotics which, in early studies, also appear to be able to bypass the usual AMP resistance mechanisms and routes to future resistance. Combinations could also offer the opportunity to deal with drug toxicity, as the concentrations of the individual drugs would be lower than in single applications.

AMPs are a fascinating group of defence molecules. It is noteworthy that they can have several other defence functions; for example, some of them are anti-inflammatory. If carefully studied and introduced, some AMPs will have the potential to help and tackle the antimicrobial resistance crisis that is accelerating in both humans and animals, as they inherently have properties that lower the risk of resistance evolution. We strongly believe that studying AMPs in their natural context can inform the development of antimicrobial drug interactions beyond AMPs.