Key points
- Antimicrobial peptides (AMPs) are part of innate immunity in virtually all living organisms. Several thousand have been described, from bacteria, plants, insects, amphibians and mammals.
- Most share two properties: a net positive charge, typically +2 to +9 at pH 7.4, and a high fraction of hydrophobic residues, often 30–50%, arranged so that the peptide becomes amphipathic when it folds.
- Many kill by disrupting bacterial membranes, which carry more negative charge than animal cell membranes. Others cross the membrane and act on internal targets.
- Resistance to AMPs develops more slowly than to conventional antibiotics, but it does develop, as the spread of colistin resistance has shown.
An ancient defence
The modern study of antimicrobial peptides began with two discoveries. In 1981 Hans Boman and colleagues isolated the cecropins from the haemolymph of the giant silk moth, showing that insects, which lack antibodies, defend themselves with small cationic peptides. In 1987 Michael Zasloff described the magainins from the skin of the African clawed frog, after noticing that frogs recovered from surgery in non-sterile water without infection.
Since then, AMPs have been found in every kingdom of life. Humans produce two main families: the defensins, small β-sheet peptides held together by three disulfide bonds, and a single cathelicidin, LL-37, released from neutrophils and epithelial cells. Plants produce defensins and thionins; bacteria produce bacteriocins, such as nisin, to kill competing species.
What makes a peptide antimicrobial
Despite enormous sequence diversity, most AMPs converge on a similar physicochemical profile. The table uses the tools on this site to characterise six well-known examples, taking into account the C-terminal amidation of those that carry it.
| Peptide | Source | Length | Net charge, pH 7.4 | GRAVY | Hydrophobic moment* | Structure |
|---|---|---|---|---|---|---|
| Magainin 2 | Frog skin | 23 | +2.8 | +0.08 | 0.45 | Amphipathic α-helix in membranes |
| Cecropin A (amide) | Silk moth | 37 | +6.8 | −0.07 | 0.27 | Helix–hinge–helix |
| Melittin (amide) | Honeybee venom | 26 | +5.8 | +0.27 | 0.35 | Kinked α-helix |
| LL-37 | Human | 37 | +5.8 | −0.72 | 0.56 | Amphipathic α-helix |
| Buforin II | Toad stomach | 21 | +5.8 | −0.64 | 0.46 | Helix with a proline kink |
| Indolicidin (amide) | Bovine neutrophils | 13 | +3.8 | −1.07 | — | Extended, Trp- and Pro-rich; not helical |
* Mean hydrophobic moment per residue for an ideal α-helix (100° per residue), calculated with the Eisenberg consensus scale over the full sequence. Not meaningful for indolicidin, which does not form a helix.
Two numbers do most of the work. The positive charge, calculated as described in the isoelectric point guide, draws the peptide to negatively charged bacterial surfaces. The amphipathicity, measured by the hydrophobic moment, lets it insert into the membrane once there. GRAVY, the overall hydrophobicity, is surprisingly uninformative on its own: LL-37 has a strongly negative GRAVY yet is highly membrane-active, because its hydrophobic residues are concentrated on one face.
Amphipathicity and the helical wheel
An α-helix turns about 100° per residue, so residues three or four positions apart end up on the same side of the helix. A helical wheel projects the helix along its axis and makes this arrangement visible:
The segregation is striking, and it is invisible to a standard hydropathy plot, which averages across both faces. This is exactly the limitation described in the guide to reading a hydropathy plot: the Kyte–Doolittle profile of magainin 2 is almost flat, while its hydrophobic moment is among the highest in the table above.
Most helical AMPs are disordered in water and fold only when they encounter a membrane, as explained in the guide to peptide secondary structure. The membrane both induces the helix and provides the environment in which its two faces make sense.
Why bacteria and not us
The selectivity of AMPs rests largely on differences in membrane composition. The outer leaflet of a bacterial cytoplasmic membrane is rich in anionic lipids such as phosphatidylglycerol and cardiolipin, and Gram-negative bacteria are additionally coated with negatively charged lipopolysaccharide; Gram-positive bacteria carry anionic teichoic acids. The outer leaflet of an animal cell membrane is instead dominated by zwitterionic phosphatidylcholine and sphingomyelin, and contains cholesterol, which makes it stiffer and harder to disrupt.
A cationic peptide is therefore concentrated at bacterial surfaces by electrostatic attraction, while animal cells present a far weaker target. The selectivity is relative, not absolute. Melittin, highly charged and strongly hydrophobic, lyses red blood cells as readily as bacteria, which is why it is a venom and not a drug. Increasing hydrophobicity generally increases potency against bacteria and toxicity to host cells together.
This trade-off is usually summarised by a therapeutic index: the ratio of the concentration that lyses half of a suspension of red blood cells to the minimal concentration that inhibits bacterial growth. Much AMP design is an effort to raise that ratio.
How they kill
Several models describe how membrane-active AMPs disrupt a bilayer. They are not mutually exclusive, and the same peptide may behave differently at different concentrations or on different membranes.
- Barrel-stave pores. Helices insert perpendicular to the membrane and assemble like the staves of a barrel around a water-filled channel, hydrophobic faces towards the lipids. Alamethicin, a fungal peptide, is the best-documented example.
- Toroidal pores. The peptides insert together with lipid head groups, which bend continuously from one leaflet to the other so that the pore is lined partly by peptide and partly by lipid. Magainin and melittin are thought to act this way at intermediate concentrations.
- Carpet mechanism. Peptides accumulate flat on the membrane surface until they cover it like a carpet; above a threshold concentration the membrane disintegrates in a detergent-like manner, forming micelles.
Not all AMPs act on the membrane. Buforin II, derived from a histone, crosses bacterial membranes without permeabilising them and binds nucleic acids inside the cell. Proline-rich AMPs from insects and mammals enter bacteria through transporters and inhibit the ribosome or chaperones. Indolicidin both perturbs membranes and interferes with DNA synthesis. In many cases the membrane is where the peptide first acts rather than the only thing it acts on.
Beyond direct killing, human AMPs such as LL-37 and the defensins act as signalling molecules: they recruit immune cells, modulate inflammation, and influence wound healing. In mammals this immunomodulatory role may be as important as their antimicrobial activity.
Resistance
Because many AMPs target the membrane as a whole rather than a single enzyme, bacteria cannot escape them with a single point mutation in a drug target. This led to the early hope that resistance would never appear. It does, through several strategies:
- Reducing surface negative charge. Adding positively charged groups to lipid A in Gram-negative bacteria, or D-alanine to teichoic acids in Gram-positive ones, weakens the electrostatic attraction.
- Proteases. Some pathogens secrete enzymes that degrade linear AMPs.
- Efflux pumps and trapping. Peptides can be pumped out or bound by secreted decoy molecules.
The clearest warning comes from the polymyxins, cyclic lipopeptides that bind lipopolysaccharide and are used as a last-resort treatment for multidrug-resistant Gram-negative infections. In 2015 a plasmid-borne gene, mcr-1, was reported that modifies lipid A and confers transferable colistin resistance; it has since been found worldwide.
Antimicrobial peptides in use
Several peptide antibiotics are long established, though most are nonribosomal cyclic or lipopeptides rather than the linear, ribosomally made AMPs described above:
| Peptide | Class | Use |
|---|---|---|
| Polymyxin B, colistin | Cyclic lipopeptides | Last-resort systemic antibiotics against Gram-negative bacteria |
| Daptomycin | Cyclic lipopeptide | Serious Gram-positive infections |
| Vancomycin | Glycopeptide | Gram-positive infections including MRSA; binds cell-wall precursors rather than membranes |
| Gramicidin, bacitracin | Cyclic and linear peptides | Topical preparations |
| Nisin | Lantibiotic | Food preservative, used for decades |
Linear, host-defence-type AMPs have proved harder to develop into systemic drugs. They are rapidly degraded by proteases in serum, bind to serum proteins, and can be toxic at the concentrations needed for systemic effect. Clinical development has therefore concentrated on topical uses, such as skin and wound infections, and on engineered analogues that use D-amino acids, cyclisation or non-natural residues to improve stability.
Designing and analysing AMPs
The tools on this site cover the first screening steps in AMP analysis:
- Calculate the net charge at pH 7.4 with the pI calculator. Remember that a C-terminal amide, common in natural AMPs, adds roughly +1.
- Check the hydropathy profile for long hydrophobic stretches, which predict toxicity to host cells and aggregation.
- Draw a helical wheel and look for segregation of hydrophobic and charged residues.
- Consider protease sites: Lys and Arg are both essential for charge and the cleavage sites of trypsin-like proteases, which is one reason D-amino acid analogues are attractive.
Large curated resources, such as the Antimicrobial Peptide Database (APD) maintained at the University of Nebraska, collect sequences, activities and structures and are the usual starting point for comparison.
Frequently asked questions
Are antimicrobial peptides the same as antibiotics?
Some antibiotics are peptides, and some antimicrobial peptides are used as antibiotics, but the terms are usually distinguished. "Antimicrobial peptide" typically refers to gene-encoded host-defence peptides; many clinical peptide antibiotics are nonribosomal products of bacteria and fungi.
Why are so many AMPs amidated at the C-terminus?
Amidation removes the negative charge of the C-terminal carboxyl, raising the net positive charge, and often stabilises the helix and protects against carboxypeptidases. The biosynthesis of the amide is described in the guide to post-translational modifications.
Do AMPs work against viruses and fungi?
Many have activity against fungi and against enveloped viruses, whose lipid envelopes can be disrupted in a similar way to bacterial membranes. Activity varies greatly between peptides.
Is melittin an antimicrobial peptide?
It has potent antimicrobial activity and the typical AMP profile, but it is equally toxic to animal cells. It is usually treated as a model membrane-active peptide and a venom toxin rather than as a selective AMP.
References
- Steiner H, Hultmark D, Engström Å, Bennich H, Boman HG (1981) Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature 292:246–248.
- Zasloff M (1987) Magainins, a class of antimicrobial peptides from Xenopus skin. PNAS 84:5449–5453.
- Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415:389–395.
- Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature Reviews Microbiology 3:238–250.
- Liu Y-Y, Wang Y, Walsh TR, et al. (2016) Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China. Lancet Infectious Diseases 16:161–168.