Antimicrobial peptides: Application informed

 

 

RES EARCH

R E V I E W S U M M A R Y

◥

ANTIMICROBIAL PEPTIDES

Antimicrobial peptides: Application informed
by evolution

Brian P. Lazzaro, Michael Zasloff, Jens Rolff*

BACKGROUND: Antimicrobial peptides (AMPs)
are small proteins with potent antibacterial,
antiviral, and antifungal activity. AMPs are
ubiquitous among multicellular eukaryotes,
with most plant and animal species expressing
dozens of distinct AMP genes in epithelial tis-
sues and in response to infection. The diversity
and potency of AMPs make them attractive
candidates for translational application, and
several are already in clinical trials. However,
if AMPs are to be used effectively and sus-
tainably, it will be imperative to understand
their natural biology and evolution in order to
lessen the risk of collateral harm and avoid the
resistance crisis currently facing conventional
antibiotics.

ADVANCES: For most of the past 25 years, the
prevailing wisdom has been that AMPs are gen-
erally nonspecific and functionally redundant—
largely interchangeable provided that they were

produced quickly enough to a threshold that
could contain infection. Support for this model
was drawn from molecular evolutionary obser-
vations that AMP genes are rapidly duplicated
and pseudogenized within and between species,
often with little evolution at the level of the
primary amino acid sequence. Furthermore,
it was believed that the biochemical simplicity
of AMPs reflected fundamentally irresistible
modes of action, including permeabilization of
the cell envelope through the formation of open
pores, which was assumed to largely prevent
bacterial evolution of resistance.

New evidence within the past 5 years, how-
ever, has begun to overturn that model. We
now know that AMPs can exhibit remarkable
levels of specificity and that some of the evo-
lutionary degradation of AMP gene families
may be adaptive. We are learning that genetic
variability in AMPs, even at the level of single
amino acids, can dramatically alter resistance

In vivo

In vitro

Synergism
on skin

Trait

Effect on bacteria

Synergies

Frequent

Bacterial killing

Fast

Dose-dependent killing

Yes

Synergism
in hemolymph

Mutagenesis

Neutral

Probability of resistance
evolution

Low

Inform

Understanding of evolution of AMP combinations and therapeutic applications

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to infection. There are now multiple documen-
tations of convergent evolution of identical
amino acid variants between species and of
shared allelic diversity between species. It is in-
creasingly clear that AMPs are highly function-
ally diversified and that
they play roles in varied
biological processes, in-
cluding the regulation of
symbiotic communities. It
is also becoming apparent
that bacteria can evolve
resistance to AMPs, although the pharmaco-
dynamics and mechanisms of killing of AMPs
are much more favorable than those of con-
ventional antibiotics for the prevention of
resistance evolution.

Read the full article
at https://dx.doi.
org/10.1126/
science.aau5480
…………………………………………..

OUTLOOK: AMPs hold considerable promise
for translational applications, but developing
their potential will require more sophisticated
foundational understanding. AMPs function
synergistically in vivo, and emerging evidence
indicates that their activities in biological con-
texts may not be fully captured with classical
in vitro assays. Further development of math-
ematical approaches to study synergies will be
required, especially for higher-order interac-
tions, in order to rationally develop cocktails
that have high efficacy at low concentrations.
Synergies between AMPs and conventional
antibiotics should be exploited to rescue drugs
that are currently lost to resistance. AMPs
should be mined from all domains of life:
Although more than 3100 naturally occurring
AMPs have been described from taxa repre-
senting the breadth of life on earth, almost
40% of AMPs under clinical trial are of hu-
man origin. This is potentially risky because
any evolved resistance to those AMPs may
result in collateral resistance to endogenous
human immunity. The biochemical proper-
ties and pharmacodynamics of AMPs make
them far more refractory to resistance evolu-
tion than conventional antibiotics, but care
should still be taken to deploy them respon-
sibly. Translational use of AMPs in clinical
and other applied settings will be greatly en-
hanced by understanding how specific AMPs
function in their natural contexts and how
their evolutionary history may predict their
future utility. If we combine the insights from
the evolutionary diversification of the AMPs,
their activity in the context of synergistic cock-
tails, and our growing understanding of how to
limit resistance evolution, we may avoid re-
peating the mistakes that have resulted in the

current crisis of antibiotic resistance.▪

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The combined insight from studying AMPs across the tree of life and the adaptive evolution of AMPs
will inform their application and the understanding of AMPs in their natural context. In nature, AMPs
are highly diverse, with most AMPs (more than 1000) described in Amphibia. They are released as
synergistic cocktails in vivo. In vitro studies found that synergies are frequent and that other traits of AMPs
result in a low probability of resistance evolution compared with conventional antibiotics.

The list of author affiliations is available in the full article online.
*Corresponding author. Email: jens.rolff@fu-berlin.de
Cite this article as B. P. Lazzaro et al., Science 368,
eaau5480 (2020). DOI: 10.1126/science.aau5480

Lazzaro et al., Science 368, 487 (2020)

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RES EARCH

R E V I E W

◥

ANTIMICROBIAL PEPTIDES

Antimicrobial peptides: Application informed
by evolution

Brian P. Lazzaro1, Michael Zasloff2, Jens Rolff3,4*

Antimicrobial peptides (AMPs) are essential components of immune defenses of multicellular organisms
and are currently in development as anti-infective drugs. AMPs have been classically assumed to have
broad-spectrum activity and simple kinetics, but recent evidence suggests an unexpected degree of
specificity and a high capacity for synergies. Deeper evaluation of the molecular evolution and population
genetics of AMP genes reveals more evidence for adaptive maintenance of polymorphism in AMP genes
than has previously been appreciated, as well as adaptive loss of AMP activity. AMPs exhibit
pharmacodynamic properties that reduce the evolution of resistance in target microbes, and AMPs may
synergize with one another and with conventional antibiotics. Both of these properties make AMPs
attractive for translational applications. However, if AMPs are to be used clinically, it is crucial to
understand their natural biology in order to lessen the risk of collateral harm and avoid the crisis of
resistance now facing conventional antibiotics.

A ntimicrobial peptides (AMPs) are small

proteins with antibacterial, antiviral, and
antifungal activity. Sometimes referred
to as “host-defense peptides,” AMPs are
ubiquitous in the epithelial barriers and
systemic induced defenses of multicellular eu-
karyotes (1). They are highly diverse within and
across species, with most plant and animal
genomes encoding 5 to 10 distinct AMP gene
families that range in size from one to more
than 15 paralogous genes. The diversity and
potency of AMPs make them attractive tar-
gets for development as antimicrobial drugs
(2) and surface antiseptics (3), and dozens of
AMPs are currently being evaluated in clinical
trials (4). More than 3100 AMPs have been de-
scribed from varied plant and animal sources
(5), diversified by rapid evolution between spe-
cies as aptly illustrated by the diversity of
AMPs in the most speciose groups of animals,
the insects [several AMP families are specific
to particular insect orders (6)].

The classically understood model of AMP ef-
ficacy (1) was that they exert microbial killing at
threshold doses through simultaneous target-
ing of diverse aspects of microbial biology.
Their inferred simplicity and redundancy were
assumed to prevent evolution of resistance, and
their lack of specificity was assumed to establish
blanket protection against microbes. However,
more recent findings are forcing a reevaluation
of that model. New evidence indicates unex-

1Department of Entomology, Cornell Institute of Host-
Microbe Interactions and Disease, Cornell University, Ithaca,
NY, USA. 2MedStar Georgetown Transplant Institute,
Georgetown University School of Medicine, Washington, DC,
USA. 3Freie Universität Berlin, Evolutionary Biology, Institut
für Biologie, Königin-Luise-Strasse 1-3, 14195 Berlin,
Germany. 4Berlin-Brandenburg Institute of Advanced
Biodiversity Research (BBIB), 14195 Berlin, Germany.
*Corresponding author. Email: jens.rolff@fu-berlin.de

pectedly high degrees of specificity and syner-
gisms among AMPs. We also now realize that
resistance evolution is possible. Although some
AMPs have evolved functions such as modula-
tion of the immune system (7), deterrence of
herbivores (8) and anticancer effects (9), we
focus here on antimicrobial activity. However,
additional functions can lead to complex and
sometimes contradictory natural selection on
AMP genes. We argue that it is essential to
understand AMP biology in natural contexts
before pursuit of translational application in
order to maximize effectiveness and to avoid
repeating the tragic mistakes of misuse that
have led to widespread bacterial resistance
to conventional antibiotics.

Classical perspective on AMPs

The basic design of antimicrobial peptides is
simple (1). They are short peptides with a net-
positive charge that attracts them to the gen-
erally negatively charged membranes of bacteria.
The hydrophilic and hydrophobic amino acids
of AMPs are structurally segregated to provide
solubility in both aqueous and lipid-rich en-
vironments. The shortest AMPs may be as
small as 15 to 20 amino acids in length, and
even the largest are not more than ~150 amino
acids (5). The biochemical simplicity of AMPs
allows them to be easily evolved de novo, and
certain three-dimensional structures, such as
those of defensins, have independently evolved
repeatedly across plants, insects, and vertebrate
animals (10, 11).

The stereotypical mechanism of AMP action
is to integrate into the bacterial cell membrane
and disrupt its integrity, resulting directly or
indirectly in cell lysis, although AMPs may also
have more complex activities, including meta-
bolic and translational inhibition (12, 13) and

formation of nanonets (14, 15). Naturally co-
occurring AMPs with distinct functions can
synergize, as illustrated by an increasing num-
ber of studies showing that some AMPs per-
meabilize membranes to enable entry of other
AMPs that have intracellular targets (16). For
example, bumblebee hymenoptaecin opens pores
in bacterial membranes that allow abaecin to
enter and bind bacterial DnaK (17). In verte-
brates, perforins form pores that allow lethal
cationic cargo to reach the cytoplasm (18). Sim-
ilarly, distinct AMPs may synergize to permea-
bilize bacterial membranes, as illustrated by
the interaction between magainin 2 and PGLa
(Fig. 1) (19). Eukaryotes can rapidly deploy
multiple distinct classes of AMP simulta-
neously in response to challenge, in some cases
up-regulating AMP gene expression several
hundredfold within hours of infection, effec-
tively killing microbes through simultaneous
targeting of multiple critical cellular functions.
These observations coalesced into a classical
model of AMP function (1, 20, 21) that relied
on three key interpretations of the data. First,
it was presumed that host production of func-
tionally diverse AMPs would result in more
effective killing, in analogy to therapeutic
application of multiple distinct antibiotics.
Second, it was inferred that AMP dose was
probably more important than specific peptide
identity, making AMPs largely interchangeable
provided that they were quickly produced
above a threshold level that could rapidly over-
whelm the infection. Last, the biochemically
simple and highly efficient killing mechanism
of even single AMPs was predicted to prevent
bacterial evolution of resistance. These inter-
pretations were fully consistent with strong
up-regulation of AMP production in response to
infection (22) and were superficially consistent
with molecular evolutionary and comparative
genomic data that showed rapid duplication,
deletion, and pseudogenization of individual
AMP genes while keeping total gene family
size fairly constant (23–26), with little indication
of adaptive amino acid diversification [(21), but
also (27, 28)]. The classical model of interchange-
able AMPs at threshold concentration was con-
sistent with the data that were available over the
roughly 20 years between 1994 and 2014. How-
ever, it is probably wrong.

Rethinking AMP function and evolution

The previously widespread belief that AMPs
have generic, broad-spectrum activity has been
recently challenged by new data. For example,
in vivo disruption of individual AMP genes
in the beetle Tenebrio molitor causes differ-
ential sensitivity to infection by the bacterium
Staphylococcus aureus (29). In Drosophila
melanogaster and its sister species Drosophila
simulans, naturally occurring null alleles of
the AMP gene Diptericin A cause acute sensi-
tivity to infection by the bacterium Providencia

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Fig. 1. Analyzing and depicting synergism. (A and B) Synergies between Magainin 2 (M) and PGLa (P) visualized with (A) a zone of inhibition assay and (B) a model
of the interaction on the membrane. (A) Identical molar amounts of Magainin 2 and PGLa were applied to a freshly inoculated lawn of E. coli and photographed after a
24-hour incubation at 37°C. The sharp zones of bacterial killing reflect the steep concentration dependence of bactericidal activity. Arrows highlight the zones of
activity resulting from synergy. (B) The synergistic interaction between PGLa and Magainin 2. Antiparallel PGLa dimers (red) span the membrane. Magainin 2
monomers (blue) lie on each surface of the membrane and contact each PGLa dimer tail to tail. [Redrawn from (19).]

rettgeri but not to other bacteria, including
close relatives of P. rettgeri (Fig. 2) (30). Fur-
thermore, a single polymorphic amino acid
substitution in the Diptericin A peptide is suf-
ficient to specifically alter resistance to P. rettgeri,
and this susceptible mutation has arisen at
least five independent times across the genus
Drosophila (31). These findings suggest a pre-
viously unsuspected specificity in AMP activity.
Expanding the work in D. melanogaster,
Hanson and colleagues systematically deleted
individual AMP genes and gene families and
showed that distinct small subsets of the fruit
fly AMP repertoire are wholly responsible for
the control of diverse bacterial infections (32).
Those observations were surprising because
bacterial infections induce transcriptional ex-
pression of broad suites of AMP genes (33),
but most of these appear to be functionally
irrelevant to suppressing the pathogen in ques-
tion. However, broad transcriptional induction
makes sense in natural contexts, particularly if
infections in nature are typically polymicrobial
or if the host is unable to finely discriminate
pathogens at the recognition stage (34). In these
cases, comprehensive induction of AMPs at
early stages of infection, including as a prophy-
lactic response to wounding (33), would ensure
activation of the subset with specific activity and
would guarantee the most effective protection.
The high rate of duplication, deletion, pseu-
dogenization, and de novo origin of AMP genes
(23–26) had previously been interpreted as evi-
dence that individual genes were superfluous if
a threshold dose of AMPs was produced. How-
ever, the observations that laboratory mutants,
as well as naturally occurring null alleles and
point mutations, have profound and specific

effects on resistance to infection are in complete
contrast to the classical threshold model of
interchangeable AMPs. An alternative inter-
pretation is that the significantly elevated
rates of gene duplication and diversification
reflect adaptation to suites of microbes (35)
and that AMP gene loss may be adaptive if
the physiological costs of producing a given
AMP outweigh its ecological benefit (36). An
adaptive diversification model might predict
that gene family expansions should be coupled
with amino acid diversification of the encoded
peptides. Consistent with this prediction, there
are many reports of AMP gene family radiations
coupled with sequence diversification, particu-
larly in vertebrates (27, 28, 37). Furthermore,
an adaptive diversification model might predict
that newly duplicated genes would acquire
distinct expression patterns, and this has been
observed where it has been examined (38, 39).
Adaptation is also revealed in the parallel
and/or convergent evolution of the same amino
acid sequence in different species. Natural se-
lection may promote evolutionary convergence
in species that share ecological pressures, or
adaptive maintenance of polymorphism within
species if alternative alleles are more effective at
killing specific pathogens that are commonly
present at different times or places in the en-
vironment. Recent analyses have revealed a sur-
prisingly high rate of convergent evolution in
AMP genes sampled from organisms as diverse
as mussels (40), birds (41), and multiple species
of Drosophila (31). In some cases, multiple spe-
cies are polymorphic for the same or similar al-
leles of AMP genes. For example, D. melanogaster
and D. simulans have each independently
evolved a Ser/Arg polymorphism at the same

Fig. 2. Small evolutionary changes matter. Small
evolutionary changes in amino acid composition of
AMPs can have major consequences for host
survival during infection (30). In D. melanogaster,
different alleles of Diptericin (arginine and serine)
show pathogen-specific activity here against
P. rettgeri. Lines of D. melanogaster with null alleles
(black) show higher mortality than do lines homo-
zygous for arginine (red). Lines homozygous for
serine (blue) show the highest survival.

codon in Diptericin A, and the alternative al-
leles are demonstrably different in resistance
to infection in both species (Fig. 2) (30). Such
allele sharing has been interpreted as evi-
dence that AMP polymorphism is adaptively
maintained in populations. Allele sharing in
AMP genes has been found in humans (42),
frogs (43), passerine birds (41), waterfowl (44),
codfish (45), mussels (40), and Drosophila
(46), although typically without phenotypic

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analysis of the alternative alleles. These mul-
tiple examples may represent just the tip of the
iceberg because population sequence samples
from multiple species are required to detect
shared polymorphisms, and sequences from
constellations of related species are required to
detect convergent evolutionary fixations, but
these are rarely available. Additionally, molec-
ular evolutionary signatures of adaptively main-
tained polymorphisms can be difficult to detect
(47, 48), particularly in species with large pop-
ulation sizes (31).

Symbionts and coevolution

In addition to combatting infectious pathogens,
AMPs are used to regulate bacterial symbionts
and communities in the gut and other tissues
(49). This is true even for organisms with fairly
simple body plans, such as the cnidarian Hydra
(50). In the mammalian gut, a growing body of
work shows the importance of AMPs secreted
by the Paneth cells in shaping the gut micro-
biota and hence determining healthy or path-
ological phenotypes (49). Many members of the
gut microbiota display high intrinsic AMP re-
sistance (51), suggesting that AMPs may be an
important tool for establishment and mainte-
nance of healthy communities. AMPs are also
important for the regulation of highly co-
adapted mutualists. For example, the weevil
Sitophilus zeamais carries an obligate symbi-
ont, Candidatus Sodalis pierantonius str. SOPE,
in specialized cells called bacteriocytes. The
symbiont shows hallmarks of tightly coevolved
mutualism, including gene loss as it becomes
more dependent on its partner (52). S. zeamais
expresses one AMP, coleoptericin-A (ColA),
exclusively in the bacteriocytes, and knocking
down ColA expression results in symbiont es-
cape into surrounding tissues (53). Similarly,
the specialized plant nodules that harbor
nitrogen-fixing symbionts in the leguminous
plant Medicago truncatula express more than
700 peptides, many of which are cysteine-
rich AMPs (54). The coevolved symbiont
Sinorhizobium meliloti has evolved a pep-
tidase that protects the symbionts against
harmful effects of these AMPs and allows
them to gain a competitive advantage over
other microbes in that niche (54).

AMPs are additionally crucial to the well-
documented example of bobtail squid, which
obtains a bioluminescent bacterial symbiont,
Vibrio fischeri, from the water column and
sequesters it in a specialized light organ. The
squid diurnally flushes its bacterial symbionts
and then allows recolonization of the light
organ from bacteria in the surrounding water.
Specific colonization is ensured by AMPs that
prevent V. fischeri from colonizing inappro-
priate tissues and that block colonization by
undesirable bacteria. Specificity in this remark-
able mutualism is further guaranteed by acidic
mucus on the surface of the light organ that

primes V. fischeri to become resistant to the
AMPs and allows it to occupy this niche (55).

do not accurately capture biological conditions
may be misleading (20, 68).

Sublethal concentrations of AMPs are de-
ployed in certain host-symbiont interactions.
For example, obligate microbial symbionts often
undergo genome erosion (56) that makes them
dependent on the host for exchange of metab-
olites. In many cases, genes that code for mem-
brane transport are vulnerable to genome
erosion. However, expression of AMPs at sub-
lethal doses may substitute for the transport
function by permeabilizing bacterial mem-
branes without killing the cells in their sym-
biotic compartments (57). Similarly, low
concentrations of AMP-like peptides that
would be lethal at higher concentrations
also stimulate terminal differentiation and
metabolic specialization of the nitrogen-fixing
bacteria in leguminous plants (58). How low
concentrations of host AMPs influence bac-
terial physiology is poorly understood, and
whether they contribute to bacterial evolu-
tion of resistance or induction of phenotypic
resistance in other contexts is not known.

Synergism

Accumulating evidence shows marked func-
tional synergism occurs among distinct AMPs
(Fig. 1) (59–62). Synergism in vivo may reduce
the chances of resistance evolving (63), espe-
cially by generalist pathogens that infect multi-
ple hosts that express different combinations of
AMPs. Yet, determining synergy from dose-
response curves can be quite challenging, par-
ticularly because they are not linear. Currently,
two main models are used to calculate synergies
(64–66), and further development of these ref-
erence models will be crucial to our understand-
ing of AMP interactions in both natural systems
and drug applications, especially for higher-
order interactions.

In vitro estimation of individual AMP ac-
tivities may not reflect in vivo efficacy when
synergisms among AMPs are common. For
example, a recent report highlighted mis-
matches between the in vitro activities of the
T. molitor AMPs Tenecin 1, 2, and 3, compared
with infection resistance profiles observed
when each of the genes that encode them was
disrupted in vivo by means of RNA interfer-
ence (29). Tenecin 2 showed no effect against
S. aureus in vitro (67), but beetles deficient of
Tenecin 2 suffered measurably increased mor-
tality upon S. aureus infection. There are nu-
merous reasons why in vitro assays may not
reflect in vivo activities, including differences
in local pH or salt concentrations, nutritional
or osmotic stress on microbes, and synergisms
among AMPs or between AMPs and other
components of the immune system. This is an
important problem because in vitro assays
have been used as a tool for estimating the
efficacy of particular AMPs against specific
microbes, but results from experiments that

Translational challenges for AMPs

Against the backdrop of accelerating antibiotic
resistance (69, 70), antimicrobial peptides hold
promise for use in clinical and veterinary set-
tings. However, to effectively deploy AMPs and
sustain their value, we need to learn from both
the historical misuse of antibiotics (69, 70) and
the evolutionary biology and natural pharma-
cology of AMPs.

Instead of reflexively relying on the current
standard of testing minimum inhibitory con-
centration (MIC) of individual components
in vitro (68), we need to understand and mea-
sure synergisms among AMPs in vivo so that
we can exploit them effectively for microbial
control at low concentrations. In addition,
AMPs can synergize with conventional anti-
biotics (68, 71, 72), which raises the prospect
that antibiotics that have been lost to resist-
ance could be resurrected. For example, a
recent study on multidrug-resistant Gram-
negative bacteria showed strong synergisms
between the antibiotic azithromycin—which
showed no activity against Gram-negative
bacteria in standard MIC tests—and the AMPs
colistin and LL-37 (68). In another example,
the AMP known as SAAP-148 was shown to be
effective at killing drug-resistant bacteria even
within biofilms in vivo on mouse skin (73).
Host-directed therapies to boost natural AMP
production can improve infection control. Ap-
plication of compounds such as phenylbuty-
rate and aroylated phenylenediamines have
been shown to boost LL-37 induction by 20- to
30-fold, and orally treated rabbits showed a
decrease of bacterial load by up to five orders
of magnitude (74). Synergisms can be synthet-
ically generated very successfully. Chimeric
peptide antibiotics that link polymyxin and
murepavidin have been demonstrated to be ac-
tive against multidrug-resistant Gram-negative
ESKAPE pathogens (Enterococcus faecium,
S. aureus, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa, and
Enterobacter species) (75). Combination ther-
apies can be envisaged that integrate synthetic
AMPs, stimulate natural AMPs, and deploy con-
ventional antibiotics for the treatment of recalci-
trant multidrug-resistant bacterial infections.
The low rates of evolved bacterial resistance
to AMPs need to be maintained as peptide
drugs are rolled out in the clinic. One route
may be through collateral sensitivity (76), in
which evolved resistance to one antibacterial
renders bacteria sensitive to another. Collat-
eral sensitivity to AMPs has been observed in
Escherichia coli strains experimentally selected
to be resistant to a variety of antibiotics (77).
By contrast, independent studies in which
S. aureus was selected for resistance to AMPs
resulted in cross-resistance to several AMPs,

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including the peptide antibiotic Daptomycin,
although not to other antibiotics (78, 79). Both
collateral sensitivity and cross-resistance have
been reported in multiple studies of E. coli
selected for resistance against AMPs (80).
Thus, active monitoring of target microbe
populations and management of therapeutic
AMP deployment will be essential.

The pharmacodynamics of AMPs reduces
the probability of resistance evolution (Fig. 3)
(81). Most AMPs interact with the bacterial cell
surface and are not directly mutagenic, where-
as many antibiotics can elevate bacterial mu-
tation rates by triggering bacterial stress
responses such as the SOS and rpoS pathways
(82). AMPs kill faster than antibiotics—within

minutes instead of hours (83)—allowing many
fewer bacterial generations in which resistance
could evolve. Because resistance to AMPs tends
to be by nonspecific mechanisms, there may be
fewer mutational routes by which resistance to
AMPs can evolve (84) and lower likelihood of
horizontal gene transfer that confers resistance
(85). Perhaps most importantly, the concentra-
tion range of intermediate efficacy, in which
resistance can evolve, is smaller for AMPs than
for antibiotics. This phenomenon is captured
by the Hill coefficient (the slope of the phar-
macodynamic curve), which describes the win-
dow between concentrations that have no effect
and concentrations that result in complete
killing (Fig. 3A) (63). AMPs have high Hill

coefficients, which means that there is a small
window in which there is selective pressure on
the bacteria to evolve resistance while they are
still viable enough to do so. Combinations of
AMPs have even higher Hill coefficients and
hence reduce the risk of resistance evolution
even further (81).

Another advantage of AMPs over antibiotics
is that AMPs tend to be less stable than anti-
biotics and have much lower environmental
persistence (86). This is partly a function of
synthetic engineering of clinical antibiotics for
extended shelf lives. The persistence of anti-
bacterials in the environment at sublethal con-
centrations continues to select for antibiotic
resistance and constitutes an important driver

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Fig. 3. Resistance evolution against AMPs. (A) Pharmacodynamics of AMPs
differ from those of conventional antibiotics (63). The solid curved line
depicts a susceptible bacterial strain, and the dashed line depicts a resistant
strain. The respective MICs are shown at the intersections by the solid
horizontal line. The Hill coefficient k, depicting the slope, represents an
antibiotic (top, k = 1), and k = 4 represents an AMP (bottom) [values are
based on (63); typically k values are higher for AMPs than for antibiotics]. The
dose-response curve for the AMP is correspondingly steeper for an AMP,

which results in a narrow mutant selection window (light blue) in which
genetically resistant mutants are favored. (B) Combining the pharmacody-
namical properties of AMPs and comparing them with those of antibiotics,
computer simulations predict a lower probability of resistance evolution
against AMPs compared with antibiotics. [Adapted from (81).] (C) Experi-
mental resistance evolution of E. coli against 15 AMPs in vitro yields a
significantly lower degree of resistance compared with the results for
12 antibiotics, with the exception of two AMPs. [Data are from (80).]

lairt lacinilc rednu yltnerrucsPMA

esabatadPMA ehtni sPMA fonigirO

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Human

Cattle

Pig

Frog

Bacteria

Synthetic

Mammals

Amphibia Sauropsida

Fish

Arthropods Molluscs

Fig. 4. Evolution of AMPs and origin of AMPs as drugs. (A) The number of AMPs currently undergoing clinical trials [data are from (4)] and the organisms
from which they are derived. (B) Relative representation of animal taxa in the antimicrobial peptide database (5). This representation is not correlated with the number
of species in each of the groups because, for example, insects are by far the most species-rich taxon.

Lazzaro et al., Science 368, eaau5480 (2020)

1 May 2020

4 of 7

RES EARCH | R E V I E W

of antibiotic resistance evolution (69, 87). Be-
cause many antibiotics were originally evolv