REVIEW
published: 27 December 2016
doi: 10.3389/fcimb.2016.00194
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 December 2016 | Volume 6 | Article 194
Edited by:
Matthew C. Wolfgang,
University of North Carolina at Chapel
Hill, USA
Reviewed by:
Charles Martin Dozois,
Institut National de la Recherche
Scientifique, Canada
Mathias Schmelcher,
ETH Zurich, Switzerland
*Correspondence:
Margit Mahlapuu
margit.mahlapuu
@promorepharma.com
Received: 23 October 2016
Accepted: 12 December 2016
Published: 27 December 2016
Citation:
Mahlapuu M, Håkansson J,
Ringstad L and Björn C (2016)
Antimicrobial Peptides: An Emerging
Category of Therapeutic Agents.
Front. Cell. Infect. Microbiol. 6:194.
doi: 10.3389/fcimb.2016.00194
Antimicrobial Peptides: An Emerging
Category of Therapeutic Agents
Margit Mahlapuu
1, 2
*
, Joakim Håkansson
3
, Lovisa Ringstad
3
and Camilla Björn
2, 3
1
Promore Pharma AB, Karolinska Institutet Science Park, Solna, Sweden,
2
The Lundberg Laboratory for Diabetes Research,
Department of Molecular and Clinical Medicine, The Sahlgrenska Academy at University of Gothenburg, Gothenburg,
Sweden,
3
SP Technical Research Institute of Sweden, Chemistry, Materials, and Surfaces, Borås, Sweden
Antimicrobial peptides (AMPs), also known as host defense peptides, are short and
generally positively charged peptides found in a wide variety of life forms from
microorganisms to humans. Most AMPs have the ability to kill microbial pathogens
directly, whereas others act indirectly by modulating the host defense systems. Against
a background of rapidly increasing resistance development to conventional antibiotics
all over the world, efforts to bring AMPs into clinical use are accelerating. Several AMPs
are currently being evaluated in clinical trials as novel anti-infectives, but also as new
pharmacological agents to modulate the immune response, promote wound healing, and
prevent post-surgical adhesions. In this review, we provide an overview of the biological
role, classification, and mode of action of AMPs, discuss the opportunities and challenges
to develop these peptides for clinical applications, and review the innovative formulation
strategies for application of AMPs.
Keywords: AMP, antimicrobial peptide, anti-infectives, antibiotic resistance, therapeutic agents
INTRODUCTION
The rapidly increasing resistance toward conventional antibiotics suggests t hat, without urgent
action, we are heading for a “post-antibiotic era,” in which the previously effective therapeutic
strategies are no longer relevant. Due to the limited number of available antibiotics, and the
similarities in their activity spectrum as well as mode of action, intensive nonclinical and clinical
research is now invested into identification of new and non-conventional anti-infective therapies,
including adjunctive or preventive approaches such as antibodies targeting a virulence factor,
probiotics, and vaccines (
Czaplewski et al., 2016). Interestingly, the antimicrobial peptides (AMPs)
have rapidly captured attention as novel drug candidates (Figure 1). AMPs have been found
virtually in all organisms and they display remarkable structural and functional diversity. Besides
direct antimicrobial activity, AMPs carry immunomodulatory properties (
Fjell et al., 2012), which
make them espe cially interesting compounds for the development of novel therapeutics. There are
encouraging examples of AMPs already introduced into the market, and many AMPs are currently
being tested in clinical trials (Fox, 2013), which provide a reason for optimism for introduction of
novel AMP-based drugs in several indication areas.
With no attempt to provide a comprehensive overview with regards to all types of AMPs
identified from different sources, this review focuses on applied therapeutic aspects with t he
emphasis of AMPs being evaluated as potential pharmacological agents.
Mahlapuu et al. Antimicrobial Peptides As Therapeutic Agents
FIGURE 1 | Published research on AMPs identified from 2004 until
September 2 016. Article counts were carried out after searching in PubMed
using the f ollowing key words: antimicrobial peptides, AMPs, and/or host
defense peptides. The search results d emonstrate that in the last decade the
AMP research field has progressively expanded as represented by the
continuous increase in the number of articles. Q, quarter.
BIOLOGICAL ROLE AND CLASSIFICATION
OF AMPS
AMPs are evolutionary conserved in the genome and produced
by all life forms, from prokaryotes to humans (Hancock, 2000).
In higher organisms, AMPs constitute important components
of the innate immunity, prote c ting the host against infections.
In contrast, bacteria produce AMPs in order to kill other
bacteria competing for the same ecological niche (
Hassan
et al., 2012). Many AMPs exhibit an extraordinarily broad
range of antimicrobial activity covering both Gram-positive
and Gram-negative bacteria as well as fungi, viruses, and
unicellular protozoa (Hancock and Diamond, 2000; Reddy et al.,
2004; Marr et al., 2006). Besides having a dire ct antimicrobial
activity, several AMPs display ability to modulate the innate
immune responses of the host and thereby indire ctly promote
pathogen clearance (Hancock and Sahl, 2006; Yeung et al., 2011).
The widespread distribution and abundance of AMPs in all
multicellular organisms underscores their critical role in innate
immunity (
Zasloff, 2002; Hancock et al., 2012). Their importance
is further demonstrated by the increased infection susceptibility
of mice genetically modified to lack the gene encoding for the
mouse analog of the human AMP LL-37 (Nizet et al., 2001) and of
humans with diseases associated with reduced AMP production
such as atopic dermatitis (Ong et al., 2002).
AMPs in nature are produced either by ribosomal translation
of mRNA or by nonribosomal peptide synthesis (Hancock and
Chapple, 1999). While nonribosomally synthesized peptides are
mainly produced by bacteria, the ribosomally synthesized AMPs
are genetically encoded and produced by all species of life,
bacteria included (Hancock and Chapple, 1999). Compared to
peptides of nonribosomal origin that have been known for
several decades and whereof many are used as antibiotics (e.g.,
polymyxins and gramicidin S), the ribosomally synt h esized
AMPs have more recently been recognized for their critical role
FIGURE 2 | Peptides representing the three main categories of the
secondary structures of AMPs. LL-37 and human lactoferricin represent
α-helical peptides, human β-defensin 1 represents β-sheet peptides, and
indolocidin represents extended/random-coil structures. Structures are from
Protein Data Bank in Europe (PDB id codes 2k6o, 1z6v, 1kj5, and 1g89).
in innate immunity and for their therapeutic potential (Hancock
and C happle, 1999; Hancock, 2000).
In mammals, AMPs are found primarily within granules of
neutrophils and in secretions from epithelial cells covering skin
and mucosal surfaces (
Boman, 1995; Hancock and Chapple,
1999). In many cases, A MPs are encoded in clusters in
the genome and co-expressed, resulting in multiple AMPs
accumulating at a single site (Lai and Gallo, 2009). Notably, many
AMPs are produced as inactive precursors requiring proteolytic
cleavage to be c ome active (Bals, 2000). Their regulation is
therefore not only dependent on their own expression but also on
the abundance of appropriate proteases (Lai and Gallo, 2009). In
multicellular organisms, some AMPs are constitutively expressed,
stored at high concentrations as inactive precursors in granules
and released locally at infection and inflammation sites, whereas
the expression of others is induced in response to pathogen-
associated molecular patterns (PAMPs) or cytokines (
Hancock
and Diamond, 2000; Lai and Gallo, 2009).
Several databases exist for natural AMPs, today covering more
than 2000 peptides (Wang, 2015). Most AMPs are relatively
short, commonly consisting of 10–50 amino acids, display an
overall positive charge ranging from +2 to +11, and contain a
substantial proportion (typically 50%) of hydrophobic residues
(Yeaman and Yount, 2003; Hancock and Sahl, 2006; Pasupuleti
et al., 2012). AMPs are commonly classified based on their
secondary structure into α-helical, β-sheet, or peptides with
extended/random-coil structure (Takahashi et al., 2010; Nguyen
et al., 2011; Pasupuleti et al., 2012
), with most AMPs belonging
to the first two categories (Figure 2). α-helical peptides are often
unstructured in aqueous solution, but adopt an amphipathic
helical structure in contact with a biological membrane (
Yeaman
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Mahlapuu et al. Antimicrobial Peptides As Therapeutic Agents
and Yount, 2003; Pasupuleti et al., 2012). Two of the most
studied peptides in this group are: (i) LL-37 (Epand and
Vogel, 1999; Pasupuleti et al., 2012
), which is produced
as an inactive precursor in the 18-kDa human cathelicidin
antimicrobial protein (hCAP18), present in neutrophils and
epithelial cells (
Lai and Gallo, 2009), and (ii) human lactoferricin,
which is derived by proteolytic cleavage of the antimicrobial
and immunomodulatory iron-binding glycoprotein lactoferrin,
present in milk and exocrine secretions (Hunter et al., 2005;
Legrand et al., 2005). β-sheet peptides are stabilized by disulphide
bonds (Powers and Hancock, 2003; Yount et al., 2006) and
are organized to create an amphipathic molecule (Yeaman
and Yount, 2003). Due to their rigid structure, the β-sheet
peptides are more ordered in aqueous solution and do not
undergo as drastic conformational change as helical peptides
upon membrane interaction (Yeaman and Yount, 2003). The
best-studied β-sheet peptides are the defensins—a large group of
AMPs, which are produced as inactive precursors in neutrophils,
macrophages, and epithelial cells (
Lai and Gallo, 2009; Pasupuleti
et al., 2012). A small portion of the natural AMPs belong to
the third class of extended/random-coil peptides which lack
secondary structure and often contain a high content of arginine,
proline, tryptophan, and/or histidine residues (Takahashi et al.,
2010; Nguyen et al., 201 1). Similarly to other AMPs, many of
the extended peptides fold into amphipathic structures after
contact with a membrane (Nguyen et al., 2011). One of the best
studied peptides in this group is indolicidin, produced by bovine
leukocytes (Powers and Hancock, 200 3).
MECHANISM OF ACTION OF AMPS
Interaction with Bacterial Membrane
Many AMPs display a direct and rapid antimicrobial activity
by causing disruption of the physical integrity of the microbial
membrane and/or by translocating across the membrane into t h e
cytoplasm of bacteria to act on intracellular targets (
Hancock and
Sahl, 2006). It is widely accepted that membrane interaction is
a key factor for th e direct antimicrobial activity of AMPs, both
when the membrane it self is targeted and when an intracellular
target must be reached by means of translocation (Jenssen et al.,
2006; Nguyen et al., 2011; Yeung et al., 2011; Malmsten, 2016
).
Electrostatic forces between the cationic AMPs and the negatively
charged bacterial surface are critical determinants for this
interaction between peptides and microbial membrane (
Yeaman
and Yount, 2003; Giuliani et al., 2007; Y eung et al., 2011; Ebenhan
et al., 2014). Bacteria are commonly divided into two families,
Gram-positive and Gram-negative, based on the differences
in cell envelope structure. In Gram-positive bacteria, the
cytoplasmic membrane is surrounded by a thick peptidoglycan
layer, whereas the cytoplasmic membrane of Gram-negative
bacteria is surrounded by a th in peptidoglycan layer as well as
an outer membrane (Lin and Weibel, 2016). The cytoplasmic
membranes of both Gram-positive and Gram-negative bacteria
are rich in the phospholipids phosphatidylglycerol, cardiolipin,
and phosphatidylserine, which have negatively charged head
groups, hig h ly attractive for positively charged AMPs (
Yeaman
and Yount, 2003; Ebenhan et al., 2014
). The presence of
teichoic acids in the cell wall of Gram-positive bacteria and
lipopolysaccharides (LPS) in the outer membrane of Gram-
negative bacteria provide additional electronegative charge to the
bacterial surface (
Lai and Gallo, 200 9; Ebenhan et al., 2014).
The fundamental differences between microbial and
mammalian membranes protect mammalian cells against
AMPs and enable selective action of these peptides (
Yeaman
and Yount, 2003). In contrast to bacteria, the cytoplasmic
membrane of mammalian cells is rich in the zwitterionic
phospholipids phosphatidylethanolamine, phosphatidylcholine,
and sphingomyelin, providing a membrane with a neutral
net charge (Yeaman and Yount, 200 3; Ebenhan et al., 2014).
There is also an asymmetric distribution of phospholipids in
mammalian membranes, with the zwitterionic phospholipids
being present in the outer leaflet, while phospholipids with
negatively charged head groups, if present, are localized in
the inner leaflet facing the cytoplasm (Zasloff, 2002; Yeaman
and Yount, 2003; Lai and Gallo, 2009
). Therefore, interactions
between AMPs and mammalian cell membrane occur mainly via
hydrophobic interactions, which are relatively weak compared
to the electrostatic interactions taking place between AMPs and
bacterial membranes. Furthermore, mammalian cell membranes,
unlike those of microbes, have a high content of cholesterol
(Yeaman and Yount, 2003; Lai and Gallo, 2009). The cholesterol
is proposed to reduce the activity of AMPs via stabilization
of the phospholipid bilayer (Zasloff, 2002). Notably, bacterial
cells typically have an inside-negative transmembrane potential
between −130 and −150 mV in contrast to mammalian cells,
where t he potential ranges from −90 to −110 mV (
Yeaman and
Yount, 2003; Matsuzaki, 2009; Ebenhan et al., 2014
). A stronger
negative membrane potential in bacteria may also contribute
to selectivity of AMPs between bacterial vs. mammalian cells
(Yeaman and Yount, 2003).
Membrane Disruption and Intracellular
Targets in Bacterial Cells
In order to reach the cytoplasmic membrane of Gram-negative
bacteria, AMPs have to first translocate through the outer
membrane. This outer membrane constitutes a permeability
barrier for many macromolecules, partly due to the divalent
cations Ca
2+
and Mg
2+
that bind to the phosphate groups of
the inner core of LPS and thereby provide stabilization of the
outer leaflet (
Clifton et al., 2015). AMPs are proposed to be
translocated through this outer membrane via so called self-
promoted uptake (Hancock, 1997; Hancock and Chapple, 1999;
Giuliani et al., 2007). This model suggests that, due to greater
affinity for the LPS, AMPs displace the divalent cations and
bind to the LPS. By being bulky, the A MPs then cause transient
cracks and permeabilize the outer membrane, thereby permitting
passage of the peptide itself across the membrane.
In contact with the cytoplasmic membrane, the AMPs form
an amphipathic secondary structure (if not already present)
essential for interaction with the cell membrane (Ebenhan et al.,
2014
). The charged domains of the peptide allow for interaction
with the hydrophilic head groups of the phospholipids, while
the hydrophobic domains of the peptide interact with the
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Mahlapuu et al. Antimicrobial Peptides As Therapeutic Agents
hydrophobic core of the lipid bilayer, thereby driving th e AMP
deeper into the membrane (Ebenhan et al., 2014). Several models
have been proposed describing the next events oc curring at
the bacterial cytoplasmic membrane, which ultimately lead to
membrane permeabilization (Figure 3;
Brogden, 2005; Toke,
2005; Nguyen et al., 2011). According to the “barrel-stave
model,” the peptides insert perpendicularly into the bilayer
while recruitment of additional peptides subsequently results in
formation of a peptide-lined transmembrane pore. In this pore,
the peptides are aligned with the hydrophobic side facing the
lipid core of t h e membrane and the hydrophilic regions facing
the interior region of the pore. According to the “toroidal-pore
model,” insertion of peptides forces the phospholipid to bend
continuously from one leaflet to the other, resulting in a pore
lined by both peptides and the hea d groups of the phospholipids.
Finally, in the “ca rpet model,” accumulation of peptides on the
membrane surface causes tension in the bilayer that ultimately
leads to disruption of the membrane a nd formation of micelles.
Membrane permeabilization by AMPs is suggested to
initially lead to leakage of ions a nd metabolites, depolarization
of t he transmembrane potential with subsequent membrane
dysfunction (e.g., impaired osmotic regulation and inhibition of
respiration), and ultimately, membrane rupture and rapid lysis of
microbial c ells (Yeaman and Yount, 2003; Brogden, 2005; E ckert,
2011).
Besides leading to membrane dysfunction and disruption,
membrane permeabilization is important for translocation of
certain AMPs into the cytoplasm, where they target key cellular
processes including DNA/RNA and protein synthesis, protein
folding, enzymatic activity, and/or cell wall synthesis (Figure 3;
Yeaman and Yount, 2003; Brogden, 2005; Yount et al., 2006;
Nguyen et al., 2011
).
Notably, it is suggested that bacterial death caused by AMPs
could be a result of multiple and complementary actions, referred
to as multi-hit mechanism. This strategy helps to increase
the efficiency of AMPs and to evade resistance development
(
Zhang et al., 2000; Yeaman and Yount, 2003; Peschel and
Sahl, 2006; Nguyen et al., 2011). It i s likely that the mode
of action of individual AMPs varies depending on parameters
such as peptide concentration, target bacterial species, as
well as tissue localization and growth phase of t h e bacteria
(Yeaman and Yount, 2003; Jenssen et al., 2006). Importantly,
regardless of the exact mode of action and target site, the
antibacterial activity of AMPs is dependent on the interaction
with microbial membrane (
Jenssen et al., 2006; Yeung et al.,
2011
).
Interestingly, the membrane-destabilizing activity of
AMPs is also utilized in so called Artilysins, which have
recently shown potential to effectively target resistant and
persistent Gram-negative infections. Artilysins are engineered
fusions of bacteriophage-encoded endolysins, which degrade
peptidoglycans of the bacterial cells wall, with specific AMPs,
which facilitate the transduction of the endolysin through the
protective outer membrane of Gram-negative pathogens to reach
its substrate (Briers et al., 2014a,b; Briers and Lavigne, 2015;
Defraine et al., 2016).
FIGURE 3 | Schematic illustration of bacterial killing mechanisms by AMPs.
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Mahlapuu et al. Antimicrobial Peptides As Therapeutic Agents
Immunomodulatory Activities
Recently published analysis of the available patent information
referring to the th erapeutic use of AMPs covering the period
from 2003 to 2015 concluded that most of the claimed
AMPs were characterized not only as potent antibiotics, but
also as effective modulators of inflammation or neutralizers
of pathogenic toxins (
Kosikowska and Lesner, 2016). The
broad range of immunomodulatory activities exerted by AMPs
include stimulation of chemotaxis, modulation of immune cell
differentiation and initiation of ad aptive immunity, together
contributing to the bacterial clearance of the host (Figure 4). The
immunomodulatory activities further include suppression of toll-
like receptors (TLR)- and/or cytokine-mediated production of
proinflammatory cytokines and anti-endotoxin activity, toget h er
preventing excessive and harmful proinflammatory responses
including sepsis (
Håversen et al., 2002; Davidson et al., 2004;
Mookherjee et al., 2006; Lai and Gallo, 2009; van der Does
et al., 2010; Yeung et al., 2011; Figure 4). As an example, LL-
37 and bovine lactoferricin have been reported to inhibit the
LPS (TLR4)-induced secretion of TNF-α and IL-6, respectively,
in TH P-1 cells and, in addition, LL-37 suppresses the LTA
(TLR2)- and LPS (TLR4)-induced production of TNF-α, IL-
1β, IL-6, and IL-8 in primary monocytes (
Mattsby-Baltzer
et al., 1996; Mookherjee et al., 2006). Several mechanisms have
been proposed to explain these immunomodulatory actions
of AMPs on mammalian cells (
Lai and Gallo, 2009). In the
“alternate ligand model,” the AMPs bind directly to specific cell
surface receptors thereby inducing receptor signaling. In the
“membrane disruption model,” the AMPs locally modify the part
of membrane that contains the receptor and thereby indirectly
alter the activation state and function of the receptor. In the
“trans-activation model,” the AMPs cause release of a membrane-
bound factor, which could then bind to its receptor (
Lai and
Gallo, 2009). In addition, scavenging of the endotoxin LPS by
AMPs has been suggested, preventing LPS from binding the
TLR4 and triggering inflammation (Lai and Gallo, 2009).
POTENTIAL FOR DEVELOPMENT OF
BACTERIAL RESISTANCE TO AMPS
The widespread bacterial resistance development toward AMPs
has generally been considered to be unlikely due to the AMPs’
mechanism of action involving at tacking multiple low-affinity
targets rather than one defined, high-affinity target characteristic
for conventional antibiotics, which makes it more difficult for
FIGURE 4 | Schematic illustration of immunomodulatory activities of AMPs. Pathogen recognition via pathogen recognition receptors (PRRs), such as T LRs,
by epithelial cells, macrophages, and dendritic cells, leads to killing via phagocytosis as well as release of proinflammatory cytokines and chemokines by these cells,
that subsequently stimulates the recruitment of additional immune cells to the site of infection. In addition, pathogen insult will lead to maturation of dendritic cells and
subsequent initiation of adaptive immunity. AMPs indirectly promote pathogen clearance by stimulating chemotaxis and immune cell differentiation, while also
preventing harmful inflammation and sepsis by inhibition of proinflammatory cytokine release and direct scavenging of bacterial endotoxins such as LPS. Up- or
down-regulation of responses by AMPs is indicated b y green arrows.
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