ANTIMICROBIAL PEPTIDES

Bruno Maresca
University of Salerno
Dept. of Pharmaceutical Sciences
Antimicrobial Peptides

Within the last two decades, several peptides have been discovered on the basis of their
ability to inhibit the growth of microbial pathogens (antimicrobial peptides, AMPs).
The first AMPs, generally defined as magainins, were discovered in Xenopus laevis by
studying the oocyte system to analyze RNA expression (Zasloff, 1987).

“Ovaries used in these studies were removed surgically from anesthetized adult females.
Incisions were made through the skin and the muscular layer of the abdomen into the
peritoneum. After removal of the ovaries, the muscular wall and the skin were separately
repaired with sutures. Despite the non-sterile surgical procedure and the microbially
contaminated water-filled tanks to which the animals were returned immediately after
surgery, it was extremely rare for these surgical wounds to develop infection. Indeed, sutures
dissolved after several weeks, and normal healing of the scar almost always occurred.
Infections were not seen on the cut margins of the wound, at the sites of suture placement,
or within the communicating subdermal space or peritoneum. Healing occurred with little
gross evidence of inflammation or cellular reaction at the wound sites. The manner in which
wound healing occurs in this animal suggested that there might be a "sterilizing" activity in
the skin. I report here the characterization of a family of potent antimicrobial peptides purified
from female X. laevis skin. These peptides may be responsible for the extraordinary freedom
from infection characteristic of wound healing in this animal and appear to constitute a
previously unrecognized antimicrobial host-defense system” (from Zasloff , 1987 p.5449).

Following this discovery, over 500 AMPs have been identified, all of which are of animal or
plant origin. AMPs participate in the innate immune response by providing a rapid first-line
defense against infection. Generally, endogenous AMPs consist of 7 to 18 amino acids
(though some are up to 64 aa long). They are ideal for fast and efficient defense against
microbes (Nissen-Meyer & Nes 1997). AMPs are specific for microbial membrane and are
supposed to cause permeabilization and lysis of invading microbes. AMPs are derived from
larger precursors that can be obtained by post-translational modifications such as proteolytic
processing, glycosylation, carboxy-terminal amidation and amino-acid isomerization.
Cyclization of two short peptides leading to the fully circular θ-defensin has also been
observed. Due to the great diversity of AMPs discovered it is difficult to categorize them
except broadly on the basis of their secondary structure. Most multicellular organisms
express a cocktail comprising multiple peptides from several structural classes.

Although the exact mechanism of action of AMPs is not clear yet, there is an agreement
on that these peptides disrupt selectively cell membranes and that the structural
arrangement of the peptides plays a significant function. The outermost leaflet of the bilayer
of bacterial membranes, the surface exposed to the outside, is heavily occupied by lipids with
negatively charged phospholipids headgroups. In contrast, that of plants and animals is
composed primarily of lipids with no net charge. The phospholipids head group charge on
cell membranes and peptide charge distribution appears to have an important role in the
peptide/membrane interactions (Anderson et al 2004). There is growing evidence that
antibacterial or self-defense peptides (usually highly basic), recognize acidic phospholipids
exposed on the surface of the bacterial membrane (Pokorny & Almeida 2004). In bacteria,
the anionic lipids are present on the outer surface of the membrane (the inner membrane in
Gram- bacteria) whereas in mammalian cells, anionic lipids are present along the
cytoplasmic side of the membrane. This feature might account for their preferential activity
against bacteria (Sitaram & Nagaraj 2002). Studies on the mechanism of action of AMPs
have shown that they may interact directly with the membrane of pathogens causing a
dislocation of important lipid membrane and/or interference with specific lipid domains. AMPs
have been studied deeply for their use in human and animal infections (currently there are
several AMPs under clinical trials, all of natural origin). A general model explains the activity
of most AMPs suggesting that the peptide interacts with the membrane causing displacement

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of lipids, alteration of membrane structure, and in certain cases entry of the peptide inside
the cell.
A consensus sequence from the over 500 AMPs known has not been identified
(http://aps.unmc.edu/AP/main.html; Antimicrobial Peptide Database, APD). Thus, the possibility to predict a
sequence with potential antimicrobial (AM) activity has so far failed, also considering that a
15 aa-long AMP is one of 2015 different possible peptide sequences.
The diversity of sequences is such that the same peptide sequence is rarely recovered
from two different species of animal, even in those evolutionary related. Dissimilarity is such
that the 5,000 living anuran frogs may produce 100,000 different AMPs. Since single
mutations can dramatically alter the biological activity of each peptide, the diversity probably
reflects the species' adaptation to their unique microbial environment. Natural peptides
composed of all D-amino acids, in place of L-amino acids, typically retain full antibiotic
potency while exhibiting expected resistance to enzymatic proteolysis. These peptides
exhibit broad-spectrum activity against a wide range of Gram - and Gram+ bacteria, protozoa,
yeasts, fungi and viruses. AMPs are universal multifunctional molecules to which so far
resistance has not developed.

While to date all AMPs identified have been obtained by purification of animal or plant cell
extracts, we developed an exclusive genomic procedure by which we have identified, 4
peptides that have AM activity (Patent Appl. No. 05017198.2). This approach allowed, for
the first time, to predict an active AMP sequence on the basis of their
nucleotide/amino acid sequence.

We had previously shown that expression in Salmonella of heterologous Synechocystis

membrane-bound ∆12-desaturase gene caused impairment of heat shock response and loss
of virulence of this pathogen in macrophage infection (Colonna-Romano et al., submitted).
Insertion of ∆12-desaturase in Salmonella membrane caused a change in membrane physical
state but not a change in saturated/unsaturated fatty acid profile, since the expressed
Synechocystis protein does not exert enzymatic activity in Salmonella (this bacterium does
not have a corresponding desaturase nor its substrate). We tried then to express separately
in Salmonella only the two membrane-spanning regions of this protein to obtain a similar
effect. Unexpectedly, we could clone the two fragments only under an inducible promoter,
thus, by serendipity, we identified two fragments of ca. 60 amino acids each that had AM
activity. Both aa sequences are not present in AMP databases and have no homology with
any of the described AMPs.
We reasoned that likely reason why the expression of Synechocystis ∆12-desaturase (or
its membrane spanning regions) had a toxic effect in Salmonella and E. coli was that in
nature this protein is bound to a cyanobacterial membrane that has a very different
protein/lipid composition compared to those of eubacteria. Each of the corresponding DNA
portions of the two membrane portions was split in two shorter fragments of 15 nt each
producing 4 nucleotide sequences that were cloned independently. All four corresponding
peptides were synthesized and tested in Salmonella and E. coli and proven to inhibit
bacterial growth.
This surprising result was corroborated by a recent paper by Watt who hypothesized
screenings for peptide drugs from the natural repertoire of biodiverse protein folds (Watt,
2006). In that article Watt “…outlined an alternative approach based on libraries of natural,
highly structured peptides that offers new opportunities for identifying effective, specific
inhibitors. Libraries of such peptides comprise both random and structured peptides encoded
by natural genes of diverse bacterial genomes. ... these diverse libraries containing millions
of phylomers constitute virtually all of the available classes of protein fold structures,
providing a rich source of peptides that interact specifically and with high affinity to human
proteins. This approach may help not only in understanding the implications of each
interaction identified within the interactome but also in the development of effective drugs… “.

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The widespread resistance of bacterial pathogens to conventional antibiotics has renewed
interest in the use of alternative natural microbial inhibitors such as AMPs. Recently, AMPs
have been shown to display both antiviral and antifungal activities in vitro and have also been
shown to be effective in experimental infections with multidrug resistant Staphylococcus
aureus and Mycobacterium tuberculosis. Recent papers (Gordon et al., 2005; Andres &
Dimarcq, 2004) have reviewed recent progress on AMPs as commercial drugs.

The pharmaceutical industry has continuously met this need by modifying existing
antibiotics and developing newer antibiotics in a timely fashion. These successful efforts
have produced the wide variety of currently available drug classes of antibiotics [β-lactams
(penicillins, carbapenems, cepahalosporins), glycopeptides, macrolides, ketolides,
aminoglycosides, fluoroquinolones, oxazolidinones, and others]. A novel approach to design
synthetic peptides is thus necessary for the development of AMPs as a new class of drugs to
prevent and treat systemic and topical infections that do not easily generate resistance.

For AMPs, there are several different potential strategies for their general therapeutic
application:

1. as single anti-infective agents,

2. in combination with conventional antibiotics or antivirals to promote any additive or
synergistic effects,
3. as immunostimulatory agents that enhance natural innate immunity, and
4. as endotoxin-neutralizing agents to prevent the potentially fatal complications
associated with bacterial virulence factors that cause septic shock. Table 1
summarizes the theoretical advantages and disadvantages of developing
antimicrobial peptides as single therapeutic antimicrobial agents.

TABLE 1
Development of Antimicrobial Peptides as Anti-Infective Drugs

Advantages Disadvantages
Broad-spectrum activity (antibacterial, antiviral, Discovery costs of synthesis and screening
antifungal) Patent exclusivity for economic viability
Rapid onset of killing Systemic and local toxicity
Cidal activity Reduced activity based on salt, serum, and pH
Potentially low levels of induced resistance sensitivity
Concomitant broad anti-inflammatory activities Susceptibility to proteolysis
Pharmacokinetic (PK) and pharmacodynamic (PD)
issues
Sensitization and allergy after repeated application
Natural resistance
Confounding biological functions (e.g., angiogenesis)
High manufacturing costs

Several peptides have shown promise for possible drug development in preclinical studies.
Helix Biomedix Inc, (Bothell, WA, USA) is developing a broad-spectrum microbicide to be
used against bacteria (Garnerella, Prevatella, Peptostretococcus, and Bacteroids), fungi
(Candida sp.), and viruses (HSV-2, HSV-1) that cause sexually transmitted diseases
(STDs).31 Plectasin, (Novozymes, Bagsvaerd, Denmark) is a recently discovered fungal
defensin from Pseudoplectania nirgrella that is effective against Gram+ bacteria in vitro and
Streptococcus pneumoniae in mouse peritonitis and pneumonia models.

The entire genome of the blue green alga Synechocystis codes for ca. 3,000 genes
(entirely sequenced Synechocystis sp. PCC 6803; http://www.kazusa.or.jp/cyanobase/
Synechocystis/). We carried out a preliminary analysis of all membrane spanning proteins of

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Synechocystis genome that allowed us to identify over 300 short peptide regions of
membrane proteins with potential AM activity. Tests were performed using an available
(though unsatisfactory) algorithm that may recognize an aa sequence with potential AM
activity.

Our finding opened for the first time the possibility to predict specific aa sequences
with potential AM activity without the need to assay metazoan protein extracts for AM
activity.

We intend to expand further the Synechocystis genomic analysis with the aid of a new
dedicated algorithm.

A general understanding of the mechanism of action of AMPs is still inadequate. While

most models so far presented consider the charge and the hydrophobicity of a peptide as
key factors to assess AM activity of a peptide sequence, they still represent an over-
simplification. Thus, a more robust theoretical model is still not on hand. We will use an
innovative and more complex approach based on highly specific peptide/membrane
interactions to determine essential physical/chemical factors responsible for AM activity.
The investigation of the interaction between a peptide and a lipid membrane provides the
basis for the understanding of the AM activity of natural peptides. Using an integrated
approach, comprising genomics, molecular biology, membrane biophysics, computer
modeling and microbiology, new more active and safer AMPs can be produced.

We will use both the sequences obtained through the genomic analysis and new
sequences that will emerge from the computer analysis. To date, algorithms predicting aa
sequences with AM activity have been based on two physical parameters only: net charge
and 3D-spatial organization. Yet this approach has been unsuccessful. We have elaborated a
more sophisticated and comprehensive algorithms which considers over 300 physical-
chemical parameters

• The simulation of lipids membranes will be carried out by Molecular Dynamics (MD),
Dissipative Particle Dynamics (DPD) and Interface Dynamics (ID). Specifically, lipid
membranes will be simulated upon the effect of several physical (such as different
temperatures and pressure) and chemical (such as water content, and interaction with
small molecules) perturbation. Recently, we have parameterized several natural lipids for
DPD simulations (among others POPC, DMPC, DPPC, cholesterol, DOPE and OA) and
have prepared virtual membranes corresponding to different prokaryotic cells. DPD is
employed to simulate the temporal evolution of lipid membranes upon the insertion of
small molecules.

• Parameterization of lipid molecules permits the design of specific membranes and the
investigation of the interaction between a parameterized molecule with the target
membrane. This method introduces a higher degree of specificity in the interaction with
small molecules and a given peptide, since the membrane is no longer considered, not
even in computer simulation, an isotropic medium. Most models consider the charge and
the hydrophobicity of a peptide to evaluate its AM activity. These methods will be further
developed to provide a detailed understanding of the interactions of a candidate peptide
with target membranes.

• The theoretical screening of potential new peptides exceeds largely the computing
capabilities of the fastest computer. Therefore we will set up a further computational step.
Selected short peptides will be analyzed and their topological, electronic, structural
properties extracted using advanced Quantitative Structure-Property Relationships
(QSPRs). These data will be combined with the results of the DPD calculations and with

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 the experimental data on AMP activity.

• Finally, a novel and efficient genetic algorithm (GA) analysis will be employed to extract a
new model for AMP activity. In a GA, a population containing a number of trial solutions
each of which is evaluated (to yield a fitness) and a new generation is “created” from the
best of them. The process continues through a number of generations with the aim that
the population will evolve to contain an acceptable solution

The computer will solve the algorithm based on the chemical structure of different
peptides such as dipolar moment, molecular density, inertial moment, volume and surface of
molecules, atomic charge, average polarization, orbital analysis, and, as well as with output
from standardized DPD simulations. Subsequently, membrane perturbation by a peptide will
be included in the QSAR analysis by adding stress tensor and the surface tension. Finally,
the best peptide candidates can be synthesized and characterized chemically and physically
and assayed in microbiological tests.

In the latest issue of Nature (Oct 19, 2006), Loose et al. (2006) have reported a rational
strategy to generate new AMPs. Peptides were designed using a linguistic model of available
natural AMPs using aa sequences of natural AMPs as a formal language and built a set of
regular grammars to describe this language. These authors used this set of grammars to
create new synthetic AMP sequences. However, as emphasized by the same authors, this
approach has a number of limitations, such as sequence families that are poorly conserved
on an amino-acid level would not benefit from this approach. Second, due to the simple
nature of regular grammars used, they would be less useful for designing larger sequences
with complex tertiary or quaternary structures. Yet this approach is poorly effective in
designing active AMPs since only the 1 of 40 of the designed AMPs is active with a MIC 50 at
concentration lower than 16 µg/ml. The major limit of this approach is due to the intrinsic
methodology used that is based on the identification of short consensus sequences among
the 500 AMPs and grouping them in subclasses. These represent “words” of the AMP
language and not a procedure applicable to all natural active AMPs. In addition, to obtain
new AMP sequences, the rules used to fill in the remaining aa are still based on the charge
of aa and spatial arrangements. This method, though represents a significant step to
generate new AMP sequences has the strong limitation that can be improved only by
including in the test new AMP sequences not yet available.

In summary, our approach for the discovery of new active and safe AMPs is based on the
recognition that AMPs acts specifically with specific components of membrane domains and
that this interaction is responsible for their mode of action. Further, our previous finding that
intra-membrane spanning regions of proteins of cyanobacteria have AM activity in
eubacterial human pathogens suggests that specific DNA fragments coding for membrane
spanning regions of evolutionary distant genomes, such as those of cyanobacteria, may
represent a reservoir of additional AM sequences active on pathogenic eubacteria. These
findings combined with our novel computational analyses provide a very innovative approach
for the identification of AMPs.

References

Anderson RC, Hancock RE, Yu PL. Antimicrobial activity and bacterial-membrane interaction of ovine-
derived cathelicidins. Antimicrob Agents Chemother, 48: 673-6, 2004
Andres E, Dimarcq JL. Cationic antimicrobial peptides: update of clinical development. J Intern Med.
255: 519-20, 2004
Gordon, EG. Romanowski, AM. McDermott A. Review of Antimicrobial Peptides and Their Therapeutic
Potential as Anti-Infective Drugs. Curr Eye Res. 30: 505–15, 2005
Loose C, Jensen K, Rigoutsos I, Stephanopoulos G. A linguistic model for the rational design of

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 antimicrobial peptides. Nature 443: 867-9, 2006
Nissen-Meyer, J & Nes IF. Ribosomally synthesized antimicrobial peptides: their function, structure,
biogenesis, and mechanism of action. Arch Microbiol. 167:67-77, 1997
Pokorny A, Almeida PF. Kinetics of dye efflux and lipid flip-flop induced by delta-lysin in
phosphatidylcholine vesicles and the mechanism of graded release by amphipathic, alpha-helical
peptides. Biochemistry, 43: 8846-57, 2004
Sitaram N, Nagaraj R. Host-defense antimicrobial peptides: importance of structure for activity. Curr
Pharm. Design, 8: 727-42, 2002
Watt PM. Screening for peptide drugs from the natural repertoire of biodiverse protein folds. Nature
Biotech. 24: 177-83, 2006
Zasloff M Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of
two active forms, and partial cDNA sequence of a precursor. Proc Natl Acad Sci U S A. 84:
5449–5453, 1987 p.5449