Post Graduate Seminar Presentation

on

ANTIMICROBIAL PEPTIDES POTENTIALS AND APPLICATION: A PROMISING

ALTERNATIVE TO ANTIBIOTICS.

By

Ajoseh, Samuel Oluwasegun (B.Sc, CPM).

Department of Microbiology, Faculty of Science, Lagos State University, Ojo.

1
 OUTLINES
• Classes of antibiotics
• Antibiotic and their mechanism of action
• Resistance mechanisms of bacteria to antibiotics
• Trends of resistance to antibiotics
• Diversity of antimicrobial peptides
• Properties of antimicrobial peptides
• Mechanisms of actions of antimicrobial peptides
• Applications of antimicrobial peptides
• Challenges with AMP
• Strategies to Improving AMP
• Conclusion
• References

2
3
 Antibiotics and their mechanisms of
Actions

Antibio
tics are
classifie
d base
o their
mecha
nism of
actio

Garima et al. 2017 4

 Mechanisms of Resistance to
Antibiotics

Fig.2

Three mechanisms of antibiotic resistance in bacteria. Most, but not all,

resistance mechanisms are encoded by plasmids, which are potentially
Fig.1. Kenneth, 2018
transmissible to other bacteria.
5
 Antibiotics Resistance Trend
Sources of data on nontyphoidal Salmonella: Resistance to fluoroquinolonesa

6
Figure.3 WHO, 2014
 preamble

All bacteria have developed and still developing resistance to antibiotics, the
reason for urgent need to think out of the box.

An evidence that birth critical novel research on antimicrobial peptides as

promising alternative to antibiotics.

7
 What are Antimicrobial Peptides
• Antimicrobial peptides (AMPs) are small (about
10kda), mostly cationic proteins that exhibit
activity against bacteria, fungi as well as certain
parasites and viruses (Józefiak and Engberg, 2017).
• Antimicrobial peptides are also known as small
oligopeptide with both antimicrobial properties and
Immunodulatory properties having poor
antimicrobial resistance tendencies (Peschel and
Sahl, 2006).
Note:
Antibiotic resistance has become
an increasingly serious problem
with global human deaths due to
antibiotic resistant infections
predicted to reach 10 million by
2050, with global economic
burden of $100 trillion by 2050.
Hence the need to think out of
the box.
8
Diversity of Antimicrobial Peptides

Archeal
Synthetic Diversity of AMP 0%
3% Kingdom
Protist
0% animal is
Fungi the most
Bacteria 1%
11% diversified
Plants with frog
11%
giving the
largest
Animals
74%

Figure.4
Adapted from Numbers obtained from http://aps.unmc.edu/AP/,
accessed on 5TH March., 2019. 9
 To understand mech. Of action novel
Properties of Antimicrobial Peptides AMP dev.
• Size
• The size of antimicrobial peptides varies from 6 amino acid residues for anionic peptides to greater than 59 amino acid residues
for Bac7. Even di- and tripeptides with antimicrobial activity have been reported.

• Sequence
• Peptides often contain the basic amino acid residues lysine or arginine, the hydrophobic residues alanine, leucine, phenylalanine
or tryptophan, and other residues such as isoleucine, tyrosine and valine. Some peptides contain amino acid repeats. Ratios of
hydrophobic to charged residues can vary from 1:1 to 2:1.

• Charge
• Anionic peptides are rich in aspartic and glutamic acids and cationic peptides are rich in arginine and lysine. Anionic peptides
that are complexed with zinc, or highly cationic peptides, are often more active than neutral peptides or those with a lower
charge.

• Conformation and structure

• Antimicrobial peptides can assume a variety of secondary structures including α-helices, elaxed coils and antiparallel β-sheet
structures.
– Amphipathic α-helical peptides are often more active than peptides with less-defined secondary structures. Peptides with a
γ-core motif (two antiparallel β-sheets with an interposed short turn in defensin-like molecules) are often very active.

• Hydrophobicity
• This characteristic enables water-soluble antimicrobial peptides to partition into the membrane lipid bilayer.

• Amphipathicity
• A trait by which peptides contain hydrophilic amino acid residues aligned along one side and hydrophobic amino acid residues
aligned along the opposite side of a helical molecule. For α-helical peptides, amphipathicity is often expressed as a hydrophobic
moment, which is the vector sum of hydrophobicity indices, treated as vectors normal to the helical axis. Other peptides often
show spatial separation of polar and hydrophobic residues that is less easy to quantify.

10
Michael et al., 2003; Tzong-Hsien Lee et al., 2016;
 Structural Characteristics of AMP
Take Home
The structural conformation of an AMP
Overview
determines its Specificity, Reactivity, affinity
and avidity on pathogens and
Immunodulatory responses
• Structure activity relationship studies of
antimicrobial peptides have revealed that there
are many factors that affect the specificity and
biological activity of these peptides.
• Factors such as charge, secondary structure,
hydrophobicity, amphipathicity and
hydrophobic moment are all critical to function
and are so interdependent that, altering one
property will often result in significant changes
to one or more of the others.
Zasloff and Magainins, 1987; Bulet et al., 1999; Bhunia et al., 2007; Thomas et al., 2010.
Charge
• Based on charge, antimicrobial peptides are mostly
Cationic while some are Anionic.
• Cationic antimicrobial peptides are of particular
interest as one face of the helix structure contains
majority of hydrophobic residues whereas the
opposite face contains mainly polar amino acids
allowing efficient solubilisation of microbial
membranes.
• Cationic antimicrobial peptides, as a class of
compounds, have broad structural diversity and
antimicrobial spectrum influenced primarily by the
amino acids that constitute the primary sequence of
the peptide.
• Examples in nature include mellitin derived from
honeybee venom, magainin obtained from the skin
secretions of the frog species Xenopus laevis, the
cecropins, a group of the dipteran insect defence
peptides.
11
 Structural Characteristics of AMP
Cont’d
Charge cont’d Amphipathicity
• Despite the vast majority of antimicrobial peptides • Amphipathicity of a peptide is a measure for the
being cationic in nature, a significant number of
anionic AMPs have been reported; serving as spatial separation between hydrophilic and
important weapons in the eukaryotic innate immune hydrophobic side chains.
response .

• Peptides that are anionic in nature tend to be rich in • The concept that amphipathicity is a key feature
glutamic and aspartic acids and include the of antimicrobial peptides has initiated the de
amphibian peptide Maximin-H5 and Dermcidin, a novo design of a number of simple antimicrobial
peptide derived from human sweat
peptides that consist of repeating sequences of
• Anionic antimicrobial peptides commonly consist of alternating hydrophobic and positively charged
5 to 70 amino acid residues, possessing a net charge stretches.
of −1 or −2 although structural characterization
demonstrated that the truncated form of bovine
peptide B, termed enkelytin, can possess a net • For instance, an increase of this parameter in
charge as high as −7.
magainin 2 analogs causes enhanced
permeabilization efficiency on
• A disadvantage of many anionic antimicrobial
peptides is that they often require cations, for phosphatidylcholine-rich bilayers, whereas the
example zinc (Zn2+), as cofactors for biocidal negatively charged model membranes are less
activity affected.

Harris et al., 2009; Prochazkova et al., 2006; Javadpour et al., 1996 12

 Structural Characteristics of AMP
Cont’d
Hydrophobicity
• Hydrophobicity of the peptide is usually defined
as the average of the numeric hydrophobicity
values of all residues and is a measure of the
ability of a peptide to move from an aqueous to a
hydrophobic phase.
• This parameter measures the degree of peptide
affinity for the lipid acyl chains at the core of
model and biological membranes.
• Most data are in agreement to associate an
increase in hydrophobicity with an enhanced
permeabilizing activity on zwitterionic bilayers.
• High measures of hydrophobicity in AMP have
been correlated with high cytotoxic activities
against the neutral membranes of mammalian
cells.
Eisenberg et al., 1984; Javadpour et al., 1996 ; Dathe and Wieprecht, 1999;
Hydrophobic and Hydrophilic
Angles
• The angles subtended by the hydrophilic and
hydrophobic faces of the peptide modulate the
manner in which amphipathic peptides bind to
membranes.
• Peptides with small hydrophilic angles and high
mean hydrophobicities tend to form
transmembrane pores, whereas peptides with
equivalent hydrophobic and hydrophilic faces
rather adopt a parallel orientation upon binding
to membranes.
• Changes in the hydrophilic angle had little effect
on the neutral lipid membranes of erythrocytes,
whereas the permeabilisation of highly charged
model membranes was decreased upon increase
of the polar angle.
13
 Mechanisms of Action of AMP
• Antimicrobial peptides mechanisms of actions
are:
– Pathogen killing mechanism and
– Immunodulatory mechanism.

• The killing mechanism is further divided into 2

• Membrane perforation mechanism
• Intracellular killing mechanism

14
 Membrane Perforation Mechanism of AMP in the Killing of Pathogens

•The barrel-stave model

The ‘barrel stave’ mechanism
describes the formation of transmembrane
channels/pores by bundles of peptides.

A crucial step in the barrel stave

mechanism requires peptides to recognize
one another in the membrane bound state.
Peptide assembly can occur on the surface or
within the hydrophobic core of the
membrane, since hydrophobic peptides can
span membranes as monomers.

•The carpet model

•According to the carpet model, the peptides
first bind onto the surface of the target
microbial cell membrane and subsequently
the membrane is covered by a ‘carpet-like’
cluster of peptides.

•Initial interaction with the negatively

charged target membrane is electrically Figure. 5
driven. In the second step, after a threshold
concentration has been reached, the peptides 15
cause membrane permeation Matsuzaki et al.,1991; Ben Efraim and Shai, 1997; Prashant et al, 2018
 Membrane Perforation Mechanism of AMP in the Killing of Pathogens
Contd
•Toroidal pore model
Furthermore, in the toroidal pore model, the
peptides also insert perpendicularly in the lipid
bilayer but specific peptide-peptide interactions
are not present .

Instead the peptides induce a local curvature of

the lipid bilayer with the pores partly formed by
peptides and partly by the phospholipid head
group.
The dynamic and transient lipid-peptide
supramolecule is known as the “toroidalpore”.

The distinguishing feature of this model as

compared to the barrel-stave pore is the net
arrangement of the bilayer:

in the barrel-stave pore, the hydrophobic and

hydrophilic arrangement of the lipids is
maintained, whereas in toroidal pores the
hydrophobic and hydrophilic arrangement of the
bilayer is disrupted.

Figure. 6

Wimley, 2010 Prashant et al, 2018

16
Intracellular Killing
Mechanism

Several intracellular mechanisms have

been demonstrated by AMP in the
killing of intracellular pathogens such
as viruses and some bacteria.

This is illustrated in the diagram by

the right

Mode of action for intracellular antimicrobial peptide activity. In this

figure Escherichia coli is shown as the target microorganism.

17
Figure. 7 Kim, 2005
 Mechanism of Action of killing Pathogens- models presented for
Immunodulatory function

Immunodulatory Mechanism

AMPs are produced by many immune cells

such as neutrophils and macrophages; they
are one of the first molecules that encounter
invading microbes.

Interestingly, some AMPs can produce a

variety of immune responses: activation,
attraction, and differentiation of white blood
cells; stimulation of angiogenesis; reduction
of inflammation by lowering the expression
of pro inflammatory chemokines; and
controlling the expression of chemokines and
reactive oxygen/nitrogen species.

Schematic illustration of immunomodulatory activities of AMPs. Pathogen recognition via

pathogen recognition receptors (PRRs), such as TLRs, by epithelial cells, macrophages, and
dendritic cells, leads to killing via phagocytosis as well as release of proinflammatory cytokines Figure. 8
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 Matsuzaki et al.,1991; Ben Efraim and Shai,
and subsequent initiation of adaptive immunity. AMPs indirectly promote pathogen clearance by
1997
stimulating chemotaxis and immune cell differentiation, while also preventing harmful
inflammation and sepsis by inhibition of proinflammatory cytokine release and direct scavenging 18
of bacterial endotoxins such as LPS. Up- or down-regulation of responses by AMPs is indicated by
 Molecular bases for AMP cell specificity

• The most interesting property of antimicrobial peptides is their cell specificity by which they
kill microbes but are nontoxic to mammalian cells (Matsuzaki, 1999).

• The relative insensitivity of eukaryotic cells to antimicrobial peptides is generally ascribed to

differences in lipid composition between eukaryotic and prokaryotic cell membranes (Kim,
2005).

• It has been proposed that the net positive charge of the antimicrobial peptides accounts for
their preferential binding to the negatively charged outer surface of bacteria, which is
different from the predominantly zwitterionic surface of normal mammalian cells (Utsugi et
al., 1991).

• Also a less negative membrane potential in eukaryotes than in prokaryotes plays an important
role in their selectivity.

• Tumor cells have lost part of their lipid asymmetry and therefore exhibit a more anionic
character on the external leaflet of their plasma membrane, which thus preferentially binds
cationic antimicrobial peptides (Michael et al., 2003).

19
 Antimicrobial Properties of Some AMPs– Plants peptides

Table showing the microbial activities of some plants peptides on some microbes

20
Adapted from Terra et al., 2015
 Antimicrobial Properties of Some
AMPs Cont’d
Comparison between antimicrobial peptide and Table showing Tet213 and bacteriocin zone diameters from S.
bacteriocins aureus, S. sanguis and P. aeruginosa using a 30 µg disk read at 24
h of incubation (means ± SD)

Bacterial P. aeruginosa S. aureus S. sanguis

species

Tet213 12.5 ± 3.1a 11.7 ± 1.1a,b 17.5 ± 3.5a,b

Bacteriocin 17.5 ± 2.9a 18.5 ± 4.4a 10.3 ± 1.7a

Tinidazole 7.9 ± 1.9 7.6 ± 2.4 8.7 ± 1.6

(control)
Figure .showing the Response of Tet213 and
bacteriocin with disks containing 30 mg of Tet213 (1) Note: a means P < 0.05 compared with bacteriocin, b means P
and 30 mg of bacteriocin (2) and 30 mg of ceftazidime
< 0.05 compared with control.
(C). A: P. aeruginosa, B: S. aureus and C: S. sanguis.

Liu Ming et al., 2015 21

Several other Antimicrobial activities of AMPs

Hilpert et al., 2015 22

Several other Antimicrobial activities of AMPs

Hilpert et al., 2015 23

Antiviral activities of Amp
• Examples include:
– Indolicidin
– Cathelicidin
– LL-37
– α-defensing; HNP-1,
HD-5
– Tilapia hepcidin etc

Avila, 2017
24
 Immunodulatory Activities of AMP

– Antimicrobial Peptides Chemoattract

and Activate Cells that Participate in
Adaptive Immunity eg Human α-
defensins 1 and 2 , β-defensins 1, 2,3
etc.

– Wound Repair and Angiogenesis eg

rat cathelicidin rCRAMP, LL-37, β-
defensins 2 etc.

Avila, 2017 25
 AMP role as growth promoter and as antimicrobial agents.

AMP Testing Effect

Species system
swine AMP A3, A5 In vivo Dietary supplement with potential
improve growth performance nd
apparent digestibility with
significant reduction in coiforms
Swine Lactoferrin In vivo Increased ADG and ADFL, diarrhea
reduction with significant reduction
I E. coli and Salmonella.
Chicken AMP-P5 In vivo Improved growth performance,
nutrient retention, intestinal
morphology and significant
reduction in excreta and coliforms
Chicken BT In vivo Up regulated chicken intestinal
immune gene expression.
Juvenie AMP P5 IV In vivo Significantly enhanced growth with
goat significantly retention of nutrients.

26
LuLu, 2014
General Overview of AMP Activities
Series1

Surface Immobilized 47

Wound healing 19

Insecticidal 33

Antifungal 1083

Antiparasitic 103

Spermicidal 13

Proteose inhibitor 26

Chemotactic 59

Antioxidant 22

Anticancer 217

Antiprotist 4

Antiviral 182

Antibacteria 34

Adapted from (http://aps.unmc.edu/AP/).

27
 Novel production and application of AMP

• Because most antimicrobial peptides are simple gene translation products, it is relatively
simple to produce them by recombinant expression methods, thus avoiding problems
associated with proteolysis and rapid clearing (De Bolle et al., 1996).

• However, this can be achieved if the producing microorganisms are resistant to the produced
peptide antibiotics.

• A promising new alternative with a lower cost is large-scale production of biologically active
proteins in transgenic plants or animals (Morrassutti et al., 2002).

• In recent years, remarkable results have been obtained by producing transgenic plants that
express elevated levels of antimicrobial peptides in leaves and seeds, thus potentially reducing
the need for using environmentally hazardous antibacterial or antifungal crop protecting
agents.

• An alternative for transgenic techniques that may be applicable in animals or humans is gene therapy .

• In specific applications, the rapid clearing of peptides can be reduced by mixing them with a
muco-adhesive polymer or with acrylic bone cement (Ruissen et al., 1999).

• In this respect, the development of vehicles delivering an efficiently high concentration of

peptide therapeutics at the right place, right time, and reasonable costs, has been becoming
non-challenging for pharmacologists (Avira, 2017).
28
 Challenges with AMP
Cytotoxicity, proteolytic digestion, kidney
clearance, immungenicity and low resistance are
mitigating factors to AMP global endorsement

• Although many eukaryotic AMPs have been identified and characterized, not many have
made it to clinical trials and only a few have been approved by the US Food and Drug
Administration (FDA).

• Most AMPs in clinical trials are analogues of natural AMPs, but there are some that are
completely synthetic (e.g., IMX942) .

• The majority of AMPs in clinical trials are limited to topical applications, due to systemic
toxicity, susceptibility of the peptides to protease degradation and rapid kidney clearance.

• Oral administration of AMPs can lead to proteolytic digestion by enzymes in the digestive
tract such as trypsin and pepsin (Honey and Hancook, 2013). .

• Moreover, systemic administration results in short half lives in vivo, protease degradation and
cytotoxic profiles in blood (Honey and Hancook, 2013). Some of these limitations or
challenges are briefly discussed below:

Jenssen et al., 2006; Honey and Hancook, 2013 29

 Challenges with AMP Cont’d

• Immunogenicity
The limited knowledge of amp pharmacology, and
pharmacokinetics, demand further finding for
global acceptability and commercialization

• Preliminary results in vivo suggest that antimicrobial peptides may work as single- shot therapeutics, so that
the risk of prolonged systemic concentrations high enough to induce an IgG antibody response may be
insignificant.

• However, certain cationic peptides are known to have a direct effect on mast cells, leading to histamine
release without an allergenic response. Guinea pig antibacterial polypeptide CAP11, neutrophil defensins,
and histatin 5 induce a strong histamine release.

• Furthermore, antimicrobial peptides may cross-react with other receptors despite being specific in
reactivity.

Jenssen et al., 2006; Honey and Hancook, 2013 30

 Challenges with AMP Cont’d
Resistance to AMP
• Alteration of net surface charges
Staphylococcus aureus transports D-alanine from the cytoplasm to the surface teichoic acid to reduce the net negative charge by
introducing basic amino groups. S. aureus also modifies its anionic membranes via MprF with L-lysine, increasing the positive net
charge. Both of these mechanisms are likely to repulse host defence peptides, which indicate a central role for antimicrobial peptide
resistance in the pathogenicity of S. aureus.

Capsule polysaccharide of Klebsiella pneumoniae limits the interaction of antimicrobial peptides with membrane targets and acapsular
mutants are more susceptible to peptide-mediated killing
In Salmonella species, the phosphate group linked to glucosamine I of Lipid A is substituted by a phosphorylethanolamine residue with a
free amino group and aminoarabinose is added to the negatively charged phosphate bound in ester linkage to the carbon-4 of
glucosamine II of Lipid A.

• Alterations in Lipid A
Salmonella species reduce the fluidity of their outer membrane by increasing hydrophobic interactions between an increased number of
Lipid A acyl tails by adding myristate to Lipid A with 2-hydroxymyristate and forming hepta-acylated Lipid A by adding
palmitate.The increased hydrophobic moment is through to retard or abolish antimicrobial peptide insertion and pore formation.

• Changes in membrane proteins

In some Gram-negative bacteria, for example Yersinia enterocolitica,alteration in the production of outer membrane proteins correlates
with resistance to killing by antimicrobial peptides.

• Role of transporters
ATP-binding cassette transporters import antimicrobial peptides and the resistance-nodulation cell-division efflux pump exports
antimicrobial peptides. Both transporters have been associated with antimicrobial peptide resistance.

• Proteolytic enzymes
Bacteria produce proteolytic enzymes,which may degrade antimicrobial peptides leading to their resistance, For example, LL-37 is
cleaved and inactivated by a S. aureusmetalloproteinase named aureolysin.
31
(Peschel, 2000; LuLu et al., 2014).
 Strategies to Improve AMP
Chemical Modification of AMPs
• Various chemical modifications of AMPs have been
utilized to improve the stability of peptides against
proteolytic digestion including the use of D-amino
acids, cyclization, acetylation and peptidomimetics.
• . Incorporation of non-natural D-amino acids into
AMP sequences reverses the stereochemistry of the
peptide and hence prevents protease degradation, as
enzymes are stereo specific.
• Generally, the use of D-amino acids in AMPs leads
to retention of the antimicrobial activity, while
preventing proteolysis. This confirms that these
AMPs interact with the bacterial membrane without
making use of specific receptors .
• Alternative strategies are thus important to reduce
the economic impact. These alternative approaches
are many and varied. For instance, the substitution of
positively-charged arginine in a sequence with other
charged non-natural amino acids, such as L-
ornithine and L-homoarginine, also increases
proteolysis stability of AMPs .
Zhao et al. 2016; Kanchanapally et al., 2015; Nordstrom and Malmsterm, 2017; 32
Delivery Systems for AMPs
• Another important strategy to improve the
properties of AMPs is to use delivery systems
which improves the stability, toxicity, half-life
and release profile of AMPs
• The AMPs can be covalently attached to the
delivery system or non-covalently encapsulated
by these different types of systems which
include:
Many inorganic materials such as mesoporous silica
particles, titanium, metalnanoparticles, quantum
dots , graphene, carbon nanotubes , silica
nanoparticles etc.
Other inorganic materials e.g., graphene oxide
nanotubes can be used to covalently attach
AMPs such as nisin to enhance the antimicrobial
activity against methicillin resistant
Staphylococcus aureus (MRSA)
 Examples of AMP in clinical trial and approved by USA FDA

peptides Progress Application AMP Analogue (Host)

Pexiganan (MSI- Phase III Topical cream for Infected diabetic Analog of magainin
78) foot ulcers (skin of African clawed
frog)
Omiganan Phase III Topical gel for Catheter infections and rosacea Derived from indolicidin (bovine)

Lytixar (LTX-109) Phase IIb Topical hydrogel for Uncomplicated Gram-positive skin Synthetic antimicrobial
infections, impetigo, and nasal colonization with S. Aureus
peptidomimetic

Novexatin (NP-213) Phase IIa Topical brush-on-treatment for Onychomycosis (fungal Derived from defensins (human)
nail infection

Leuprolide Approved To treat prostate cancer, endometriosis, uterine fibroids Leuprolide is a

and premature puberty
synthetic 9 residue
peptide analog of
gonadotropin
Adapted from Prashant et al., 2018 and Margit et al., 2016 33
 Conclusion
• Antimicrobial peptides are unique biomolecules with promising potential to treat
multidrug-resistant organisms. Interestingly, AMPs have many mechanisms of
action and can also display antiviral and anticancer activities. Over the last decade,
different strategies have been utilized to improve the efficacy of AMPs to push
forward their development as therapeutic agents. Overall, due to the emergence of
antibiotic resistance and the fact that many AMP-based drugs are in clinical trials or
under development, the next decade will reveal the benefits of these novel
compounds and lead to their commercialization.

34
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