ANTIMICROBIAL PEPTIDES

DEPARTMENT OF PERIODOTOLOGY
 INTRODUCTION
HISTORY
CLASSIFICATION
MECHANISM OF ACTIONS
OF AMPS
FUNCTIONS OF AMPS
CONTENTS
ANTIMICROBIAL PEPTIDES
AND THE ORAL CAVITY
ANTIMICROBIAL PEPTIDES
IN PERIODONTAL HEALTH
AND DISEASE
DISORDERS ASSOCIATED
WITH AMPS
CLINICAL
APPLICATIONS/FUTURE
PERSPECTIVES

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 INTRODUCTION
1. Periodontal disease is an inflammatory disease initiated by the formation
of mixed biofilms on the teeth.
2. Biofilm bacteria and their toxins perturb gingival epithelial cells leading to
a sequence of inflammatory and immune processes that may result in the
destruction of gingival tissues, attachment loss and periodontal pockets.
3. The host-response consists of a cascade of events by the innate and
acquired immune systems.
4. AMPs are components of innate immunity, forming the first-line of
defense used by many organisms against the invading pathogens (Jenssen
et al., 2006).
Bartold PM, Van Dyke TE. Periodontitis: A host-mediated disruption of microbial homeostasis. Unlearning Contoso 3
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learned concepts. Periodontol 2000 2013;62:203-17.
 Gingival epithelium works in many ways for the protection of underlying
connective tissue.

First, it provides a physical barrier which does not allow microbes to invade while
permitting selective transaction with oral environment.
Second, upon interaction with microbes it secretes cytokines as well as chemokines to
activate the influx of neutrophils and other immune cells into the sulcular area.
Third, the gingival epithelium has been found to be a source of antimicrobial products
termed antimicrobial peptides (AMPS).
Altman H, Steinberg D, Porat Y, Mor A, Fridman D, Friedman M, et al. In vitro assessment of antimicrobial Contoso 4
peptides as potential agents against several oral bacteria. J Antimicrob Chemother 2006;58:198-201. S u i t e s
 • These AMPs are considered to be important for the innate immunity of
the host.
• These peptides are known to possess a wide spectrum of antimicrobial
activity acting against Gram-positive and Gram-negative bacterial
species as well as yeast and some viruses.
• Thereby, AMPS have the potential to prevent various oral diseases of
microbial origin such as periodontitis, dental caries, candidiasis, and
herpetic gingivostomatitis.

G. Diamond, N. Beckloff, and L. K. Ryan, “Host defense peptides in the oral cavity and the lung: similarities and differences,” Journal of Dental Research, vol. 87, no.
10, pp. 915–927, 2008.
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• The amps are biological defense molecules encoded by specific genes exressed

by the host cells as an active participant in the innate immune system

keeps natural microflora in a steady state.
GENERAL FEATURES OF AMP'S:
positive charge
peptide chain of less than 60-100 amino acid units
broad spectrum of antimicrobial action
ability to adopt an amphipathic shape

Gorr SU. Antimicrobial peptides of the oral cavity. Periodontol 2000 2009;51:152-80. Contoso 6
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 Properties such as a-
Net positive charge and the ability to adopt an amphipathic structure attract the
AMP’s to the negatively charged phospholipids at the outer surfaces of
bacterial membranes.

On disrupting the membrane integrity, they target the cytoplasmic elements

ultimately causing

bacterial cell death

Hence, they help maintain the complex ecological homeostasis between the
host and bacteria in the oral environment.
Diamond G, Beckloff N, Weinberg A, Kisich KO. The roles of antimicrobial peptides in innate host defense. Contoso 7
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Curr Pharm Des 2009;15:2377-92.
• AMPs are produced by a variety of cells in the body, including
1. epithelial cells,
2. lymphocytes,
3. phagocytes,
4. neutrophils, and
5. keratinocytes in places including the -
lymphatic system,
genitourinary tract,
gastrointestinal tract, and
immune systems.

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 • Nearly 45 known AMP gene products are secreted by the salivary glands with
some secreted in the GCF as well.
• The neutrophils being an important part of the innate defense also secrete
AMPs.
• Resistance against AMPs is developed at a relatively slower pace and is a less
common phenomenon.

G. Diamond, N. Beckloff, and L. K. Ryan, “Host defense peptides in the oral cavity and the lung: similarities and differences,” Journal of Dental Research, vol. 87, no.
10, pp. 915–927, 2008.
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 HISTORICAL TRACK ON ANTIMICROBIAL
PEPTIDES

• 1922-ALEXANDER FLEMMING -isolated first human antimicrobial peptide from nasal

mucous
• 1939-Dubos extracted an antimicrobial agent from a soil Bacillus strain.
• HOTCHKISS AND DUBOS fractionated this extract and identified an AMP which was
named gramicidin.
• The first reported animal-originated AMP is defensin, which was isolated from rabbit
leukocytes in 1956.
• 1966 ZEYA AND SPITZNAGEL first decribed Peptides with anti microbial activity
• To date around 106 Human host defense peptides have been identified (Wang, 2014)

Mukesh Pasupuleti, Artur Schmidtchen & Martin Malmsten. Antimicrobial peptides: key components of the innateContoso 10
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immune system. Critical Reviews in Biotechnology.2012;32:2, 13-171
classification

Huan Yuchen, Kong Qing, Mou Haijin, Yi Huax .Antimicrobial Peptides: Classification, Design, Application Contoso 11
and Research Progress in Multiple Fields.Frontiers in Microbiology .2020 S u i t e s
 Mechanism of action

• AMPs are positively charged amphipathic molecules

that kill bacteria by using two major MOAs.
• In the first MOA, AMPs cause membrane disruption,
leading to cell lysis and finally cell death.
• In the second mechanism, AMPs traverse through the
cell membrane without disrupting it and inhibit critical
intracellular functions by binding to DNA, RNA, or
intracellular proteins.

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 1. Attraction-
the electrostatic interaction between the cationic peptides and the anionic
moieties on bacterial membrane (Gram-negative bacteria: lipopolysaccharide
[LPS] phosphate group and anionic lipids; Gram-positive bacteria: teichoic
acids) is presumed to cause attraction.

Mallapragada S, Wadhwa A, Agrawal P. Antimicrobial peptides: The miraculous biological molecules. J Contoso 13
Indian Soc Periodontol 2017;21:434-8 S u i t e s
 2. Attachment –
After getting bound to the bacterial membrane surface the AMPs

get transformed into a secondary structure

causes them to orient either parallel or perpendicular to the membrane.

The initial low and later high peptide/lipid ratios

allow the bacterial membrane to stretch causing it to thin down.

.
Mallapragada S, Wadhwa A, Agrawal P. Antimicrobial peptides: The miraculous biological molecules. J Contoso 14
Indian Soc Periodontol 2017;21:434-8 S u i t e s
 • This is followed by subsequent pore formation with the peptides getting
oriented in a perpendicular fashion to insert into the bilayer

Mallapragada S, Wadhwa A, Agrawal P. Antimicrobial peptides: The miraculous biological molecules. J Contoso 15
Indian Soc Periodontol 2017;21:434-8 S u i t e s
3.Models of insertion –
the penetration of the AMPs across the bacterial membrane takes place by either the carpet
model, the barrel-stave model, or the toroidal pore model causing the ultimate bacterial cell
death.

Mallapragada S, Wadhwa A, Agrawal P. Antimicrobial peptides: The miraculous biological molecules. J Contoso 16
Indian Soc Periodontol 2017;21:434-8 S u i t e s
• Based on the ability to form pores the models are divided into two groups:
pore-forming models and non-pore forming models.
• The two main pore formation models are barrel-stave and toroidal pore.
• The non-pore formation model is the carpet mechanism.
• The mechanism can be divided further into two based on cellular absorption
processes: ATP dependent and ATP independent uptake process. 
• The barrel-stave model, carpet model, or toroidal model are all ATP-
independent uptake mechanisms, while macropinocytosis is an energy-
dependent uptake mechanism 

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 BARREL-STAVE
MODEL
• In the barrel-stave model, AMPs bind with the membrane outer surface via electrostatic
interaction following it then undergo a conformational change attaining an amphipathic
structure.

• The “barrel-stave” model indicates that peptides insert into the membrane and direct their
hydrophobic regions toward the lipid core of the bilayer, forming a transmembrane pore.

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 Toroidal pore model
• The “toroidal pore” model implies that
AMPs insert themselves perpendicularly into
the membrane through interactions between
the lipid bilayer and the hydrophilic region
of the peptides.
• This generates a rupture in the hydrophobic
part of the membrane, resulting in a strain.
• The strain, as well as membrane thinning,
creates the surface of bilayer fragile to the
AMPs by destructing the composition of the
membrane 
• In doing so, the membrane distorts and thus
forms a “toroidal pore

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 CARPET
MODEL
• .
• The “carpet” model suggests that AMPs
accumulate parallel to the membrane
surface, forming a “carpet” . Once a
threshold concentration of peptides has
been reached, they exert a “detergent”
effect, leading to the rupture of the cell
membrane.

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 role of antimicrobial peptides

The terms “antimicrobial peptide” or

Insert or Drag & Drop your photo
“natural antibiotic” have biased the
interpretation of the function of AMPs, and
most likely delayed the discovery of other
functions (Lai and Gallo, 2009).
Significance and other functions of AMPs
• microbicidal activities in host tissues,
• stimulating cell proliferation,
• activating the immune system, and
• exhibiting a cytotoxic effect on tumor cells
(McDermott, 2009).
• antiviral,
• antitumor (Bhutia and Maiti, 2008; Rodrigues
et al., 2009),
• antiobesity (Dodd et al., 2010),
• angiogenesis, and vasculogenesis properties
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• Immuno-modulatory activity such as promotion of wound healing (Hancock,
2001; Bowdish et al., 2005c; Tokumaru et al., 2005),
• inhibition of proinflammatory responses induced by LPS (Sawa et al., 1998;
Bals et al., 1999a; Bals et al., 1999b),
• recruitment of leukocyte (Welling et al., 1998),
• chemokine production (Territo et al., 1989; Chertov et al., 1996; Scott et al.,
2002; Grutkoski et al., 2003), and
• anti-inflammatory properties (Turner et al., 1998; Scott and Hancock, 2000;
Scott et al., 2000a; Scott et al., 2000b; Nagaoka et al., 2001).
• antibioflim

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 ANTIMICROBIAL PEPTIDES OF ORAL CAVITY
Antimicrobial proteins and peptides (AMPS) are expressed by salivary glands,
oral epithelial cells and neutrophils.
✔A complex mixture of over 45 antimicrobial proteins and peptides are found
in oral fluids (Denny et al., 2008);
13 are upregulated in periodontal disease, while 11 are down-regulated
✓Defensins and cathelicidin are important contributors for maintaining the
balance between the healthy and diseased conditions of the oral cavity

G. Diamond, N. Beckloff, and L. K. Ryan, “Host defense peptides in the oral cavity and the lung: similarities and differences,” Journal of Dental Research, vol. 87, no.
10, pp. 915–927, 2008.
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• Based on the site of expression, size, structure, and pattern of disulphide bridges,
1.Defensins are classified into 2 types;α, β,
• 2.calprotectin,
• 3.adrenomedullin,
• 4.histatins, and
• 5. cathelicidin
• 6.cystatin
• Of these 𝛼-defensins are of nonepithelial origin, with their major source being the
neutrophils migrating into the gingival sulcus as primary host response, whereas histatins
are secreted in saliva, produced by parotid and submandibular salivary gland duct cells.
• Defensins and cathelicidins are supposed to be the most abundant human AMPs.

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DEFENSINS
• The defensins chiefly constituted two families:
1.The alpha-defensins (α-defensins) predominantly found in neutrophils and
the intestine, and
2.The β-defensins predominantly found in epithelial cells.
• In the oral cavity, saliva has been found to be associated with the expression
of defensins alongside the oral, sulcular/pocket, junctional epithelia, as well
as the dentogingival junction.
• The polymorphonuclear leukocytes found in discrete areas of the gingival
connective tissue are also associated with the expression of defensins.

Khurshid Z, Naseem M, Sheikh Z, Najeeb S, Shahab S, Zafar MS, et al. Oral antimicrobial peptides: Types Contoso 28
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and role in the oral cavity. Saudi Pharm J 2016;24:515-24.
ALPHA -DEFENSIN
• First group of defensins isolated from human, rat, guinea pig, and rabbit
phagocytes.
• They comprise a large subfamily of arginine-rich, and therefore cationic
• They make up as much as 7% of all proteins in human PMNs
• They act on a wide variety of bacteria and some fungi.
• However, they are also cytotoxic to various host cells
• The mature peptides are stored in azurophilic granules of human PMNs, and
in the granules of Paneth cells found at the base of the crypts of Lieberkiihn
in the small intestine of mice, rats, and humans

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 • α-defensins isolated from neutrophils in the oral cavity include human
neutrophil peptide (hNP)-1, hNP-2, hNP-3, and hNP-4.

• hNP-1–3 are abundantly expressed in saliva (90%) and hNP-4 are roughly
100-fold lower.

• hNP-5 and 6 are expressed in the enteric system and not the oral cavity.

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Jung S, Mysliwy J, Spudy B, Lorenzen I, Reiss K, Gelhaus C, et al. Human beta-defensin 2 and beta-defensin 3 chimeric peptides
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reveal the structural basis of the pathogen specificity of their parent molecules. Antimicrob Agents Chemother 2011;55:954-60.
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β-defensin
• They were first described in columnar cells of bovine tracheal pseudostratified epithelium,
and are now known to be expressed, at least at the mRNA level, in various mucosa and
epithelial tissues.
• β-defensins are a bit larger than α-defensins, with 36-42 residues, are very basic, and have
the characteristic 3-sheet structures stabilized by three intramolecular disulfide bonds.
• Six members of the β-defensin family are expressed in humans, that is, human β-defensin
(hBD)-1–6 principally by all epithelial surfaces (skin, oral mucosa, respiratory tract,
gastrointestinal tract, genitourinary tract, and the kidney).
• Studies reveal that only hBD-1–3 are expressed in the oral cavity (gingival epithelia,
tongue, palate, and buccal mucosae, salivary glands/ducts, and saliva).

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Cathelicidins

• Cathelicidins are AMP’s belonging to the α-helical peptides

without cysteine
• LL-37 is the only cathelicidin expressed in humans.
• The number 37 denotes the length of the peptide and “LL” stands
for the two amino acids formed by leucine.
• Other names such as human cathelicidin antimicrobial proprotein
18, CAMP, and FALL-39 have been used to describe LL-37.
• They have also been found to be expressed in both steady state and
disease state of the periodontium

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• Active against gram positive and gram negative bacterias, it directly binds to
the LPS of bacterial cells, and is native to the oral cavity in several sites,
including the buccal and tongue mucosa, gingival crevicular fluid.
• By activating antigen-presenting cells, it presents as a chemoattractant for
immune cells, including monocytes, T cells, etc
• Their mode of action involves intracellular killing of the phagosomes after
phagocytosis of the bacteria, where the AMP in the neutrophil is severed into
a fully developed peptide

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Histatins
• These AMPs are exclusively resided in the salivary glands, i.e.
parotid and submandibular salivary duct cells.
• Histamine 1, 3 and 5 are found to be predominant of the total
histatin proteins (85%) in the saliva.
• They have a major role in fungicidal activity, having a noteworthy
role in oral candidiasis restraint, especially histatin-5, which is the
most significantly active against candida species as well as
bactericidal activities against Porphyromonas gingivalis and
Streptococcus mutans, which play a key role in the
etiopathogenesis of periodontal disease and dental
caries ,respectively.
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 ANTIMICROBIAL PEPTIDES IN
PERIODONTAL HEALTH AND
DISEASE
• The oral cavity is unique in that the microorganisms and pathogens have an
easy access to it and the rest of the body through the epithelium and the
gastrointestinal tract.
• The gingival epithelial cells on exposure to bacteria related to periodontitis
actively express AMPs.
• Nearly twenty genetic disorders connected to periodontal disease have been
identified so far and are associated with the expression of AMPs.
• GCF analysis for the content of LL-37 was carried out by Puklo et al. in
2008.
• They concluded that LL‑37 was chiefly produced by neutrophils in the
healthy periodontium and helped maintain it in a steady state.
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 Cathelicidins and Periodontal Disease
• In patients with chronic or aggressive periodontitis cells other
than neutrophils were responsible for the production of LL-37.
• Therefore, in case of a state of deficiency in LL‑37 secretion,
as noted in morbus Kostmann syndrome and Papillon–Lefevre
syndrome, the propensity to infection including chronic
periodontal infections increases.
• In addition, LL-37 could also modulate neutrophil response to
bacterial challenge, especially in aggressive forms of disease
• Children with Down’s syndrome commonly suffer from
periodontal disease as well and are related to LL-37 deficiency.

Okumura K. Cathelicidins-therapeutic antimicrobial and antitumor host defense peptides for oral diseases. Jpn Contoso 42
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Dent Sci Rev 2011;47:67-81.
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• LL-37 possesses a broad spectrum of activity against both Gram-positive and
Gram-negative bacteria including periodontopathogens such as
Porphyromonas gingivalis, Prevotella intermedia, and Aggregatibacter
actinomycetemcomitans alongside fungi and viruses.
• It miraculously neutralizes the activity of LPS and thus help protect the
tissues from their harmful effects. They maintain a balance between pro‑ and
anti‑inflammatory mediators at the time of LPS attack.
• The LL-37 peptides possess a direct bactericidal action against LPS of Gram-
negative and teichoic acid of Gram-positive bacteria.

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 • LL‑37 has also emerged as having novel antibiofilm properties at low
concentration as well helping fight the biofilm bacteria such as Pseudomonas
aeruginosa,Streptococcus mutans, and Staphylococcus epidermidis.
• It prevents the adherence of bacteria to the tooth surface by controlling the
genes responsible for quorum sensing, which are necessary for the formation
of biofilm.

Bai LL, Takagi S, Guo YK, Kuroda K, Ando T, Yoneyama H, et al. Inhibition of Streptococcus mutans biofilm Contoso 45
by LL‑37. Int J Med Sci Biotech 2013;1:56-64 S u i t e s
 • A metalloproteinase, karilysin found to be released by the red complex
bacteria such as Tannerella forsythia, downregulates the antibacterial and
anti‑inflammatory properties of LL‑37.
• The maximum inhibitory effect of LL-37 has been exerted against primary
colonizers belonging to the yellow-orange complex.

Vallaban CG, Sivarajan S, Ahamed M, Chandramohan S, Thomas BR, Geetha S. A comprehensive review Contoso 46
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of LL-37 in periodontal disease. J Int Oral Health 2016;8:147-52.
ADVANTAGES AND DISADVANTAGES

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Limitations of AMPs and possible solutions for their therapeutic use.

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CLINICAL APPLICATIONS/FUTURE PERSPECTIVES
• In the use of AMPs as biomarkers for periodontal disease assessment, the
ratios between up- and downregulated proteins from individual patients may
overcome individual variation and potentiate the observed differences to
allow prospective assignment of any given sample as “healthy” or
“diseased.”
• Thus, treatment outcomes may be monitored by changes in AMP levels pre-
and post-treatment.
• Systems which specifically target the AMPs to the bacteria are being
developed.
• Their antimicrobial activity and selectivity help kill bacteria in mixed
cultures.

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• To overcome the problems of toxicity, peptide mimetics with the ability to
retain their properties as AMPs are being developed with a favorable
therapeutic index and stability; one such mimetic being XOMA 629.
• Topical and local applications under the names of Pexiganan (MSI-78),
Iseganan (IB-367), P113, and KSL have successfully been used as adjunct to
standard oral hygiene care, mouthrinse for oral candidiasis in HIV patients,
prevention of plaque-mediated dental diseases, and control of biofilms

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• Furthermore, coated AMPs are used to prevent the biofilm
formation on implants.
• Additional studies into new biological activities of AMPs are
needed and will be beneficial for us to better understand and gain
deep insight into the importance of these multifunctional molecules
in maintaining tissue homeostasis during the healthy and diseased
states of the periodontium.

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 Potential use of AMPs.

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references
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differences,” Journal of Dental Research, vol. 87, no. 10, pp. 915–927, 2008.
• [6] T. Ganz, M. E. Selsted, D. Szklarek et al., “Defensins. Natural peptide antibiotics of human neutrophils,” The
Journal of Clinical Investigation, vol. 76, no. 4, pp. 1427–1435, 1985.
• [7] F. G. Oppenheim, T. Xu, F. M. McMillian et al., “Histatins, a novel family of histidine-rich proteins in human
parotid secretion. Isolation, characterization, primary structure, and fungistatic effects on Candida albicans,” Journal of
Biological Chemistry, vol. 263, no. 16, pp. 7472–7477, 1988.

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Thank You!!!!
THANK
YOU

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