amp in oral cavity

Antimicrobial peptides of theoral cavity

SV E N-UL R I K GO R R

Antimicrobial proteins and peptides constitute a

diverse class of host-defense molecules that act early

to combat invasion and infection with bacteria and

other microorganisms. These peptides have engen-

dered considerable interest in the past decade as a

biological paradigm in innate immunity and as a

potential source of novel antibiotics. Many recent

reviews have explored the biological diversity and

function of this class of peptides [e.g. (36, 50, 73, 80,

103, 126, 127, 191, 209, 221, 226, 255)]. This review

focuses especially on antimicrobial proteins and

peptides of the oral cavity and on their role in perio-

dontal disease. The potential use of antimicrobial

peptides as templates for novel antibiotics is also

discussed.

Oral bacteria and infection

Oral infections and attendant inflammatory diseases

are among the most common human infections. The

oral cavity is a major entry point for bacteria and

other microorganisms and is typically considered

host to over 700 species of bacteria, including known

pathogens and commensals, and over 400 of these

bacterial species are found in the periodontal

pocket. Not all bacteria are found in every individ-

ual. For example, only 69 of the 400 species of

periodontal bacteria were detected in multiple sub-

jects in one study (188). A recent study, using

pyrosequencing of short sequence tags for the 16S

ribosomal DNA V6 region, led to much higher esti-

mates of microbial diversity in saliva and plaque

(124). A preliminary estimate identified 5669 and

10,052 phylotypes (species) in saliva and plaque,

respectively, using operational taxonomic units at

3% difference. This may represent about 50% of the

total species present (124). However, 95% of the

sequences were represented by the 1000 most

abundant operational taxonomic units, which is

similar to previous estimates.

Although there are a large number of oral mi-

crobes, only a relatively small number of species

have been consistently associated with periodontal

disease (190, 218, 230) (Table 1). It should be noted

that most of the bacteria associated with disease are

found in the gingival crevices and periodontal

pockets of both healthy and diseased sites (45, 230).

However, an age difference has been noted in the

types of bacteria associated with periodontal

disease, whereby Aggregatibacter actinomycetem-

comitans is more prevalent in the young while Por-

phyromonas gingivalis is the dominant bacterial

agent later in life (217). It is probable that differ-

ences in environmental factors and in the host�sacquired and innate immune responses, including

antimicrobial peptides, pattern recognition recep-

tors, chemokines and cytokines, play a role in

determining the microbial balance of oral tissues

and the subsequent outcome of bacterial coloniza-

tion. Consistent with this view, population studies

have identified population subgroups with high,

moderate or low susceptibility to inflammatory

diseases, including periodontitis (151).

In general, the unique profiles of antimicrobial

proteins found in different mucosal secretions pro-

vide an appropriate response to the microorganisms

invading each epithelium. As an example, the oral

cavity and airways share exposure to similar envi-

ronments, and many antimicrobial proteins are

common to both locations. However, differences in

gene expression and regulation suggest that the in-

nate immune responses differ in these locations (57).

Table 1 lists the expression of antimicrobial peptides

produced by host cells in response to periodontal

pathogens. Because of the lack of available data, the

absence of an antimicrobial peptide from the host

response may simply be a result of limited data

152

Periodontology 2000, Vol. 51, 2009, 152–180

Printed in Singapore. All rights reserved

� 2009 John Wiley & Sons A/S

PERIODONTOLOGY 2000

availability, rather than a lack of response. Several of

the antimicrobial proteins and peptides are cationic

and it has been found that depletion of cationic

proteins in human airway fluid also removes the

antibacterial activity (44). A similar experiment has

not been reported for saliva. However, we recently

Table 1. Effect of periodontal pathogens on antimicrobial peptide expression in human gingival epithelial cells

Periodontal pathogens Antimicrobial peptide Expression References

Aggregatibacter

(Actinobacillus)

actinomycetemcomitans1

hBD-2 Up4 140, 186

hBD-3 Up 186

hCAP18 Up 186

Fibronectin Down5 246

Porphyromonas gingivalis1,3 PSP ⁄ SPLUNC2 Up 210

hBD-2 Up 43

hBD-1 Up6 240

hBD-3 Up 240

Adrenomedullin Up 122

LL-37 = 114

Calgranulin Up7 170

Cystatin C Down 64

Tannerella forsythia

(T. forsythensis)1,3hBD-3 Down 114

hBD-1 = 114

hBD-2 = 114

LL-37 = 114

Treponema denticola2,3 hBD-1 Down6 114

hBD-3 Down 114

hBD-2 = 35

LL-37 = 114

Fusobacterium nucleatum2 hBD-1 = 114

hBD-2 Up 136

hBD-3 Up 114

LL-37 Up7 114

Calgranulin 170

Eubacterium nodatum2

Prevotella intermedia2 hBD-1 Up6 114

hBD-2 Up 114

hBD-3 Up 114

LL-37 Up 114

Prevotella nigrescens2

PubMed was searched for the listed bacterial species AND each antimicrobial peptide listed in Table 2 AND the keyword �epithelial� to identify relevant references.Gene expression in response to each pathogen is shown as up-regulated (Up), down-regulated (Down) or unchanged (=).1Consensus pathogens (190).2Suspected periodontal pathogens with �strong supporting data� (230).3Bacteria in the �red complex� described by Socransky et al. (218).4A. actinomycetemcomitans has no effect on hBD-2 expression in cells from a patient with localized aggressive periodontitis (140).5Cells treated with A. actinomycetemcomitans protease.6Some studies found that hBD-1 is expressed constitutively in HGEC (56, 114, 137).7Expression was tested in the oral squamous cell carcinoma cell line H400 (194).hBD, human b-defensin; PSP, parotid secretory protein.

153

Antimicrobial peptides of the oral cavity

found that ion-exchange fractionation of whole saliva

revealed anti-pseudomonal activity in some fractions

that was not apparent in the starting material (S. U.

Gorr, unpublished data).

Functional families ofantimicrobial proteins in the oralcavity

Despite the rich microbial environment of the oral

cavity, cuts, abrasions and invasive procedures, such

as tooth extraction, rarely lead to infection, indicating

the effectiveness of host-defense mechanisms (266).

Indeed, oral epithelial cells, neutrophils and salivary

glands secrete at least 45 known antimicrobial gene

products that are found in saliva (Table 2). A subset

of these antimicrobial peptides is also found in gin-

gival crevicular fluid (Table 3). Indeed, all of the

antimicrobial peptides found in gingival crevicular

fluid are also found in saliva. Several antimicrobial

peptides are more highly concentrated in gingival

crevicular fluid than in saliva. The concentrations of

calgranulins, fibronectin, substance P and calcitonin

gene related peptide are 100–10,000-fold higher

in gingival crevicular fluid than in whole saliva.

Adrenomedulin and beta-2-microglobulin are en-

riched about 30-fold in gingival crevicular fluid. Of

the antimicrobial peptides for which gingival crevic-

ular fluid concentrations are shown in Table 3, only

the alpha-defensins are found at a concentration

1000-fold lower in gingival crevicular fluid than in

saliva (Tables 2 and 3). The antimicrobial peptides

that are highly expressed in gingival crevicular fluid

are unlikely to be caused by contamination of the

gingival crevicular fluid samples with saliva (83).

Several of these antimicrobial peptides are

post-translationally processed (167, 199) or exhibit

polymorphisms (182, 257) that further diversify the

antimicrobial response of oral tissues and saliva.

Antimicrobial peptides are early responders of the

innate immune system that �search and destroy�invading pathogens. The large variety of antimicro-

bial peptides presumably allows for an effective

response to the large variety of microorganisms that

invade the mouth and airways. In addition, a host

response that involves multiple antimicrobial pep-

tides to a single pathogen is less likely to be met with

antimicrobial resistance. Thus, multiple antimicro-

bial peptides with different minimal inhibitory

concentrations act on oral microbes (49). Table 4A,B

shows representative antimicrobial activities for

antimicrobial peptides found in the oral cavity.

Comparison with the data shown in Table 3 suggests

that the concentration of these antimicrobial pep-

tides in gingival crevicular fluid is well below the

minimal inhibitory concentration for most microbes.

However, we do not have sufficient data to determine

if multiple antimicrobial peptides are involved in the

killing or elimination of individual microorganisms

in vivo.

Cationic peptides

Cationic peptides are typically bactericidal and ⁄ or

bacteriostatic. Their activities against typical oral

pathogens are listed in Table 4A.

Adrenomedullin

This 185 amino acid protein is proteolytically pro-

cessed and C-terminally amidated to produce the

52 amino acid mature adrenomedullin, a cationic

amphipathic peptide with one disulfide bond. It is

found in gingival crevicular fluid and glandular and

whole saliva. Whole saliva contains higher concen-

trations of adrenomedullin than glandular saliva,

suggesting that oral epithelial cells contribute to the

salivary expression (123). In gingival crevicular fluid,

the amount of adrenomedullin is about twice as high

in periodontal disease sites than in healthy sites

(158).

Alpha-defensins

Human neutrophil peptides 1–4 are 94-amino acid

cationic pre-pro-peptides. Human neutrophil pep-

tide-1 (amino acids 65–95 of the DEFA1 product) and

human neutrophil peptide-3 (amino acids 65–94 of

the DEFA3 product) differ only at the N-terminal

residue. Human neutrophil peptide-2 does not

contain this residue and can be derived from both

precursor proteins. Human neutrophil peptide-4 is

a product of the DEFA4 gene, which contains an

83-base segment not found in the other genes. The

mature peptides contain three disulfide bridges and

are mainly expressed in neutrophils and saliva (79).

Alpha-defensins are expressed in neutrophils and

have been identified in the gingival crevicular fluid of

both healthy and diseased sites, although different

experimental methodologies lead to somewhat dif-

ferent conclusions on the relative expression in

healthy and diseased individuals (56, 159, 193, 195).

Defensins exhibit broad antibacterial activity to both

gram-positive and gram-negative bacteria (143). The

peptides bind to or are inserted into the bacterial cell

membrane, causing membrane permeabilization and

cell lysis. Defensins are most active against negatively

154

Gorr

Table 2. Antimicrobial proteins and peptides found in saliva

Protein Gene Whole saliva

(lg ⁄ ml)

Parotid saliva

(lg ⁄ ml)

SM ⁄ SL

saliva (lg ⁄ ml)

References

Adrenomedullin ADM 0.06 0.02 0.035 123

HNP-1 DEFA1 8.6 MS MS 79

HNP-2 DEFA1 ⁄ DEFA3 5.6 79

HNP-3 DEFA3 0–2.7 74

hBD-1 DEFB1 0.15 MS MS 164

hBD-2 DEFB4 0.150 164

hBD-3 DEFB103A 0.31 231

Cathelicidin ⁄LL-37

CAMP 1.6 229, 16

Histatin 1 HTN1 MS 10.1 34.7 116

Histatin 3 HTN3 7.3 10.2 116

Statherin STATH 26.5 69 75 46

99

CCL28 CCL28 0.9 2.7 102

Azurocidin ⁄CAP37 ⁄heparin-binding

protein

AZU1 MS MS

Substance P TAC1 7.5 · 10)6 6.2 · 10)6 52

Calcitonin gene

related peptide

CALCA 23.5 · 10)6 1.5 · 10)6 52

Neuropeptide Y NPY 41.4 · 10)6 44.4 · 10)6 52

Vasoactive

intestinal

peptide

VIP 39.9 · 10)6 29.9 · 10)6 52

Mucin 7 MUC7 40 MS 116–157 189

21

GP340 ⁄ salivary

agglutinin ⁄DMBT1

DMBT1 MS 6.31 67

Surfactant

protein-A

SFTPA1 0.9 215

Beta-2-micro-

globulin

B2M 0.38 MS MS 169

Proline-rich

proteins

PRH1 MS 512 652 111

PRH2 MS 812 1032 111

PRB1 MS MS MS

PRB3 MS MS

PRB2 MS MS

PRB4 MS MS MS

Fibronectin FN1 1.2–0.13 MS 0.5 150, 236

Calgranulin A S100A8 1.93 MS MS 129

155


charged phospholipids and their activity is inhibited

by increased salt concentrations, suggesting that

ionic interactions between membrane lipids and the

cationic peptide are involved in their activity (71, 72).

Beta-defensins

hBD1, hBD2 and hBD3 are cationic peptides with

three disulfide bonds. Alpha- and beta-defensin differ

in the spacing and the pairing of the cysteine residues

(72). They are expressed in epithelial cells of the oral

cavity, lung and nasal epithelia, kidney, pancreas,

uterus and eye (1), and are found in the gingival

crevicular fluid (56) and in the saliva. In gingival

epithelial cells, hBD2 expression is correlated with

cellular differentiation and is regulated by calcium

and phospholipase D (135).

Cathelicidin

Cathelicidin is the 18 kDa precursor of the antimi-

crobial peptides FALL-39 and LL-37. LL-37 is a

cationic alpha-helical peptide, expressed in neu-

trophils, epithelial cells, saliva and gingival crevic-

ular fluid (176, 195). In addition to antibacterial

activity (Table 4A), LL-37 also binds to and neu-

tralizes lipopolysaccharide from gram-negative

bacteria. In Candida albicans, the peptide causes

Table 2. Continued

Protein Gene Whole saliva

(lg ⁄ ml)

Parotid saliva

(lg ⁄ ml)

SM ⁄ SL

saliva (lg ⁄ ml)

References

Calgranulin B S100A9 1.93 MS MS 129

Psoriasin S100A7 MS

Lactoferrin LTF 20 4.7 207, 212, 228

Cystatin A CSTA MS MS MS

Cystatin B CSTB MS MS MS

Cystatin C CST3 0.9 0.4 MS 101, 238

Cystatin D CST5 3.8 MS MS 69

Cystatin S CST4 53–116 1.0 MS 18, 101

Cystatin SA CST2 78 MS MS 18

Cystatin SN CST1 39 MS MS 18

Secretory leuko-

cyte protease

inhibitor

SLPI 2.9 0.55 1.1 147, 212

SKALP ⁄ Elafin PI3 0.020 PS4 SMG4 235

142

Lactoperoxidase LPO 1.9 6.2 MS WS = Unstim (233)

(salivary

peroxidase)

PS = Stim (207)

Myeloperoxidase MPO 3 MS MS 233

185

Lysozyme C LYZ 40 4.4 MS 7, 130, 207, 212

Peptidoglycan

recognition

protein 1

PGLYRP1 MS

Common name, gene symbol, concentrations in whole saliva; parotid saliva and submandibular ⁄ sublingual (SM ⁄ SL) saliva (lg ⁄ ml). Values in bold ⁄ italic arefrom stimulated saliva. MS = mass spectrometry detection of proteins in unstimulated whole saliva (260, 264) or in stimulated glandular saliva (55). Saliva wascollected from healthy subjects, unless stated otherwise. It should be noted that the concentrations shown are representative average values only. Individualvariation and variation between studies can be significant; see references for details.1Salivary agglutinin (DMBT1) purified from parotid saliva represented 0.4% of total protein (67).2Calculation based on an average parotid salivary flow rate of 0.7 ml ⁄ min ⁄ gland (100) and SM ⁄ SL flow rate of 0.4 ml ⁄ min ⁄ gland (261).3Measured as calprotectin (calgranulin A and B complex) (129).4Found in parotid saliva and submandibular glands (SMG) (142).HNP, human neutrophil peptide, a-defensin.

156

Gorr

Table 3. Antimicrobial proteins and peptides found in gingival crevicular fluid

Protein Gingival crevicular

fluid: healthy

(lg ⁄ ml)

Gingival crevicular fluid: periodontitis

Adrenomedullin 1.8

(158)

Twofold increase in periodontitis (158)

HNP-1 MS

HNP-2 MS

HNP-3 MS

HNP(1–3) 0.0012

(195)

Up-regulated 15-fold in aggressive

periodontitis and 60-fold in chronic

periodontitis, respectively (195)

HNP-4 MS

hBD-1 (56)

hBD-2 (56)

hBD-3

Cathelicidin ⁄ LL-37 (195) Up-regulated in aggressive periodontitis

and chronic periodontitis (195)

Statherin MS

Beta-2-microglobulin 9.4

(174)

Increased threefold in mild periodontitis

and 10-fold in severe periodontitis,

respectively (174)

Proline rich protein MS

Proline rich peptide P-B MS

Fibronectin 106

(152)

Decreased twofold in periodontitis and

30-fold in gingivitis (152). Less intact FN

is found in periodontitis than in healthy

control or treated sites (227)

Calgranulin A 240

(154)

Increased 2–3-fold in periodontitis,

decreased 2–3-fold after periodontal

therapy (132, 154, 155)

Calgranulin B (132) Increased in periodontitis, decreased

2–3-fold after periodontal therapy (132)

Calprotectin 570

(125)

Calprotectin concentration increased

with the gingival index (180); 3–5-fold

increase in periodontitis (>4 mm pocket

depth) (125); 70 ng ⁄ ll detected in

children (172)

Lactoferrin 600 Concentration highly variable. No

consistent change with disease

(2, 3, 70, 112)

Cystatin A MS Main cystatin activity in gingival

crevicular fluid of periodontal

patients (30)

Cystatin C 1.15 (children)

(237)

No change in children with gingivitis

(237)

Secretory Leukocyte No data 79.7 pg ⁄ ll in periodontitis, increased

3–4-fold at 2 and 4 weeks post

treatment (179)

157


disintegration of the plasma membrane and release

of ATP, nucleotides and proteins up to 40 kDa in

size (54).

Histatin 1 and 3

Histatin 1 and 3 are histidine-rich, cationic peptide

precursors for multiple peptides that exhibit bacte-

ricidal and candidacidal activity. Histatin 5 is derived

from histatin 3. Unlike LL-37, histatin causes only

minor disruption of the Candida plasma membrane

and accumulates intracellularly. Thus, C. albicans is

killed by histatin 5 in a mechanism that involves the

nonlytic release of ATP from the fungal cells (134).

However, the efflux of nucleotides is similar with the

two peptides and may play a significant role for

candidacidal activity (54). Histatins are found in sal-

iva (55, 242) and are only expressed in salivary glands.

The histatin genes and the statherin gene map to the

same area of chromosome 4q13 as aggressive (juve-

nile) periodontitis and dentinogenesis imperfecta

(239).

Statherin

The 5.4 kDa peptide, statherin, belongs to the

histatin ⁄ statherin family. Statherin and a basic

histidine-rich peptide may have evolved from a

common ancestral gene (59). Statherin is found in

gingival crevicular fluid (193) and saliva (55, 242,

260). The peptide is secreted by the parotid and

submandibular glands and inhibits the crystallization

of calcium phosphate but also inhibits growth of

anaerobic bacteria isolated from the oral cavity. It

is the C-terminal peptide of statherin that exhibits

the antibacterial effect (131). Statherin has recently

emerged as a potential biomarker for oral infections

through proteomic analysis of saliva from patients

with high and low scores for bacterial adhesion and

agglutination (206).

C-C motif chemokine 28

This 128-amino acid peptide is preferentially ex-

pressed in various epithelial cells, including salivary

glands, and is found in saliva (55). The peptide acts

both as a chemokine and a broad-spectrum anti-

microbial peptide. Indeed, a C-terminal 28-amino

acid peptide is similar to histatin 5. This peptide in-

duces membrane permeability and is salt sensitive, as

noted for other cationic antimicrobial peptides (102).

Azurocidin

Azurocidin (a 37 kDa cationic antimicrobial protein,

CAP37) is expressed in azurophil granules of

neutrophils and was identified in human saliva by

proteomic analysis (55, 264). This 251-amino acid

protein is antibacterial to gram-negative bacteria,

presumably because of a strong affinity for lipo-

polysacchaide. Two cysteine residues in positions 52

and 68 are necessary for antibacterial activity.

Table 3. Continued

Protein Gingival crevicular

fluid: healthy

(lg ⁄ ml)

Gingival crevicular fluid: periodontitis

Protease Inhibitor

Lactoperoxidase

(Salivary Peroxidase)

Possible

(14)

Peroxidase identified in gingival

crevicular fluid is most likely

myeloperoxidase (162)

Myeloperoxidase 0.3–5.5

(185, 195)

No change in aggressive or chronic

periodontitis (195). 660 lg ⁄ ml in

>5 mm pockets. Concentration

decreased twofold after therapy (121)

Lysozyme C (70, 112) Increased in juvenile periodontitis (70)

Substance P 0.061–0.11

(15)

(148)

SP concentration was similar in healthy,

gingivitis or periodontitis (148). Others

found that concentration was decreased

sevenfold post periodontal treatment

(156)

Calcitonin Gene

Related Peptide

0.013–0.7

(15, 160)

Concentration 20-fold lower in gingivitis,

not detected in periodontitis (160)

Common name, presence in gingival crevicular fluid (concentration in lg ⁄ ml is given when known) from healthy subjects and changes in periodontitispatients.hBD, human b-defensin; HNP, human neutrophil peptide, a-defensin; MS, proteins detected by mass spectrometry of gingival crevicular fluid (159, 193).

158

Gorr

Table 4A. Antimicrobial activities of peptides that are directly bactericidal or bacteriostatic

Antimicrobial

peptide

Targets Activity Reference

Adrenomedullin P. gingivalis MIC 7.75 · 10)4 lg ⁄ ml 6

S. mutans MIC 12.5 lg ⁄ ml

HNP-1 S. mutans MIC 4.1 lg ⁄ ml 157

P. aeruginosa MIC 10.3 lg ⁄ ml 157

C. albicans MIC 52.5 lg ⁄ ml 157

Enveloped viruses Inactivation 48

HIV-1 Inhibits replication 267

A. actinomycetemcomitans No activity (>500 lg ⁄ ml) 171

P. gingivalis No activity (>200 lM) 197

HNP-2 P. gingivalis No activity (>200 lM) 197


C. albicans EI90 65 lM 197

Herpes simplex virus-1 Inactivation 48


HNP-3 P. gingivalis No activity (>200 lM) 197


C. albicans No activity 143

Herpes simplex virus-1 Inactivation 48


HNP-4 E. coli LD50 0.085 lg ⁄ ml 258

C. albicans LD50 0.5 lg ⁄ ml 258

hBD-1 P. gingivalis MIC 50 lg ⁄ ml 187

A. actinomycetemcomitans MIC 50 lg ⁄ ml 187

C. albicans MFC 7 lM 138

hBD-2 P. gingivalis MIC 34.6–>250 lg ⁄ ml 117

S. mutans MIC 4–8 lg ⁄ ml

C. albicans MIC 5–59 lg ⁄ ml

HIV-1 IC50 9–19 lg ⁄ ml

hBD-3 P. gingivalis MIC 42.1 lg ⁄ ml 113

A. actinomycetemcomitans MIC 45.6 lg ⁄ ml 113

S. mutans MIC 3–5 lg ⁄ ml 117

C. albicans MIC 3–7 lg ⁄ ml 117

HIV-1 IC50 20–40 lg ⁄ ml

LL-37 P. gingivalis MIC >125 lg ⁄ ml 113

A. actinomycetemcomitans MIC 37.8 lg ⁄ ml 113

S. gordonii MIC 102.6 lg ⁄ ml 113

C. albicans LD50 0.8 lM 54

159


Table 4A. Continued

Antimicrobial

peptide

Targets Activity Reference

Histatin 5 A. actinomycetemcomitans Neutralizes leukotoxin 178

C. albicans LD50 1.6 lM 54

Statherin Oral anaerobes MIC <12.5 lg ⁄ ml

to >100 lg ⁄ ml

131

C. albicans Aggregation 115

CCL28 S. mutans IC50 1.7 lM 102

C. albicans IC50 0.7 lM

Azurocidin ⁄ CAP37 E. coli LD50 1.3 lg ⁄ ml 8

C. albicans MIC 40 lg ⁄ ml 165

Substance P P. aeruginosa MIC 15.7 lg ⁄ ml 63


C. albicans MIC 8.1 lg ⁄ ml

Calcitonin

gene related

peptide


S. mutans MIC >500 lg ⁄ ml


Neuropeptide Y P. aeruginosa MIC 134.3 lg ⁄ ml 63



Vasoactive

intestinal

peptide




Psoriasin E. coli MBC 100 lg ⁄ ml 168

Lysozyme C Gram-positive bacteria Cell wall lysis

Peptidoglycan

recognition

protein 1

S. aureus LD99 60 lg ⁄ ml 245

E. coli LD99 30 245


Peptidoglycan

recognition

protein 3


Gram-negative

bacteria

LD99 30 lg ⁄ ml 245


Peptidoglycan

recognition

protein 4


Gram-negative

bacteria

LD99 200 lg ⁄ ml 245


SKALP ⁄ Elafin P. aeruginosa LD96 2.5 lM 213

Secretory leukocyte

protease inhibitor

P. aeruginosa LF95 2.5 lM 213

Antimicrobial peptide name, microbe targets, antimicrobial activity and representative references are shown.hBD, human b-defensin; HNP, human neutrophil peptide, a-defensin; LDxx, concentration that kills XX% of bacteria; MBC, minimal bactericidal concentration;MFC, minimal fungicidal concentration; MIC, minimal inhibitory concentration.

160

Gorr

Table 4B. Antimicrobial activities of peptides that modulate microbial adhesion or activity

AMP Targets Activity References

Mucin 7 Oral streptococci Bacterial binding and

agglutination

11

A. actinomycetemcomitans Bacterial binding 84

C. albicans Binding and clearance 104

HIV Prevent cell entry 86

GP340 ⁄ salivary

agglutinin ⁄ DMBT1

Oral streptococci Agglutination 145

A. actinomycetemcomitans Binding 84

HIV-1 Blocks cell entry 263

Influenza A virus Inactivation by agglutination 97

Surfactant protein-A Bacteria Binding and clearance 133

Influenza A virus Agglutination 256

Beta-2-microglobulin S. mutans Agglutination in presence of

Ca2+66

Proline rich proteins Oral bacteria Bacterial adhesion 139

C. albicans Adhesion 115

HIV-1 Inhibits infectivity 202

Fibronectin P. gingivalis Binding to fimbrillin 177

S. mutans Adhesion and agglutination 149

Calprotectin P. gingivalis Protects epithelial cells from

infection

181

C. albicans Candidastatic by Zn chelation;

MIC 18 lg ⁄ ml

220

34

S. aureus Zn chelation, MIC 64 lg ⁄ ml 34

Lactoferrin P. gingivalis 35% growth inhibition at

2 mg ⁄ ml apoLf

4

A. actinomycetemcomitans 1.9 lM apoLf (iron-free)

kills 99.9% in 3 h

120

C. albicans Induces apoptosis 12

Cystatins Bacterial or host proteases Cysteine protease inhibitor 32

Cystatin C P. gingivalis Growth inhibition 31

Herpes simplex virus-1 Inhibits viral replication 29

Cystatin S P. gingivalis Growth inhibition 31

Secretory

leukocyte protease

inhibitor

P. gingivalis Degraded by gingipain 108

S. aureus 259

HIV Blocks virus entry

C. albicans 166

Myeloperoxidase A. actinomycetemcomitans Bactericidal OCl) 173

Oral streptococci

P. gingivalis Bactericidal OI) 105

161


Neuropeptides

The neuropeptides calcitonin gene related peptide

and substance P are found in gingival crevicular

fluid (15). These peptides, and neuropeptide Y and

vasoactive intestinal peptide, are also found in saliva

(52). However, the concentrations ranging from

2–45 pg ⁄ ml are several orders of magnitude lower

than the minimal inhibitory concentrations against

bacteria and C. albicans (63).

Bacterial agglutination and adhesion

Mucin-7 (MUC7)

The small salivary mucin MUC7 (MG2) is expressed

in the mucous acinar cells of the sublingual and

submandibular glands and is found in saliva (55). The

377-amino acid protein contains four potential N-

glycosylation sites. Two isoforms, MG2a and MG2b,

differ in their sialic acid and fucose contents (201).

Mucin-7 promotes bacterial agglutination. Binding of

mucin-7 to clinical isolates of A. actinomycetem-

comitans depends on sialic acid residues on the

mucin (84). The levels of MUC7 in stimulated saliva

are twofold lower in patients with periodontitis than

in healthy subjects (16.7 lg ⁄ ml vs. 30.6 lg ⁄ ml) (85).

In addition to interacting with bacteria, MUC7 and

the larger mucin, Muc5b, prevent entry of human

immunodeficiency virus (HIV) into host cells.

Salivary agglutinin ⁄ GP340 ⁄ deleted in malignant

brain tumors-1 (DMBT1)

This large glycoprotein contains multiple scavenger

receptor cysteine-rich repeats. The protein is found

in saliva (55, 260, 264) and is expressed in salivary

glands, ocular mucosal tissue, lacrimal glands, lung,

trachea, gastointestinal tract and macrophages.

DMBT1 polymorphisms have been associated with a

high incidence of caries (118). The protein acts in

binding and agglutination of bacteria, including oral

streptococci (145). Thus, DMBT1 associates with

Staphylococcus aureus but not with Pseudomonas

aeruginosa (119), indicating that different host-de-

fense proteins act on different microorganisms.

DMBT1 also acts as an opsonin receptor to promote

bacterial clearance. The highly glycosylated protein

acts as a mucin that binds influenza virus hemag-

glutinin via sialic acid residues and inactivates the

virus (96, 97). Entry of HIV-1 into epithelial cells is

also blocked by DMBT1 (263).

Surfactant protein-A

This 248-amino acid protein was detected in saliva

and sputum (214) and is up-regulated in patients

with chronic sialadenitis (141). Surfactant protein-A

acts like mucins by providing sialic acid residues that

bind bacteria and the influenza virus. In the latter,

binding of hemagglutinin causes inactivation of the

virus (256).

Beta-2-microglobulin

This 11.8 kDa protein is found in saliva (55, 242) and

in gingival crevicular fluid. In gingival biopsies, beta-

2-microglobulin is absent in most samples (71%)

from normal controls and in specimens from patients

with chronic severe periodontitis, but is detected in

most (82%) biopsies from patients with juvenile

periodontitis (224). Similarly, beta-2-microglobulin

levels are higher in gingival crevicular fluid from

patients with periodontal than in gingival crevicular

fluid from healthy controls (225). However, this dif-

ference was not noted in a study of juvenile perio-

dontitis saliva, where the beta-2-microglobulin level

did not differ between patients and controls, while

serum levels were higher in periodontitis patients

than in controls (5). Beta-2-microglobulin causes

agglutination of S. mutans in the presence of 1.4 mM

Ca2+ (66).

Proline-rich proteins

These proteins are found in saliva (55) and bind to

the tooth surface and affect bacterial adhesion. Thus,

while the larger proline-rich proteins promote

Table 4B. Continued

AMP Targets Activity References

Lactoperoxidase

(salivary

peroxidase)

A. actinomycetemcomitans Bactericidal I) H2O2 107

Oral streptococci Bacteriostatic OSCN) ⁄Bactericidal OI)

234

P. gingivalis Bactericidal OI) 105

These proteins are typically not bactericidal, with the exception of the peroxidases that produce bactericidal oxidation products. Antimicrobial peptide name,microbe targets, antimicrobial activity and representative references are shown.I), iodide; H2O2, hydrogen peroxide; MIC, minimal inhibitory concentration; OCl), hypochlorite; OI), hypoiodite; OSCN), hypothiocyanite.

162

Gorr

bacterial attachment to the pellicle, it has been sug-

gested that smaller proline-rich proteins reduce

attachment (139). Similarly, proteolytic processing of

proline-rich proteins by commensal bacteria can

produce peptides that limit bacterial adhesion (144).

Adhesion of Streptococcus mutans correlates with

high caries incidence and the presence of the Db

allele of PRH1 (222). Binding of bacterial fimbrillin to

proline-rich protein 1 is inhibited by C-terminal

fimbrillin peptides corresponding to amino acid

residues 266–286 and 318–337 (10). Proline-rich

proteins, fibronectin and other proteins that affect

bacterial adhesion and biofilm formation can delay

or prevent bacterial colonization of oral surfaces.

Fibronectin

Fibronectin is a large glycoprotein (2386 amino acids

in size) that is expressed in hepatocytes, epithelial

cells and other cells, and is present in saliva (150).

The protein induces bacterial agglutination and plays

a role in reducing bacterial adhesion to oral surfaces

(149). Fibronectin also binds directly to fimbrillin

from P. gingivalis and thereby inhibits the fimbrillin-

induced expression of inflammatory cytokines in

macrophages (177). Low levels of fibronectin are

correlated with high levels of S. mutans in children

(149) and periodontitis is associated with a relative

lack of fibronectin in adults (177).

Metal ion chelators

Calgranulin A (S100A8) and calgranulin B (S100A9);

calprotectin

These metal ion-binding proteins inhibit bacterial

growth by acting as divalent cation scavengers. The

dimer of calgranulin A and B, termed calprotectin, is

expressed in the cytosol of neutrophils, monocytes

and keratinocytes. The expression levels of calgran-

ulin A and B are up-regulated in oral epithelial H400

cells exposed to P. gingivalis or Fusobacterium

nucleatum (170). Accordingly, calprotectin is up-

regulated in periodontal disease and is detected at

increased levels in the gingival crevicular fluid of

patients with periodontitis (125). This is probably a

defense mechanism because the expression of

calprotectin in KB (HeLa) cells protects the cells from

invasion with P. gingivalis (181). Calprotectin levels

are also increased in saliva from patients with

candidiasis (129). Calprotectin inhibits the growth of

S. aureus by chelation of Mn2 + and Zn2 + ions, which

leads to reprogramming of the bacterial transcrip-

tome (47).

Lactoferrin ⁄ lactotransferrin

This 80 kDa iron-binding glycoprotein acts as a

scavenger of Fe3+ ions. Lactoferrin binds two Fe3+

ions in association with bicarbonate or another an-

ion. It is produced by mucosal epithelial cells and is

found in their secretions, including saliva (250) and

gingival crevicular fluid, which is a significant source

of oral lactoferrin (60). Decreased levels of lactoferrin

in granulocytes have been associated with perio-

dontal disease (219), while the saliva levels of lacto-

ferrin appear to be increased in periodontitis (85).

Gene polymorphisms of lactoferrin have recently

been associated with aggressive periodontitis (262),

suggesting that the antibacterial activity of this pro-

tein plays a role in the development of the disease.

Lactoferrin acts on bacteria, viruses, fungi and para-

sites. Lactoferrin kills A. actinomycetemcomitans

(120), inhibits the growth of P. gingivalis but has no

effect on the growth of Prevotella intermedia and

Prevotella nigrescens (4). However, lactoferrin inhibits

the adhesion of P. intermedia, P. nigrescens and

A. actinomycetemcomitans to fibroblasts (9). In

addition to its effects on periodontal pathogens, the

protein induces apoptosis in C. albicans in a process

that depends on cellular K+-channels (12). Lactoferrin

also binds to the lipid A part of lipopolysaccharide,

resulting in both anti-inflammatory activity and

bactericidal activity as a result of increased mem-

brane permeability (184).

Protease inhibitors

Cystatins

The human cystatin gene family contains 14 genes

and two pseudogenes. Seven of these genes are ex-

pressed in saliva (Table 2). The cystatins are cysteine

protease inhibitors that block the action of bacterial

proteases on target tissues (58). Based on structural

similarities it has recently been proposed that

cathelicidin gradually evolved from cystatins (270).

Indeed, Cystatins C and S inhibit the growth of

P. gingivalis (31).

Secretory leukocyte protease inhibitor

This 11.7 kDa cationic, nonglycosylated protein

contains 16 cysteine residues that form eight disul-

fide bridges. The protein is expressed in mucosal

epithelial cells and secretions, including gingival

keratinocytes and saliva. Secretory leukocyte protease

inhibitor acts as a bacterial serine protease inhibitor

and is an antibacterial and anti-inflammatory protein

163


(252). The N-terminal cationic domain of secretory

leukocyte protease inhibitor is antimicrobial with

activity against P. aeruginosa, S. aureus and C. albi-

cans (259). Secretory leukocyte protease inhibitor

expression in oral epithelial cells is induced by HIV-1

(110) and the protein blocks the entry of HIV-1 by

interaction with the host cell. This activity appears to

be independent of the protease inhibitor activity.

Secretory leukocyte protease inhibitor expression is

decreased in chronic periodontitis, and the gingival

crevicular fluid levels of secretory leukocyte protease

inhibitor are lower in P. gingivalis-infected sites,

possibly because of secretory leukocyte protease

inhibitor degradation by arginine-specific gingipains

from P. gingivalis (108).

SKALP ⁄ Elafin

Skin-derived antileukoproteinase (SKALP) ⁄ Elafin is

a 12 kDa protein expressed in the human sub-

mandibular gland (142) and in saliva (142, 235). The

protein has an N-terminal domain that acts as a

transglutaminase substrate and a C-terminal domain

that exhibits anti-elastase activity. In addition, the

protein kills both gram-negative and gram-positive

bacteria; this activity depends on the presence of

both peptide domains (213).

Peroxidases

Lactoperoxidase (salivary peroxidase) is a heme

peroxidase that is found in saliva and milk. Lactop-

eroxidase and myeloperoxidase form the principal

components of the peroxidase system of saliva (106).

The enzymes catalyse the oxidation of thiocyanate

ions (SCN)) by hydrogen peroxide, and the reaction

product hypothiocyanite (OSCN)) is bactericidal. (14).

Hydrogen peroxide-mediated oxidation of chloride

and iodide produces further bactericidal reaction

products (105, 173).

Having a similar action to lactoperoxidase, myelop-

eroxidase (a heme peroxidase) is expressed in neutro-

philic polymorphonuclear leukocytes and catalyses the

oxidation of thiocyanate ions by hydrogen peroxide to

produce hypothiocyanite. In addition, myeloperoxi-

dase can produce hypochlorite (OCl)), which is a

stronger oxidant implicated in inflammatory tissue

damage (14). Myeloperoxidase is found in gingival

crevicularfluidataconcentrationofabout5 lg ⁄ mlbut

no significant differences were found between the lev-

els of myeloperoxidase found in chronic periodontitis,

aggressive periodontitis and healthy controls (195).

However, the myeloperoxidase levels in gingival cre-

vicular fluid were significantly reduced in periodontal

patients after antibiotic treatment for 3 months (121).

Activity against bacterial cell walls

Lysozyme

1,4-Beta-N-acetylmuramidase (lysozyme) is a

bacteriolytic enzyme that hydrolyses the (1, 4)-beta-

linkages between N-acetylmuramic acid and N-ace-

tyl-D-glucosamine residues in peptidoglycans. The

enzyme is mainly active against the cell wall of gram-

positive bacteria. The 14 kDa protein is expressed

widely in mucosal epithelia and is found in saliva,

milk and tears. Lysozyme is found in gingival

crevicular fluid. The lysozyme levels do not appear to

differ between adult periodontitis and healthy con-

trols, (225) but are significantly increased in juvenile

periodontitis (70).

Peptidoglycan recognition proteins 3 and 4

These large host-defense proteins (89–115 kDa

disulfide-linked homodimers or heterodimers) are

found in species ranging from insects to mammals

(61). Interestingly, different peptidoglycan-recogni-

tion proteins have diversified to carry out different

antimicrobial functions (205). Four peptidoglycan-

recognition proteins are found in humans: peptido-

glycan recognition proteins 3 and 4 are expressed in

skin and mucosal epithelia, including salivary glands,

and peptidoglycan recognition protein 1 is found in

saliva. Peptidoglycan recognition protein 2 is an

amidase that is secreted by the liver and epithelial

cells. The proteins exert their bactericidal effect

by binding to cell wall peptidoglycans, but do not

permeabilize bacterial membranes (153). The pro-

teins are bactericidal for pathogenic and nonpatho-

genic gram-positive bacteria but not for normal

flora bacteria. Moreover, the proteins are bacterio-

static for most gram-positive and gram-negative

bacteria but not for C. albicans (153).

The role of antimicrobial proteinsin periodontal disease

Striking differences in susceptibility to the antimicro-

bial peptides LL-37 and hBD3 have been observed

between different species of oral bacteria and different

strains of the same species (113). As an example,

Streptococcus gordonii M5 is weakly susceptible to

both antimicrobial peptides, while S. gordonii 10558 is

highly susceptible. P. gingivalis 33277, by contrast, is

weakly susceptible to LL-37 but highly susceptible to

killing by hBD3 (113) (Table 4A). Indeed, different oral

bacteria elicit distinct transcription profiles in oral

epithelial cells (90), thus further diversifying the

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antimicrobial peptide response to bacteria. The

production of beta-defensins, and LL-37, in particular,

has been analyzed after exposure of gingival epithelial

cells to bacteria associated with periodontitis

(Table 1).

Approximately 20 genetic diseases connected with

periodontitis have been identified (92). Some of these

diseases are associated with changes in antimicrobial

peptide expression, which may play a role in the

increased susceptibility to bacterial infection.

Morbus Kostman disease is a severe congenital

neutropenia that is associated with severe perio-

dontitis (196). Patients with morbus Kostmann are

deficient in LL-37 in neutrophils, plasma and saliva.

Patients are also deficient in alpha-defensins (30% of

normal) while the plasma lactoferrin content is nor-

mal (196). Patients treated with granulocyte–colony

stimulating factor exhibit normal numbers of

neutrophils but lack LL-37 and continue to show

periodontal disease (40, 196). In a single patient, a

bone marrow transplant restored both LL-37 and

neutrophils to normal levels and no dental problems

were noted. While this study suggests that a lack of

LL-37 is associated with periodontal disease, normal

levels of LL-37 are not sufficient to prevent the

disease. Thus, periodontal disease is prevalent in

children with Down�s syndrome (trisomy 21) (183).

However, this increased occurrence of periodontitis

is not correlated with a decrease in LL-37 levels (16).

Other antimicrobial peptides associated with

periodontal disease are mucin-7 and lactoferrin. The

level of mucin-7 (MG2) is decreased threefold in

patients with A. actinomycetemcomitans-associated

periodontitis compared with healthy controls.

Lactoferrin levels were normal but the protein was

iron saturated, suggesting that its antimicrobial

properties are reduced in patients with periodontitis

(85).

Papillon–Lefevre syndrome and Haim–Munk syn-

drome are characterized by palmoplantar kerato-

derma and severe periodontitis. Both diseases are

caused by allelic mutations of the cathepsin C gene,

CTSC (94). The lysosomal enzyme cathepsin C is

responsible for the activation of neutrophil serine

proteases, which are antibacterial to S. aureus and

A. actinomycetemcomitans (93, 95, 192, 223). While

patients with Papillon–Lefevre syndrome express

normal levels of the cathelicidin precursor, little is

processed to the mature LL-37 peptides. As in

morbus Kostman, it is likely that the low levels of

LL-37 contribute to the development of periodon-

titis in patients with Papillon–Lefevre syndrome

(53).

Antimicrobial proteins and otheroral infections

Oral infections other than periodontitis have also been

linked to the levels of expression of antimicrobial

peptides. Oral candidiasis is associated with low levels

of several antimicrobial peptides, including lactofer-

rin, and beta-defensins 1 and 2. By contrast, the con-

centration of alpha-defensin 1 increased in patients,

whereas the concentration of transferrin did not differ

between patients and healthy controls (228).

The correlation of antimicrobial peptide expression

levels and caries incidence has been difficult to

establish. Caries incidence in children has been

associated with the expression of a low-level of alpha-

defensins (human neutrophil peptides 1–3) (229).

However, because caries is seen at a wide range of

alpha-defensin expression levels, it is not clear if

alpha-defensin expression is predictive for future

caries development (50). Furthermore, the host-

defense proteins lysozyme, salivary peroxidase and

lactoferrin were not correlated with caries incidence

in studies of 13-year-old children (128) and navy

recruits (82). The difficulty in linking single point

analysis of antimicrobial peptides with oral disease

(231) may be a result of the wide variety of antimi-

crobial peptide expression levels between subjects.

Salivary peptide levels can vary about 100-fold

between subjects, even when normalized to total

salivary protein (229). Thus, it is difficult to define

normal values for individual antimicrobial peptides

in saliva. Instead, it may be possible to use multiplex

analysis of antimicrobial protein expression in health

and disease to identify �antimicrobial peptide signa-

tures� that will have predictive or diagnostic power.

New antimicrobial proteins

Many types of antimicrobial peptides have been

identified in the oral cavity, as described in the

preceding sections. With the advent of saliva and

gingival crevicular fluid proteomics it is predicted

that additional antimicrobial peptides will be identi-

fied. It should be noted that not all of these peptides

will be present at biologically significant concentra-

tions. Nevertheless, identification of antimicrobial

peptides in saliva provides a starting point for the

study of their potential role in host defense. In one

study, proteomic analysis of labial salivary gland

secretions identified 56 proteins, 32% of which were

characterized as host-defense proteins (215). The

165


antimicrobial peptides listed in the preceding

sections were extracted from a data set of 633 known

salivary proteins described in PubMed. A recent

comprehensive analysis of the human glandular

salivary proteome, supported by the National Insti-

tute of Dental and Craniofacial Research, at the US

National Institutes of Health, has identified 1166 total

proteins: 914 were found in parotid saliva and 917

were found in submandibular ⁄ sublingual saliva (55).

About 25% of these proteins were �hypothetical� and

lacked annotations. It is possible that additional

antimicrobial peptides will be detected in this data

set. As in the case of the palate, lung, nasal

epithelium clone (PLUNC) proteins, described

below, sequence–sequence analyses, structural pre-

diction (75, 81) and analysis for antimicrobial

consensus motifs in peptide sequences (265) can

be used to identify potential new antimicrobial

peptides.

Bactericidal ⁄ permeability-increasing protein-like proteins

Recent proteomic analyses of saliva have identified

bactericidal ⁄ permeability-increasing protein and

several potential antimicrobial proteins related

to bactericidal ⁄ permeability-increasing protein (Ta-

ble 5). Bactericidal ⁄ permeability-increasing protein

is a 55 kDa cationic protein expressed in polymor-

phonuclear leukocytes where it is stored in azurophilic

granules (38, 65). The protein is also expressed in

mucosal epithelia (39), and its presence in saliva sug-

gests that it may have a functional role in the oral

cavity.

Bactericidal ⁄ permeability-increasing protein acts

as an anti-inflammatory protein by inhibiting the

binding of lipopolysaccharide to lipopolysaccharide-

binding protein. As both bactericidal ⁄ permeability-

increasing protein and lipopolysaccharide-binding

protein bind lipopolysaccharide, it is probable that

common structures in these proteins interact with

lipopolysaccharide, although with opposing effects

(109). Bactericidal ⁄ permeability-increasing protein

is active against gram-negative bacteria and neutral-

izes their lipopolysaccharides (65). Bactericidal ⁄permeability-increasing protein contains two func-

tional domains, namely BPI1 and BPI2. The protein

exhibits opsonic activity via binding of the N-termi-

nal BPI1 domain to bacteria and binding of the C-

terminal BPI2 domain to neutrophils and monocytes

(38).

Bactericidal ⁄ permeability-increasing protein be-

longs to the bactericidal ⁄ permeability-increasing

protein superfamily, which consists of lipopolysac-

charide-binding protein, cholesteryl ester transport

protein and phospholipid transport protein. More

recently, 10 genes related to bactericidal ⁄ perme-

ability-increasing protein have been identified on

human chromosome 20q11 (nine genes) and chro-

mosome 22q12 (one gene; BPIL2). These are termed

the PLUNC proteins after an early identified member

(26–28, 126, 175). Several of these proteins are found

in saliva (Table 5).

The PLUNC proteins are divided into the short

PLUNCs (SPLUNC), which are similar to the

N-terminal BPI1 antibacterial domain of bacterici-

dal ⁄ permeability-increasing protein, and the long

PLUNCs (LPLUNC), which exhibit similarity to both

BPI1 and BPI2 domains (126). The sequence iden-

tity of bactericidal ⁄ permeability-increasing protein

and the PLUNC proteins is low (<25%), consistent

with observations for other proteins related to the

bactericidal ⁄ permeability-increasing protein family

(20). However, bactericidal ⁄ permeability-increasing

protein and the PLUNC proteins (except for BASE)

contain conserved Cys residues that form a disul-

fide bridge in bactericidal ⁄ permeability-increasing

protein. The disulfide bridge has not been verified

in parotid secretory protein, but a parotid secretory

protein mutant lacking the Cys residues is not

secreted, suggesting that its structure is significantly

altered (C. Geetha and S.-U. Gorr, unpublished

data). Several of these human proteins have

homologs in other species, including rat and mouse

parotid secretory protein (17, 161), bovine Bsp30,

PLUNC and Von Ebner minor salivary gland protein

(198, 254), and mouse PLUNC (253). Although the

existence of some of these proteins has been known

for over 20 years, it was the identification of mouse

PLUNC (253) and the sequencing of human chro-

mosome 20 that triggered the current interest in

these proteins (27). The PLUNC proteins are

primarily expressed in oral and airway epithelia and

it is thought that they play antimicrobial roles in

these tissues. The difference in expression between

different PLUNC proteins suggests that each occu-

pies an epithelial niche (126).

Latherin (breast cancer and salivary gland expres-

sion gene) is a member of the PLUNC protein family

(27) that is specifically expressed in normal salivary

glands, as determined by messenger RNA hybridiza-

tion of 61 human tissues (62). Surprisingly, latherin

was not detected in proteomic studies of saliva (55,

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Gorr

264). It is possible that the unusual surface-adhesion

properties of latherin prevented its detection (23). To

date, none of the PLUNC proteins have been detected

in gingival crevicular fluid (193).

The predicted structural similarity of PLUNC

proteins and bactericidal ⁄ permeability-increasing

protein, suggested that the PLUNC proteins could

play a role as antibacterial and ⁄ or anti-inflammatory

Table 5. Bactericidal ⁄ permeability-increasing protein-like proteins in saliva

Protein Gene Accession Parotid saliva SMG ⁄ SLG saliva Whole saliva

Protein Plunc PLUNC Q9NP55 X X X1

Bactericidal ⁄permeability-

increasing

protein-like 1

BPIL1 Q8N4F0 X X X1,2


increasing

protein-like 2

BPIL2 Q8NFQ6


increasing

protein-like 3

BPIL3 Q8NFQ5

Long palate, lung

and nasal

epithelium

carcinoma-

associated

protein 1

LPLUNC1 Q8TDL5 X X X1,2

Long palate, lung

and nasal

epithelium

carcinoma-

associated

protein 3

LPLUNC3 P59826

Long palate, lung

and nasal

epithelium

carcinoma-

associated

protein 4

LPLUNC4 P59827

Short palate, lung

and nasal

epithelium

carcinoma-

associated

protein 2

(parotid secretory

protein) (PSP)

SPLUNC2 Q96DR5 X X X1,2

Shortpalate, lung

andnasal

epithelium

carcinoma-

associated

protein 3

SPLUNC3 Q9BQP9

Latherin (BASE) LATH Q86YQ2

Protein names, Gene symbols and accession numbers are from the UniProt database. Parotid and submandibular gland (SMG) ⁄ sublingual gland (SLG) saliva dataare from a previous publication (55). Whole saliva data are from 1(264); and 2(260). �X� indicates proteins that are found in saliva. None of the proteins listed havebeen found in gingival crevicular fluid.

167


host-defense proteins (26, 76). Consistent with this

proposal, we found that parotid secretory protein

expression in human gingival epithelial cells is in-

duced by P. gingivalis, tumor necrosis factor-alpha

and estradiol (210). Mouse parotid secretory protein

binds bacteria (203), and human parotid secretory

protein exhibits moderate antibacterial activity to

P. aeruginosa (76). Similarly, PLUNC shows moderate

activity against Mycoplasma pneumoniae (60–70%

killing in 2 h) (41) and one group reported 60–70%

killing of P. aeruginosa (268, 269), while others failed

to detect killing of P. aeruginosa, Escherichia coli or

Listeria monocytogenes in 3-h kill assays based on

colony-forming unit counts (19). Importantly, none

of these experiments report more than 90% (one

order of magnitude) killing. Thus, it remains to

be determined if the bactericidal ⁄ permeability-

increasing protein-like proteins function as bacteri-

cidal proteins in vivo. Indeed, we have recently found

that a parotid secretory protein-derived peptide

causes bacterial agglutination and lowers colony-

forming unit counts in bacterial killing assays (81).

However, the peptide did not result in decreased

bacterial viability in biochemical assays, suggesting

that the reduced colony-forming unit counts could be

attributed to bacterial agglutination. Based on these

results, we speculate that parotid secretory protein is

functionally related to salivary agglutinin (DMBT1)

and to surfactant protein D. As such, parotid secre-

tory protein could play a role in bacterial binding,

agglutination and clearance by phagocytic cells (81).

Consistent with the proposed bacterial binding

function of bactericidal ⁄ permeability-increasing

protein-like proteins, the members of this family

bind lipids and lipopolysaccharide. Similarly, hu-

man PLUNC (77) and parotid secretory protein

(S.-U. Gorr et al., unpublished data) bind immobilized

lipopolysaccharide. Immobilized BSP30, by contrast,

does not bind soluble lipopolysaccharide (87) but it

is not clear if this is a result of structural or

methodological differences. While PLUNC does not

compete with lipopolysaccharide-binding protein

for lipopolysaccharide binding (37), the identifica-

tion of anti-inflammatory peptides in the parotid

secretory protein sequence (75) suggests that par-

otid secretory protein may play a role in neutral-

izing lipopolysaccharide at mucosal surfaces. The

relatively recent identification of human parotid

secretory protein and its related proteins in human

saliva, and the large number of �unknown� proteins

present in saliva, suggest that more potential

antimicrobial proteins can be identified in this

biofluid.

Peptides derived from host-defenseproteins

Antimicrobial peptides constitute an interesting new

class of compounds that can augment a decreasing

arsenal of effective antibiotics to many bacterial

species and fungi. Microbial resistance to antibiotics

was recognized early and the problem was pointed

out in Fleming�s 1945 Nobel lecture (68). As

resistance to known antibiotics is on the rise, the

search for new classes of antibiotics continues to be

of importance (80). The development of new

antibiotics based on host-defense proteins is a par-

ticularly attractive option (88). Antimicrobial pep-

tides have functioned in host defense for millions of

years and it is thought that the co-evolution of anti-

microbial peptides and pathogens is a factor in the

relatively low resistance of bacteria to these com-

pounds (191).

To date, over 1000 antimicrobial peptides have

been identified and are accessible in online databases

(33, 42, 211, 244, 247) (http://www.bbcm.univ.trieste.

it/~tossi/pag1.htm). A comprehensive list of known

and potential antimicrobial peptides has also been

reported: http://aps.unmc.edu/AP/main.php. This

list contains over 1100 peptides. These databases are

a rich resource for the identification of clinically

useful peptides that could exhibit both high in vivo

efficacy and low host toxicity. Both factors have been

concerns in clinical trials of antimicrobial peptides

(80).

While naturally occurring host-defense proteins

are effective endogenous antimicrobial agents,

often their size, folding and other physico-

chemical properties make them less ideal as

synthetic therapeutics. Instead, the active regions

of several host-defense proteins have been iden-

tified and peptides based on these regions have

been designed. Examples of such antimicrobial

peptides derived from human salivary proteins are

provided below. The sequences of representative

peptides from each parent protein are listed in

Table 6.

Bactericidal ⁄ permeability-increasingprotein

Bactericidal ⁄ permeability-increasing protein exhib-

its both antibacterial and anti-inflammatory activities

(251). A recombinant N-terminal fragment (rBPI21)

retains the biological activity of bactericidal ⁄ per-

meability-increasing protein and has been evaluated

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Gorr

in clinical trials (78). The bactericidal ⁄ permeability-

increasing protein molecule has also served as a

template for the design of anti-inflammatory pep-

tides. Chimeric peptides consisting of bactericidal ⁄permeability-increasing protein and the Limulus

anti-lipopolysaccharide factor were especially effec-

tive in blocking lipopolysaccharide action in vitro

and in vivo (51).

Mucins

The small salivary mucin MUC7 (MG2, 357 amino

acid residues) is a host-defense protein with a role in

binding and clearance of microorganisms. Peptides

(12–50 amino acid residues) from the N-terminal

domain of MUC7 exhibit antifungal activities (216).

The optimal 12-mer peptide also exhibits activity

against oral bacteria in either planktonic growth or

in biofilms (248, 249). A 23-amino acid proline-

rich tandem repeat, when used at concentrations of

1.5–3.0 lM, exhibits bactericidal activity to the oral

bacteria A. actinomycetemcomitans, P. gingivalis,

S. gordonii and S. mutans (13).

Histatins

The histatin family is a naturally rich source of

antimicrobial peptides found in the oral cavity but

not in the airways (57). Histatins are active against

C. albicans and other fungi, which they kill by

nonlytic release of ATP (243). Proteomic analysis of

glandular saliva has identified a vast array of histatin

peptides (91). Several histatin 3 peptides were newly

identified and one of these also exhibited anti-

candidal activity. A histatin 5 12-mer peptide (P113)

exhibits candidacidal activity (204) and is bactericidal

against several species of oral bacteria (248).

Defensin

The defensin family of antimicrobial peptides con-

sists of alpha and beta (and theta) defensins, which

are characterized by the location of three character-

istic disulfide bonds (72). Chemical synthesis of de-

fensins is complicated by the correct formation of

disulfide pairs. Truncated alpha-defensins have been

produced to overcome this obstacle. While linear

peptides were not antimicrobial (163), the incorpo-

ration of a 2-aminobenzoyl derivative restored anti-

microbial activity (157). The most promising of these

truncated peptides, 2Abz23S29, killed both gram-

positive and gram-negative bacteria with minimal

inhibitory concentrations of 2–10-fold those of the

native alpha-defensin 1 peptide (157).

Lactoferrin

Proteolytic digestion of lactoferrin produces lacto-

ferricin, which has been used as a template to model

pharmacologically effective antimicrobial peptides

with high antibacterial activity (98). Two additional

antimicrobial peptides derived from lactoferrin have

recently been described. Lfpep (Lf 18–40) has a net

charge of +7, while Kaliocin-1 (Lf 153–183) has a net

charge of +1. The latter peptide is highly similar to the

frog antimicrobial peptide brevinin-1Sa (241). Both

peptides kill C. albicans, but only Lfpep is able to

permeabilize the cell membrane (241). Lactoferricin

B induced resistance in S. aureus (208), raising dis-

cussion over the use of this or similar peptides in

human therapy (24, 89).

Table 6. Examples of antimicrobial peptides derived from salivary host-defense proteins (parent protein) (see the textfor functional details and references)

Parent protein Peptide Sequence

BPI XOMA 629 KLFR-[3-(1-naphthyl)-A]-QAK-[3-(1-

naphthyl)-A]-NH2

MUC7 12-mer-L RKSYKCLHKRCR

Histatin Hsn5 12-mer AKRHHGYKRKFH

Defensin 2Abz23S29 RYGTC(Acm)IYQ-2Abz-RLWAFS

Cathelicidin LL-37 K-18-mer KLFKRIVKRILKFLRKLV

Lactoferrin Lfpep TKCFQWQRNMRKVRGPPVSCIKR

DMBT1 SRCRP2 QGRVEVLYRGSWGTVC

PSP GL13-NH2 GQIINLKASLDLL-NH2

BPI, bactericidal ⁄ permeability increasing protein; DMBT1, deleted in malignant brain tumor 1,salivary agglutinin; MUC7, mucin 7; PSP, parotid secretoryprotein.

169


Salivary agglutinin (DMBT1)

A consensus peptide based on the SRCR domain of

DMBT1 was found to cause bacterial agglutination

and block the agglutination induced by DMBT1.

Thus, this peptide captures an important biological

activity of the native protein. The bacteria aggluti-

nated by this peptide include several oral bacteria

such as S. gordonii (25).

Parotid secretory protein

Based on the predicted structural similarity of parotid

secretory protein and bactericidal ⁄ permeability-

increasing protein, and the location of active peptides

in the bactericidal ⁄ permeability-increasing protein

sequence, we identified potential antimicrobial se-

quences in parotid secretory protein. The peptides

designed initially inhibited the binding of lipopoly-

saccharide to lipopolysaccharide-binding protein and

inhibited lipopolysaccharide-stimulated secretion of

tumor necrosis factor-alpha from macrophages (75).

Recent results suggest that a modified parotid

secretory protein peptide is also active against lipo-

polysaccharide in a mouse model of sepsis (S. U.

Gorr, unpublished data). The peptide GL-13 caused

agglutination of both gram-positive and gram-nega-

tive bacteria in a dose-dependent manner. Aggluti-

nation was not affected by salt concentration, but

nonionic detergent reduced agglutination, suggesting

that hydrophobic interactions play a role. Agglutina-

tion limited the spread of P. aeruginosa infection in

Romaine lettuce leaves and increased the uptake of

P. aeruginosa by macrophages (81).

Antimicrobial peptide mimetics

The synthesis, purification, delivery and in vivo effi-

cacy of antimicrobial peptides remain areas of con-

siderable challenge, as evidenced by the limited

number of antimicrobial peptides in clinical trials. A

promising alternative to natural or synthetic peptides

is the use of peptide mimetics that capture the bio-

logical activity of host-defense peptides but in a

chemical format that is more readily produced and

delivered (232). Mimetics based on the structure of

defensin have shown a high therapeutic index in

preclinical studies (22). Similarly, XOMA 629 is a

modified D-enantiomer peptide derived from

functional domain II of human bactericidal ⁄ perme-

ability-increasing protein (amino acid residues

65–99) and is highly active against a wide variety of

bacteria and fungi (146). Given the large variety of

antimicrobial peptides, it may be possible to design

both broad-range mimetics and some that are limited

to specific infectious agents without the unintended

elimination of beneficial commensals.

Clinical applications

The promise of antimicrobial peptides lies in their

broad efficacy and potentially low rates of induced

resistance resulting from the co-evolution of patho-

gens and host antimicrobial peptides. Indeed, a

number of antimicrobial peptides or synthetic

derivatives have reached clinical trials (Table 7).

However, a recent review indicates that the results

have been mixed and none has been approved by the

US Food and Drug Administration (80). The most

promising antimicrobial peptide appears to be the

histatin 5 12-mer P113 (Demegen), which completed

Phase II clinical trials as a mouth rinse for oral can-

didiasis in patients with HIV. The bactericidal ⁄ per-

meability-increasing protein derivative XOMA 629

(XOMA) is in Phase 2a trials for the skin infection

impetigo, while the defensin mimetic PMX-30063

(Polymedix Inc.) has passed Phase I safety evaluation.

Table 7. Clinical trials of antimicrobial peptides

Type Compound Function Condition Reference

BPI peptide XOMA629 Antibacterial Impetigo XOMA

Histatin 5 P113 Antifungal Candidiasis Demegen

Defensin mimetic PMX-30063 Antibacterial Infection Polymedix

Lactoferrin hLf1-11 Antimicrobial Infections in transplant

recipients

NCT00430469

Short fatty acid Sodium butyrate Stimulate LL-37 and

hBD-1 expression

Shigellosis NCT00800930

Antimicrobial peptides discussed in the text that have been or are in clinical trials are listed. The References are the ClinicalTrials.com ID number or the name of thecompany developing the drug.BPI, bactericidal ⁄ permeability-increasing protein.

170

Gorr

While the initial promise of this class of antimicro-

bials remains to be confirmed, continued efforts in

this area appear justified as resistance to existing

antibiotics and emerging infections are an ongoing

problem (80, 226).

As an alternative to antimicrobial peptide therapy,

the stimulation of expression of endogenous antimi-

crobial peptides may be used to fight infections. As an

example, reduced intestinal levels of LL-37 and beta-

defensin-1 are associated with shigellosis. In a rabbit

model of the disease, induction of CAP-18 (the rabbit

homologue of LL-37) with sodium butyrate reduced

clinical illness and the bacterial load in the stool (200).

A clinical trial is underway to determine if butyrate

treatment is effective in human shigellosis (Table 7).

By using similar approaches it may be possible to

regulate the expression of antimicrobial peptides in

oral tissues, saliva and gingival crevicular fluid.

Conclusions

A broad array of antimicrobial proteins and peptides

with diverse functions has been identified in oral

tissues and fluids. Most of the antimicrobial peptides

found in gingival crevicular fluid are also found in

saliva, while saliva contains additional (as yet unde-

tected) antimicrobial peptides in gingival crevicular

fluid. These antimicrobial peptides play a protective

role, and their diversity is thought to protect against

the many microbes entering the oral cavity and to

combine effectively to fight individual organisms.

Several antimicrobial peptides are active against the

main bacteria associated with periodontal disease,

and the absence of antimicrobial peptides has been

linked with inherited forms of periodontitis. It is

predicted that additional new antimicrobial peptides

will be detected in oral tissue and fluids and serve as

templates for the design of effective antibiotics

against oral microbes.

Acknowledgments

The author�s work on parotid secretory protein was

supported by grant number 2 R01 DE012205 from the

National Institute for Dental and Craniofacial Re-

search at the National Institutes of Health. Work on

antimicrobial PSP peptides was supported by a Proof

of Concept Grant from the Office of the Executive

Vice President for Research of the University of

Louisville. The University of Louisville School of

Dentistry is gratefully acknowledged for support.

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