AMP in Oral Cavity

Periodontology 2000, Vol. 51, 2009, 152180 Printed in Singapore.
All rights reserved
2009 John Wiley & Sons A/S
PERIODONTOLOGY 2000
Antimicrobial peptides of the oral cavity

S V E N -U L R I K G O 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 engendered 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 periodontal disease. The potential use of antimicrobial peptides as templates for novel antibiotics is also discussed.
Oral bacteria and infection

Oral infections and attendant inammatory 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 individual. For example, only 69 of the 400 species of periodontal bacteria were detected in multiple subjects in one study (188). A recent study, using pyrosequencing of short sequence tags for the 16S ribosomal DNA V6 region, led to much higher estimates of microbial diversity in saliva and plaque (124). A preliminary estimate identied 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 microbes, 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 actinomycetemcomitans is more prevalent in the young while Porphyromonas gingivalis is the dominant bacterial agent later in life (217). It is probable that differences in environmental factors and in the hosts acquired and innate immune responses, including antimicrobial peptides, pattern recognition receptors, chemokines and cytokines, play a role in determining the microbial balance of oral tissues and the subsequent outcome of bacterial colonization. Consistent with this view, population studies have identied population subgroups with high, moderate or low susceptibility to inammatory diseases, including periodontitis (151). In general, the unique proles of antimicrobial proteins found in different mucosal secretions provide an appropriate response to the microorganisms invading each epithelium. As an example, the oral cavity and airways share exposure to similar environments, and many antimicrobial proteins are common to both locations. However, differences in gene expression and regulation suggest that the innate 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
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Table 1. Effect of periodontal pathogens on antimicrobial peptide expression in human gingival epithelial cells
Periodontal pathogens Aggregatibacter (Actinobacillus) actinomycetemcomitans1 Antimicrobial peptide hBD-2 hBD-3 hCAP18 Fibronectin Porphyromonas gingivalis
1,3
Expression Up
4
References 140, 186 186 186 246 210 43 240 240 122 114
Up Up Down5 Up Up Up
6
PSP SPLUNC2 hBD-2 hBD-1 hBD-3 Adrenomedullin LL-37 Calgranulin Cystatin C
Up Up = Up
7
170 64 114 114 114 114 114 114 35 114 114 136 114 114 170
Down Down = = = Down6 Down = = = Up Up Up

7
Tannerella forsythia (T. forsythensis)1,3
hBD-3 hBD-1 hBD-2 LL-37
Treponema denticola2,3
hBD-1 hBD-3 hBD-2 LL-37
Fusobacterium nucleatum
hBD-1 hBD-2 hBD-3 LL-37 Calgranulin
Eubacterium nodatum2 Prevotella intermedia2 hBD-1 hBD-2 hBD-3 LL-37 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 (=). 1 Consensus pathogens (190). 2 Suspected periodontal pathogens with strong supporting data (230). 3 Bacteria in the red complex described by Socransky et al. (218). 4 A. actinomycetemcomitans has no effect on hBD-2 expression in cells from a patient with localized aggressive periodontitis (140). 5 Cells treated with A. actinomycetemcomitans protease. 6 Some studies found that hBD-1 is expressed constitutively in HGEC (56, 114, 137). 7 Expression was tested in the oral squamous cell carcinoma cell line H400 (194). hBD, human b-defensin; PSP, parotid secretory protein.
Up6 Up Up Up
114 114 114 114
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 uid also removes the antibacterial activity (44). A similar experiment has not been reported for saliva. However, we recently
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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 of antimicrobial proteins in the oral cavity

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 gingival crevicular uid (Table 3). Indeed, all of the antimicrobial peptides found in gingival crevicular uid are also found in saliva. Several antimicrobial peptides are more highly concentrated in gingival crevicular uid than in saliva. The concentrations of calgranulins, bronectin, substance P and calcitonin gene related peptide are 10010,000-fold higher in gingival crevicular uid than in whole saliva. Adrenomedulin and beta-2-microglobulin are enriched about 30-fold in gingival crevicular uid. Of the antimicrobial peptides for which gingival crevicular uid concentrations are shown in Table 3, only the alpha-defensins are found at a concentration 1000-fold lower in gingival crevicular uid than in saliva (Tables 2 and 3). The antimicrobial peptides that are highly expressed in gingival crevicular uid are unlikely to be caused by contamination of the gingival crevicular uid 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 antimicrobial 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 peptides to a single pathogen is less likely to be met with antimicrobial resistance. Thus, multiple antimicrobial 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 peptides in gingival crevicular uid is well below the minimal inhibitory concentration for most microbes. However, we do not have sufcient 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 processed and C-terminally amidated to produce the 52 amino acid mature adrenomedullin, a cationic amphipathic peptide with one disulde bond. It is found in gingival crevicular uid and glandular and whole saliva. Whole saliva contains higher concentrations of adrenomedullin than glandular saliva, suggesting that oral epithelial cells contribute to the salivary expression (123). In gingival crevicular uid, the amount of adrenomedullin is about twice as high in periodontal disease sites than in healthy sites (158). Alpha-defensins Human neutrophil peptides 14 are 94-amino acid cationic pre-pro-peptides. Human neutrophil peptide-1 (amino acids 6595 of the DEFA1 product) and human neutrophil peptide-3 (amino acids 6594 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 disulde bridges and are mainly expressed in neutrophils and saliva (79). Alpha-defensins are expressed in neutrophils and have been identied in the gingival crevicular uid of both healthy and diseased sites, although different experimental methodologies lead to somewhat different 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
Table 2. Antimicrobial proteins and peptides found in saliva

Protein Adrenomedullin HNP-1 HNP-2 HNP-3 hBD-1 hBD-2 hBD-3 Cathelicidin LL-37 Histatin 1 Histatin 3 Statherin CCL28 Azurocidin CAP37 heparin-binding protein Substance P Calcitonin gene related peptide Neuropeptide Y Vasoactive intestinal peptide Mucin 7 Gene ADM DEFA1 DEFA1 DEFA3 DEFA3 DEFB1 DEFB4 DEFB103A CAMP HTN1 HTN3 STATH CCL28 AZU1 26.5 0.9 MS Whole saliva (lg ml) 0.06 8.6 5.6 02.7 0.15 0.150 0.31 1.6 MS 10.1 7.3 69 2.7 MS 34.7 10.2 75 MS MS Parotid saliva (lg ml) 0.02 MS SM SL saliva (lg ml) 0.035 MS References 123 79 79 74 164 164 231 229, 16 116 116 46 99 102
TAC1 CALCA NPY VIP
7.5 10)6 23.5 10

)6
6.2 10)6 1.5 10

)6
52 52 52 52
41.4 10)6 39.9 10

)6
44.4 10)6 29.9 10

)6
MUC7
40
MS
116157
189 21
GP340 salivary agglutinin DMBT1 Surfactant protein-A Beta-2-microglobulin Proline-rich proteins
DMBT1
MS
6.3
67
SFTPA1 B2M PRH1 PRH2 PRB1 PRB3 PRB2 PRB4
0.9 0.38 MS MS MS MS 512 81

2
215 MS 652 103

2
169 111 111
MS MS MS
MS MS MS MS 0.5 MS 150, 236 129
MS 1.20.13 1.93
MS MS MS
Fibronectin Calgranulin A
FN1 S100A8
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Table 2. Continued
Protein Calgranulin B Psoriasin Lactoferrin Cystatin A Cystatin B Cystatin C Cystatin D Cystatin S Cystatin SA Cystatin SN Secretory leukocyte protease inhibitor SKALP Elan Gene S100A9 S100A7 LTF CSTA CSTB CST3 CST5 CST4 CST2 CST1 SLPI Whole saliva (lg ml) 1.93 MS 20 MS MS 0.9 3.8 53116 78 39 2.9 4.7 MS MS 0.4 MS 1.0 MS MS 0.55 MS MS MS MS MS MS MS 1.1 101, 238 69 18, 101 18 18 147, 212 207, 212, 228 Parotid saliva (lg ml) MS SM SL saliva (lg ml) MS References 129
PI3
0.020
PS4
SMG4
235 142
Lactoperoxidase (salivary peroxidase) Myeloperoxidase
LPO
1.9
6.2
MS
WS = Unstim (233) PS = Stim (207)
MPO
MS
MS
233 185
Lysozyme C Peptidoglycan recognition protein 1
LYZ PGLYRP1
40
4.4
MS MS
7, 130, 207, 212
Common name, gene symbol, concentrations in whole saliva; parotid saliva and submandibular sublingual (SM SL) saliva (lg ml). Values in bold italic are from stimulated saliva. MS = mass spectrometry detection of proteins in unstimulated whole saliva (260, 264) or in stimulated glandular saliva (55). Saliva was collected from healthy subjects, unless stated otherwise. It should be noted that the concentrations shown are representative average values only. Individual variation and variation between studies can be signicant; see references for details. 1 Salivary agglutinin (DMBT1) puried from parotid saliva represented 0.4% of total protein (67). 2 Calculation based on an average parotid salivary ow rate of 0.7 ml min gland (100) and SM SL ow rate of 0.4 ml min gland (261). 3 Measured as calprotectin (calgranulin A and B complex) (129). 4 Found in parotid saliva and submandibular glands (SMG) (142). HNP, human neutrophil peptide, a-defensin.
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 disulde 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 uid (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 antimicrobial peptides FALL-39 and LL-37. LL-37 is a cationic alpha-helical peptide, expressed in neutrophils, epithelial cells, saliva and gingival crevicular uid (176, 195). In addition to antibacterial activity (Table 4A), LL-37 also binds to and neutralizes lipopolysaccharide from gram-negative bacteria. In Candida albicans, the peptide causes
156
Table 3. Antimicrobial proteins and peptides found in gingival crevicular uid

Protein Gingival crevicular uid: healthy (lg ml) 1.8 (158) MS MS MS 0.0012 (195) MS (56) (56) Up-regulated 15-fold in aggressive periodontitis and 60-fold in chronic periodontitis, respectively (195) Gingival crevicular uid: periodontitis
Adrenomedullin HNP-1 HNP-2 HNP-3 HNP(13)
Twofold increase in periodontitis (158)
HNP-4 hBD-1 hBD-2 hBD-3 Cathelicidin LL-37 Statherin Beta-2-microglobulin
(195) MS 9.4 (174) MS MS 106 (152)
Up-regulated in aggressive periodontitis and chronic periodontitis (195)
Increased threefold in mild periodontitis and 10-fold in severe periodontitis, respectively (174)
Proline rich protein Proline rich peptide P-B Fibronectin
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) Increased 23-fold in periodontitis, decreased 23-fold after periodontal therapy (132, 154, 155) Increased in periodontitis, decreased 23-fold after periodontal therapy (132) Calprotectin concentration increased with the gingival index (180); 35-fold increase in periodontitis (>4 mm pocket depth) (125); 70 ng ll detected in children (172) Concentration highly variable. No consistent change with disease (2, 3, 70, 112) Main cystatin activity in gingival crevicular uid of periodontal patients (30) No change in children with gingivitis (237) 79.7 pg ll in periodontitis, increased 34-fold at 2 and 4 weeks post treatment (179)
Calgranulin A
240 (154) (132) 570 (125)
Calgranulin B Calprotectin
Lactoferrin
600
Cystatin A
MS
Cystatin C Secretory Leukocyte
1.15 (children) (237) No data
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Table 3. Continued
Protein Gingival crevicular uid: healthy (lg ml) Gingival crevicular uid: periodontitis
Protease Inhibitor Lactoperoxidase (Salivary Peroxidase) Myeloperoxidase Possible (14) 0.35.5 (185, 195) Peroxidase identied in gingival crevicular uid is most likely myeloperoxidase (162) No change in aggressive or chronic periodontitis (195). 660 lg ml in >5 mm pockets. Concentration decreased twofold after therapy (121) Increased in juvenile periodontitis (70) SP concentration was similar in healthy, gingivitis or periodontitis (148). Others found that concentration was decreased sevenfold post periodontal treatment (156) Concentration 20-fold lower in gingivitis, not detected in periodontitis (160)
Lysozyme C Substance P
(70, 112) 0.0610.11 (15) (148)
Calcitonin Gene Related Peptide
0.0130.7 (15, 160)
Common name, presence in gingival crevicular uid (concentration in lg ml is given when known) from healthy subjects and changes in periodontitis patients. hBD, human b-defensin; HNP, human neutrophil peptide, a-defensin; MS, proteins detected by mass spectrometry of gingival crevicular uid (159, 193).
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 bactericidal 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 efux of nucleotides is similar with the two peptides and may play a signicant role for candidacidal activity (54). Histatins are found in saliva (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 (juvenile) 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 uid (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 expressed in various epithelial cells, including salivary glands, and is found in saliva (55). The peptide acts both as a chemokine and a broad-spectrum antimicrobial peptide. Indeed, a C-terminal 28-amino acid peptide is similar to histatin 5. This peptide induces 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 identied in human saliva by proteomic analysis (55, 264). This 251-amino acid protein is antibacterial to gram-negative bacteria, presumably because of a strong afnity for lipopolysacchaide. Two cysteine residues in positions 52 and 68 are necessary for antibacterial activity.
158
Table 4A. Antimicrobial activities of peptides that are directly bactericidal or bacteriostatic
Antimicrobial peptide Adrenomedullin Targets P. gingivalis S. mutans HNP-1 S. mutans P. aeruginosa C. albicans Enveloped viruses HIV-1 A. actinomycetemcomitans P. gingivalis HNP-2 P. gingivalis A. actinomycetemcomitans C. albicans Herpes simplex virus-1 HIV-1 HNP-3 P. gingivalis A. actinomycetemcomitans C. albicans Herpes simplex virus-1 HIV-1 HNP-4 E. coli C. albicans hBD-1 P. gingivalis A. actinomycetemcomitans C. albicans hBD-2 P. gingivalis S. mutans C. albicans HIV-1 hBD-3 P. gingivalis A. actinomycetemcomitans S. mutans C. albicans HIV-1 LL-37 P. gingivalis A. actinomycetemcomitans S. gordonii C. albicans Activity MIC 7.75 10)4 lg ml MIC 12.5 lg ml MIC 4.1 lg ml MIC 10.3 lg ml MIC 52.5 lg ml Inactivation Inhibits replication No activity (>500 lg ml) No activity (>200 lM) No activity (>200 lM) No activity (>500 lg ml) EI90 65 lM Inactivation Inhibits replication No activity (>200 lM) No activity (>500 lg ml) No activity Inactivation Inhibits replication LD50 0.085 lg ml LD50 0.5 lg ml MIC 50 lg ml MIC 50 lg ml MFC 7 lM MIC 34.6>250 lg ml MIC 48 lg ml MIC 559 lg ml IC50 919 lg ml MIC 42.1 lg ml MIC 45.6 lg ml MIC 35 lg ml MIC 37 lg ml IC50 2040 lg ml MIC >125 lg ml MIC 37.8 lg ml MIC 102.6 lg ml LD50 0.8 lM 113 113 113 54 113 113 117 117 157 157 157 48 267 171 197 197 171 197 48 267 197 171 143 48 267 258 258 187 187 138 117 Reference 6
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Table 4A. Continued

Antimicrobial peptide Histatin 5 Targets A. actinomycetemcomitans C. albicans Statherin Oral anaerobes C. albicans CCL28 S. mutans C. albicans Azurocidin CAP37 E. coli C. albicans Substance P P. aeruginosa S. mutans C. albicans Calcitonin gene related peptide Neuropeptide Y P. aeruginosa S. mutans C. albicans P. aeruginosa S. mutans C. albicans Vasoactive intestinal peptide Psoriasin Lysozyme C Peptidoglycan recognition protein 1 Peptidoglycan recognition protein 3 P. aeruginosa S. mutans C. albicans E. coli Gram-positive bacteria S. aureus E. coli C. albicans S. aureus Gram-negative bacteria C. albicans Peptidoglycan recognition protein 4 S. aureus Gram-negative bacteria C. albicans SKALP Elan Secretory leukocyte protease inhibitor P. aeruginosa P. aeruginosa Activity Neutralizes leukotoxin LD50 1.6 lM MIC <12.5 lg ml to >100 lg ml Aggregation IC50 1.7 lM IC50 0.7 lM LD50 1.3 lg ml MIC 40 lg ml MIC 15.7 lg ml MIC 171.6 lg ml MIC 8.1 lg ml MIC 5.9 lg ml MIC >500 lg ml MIC 63.1 lg ml MIC 134.3 lg ml MIC 210.9 lg ml MIC 243.2 lg ml MIC 4.1 lg ml MIC 150.7 lg ml MIC 46.5 lg ml MBC 100 lg ml Cell wall lysis LD99 60 lg ml LD99 30 No activity LD99 45 lg ml LD99 30 lg ml No activity LD99 45 lg ml LD99 200 lg ml No activity LD96 2.5 lM LF95 2.5 lM 245 245 153 245 245 153 245 245 153 213 213 168 63 63 63 8 165 63 Reference 178 54 131 115 102
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.
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Table 4B. Antimicrobial activities of peptides that modulate microbial adhesion or activity
AMP Mucin 7 Targets Oral streptococci A. actinomycetemcomitans C. albicans HIV GP340 salivary agglutinin DMBT1 Oral streptococci A. actinomycetemcomitans HIV-1 Inuenza A virus Surfactant protein-A Bacteria Inuenza A virus Beta-2-microglobulin Proline rich proteins S. mutans Oral bacteria C. albicans HIV-1 Fibronectin P. gingivalis S. mutans Calprotectin P. gingivalis C. albicans S. aureus Lactoferrin P. gingivalis A. actinomycetemcomitans C. albicans Cystatins Cystatin C Bacterial or host proteases P. gingivalis Herpes simplex virus-1 Cystatin S Secretory leukocyte protease inhibitor P. gingivalis P. gingivalis S. aureus HIV C. albicans Myeloperoxidase A. actinomycetemcomitans Oral streptococci P. gingivalis Bactericidal OI) 105 Bactericidal OCl) Blocks virus entry 166 173 Activity Bacterial binding and agglutination Bacterial binding Binding and clearance Prevent cell entry Agglutination Binding Blocks cell entry Inactivation by agglutination Binding and clearance Agglutination Agglutination in presence of Ca2+ Bacterial adhesion Adhesion Inhibits infectivity Binding to mbrillin Adhesion and agglutination Protects epithelial cells from infection Candidastatic by Zn chelation; MIC 18 lg ml Zn chelation, MIC 64 lg ml 35% growth inhibition at 2 mg ml apoLf 1.9 lM apoLf (iron-free) kills 99.9% in 3 h Induces apoptosis Cysteine protease inhibitor Growth inhibition Inhibits viral replication Growth inhibition Degraded by gingipain References 11 84 104 86 145 84 263 97 133 256 66 139 115 202 177 149 181 220 34 34 4 120 12 32 31 29 31 108 259
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Table 4B. Continued

AMP Lactoperoxidase (salivary peroxidase) Targets A. actinomycetemcomitans Oral streptococci P. gingivalis Activity Bactericidal I H2O2 Bacteriostatic OSCN Bactericidal OI) Bactericidal OI)
) )
References 107 234 105
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.
Neuropeptides
The neuropeptides calcitonin gene related peptide and substance P are found in gingival crevicular uid (15). These peptides, and neuropeptide Y and vasoactive intestinal peptide, are also found in saliva (52). However, the concentrations ranging from 245 pg ml are several orders of magnitude lower than the minimal inhibitory concentrations against bacteria and C. albicans (63).
Staphylococcus aureus but not with Pseudomonas aeruginosa (119), indicating that different host-defense 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 inuenza virus hemagglutinin 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 inuenza 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 uid. In gingival biopsies, beta2-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 uid from patients with periodontal than in gingival crevicular uid from healthy controls (225). However, this difference was not noted in a study of juvenile periodontitis 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
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 Nglycosylation 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. actinomycetemcomitans 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 immunodeciency 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
162
bacterial attachment to the pellicle, it has been suggested 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 mbrillin to proline-rich protein 1 is inhibited by C-terminal mbrillin peptides corresponding to amino acid residues 266286 and 318337 (10). Proline-rich proteins, bronectin and other proteins that affect bacterial adhesion and biolm 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 mbrillin from P. gingivalis and thereby inhibits the mbrillininduced expression of inammatory cytokines in macrophages (177). Low levels of bronectin are correlated with high levels of S. mutans in children (149) and periodontitis is associated with a relative lack of bronectin in adults (177).
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 anion. It is produced by mucosal epithelial cells and is found in their secretions, including saliva (250) and gingival crevicular uid, which is a signicant source of oral lactoferrin (60). Decreased levels of lactoferrin in granulocytes have been associated with periodontal disease (219), while the saliva levels of lactoferrin 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 protein plays a role in the development of the disease. Lactoferrin acts on bacteria, viruses, fungi and parasites. 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 broblasts (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-inammatory activity and bactericidal activity as a result of increased membrane permeability (184).
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 calgranulin A and B are up-regulated in oral epithelial H400 cells exposed to P. gingivalis or Fusobacterium nucleatum (170). Accordingly, calprotectin is upregulated in periodontal disease and is detected at increased levels in the gingival crevicular uid 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 transcriptome (47).
Protease inhibitors
Cystatins The human cystatin gene family contains 14 genes and two pseudogenes. Seven of these genes are expressed 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 disulde 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-inammatory protein
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(252). The N-terminal cationic domain of secretory leukocyte protease inhibitor is antimicrobial with activity against P. aeruginosa, S. aureus and C. albicans (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 uid levels of secretory leukocyte protease inhibitor are lower in P. gingivalis-infected sites, possibly because of secretory leukocyte protease inhibitor degradation by arginine-specic gingipains from P. gingivalis (108). SKALP Elan Skin-derived antileukoproteinase (SKALP) Elan is a 12 kDa protein expressed in the human submandibular 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. Lactoperoxidase 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, myeloperoxidase (a heme peroxidase) is expressed in neutrophilic polymorphonuclear leukocytes and catalyses the oxidation of thiocyanate ions by hydrogen peroxide to produce hypothiocyanite. In addition, myeloperoxidase can produce hypochlorite (OCl)), which is a stronger oxidant implicated in inammatory tissue damage (14). Myeloperoxidase is found in gingival crevicular uid at a concentration of about 5 lg ml but no signicant differences were found between the levels of myeloperoxidase found in chronic periodontitis, aggressive periodontitis and healthy controls (195). However, the myeloperoxidase levels in gingival crevicular uid were signicantly 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)-betalinkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycans. The enzyme is mainly active against the cell wall of grampositive 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 uid. The lysozyme levels do not appear to differ between adult periodontitis and healthy controls, (225) but are signicantly increased in juvenile periodontitis (70). Peptidoglycan recognition proteins 3 and 4 These large host-defense proteins (89115 kDa disulde-linked homodimers or heterodimers) are found in species ranging from insects to mammals (61). Interestingly, different peptidoglycan-recognition proteins have diversied to carry out different antimicrobial functions (205). Four peptidoglycanrecognition proteins are found in humans: peptidoglycan 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 proteins are bactericidal for pathogenic and nonpathogenic gram-positive bacteria but not for normal ora bacteria. Moreover, the proteins are bacteriostatic for most gram-positive and gram-negative bacteria but not for C. albicans (153).
The role of antimicrobial proteins in periodontal disease

Striking differences in susceptibility to the antimicrobial 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 proles 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 identied (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 periodontitis (196). Patients with morbus Kostmann are decient in LL-37 in neutrophils, plasma and saliva. Patients are also decient in alpha-defensins (30% of normal) while the plasma lactoferrin content is normal (196). Patients treated with granulocytecolony 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 sufcient to prevent the disease. Thus, periodontal disease is prevalent in children with Downs 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). ` vre syndrome and HaimMunk synPapillonLefe drome are characterized by palmoplantar keratoderma 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 ` vre syndrome express patients with PapillonLefe 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` vre syndrome titis in patients with PapillonLefe (53).
Antimicrobial proteins and other oral 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 lactoferrin, and beta-defensins 1 and 2. By contrast, the concentration 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 difcult to establish. Caries incidence in children has been associated with the expression of a low-level of alphadefensins (human neutrophil peptides 13) (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 hostdefense 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 difculty in linking single point analysis of antimicrobial peptides with oral disease (231) may be a result of the wide variety of antimicrobial 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 difcult to dene 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 signatures that will have predictive or diagnostic power.
New antimicrobial proteins

Many types of antimicrobial peptides have been identied in the oral cavity, as described in the preceding sections. With the advent of saliva and gingival crevicular uid proteomics it is predicted that additional antimicrobial peptides will be identied. It should be noted that not all of these peptides will be present at biologically signicant concentrations. Nevertheless, identication 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 identied 56 proteins, 32% of which were characterized as host-defense proteins (215). The
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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 Institute of Dental and Craniofacial Research, at the US National Institutes of Health, has identied 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, sequencesequence analyses, structural prediction (75, 81) and analysis for antimicrobial consensus motifs in peptide sequences (265) can be used to identify potential new antimicrobial peptides.
Bactericidal permeabilityincreasing protein-like proteins

Recent proteomic analyses of saliva have identied bactericidal permeability-increasing protein and several potential antimicrobial proteins related to bactericidal permeability-increasing protein (Table 5). Bactericidal permeability-increasing protein is a 55 kDa cationic protein expressed in polymorphonuclear leukocytes where it is stored in azurophilic granules (38, 65). The protein is also expressed in mucosal epithelia (39), and its presence in saliva suggests that it may have a functional role in the oral cavity. Bactericidal permeability-increasing protein acts as an anti-inammatory protein by inhibiting the binding of lipopolysaccharide to lipopolysaccharidebinding protein. As both bactericidal permeabilityincreasing 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 neutralizes their lipopolysaccharides (65). Bactericidal permeability-increasing protein contains two functional domains, namely BPI1 and BPI2. The protein exhibits opsonic activity via binding of the N-terminal BPI1 domain to bacteria and binding of the Cterminal BPI2 domain to neutrophils and monocytes (38).
Bactericidal permeability-increasing protein belongs to the bactericidal permeability-increasing protein superfamily, which consists of lipopolysaccharide-binding protein, cholesteryl ester transport protein and phospholipid transport protein. More recently, 10 genes related to bactericidal permeability-increasing protein have been identied on human chromosome 20q11 (nine genes) and chromosome 22q12 (one gene; BPIL2). These are termed the PLUNC proteins after an early identied member (2628, 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 bactericidal permeability-increasing protein, and the long PLUNCs (LPLUNC), which exhibit similarity to both BPI1 and BPI2 domains (126). The sequence identity 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 disulde bridge in bactericidal permeability-increasing protein. The disulde bridge has not been veried in parotid secretory protein, but a parotid secretory protein mutant lacking the Cys residues is not secreted, suggesting that its structure is signicantly 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 identication of mouse PLUNC (253) and the sequencing of human chromosome 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 occupies an epithelial niche (126). Latherin (breast cancer and salivary gland expression gene) is a member of the PLUNC protein family (27) that is specically expressed in normal salivary glands, as determined by messenger RNA hybridization of 61 human tissues (62). Surprisingly, latherin was not detected in proteomic studies of saliva (55,
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Table 5. Bactericidal permeability-increasing protein-like proteins in saliva

Protein Protein Plunc Bactericidal permeabilityincreasing protein-like 1 Bactericidal permeabilityincreasing protein-like 2 Bactericidal permeabilityincreasing protein-like 3 Long palate, lung and nasal epithelium carcinomaassociated protein 1 Long palate, lung and nasal epithelium carcinomaassociated protein 3 Long palate, lung and nasal epithelium carcinomaassociated protein 4 Short palate, lung and nasal epithelium carcinomaassociated protein 2 (parotid secretory protein) (PSP) Short palate, lung and nasal epithelium carcinomaassociated protein 3 Latherin (BASE) Gene PLUNC BPIL1 Accession Q9NP55 Q8N4F0 Parotid saliva X X SMG SLG saliva X X Whole saliva X1 X1,2
BPIL2
Q8NFQ6
BPIL3
Q8NFQ5
LPLUNC1
Q8TDL5
X1,2
LPLUNC3
P59826
LPLUNC4
P59827
SPLUNC2
Q96DR5
X1,2
SPLUNC3
Q9BQP9
LATH
Q86YQ2
Protein names, Gene symbols and accession numbers are from the UniProt database. Parotid and submandibular gland (SMG) sublingual gland (SLG) saliva data are 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 have been found in gingival crevicular uid.
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 uid (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-inammatory
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host-defense proteins (26, 76). Consistent with this proposal, we found that parotid secretory protein expression in human gingival epithelial cells is induced 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 (6070% killing in 2 h) (41) and one group reported 6070% 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 permeabilityincreasing protein-like proteins function as bactericidal proteins in vivo. Indeed, we have recently found that a parotid secretory protein-derived peptide causes bacterial agglutination and lowers colonyforming 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 secretory 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, human 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 identication of anti-inammatory peptides in the parotid secretory protein sequence (75) suggests that parotid secretory protein may play a role in neutralizing lipopolysaccharide at mucosal surfaces. The relatively recent identication 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 identied in this biouid.
Peptides derived from host-defense proteins

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 Flemings 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 particularly attractive option (88). Antimicrobial peptides have functioned in host defense for millions of years and it is thought that the co-evolution of antimicrobial peptides and pathogens is a factor in the relatively low resistance of bacteria to these compounds (191). To date, over 1000 antimicrobial peptides have been identied 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 identication of clinically useful peptides that could exhibit both high in vivo efcacy 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 physicochemical properties make them less ideal as synthetic therapeutics. Instead, the active regions of several host-defense proteins have been identied 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-increasing protein

Bactericidal permeability-increasing protein exhibits both antibacterial and anti-inammatory activities (251). A recombinant N-terminal fragment (rBPI21) retains the biological activity of bactericidal permeability-increasing protein and has been evaluated
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Table 6. Examples of antimicrobial peptides derived from salivary host-defense proteins (parent protein) (see the text for functional details and references)
Parent protein BPI MUC7 Histatin Defensin Cathelicidin Lactoferrin DMBT1 PSP Peptide XOMA 629 12-mer-L Hsn5 12-mer 2Abz S
23 29
Sequence KLFR-[3-(1-naphthyl)-A]-QAK-[3-(1naphthyl)-A]-NH2 RKSYKCLHKRCR AKRHHGYKRKFH RYGTC(Acm)IYQ-2Abz-RLWAFS KLFKRIVKRILKFLRKLV TKCFQWQRNMRKVRGPPVSCIKR QGRVEVLYRGSWGTVC GQIINLKASLDLL-NH2
LL-37 K-18-mer Lfpep SRCRP2 GL13-NH2
BPI, bactericidal permeability increasing protein; DMBT1, deleted in malignant brain tumor 1,salivary agglutinin; MUC7, mucin 7; PSP, parotid secretory protein.
in clinical trials (78). The bactericidal permeabilityincreasing protein molecule has also served as a template for the design of anti-inammatory peptides. Chimeric peptides consisting of bactericidal permeability-increasing protein and the Limulus anti-lipopolysaccharide factor were especially effective in blocking lipopolysaccharide action in vitro and in vivo (51).
exhibits candidacidal activity (204) and is bactericidal against several species of oral bacteria (248).
Defensin
The defensin family of antimicrobial peptides consists of alpha and beta (and theta) defensins, which are characterized by the location of three characteristic disulde bonds (72). Chemical synthesis of defensins is complicated by the correct formation of disulde pairs. Truncated alpha-defensins have been produced to overcome this obstacle. While linear peptides were not antimicrobial (163), the incorporation of a 2-aminobenzoyl derivative restored antimicrobial activity (157). The most promising of these truncated peptides, 2Abz23S29, killed both grampositive and gram-negative bacteria with minimal inhibitory concentrations of 210-fold those of the native alpha-defensin 1 peptide (157).
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 (1250 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 biolms (248, 249). A 23-amino acid prolinerich tandem repeat, when used at concentrations of 1.53.0 lM, exhibits bactericidal activity to the oral bacteria A. actinomycetemcomitans, P. gingivalis, S. gordonii and S. mutans (13).
Lactoferrin
Proteolytic digestion of lactoferrin produces lactoferricin, 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 1840) has a net charge of +7, while Kaliocin-1 (Lf 153183) 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 discussion over the use of this or similar peptides in human therapy (24, 89).
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 identied a vast array of histatin peptides (91). Several histatin 3 peptides were newly identied and one of these also exhibited anticandidal activity. A histatin 5 12-mer peptide (P113)
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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 agglutinated 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 permeabilityincreasing protein, and the location of active peptides in the bactericidal permeability-increasing protein sequence, we identied potential antimicrobial sequences in parotid secretory protein. The peptides designed initially inhibited the binding of lipopolysaccharide to lipopolysaccharide-binding protein and inhibited lipopolysaccharide-stimulated secretion of tumor necrosis factor-alpha from macrophages (75). Recent results suggest that a modied parotid secretory protein peptide is also active against lipopolysaccharide in a mouse model of sepsis (S. U. Gorr, unpublished data). The peptide GL-13 caused agglutination of both gram-positive and gram-negative bacteria in a dose-dependent manner. Agglutination was not affected by salt concentration, but nonionic detergent reduced agglutination, suggesting that hydrophobic interactions play a role. Agglutination limited the spread of P. aeruginosa infection in Romaine lettuce leaves and increased the uptake of P. aeruginosa by macrophages (81).
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 biological 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 modied D-enantiomer peptide derived from functional domain II of human bactericidal permeability-increasing protein (amino acid residues 6599) 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 specic infectious agents without the unintended elimination of benecial commensals.
Clinical applications
The promise of antimicrobial peptides lies in their broad efcacy and potentially low rates of induced resistance resulting from the co-evolution of pathogens 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 candidiasis in patients with HIV. The bactericidal permeability-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.
Antimicrobial peptide mimetics

The synthesis, purication, delivery and in vivo efcacy of antimicrobial peptides remain areas of conTable 7. Clinical trials of antimicrobial peptides
Type BPI peptide Histatin 5 Defensin mimetic Lactoferrin Short fatty acid Compound XOMA629 P113 PMX-30063 hLf1-11 Sodium butyrate
Function Antibacterial Antifungal Antibacterial Antimicrobial Stimulate LL-37 and hBD-1 expression
Condition Impetigo Candidiasis Infection Infections in transplant recipients Shigellosis
Reference XOMA Demegen Polymedix NCT00430469 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 the company developing the drug. BPI, bactericidal permeability-increasing protein.
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While the initial promise of this class of antimicrobials remains to be conrmed, continued efforts in this area appear justied 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 antimicrobial peptides may be used to ght infections. As an example, reduced intestinal levels of LL-37 and betadefensin-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 uid.
References
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Conclusions
A broad array of antimicrobial proteins and peptides with diverse functions has been identied in oral tissues and uids. Most of the antimicrobial peptides found in gingival crevicular uid are also found in saliva, while saliva contains additional (as yet undetected) antimicrobial peptides in gingival crevicular uid. 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 ght 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 uids and serve as templates for the design of effective antibiotics against oral microbes.
Acknowledgments
The authors work on parotid secretory protein was supported by grant number 2 R01 DE012205 from the National Institute for Dental and Craniofacial Research at the National Institutes of Health. Work on antimicrobial PSP peptides was supported by a Proof of Concept Grant from the Ofce 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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AMP in Oral Cavity

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Periodontology 2000, Vol. 51, 2009, 152180 Printed in Singapore.

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2009 John Wiley & Sons A/S

Antimicrobial peptides of the oral cavity

Oral bacteria and infection

Antimicrobial peptides of the oral cavity

PSP SPLUNC2 hBD-2 hBD-1 hBD-3 Adrenomedullin LL-37 Calgranulin Cystatin C

Down Down = = = Down6 Down = = = Up Up Up

Tannerella forsythia (T. forsythensis)1,3

hBD-3 hBD-1 hBD-2 LL-37

hBD-1 hBD-3 hBD-2 LL-37

hBD-1 hBD-2 hBD-3 LL-37 Calgranulin

114 114 114 114

Functional families of antimicrobial proteins in the oral cavity

Antimicrobial peptides of the oral cavity

Table 2. Antimicrobial proteins and peptides found in saliva

TAC1 CALCA NPY VIP

7.5 10)6 23.5 10

6.2 10)6 1.5 10

41.4 10)6 39.9 10

44.4 10)6 29.9 10

GP340 salivary agglutinin DMBT1 Surfactant protein-A Beta-2-microglobulin Proline-rich proteins

SFTPA1 B2M PRH1 PRH2 PRB1 PRB3 PRB2 PRB4

0.9 0.38 MS MS MS MS 512 81

215 MS 652 103

169 111 111

MS MS MS MS 0.5 MS 150, 236 129

Lactoperoxidase (salivary peroxidase) Myeloperoxidase

WS = Unstim (233) PS = Stim (207)

Lysozyme C Peptidoglycan recognition protein 1

7, 130, 207, 212

Antimicrobial peptides of the oral cavity

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

Adrenomedullin HNP-1 HNP-2 HNP-3 HNP(13)

Twofold increase in periodontitis (158)

HNP-4 hBD-1 hBD-2 hBD-3 Cathelicidin LL-37 Statherin Beta-2-microglobulin

(195) MS 9.4 (174) MS MS 106 (152)

Up-regulated in aggressive periodontitis and chronic periodontitis (195)

Proline rich protein Proline rich peptide P-B Fibronectin

240 (154) (132) 570 (125)

Cystatin C Secretory Leukocyte

1.15 (children) (237) No data

(70, 112) 0.0610.11 (15) (148)

Calcitonin Gene Related Peptide

0.0130.7 (15, 160)

Antimicrobial peptides of the oral cavity

Table 4A. Continued

Antimicrobial peptides of the oral cavity

Table 4B. Continued

References 107 234 105

Bacterial agglutination and adhesion

Antimicrobial peptides of the oral cavity

Metal ion chelators

Activity against bacterial cell walls

The role of antimicrobial proteins in periodontal disease

Antimicrobial peptides of the oral cavity

Antimicrobial proteins and other oral infections

New antimicrobial proteins

Bactericidal permeabilityincreasing protein-like proteins

Antimicrobial peptides of the oral cavity

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

Peptides derived from host-defense proteins

Bactericidal permeability-increasing protein