Potential applications of antimicrobial peptides and their mimics in combating caries and pulpal infections

Accepted Manuscript Review article Potential Applications of Antimicrobial Peptides and their Mimics in Combating Caries and Pulpal Infections Sui Mai, Matthew T. Mauger, Li-na Niu, Jonathan B. Barnes, Solon Kao, Brian E. Bergeron, Jun-qi Ling, Franklin R. Tay PII: DOI: Reference:

S1742-7061(16)30622-5 http://dx.doi.org/10.1016/j.actbio.2016.11.026 ACTBIO 4535

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

7 July 2016 24 October 2016 10 November 2016

Please cite this article as: Mai, S., Mauger, M.T., Niu, L-n., Barnes, J.B., Kao, S., Bergeron, B.E., Ling, J-q., Tay, F.R., Potential Applications of Antimicrobial Peptides and their Mimics in Combating Caries and Pulpal Infections, Acta Biomaterialia (2016), doi: http://dx.doi.org/10.1016/j.actbio.2016.11.026

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Potential Applications of Antimicrobial Peptides and their Mimics in Combating Caries and Pulpal Infections Sui Maia, Matthew T. Maugerb, Li-na Niuc, Jonathan B. Barnesb, Solon Kaod, Brian E. Bergeronb, Jun-qi Linga*, Franklin R. Tayb* a

Guangdong Provincial Key Laboratory of Stomatology, Department of Operative Dentistry and

Endodontics, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, PR China; b

Department of Endodontics, The Dental College of Georgia, Augusta University, Georgia, USA;

c

State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral

Diseases & Shaanxi Key Laboratory of Oral Diseases, Department of Prosthodontics, School of Stomatology, The Fourth Military Medical University, Xi’an, PR China; dDepartment of Oral and Maxillofacial Surgery, The Dental College of Georgia, Augusta University, Georgia, USA Key words: antimicrobial peptides; biofilms; immunomodulation; peptide mimics

*Co-corresponding authors: Franklin R. Tay, Department of Endodontics, The Dental College of Georgia, Augusta University, Georgia, USA. Tel: 1-706-7212606, Email: [email protected]; Junqi Ling: Guangdong Provincial Key Laboratory of Stomatology; Guanghua School of Stomatology, Sun

Yat-sen

University,

Guangzhou,

PR

China.

Tel.:

+86

020

83862621,

Email:

[email protected]

Acknowledgments The authors reported no conflicts of interest in this work. This work was supported by Foreign Science and Technology Cooperation Project of Guangdong Province (Grant 2013B051000031; PI. S. Mai), Natural Science Foundation of Guangdong Province (Grant 2014A030313068; PI. S. Mai), National High Technology Research and Development Program of China (Grant 2015AA020942; PI. L.N. Niu) and National Nature Science Foundation of China (Grant 81400555; PI. L.N. Niu). The authors thank Dr. Ya Shen for providing the reconstructed confocal laser scanning microscopy images used in the paper.

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Abstract Antimicrobial peptides (AMPs) are short cationic host-defense molecules that provide the early stage of protection against invading microbes. They also have important modulatory roles and act as a bridge between innate and acquired immunity. The types and functions of oral AMPs were reviewed and experimental reports on the use of natural AMPs and their synthetic mimics in caries and pulpal infections were discussed. Natural AMPs in the oral cavity, predominantly defensins, cathelidin and histatins, possess antimicrobial activities against oral pathogens and biofilms. Incomplete debridement of microorganisms in root canal space may precipitate an exacerbated immune response that results in periradicular bone resorption. Because of their immunomodulatory and wound healing potentials, AMPs stimulate pro-inflammatory cytokine production, recruit host defense cells and regulate immuno-inflammatory responses in the vicinity of the pulp and periapex. Recent rapid advances in the development of synthetic AMP mimics offer exciting opportunities for new therapeutic initiatives in root canal treatment and regenerative endodontics.

2

1.

Introduction Antimicrobial peptides (AMPs), a wide-ranging class of host-defense molecules, have

attracted much attention in clinical medicine due to their potent antimicrobial activities against a broad spectrum of microorganisms and their low bacterial resistance [1-4]. Natural AMP molecules are present in the oral cavity and possess antimicrobial activities against oral pathogenic bacteria and biofilms [5]. These small cationic peptides also play important roles in the development of innate immunity and possess immunomodulatory functions [6-11]. Because intracanal pathogens may evade chemomechanical debridement [12], elimination of persistent pathogens and effective control of chronic immuno-inflammatory responses represent important targets for development of novel therapeutic initiatives in root canal therapy, potentially through the use AMP-mimicking peptides [13]. Hence, the objectives of the present review are to clarify the function of oral AMPs and their peptidomimetics, and to highlight their potential applications in combating caries and pulpal infections. 2.

Classification and overall functions Since the discovery of lysozyme, the first natural antibiotic isolated from the human body a

century ago, a plethora of molecules with antimicrobial activities have been identified from animals, insects, plants and bacteria that have revolutionized clinical medicine [14]. According to the most recent antimicrobial peptide databases [15-17], more than 4,000 AMPs have been discovered to date. Natural AMPs are highly heterogeneous in length, sequence and structure, but the majority are small, cationic and amphipathic. Three important classes of AMPs are present in humans: defensins, cathelicidins and histatins [18]. Alpha-defensins and β-defensins are cationic, nonglycosylated peptides containing six cysteine residues that form three intramolecular disulfide bridges, resulting in a triple-stranded β-sheet structure. Histatins are small, cationic, histidine-rich peptides present in the saliva. They adopt a random coil conformation in aqueous solvents and form α-helices in non-aqueous solvents. Only one cathelicidin, LL-37, is identified in humans. This peptide is cleaved from the C-terminal end of the human CAP18 protein. Similar to histatins, the LL37 molecule adopts a random coil conformation in a hydrophilic environment, and forms an αhelical structure in a hydrophobic environment. Human AMPs exhibit broad spectrum activities against Gram-positive and Gram-negative bacteria, yeasts, fungi and enveloped viruses. The innate immune system augments the physical and chemical barriers of the human body (e.g. skin and mucous membranes) by producing AMPs [19]. These natural peptides have pleiotropic functions; they not only kill microbes but also control host

3

physiologic functions such as inflammation, angiogenesis and wound healing. Their activities include chemotactic functions, cytokine production, histamine release, lipopolysaccharide-binding and other immunomodulatory activities that, in concert, result in activation of the adaptive immune response [20]. Recent advances in the understanding of AMPs have been associated with their abnormal production in dermatological disorders such as psoriasis, atopic dermatitis and rosacea [20]. Expression of AMPs is also associated with viral infectious diseases such as mollusca contagiosm, condyloma acuminatum and verruca vulgaris [21,22], as well as autoimmune diseases such as cutaneous lupus erythematosus [23]. These examples illustrate how AMP induction or suppression may be adopted for management of autoimmune diseases and inflammation [24]. Commercialized AMP products are available with the potential to treat osteoporosis, diabetes, HIV, prostate, breast and bone cancer, heart failure, multiple sclerosis, neuroendocrine tumors, hereditary angioedema, pain, and idiopathic thrombocytopenic purpura [25]. 2.1

Selective cytotoxicity of AMPs to microbes Antimicrobial peptides may be described as natural microbicides that are selectively

cytotoxic to bacteria, while exhibiting minimal cytotoxicity toward mammalian cells of the host organism. The selective cytotoxicity of AMPs toward microbes is due to the fundamental differences in composition and structure of the host cells, compared to those of pathogenic bacteria and yeasts, as well as the differential expression and localization of AMPs that prevent unwanted interactions with vulnerable host cells [26].These peptides act by their relatively strong electrostatic attraction to the negatively-charged bacterial cells and relatively weak interaction to the eukaryote host cells; the latter are usually less negatively-charged than prokaryotes [27]. Regardless of their origin, AMPs share many common properties such as having net positive charges, being amphipathic and, in most cases, are membrane active [28]. The cell membranes of most pathogenic bacteria comprise mostly hydroxylated phospholipids such as phosphatidylglycerol, cardiolipin, and phosphatidylserine, which render them very electronegative. By contrast, mammalian cell membranes are rich in phosphatidylethanolamine, phosphatidylcholine or its analog, sphingomyelin. This makes the mammalian cell membranes neutral in terms of net charges [29, 30]. In addition, cholesterol and other sterols such as ergosterol are abundantly found in eukaryotic cell membranes, but are seldom identified in prokaryotic membranes/ These molecules are generally neutrally-charged [29] and increase the rigidity of membranes, which can reduce the insertion of AMPs. Differences in membrane symmetry, saturation of phospholipid bilayers, and compositional stoichiometry will influence the membrane’s fluidity 4

and phase transition [31]. This significant difference in transmembrane electrochemical potential may be another factor that enables AMPs to distinguish between host and target cells [27]. Previous studies suggest that the dynamic and/or inherent conformations of AMPs contribute to their selective cytotoxicity [27,32,33]. Moreover, AMPs may undergo conformational transition, self-association or oligomerization within the target pathogen membrane, but not within the host cell membrane to increase cell-specific toxicity [31]. 3.

Mechanisms of action The most-widely accepted mechanism of AMPs for killing microorganisms is

permeabilization followed by membrane disruption (Figure 1A), which has been classically conceptualized using the barrel-stave the carpet and the toroidal pore models that involve rearrangement of the phospholipid component of the microbial cell membrane [9,34]. In the barrelstave model, the AMPs aggregate and span the membrane, with their hydrophobic domains aligning with the lipid bilayer. In this way, they form a pore with the hydrophilic portion of the peptides lining the pore. In the carpet model, the AMPs orient parallel to the surface of the lipid layer and form an extensive carpet that act like a detergent to disrupt the membrane structure. In the toroidal model, the hydrophilic portions of the AMPs associated themselves with the lipid head groups, and induce the lipid monolayer to bend continuously until a pore lined by both the inserted peptides and the lipid head groups is formed. Other pore formation models have also been proposed. In the molecular electroporation model, the interaction of the cationic AMP with the bacterial cell membrane promotes an electrical potential difference across the membrane. A pore is purportedly created when the electrostatic potential reaches 0.2 V. In the sinking raft model, aggregation of AMPs on the outer leaflet membrane produces a mass imbalance between the two leaflets of the membrane, creating a curvature gradient that enable the peptides to sink into the membrane to create a transient pore for the leakage of intracellular contents. After membrane relaxation, AMPs reside on both leaflets of the membrane [35]. More recent research emphasized the impact of Gram-positive bacterial cell wall components on AMP activities. Killing occurs only when bacteria cell membranes are completely saturated by AMPs. Whereas interaction of AMPs with peptidoglycan may not result in reduction in antimicrobial activity, interaction of AMPs with anionic lipoteicholic acid may reduce the local AMP concentration that is required for destabilization of the cytoplasmic membrane and pore formation [36]. 5

The rupture of Gram positive bacteria by coated surfaces with AMPs may be mediated by molecular interaction between the peptides such as GL13K in the coating and teichoic acids in the bacteria wall [37]. Teicholic acids have strong affinity for cationic AMPs because of their highly anionic properties [38]. The negatively-charged phosphate groups of teicholic acid trap protons in the cell wall, creating an acidic environment that keeps autolysin activity at a low level. The cationic AMPs can strongly interact with the phosphate groups of teicholic acid, leading to the release of protons. The resulting increase in local pH activates autolysin, which weakens the cell wall by breaking down glycosidic bonds and peptide crosslinks. Apart from the transmembrane pore-forming mechanisms, the killing effects of AMPS may also be attributed to intracellular targeting (Figure 1B), which includes inhibition of microbial functional proteins, DNA and RNA synthesis or by interacting with certain intracellular targets following their uptake through direct penetration and endocystosis [34,39]. For immunomodulation (Figure 1C), membrane disruption may also be utilized by AMPs to displace/alter/inactivate host cell membrane receptors (e.g. inactivation of Toll-like receptor 4 (TLR4)). Two alternative models have also been proposed for activation of host immunomodulatory functions. In the trans-activation model, AMPs stimulate release of membrane-bound growth factors. The AMPs subsequently bind to and activate the receptors of those growth factor (e.g. activation of heparin binding epidermal growth factor (HB-EGF) and the EGF receptor). In the alternative ligand model, AMPS act as direct ligands for a specific receptor. Activation of the specific receptor following AMP binding initiates receptor signaling (e.g. C-C chemokine receptor type 6 (CCR6) and G protein-coupled formyl peptide receptor-like 1 (FPRL-1)) [7]. 4.

Potential applications of native AMPs and synthetic mimics in endodontics

4.1

AMPs in oral health and diseases Despite the high microbial load in the oral cavity, abrasions, cuts and minor surgical

procedures rarely lead to infections [40]. This indicates that highly effective host-defense mechanisms are present. A growing body of evidence supports the view that AMPs contribute to maintaining the balance between health and disease in the complex oral environment [41-43]. For example, β-defensins are expressed in the gingival epithelium and the salivary glands. Neutrophils express β-defensins in the crevicular fluid and LL-37 in the inflamed epithelium, submandibular glands and saliva. These peptides possess synergistic activity with other AMPs and are co-expressed in the saliva [44]. The predominant types of oral AMPs include α-defensins, β-defensins, histatins, adrenomedullin, statherin, C–C motif chemokine 28, neuropeptides and azurocidin [45]. In addition 6

to their role as antimicrobial agents, AMPs also serve as effective biological molecules in immune activation, inflammation and wound healing, and hence are indispensable components of the host defense system (Figure 2) [46]. Dental caries is the most common cause of pulp infections; pulp inflammation begins before direct contact of pathogens with pulpal tissues. During the initial stage of caries, chronic inflammation is initiated in the subodontoblastic region. The mineralized tissue cage that surrounds the dental pulp cannot expand to relief pressure created by edema. Compromises in blood circulation blocks host defense responses and enables infection to spread to the entire pulp, causing pulpal necrosis [47]. Spreading of infection by-products such as lipolysaccharides and lipoteicholic acid to the periapex triggers immune responses that result in bone destruction in apical periodontitis [48-50]. Although there is no untreatable oral bacteria to date that cause life-threatening diseases, antibiotic resistance does exist in infections involving the oral microbiota [51-61]. This issue is a major concern in both general and specialty dental practices. Another issue is that in the majority of chronic oral infections, microorganisms are rarely found as planktonic organisms. Rather, they increased their survival capacity by existing as biofilm communities, as a consequence of complex developmental processes emerging in response to environmental changes. Hence, AMPs have been perceived as potential candidates for treating oral infections associated with biofilm development, including caries, refractory apical periodontitis, localized aggressive periodontitis, implantassociated infection and candidiasis [62-66]. Anti-biofilm strategies mediated by AMPs may involve minimizing initial adhesion of microbes to biotic and/or abiotic surfaces, prevention of biofilm maturation by eradicating early surface colonizers, inhibition of quorum-sensing, neutralization of the local and systemic effects of endotoxins, recruitment of innate and adaptive immune cells and enhancement of the host immune response against biofilms (Figure 3) [39,67]. As defense biomolecules produced in response to infections, the advantages of AMPs include: broad activity spectrum, favorable structural characteristics, relative stability (protease resistance or activity retained in saliva), selectivity towards microbial membranes, low cytotoxicity, low frequency in selection of resistant mutants, ability to synergize with commonly-used drugs allowing their use at lower concentrations, and the ability to bind bacterial endotoxins and to neutralize their biological effects [68-71]. Because the activities of most oral AMPs are inhibited by ions/salt at physiologic levels [7274], AMPs in the oral environment tend to function optimally at surfaces (e.g. epithelium) that do not contain high salt concentrations and interfering substances derived from the blood [75]. The

7

activities of AMPs against various pathogenic microorganisms involved in caries, endodontic infections and apical periodontitis, in planktonic form and in monospecies and multi-species biofilms, as well as the cytocompatibility of those AMPs, are shown in Table I. 4.2

Natural AMPs The antimicrobial activities of most defensins against bacteria, virus and fungi have minimal

inhibitory concentrations (MIC) in the µg/mL range. Human β-defensin-1 (hBD-1) and hBD-2 are both active against Gram-negative bacteria but have limited activities against Gram-positive bacteria, while hBD-3 is highly active against Gram-positive bacteria [75-77]. These defensins all have marked antifungal activity against Candida albicans [76,78]. Among the defensins, hBD-1,3,5 are antibacterial against microbes involved in root canal infections, including Enterococcus faecalis, Fusobacterium. nucleatum, Tannerella forsythia, Eikenella corrodens and C. albicans [79-81]. Human β-defensin 3 (HBD3) peptide exhibits more antibacterial activity against mature multispecies biofilms containing A. naeslundii, L. salivarius, and E. faecalis, than either calcium hydroxide or 2% chlorhexidine solution [81]. Because defensins may act synergistically with other AMPs and antibiotics, their therapeutic potential is augmented in light of the rapidly increasing emergence of bacterial resistance against classic antibiotics. Histatins exhibit high affinity for hydroxyapatite and contribute to the acquired enamel pellicle. Statherin and histatin-1 competitively inhibit adsorption of salivary high-molecular weight glycoprotein fraction (HMWGP), and reduce adhesion of S. mutans onto hydroxyapatite surfaces [82]. Adsorption of histatin-5 on polymethyl methacrylate and hydroxyapatite surfaces effectively inhibits C. albicans colonization [83]. Derivatives of histatins have been evaluated for their therapeutic potential. P-113 (Pacgen Life Science Corp., Vancouver, BC, Canada) is a C-terminusamidated histatin-5 derivative that exhibits improved activity against a number of potential pathogens, including S. mutans and Streptococcus sobrinus [84,85]. The cathelidicin AMP, LL-37, is effective against Treponema denticola, E. faecalis, Porphyromonas gingivalis, T. forsythia and E. corrodens [74,86,87]. Apart from its role in modulating the innate immune response, LL-37 inhibits biofilm formation in an in vitro biofilm model at concentration far below the MIC required to kill or inhibit bacterial growth [88]. Apart

from

classical

AMPs,

neuropeptides,

which

are

normally classified

as

neurotransmitters, are pletotrophic molecules that also possess direct and indirect antimicrobial and anti-inflammatory activities [89]. Neuropeptides are present in human dental pulps in healthy and carious teeth. They exhibit antimicrobial activities against S. mutans, Lactobacillus acidophilus, E. 8

faecalis, P. aeruginosa, Staphylociccus aureus and C. albicans [90-93]. In addition, lactoferrin, a multifunctional glycoprotein of the transferrin family that is widely represented in secretory fluids such as milk, saliva, tears and nasal secretions, suppresses initial attachment of S. gordonii and S. gordonii coaggregates by iron sequestration. The attachment of a dual-species biofilm containing S. gordonii (i.e. S. gordonii/F. nucleatum or S. gordonii/P. gingivalis) is significantly reduced (48.7% or 62.1%, respectively), which may lead to subsequent inhibition of oral biofilm development [94]. 4.3

AMP mimics Antimicrobial peptides represent promising therapeutic agents against bacterial, fungal and

viral pathogens, but they do have some disadvantages when considered for clinical use. Natural AMPs usually have high cytotoxicity, poor tissue distribution, susceptibility to proteolysis and hydrolysis and the development of allergies to the peptides. The high cost involved in the synthesis of AMPs is another factor hampering their use as drug candidates. Therefore, contemporary research has focused on the development of modified synthetic peptides with unique properties [95,96]. The use of AMPs as potential pharmaceuticals necessitates the development of modified synthetic peptides, including AMP mimetics, hybrid AMPs, AMP congeners, cyclotides, stabilized AMPs, AMP conjugates and immobilized AMPs [97-104]. As a result, a new class of AMPs known as peptidomimetics has surfaced out of these contemporary research endeavors; these peptide mimics can imitate the bactericidal mechanism of natural AMPs, while being stable to enzymatic degradation and displaying potent activity against multidrug-resistant bacteria [105,106]. To resolve issues of hemolytic activity and cytotoxicity of some antimicrobial peptidomimetics, the use of more cationic charges, for example, may result in decreases in cytotoxicity to host cells. Fine-tuning the biological activity of peptidomimetics may possibly be achieved with the introduction of a variety of additional hydrophobic building blocks [107]. Antimicrobial peptide mimics have been developed for controlling caries progression and pulpal infections. The synthetic AMPs, VSL2 and VS2, are highly active against E. faecalis and C. albicans in infections of the root canal space. These biocompatible peptides significantly reduce dentin microbial load to a depth of 400 µm [108]. Another AMP mimic, KSL, has potent antimicrobial activity against oral bacteria and fungi, (MIC: 0.0625 mg/mL). It also inhibits one-dayold S. mutans biofilms with a minimum inhibition concentration of 0.25–0.5 mg/mL [109]. Metaphenylene ethynylene, a mimetic of the AMP magainin, exhibits anti-biofilm activity at nanomolar concentrations against bacterial and Candida species found in oral infections [110]. The peptide Lys-

9

a1, a synthetic derivative of the Hy-A1 peptide isolated from amphibian, demonstrates remarkable antimicrobial activities against planktonic and biofilm growth of many oral streptococci strains even at low concentrations [111]. Lipopeptides C16-KGGK are highly effective against E. faecalis. When C16-KGGK is formulated with one of the two polymers poly(lactic acid-co-castor oil) or ricinoleic acid-based poly(ester-anhydride), these peptide-synthetic polymer conjugates exhibit stronger and improved anti-E. faecalis biofilm activity [112]. There is increasing effort to design peptidomimetics using natural host-defense peptides as templates.

These

novel

innate

defense

regulatory peptides

(IDRs)

possess

enhanced

immunomodulatory functions. Examples include Bac2A (a derivative of the cathelicidin “bactenecin” in bovine neutrophils) [113,114], IDR-1 [115], IDR-1002 [116], IDR-1018 [117,118] and others [118,120]. Bac8c, a 12-amino acid peptide modified from Bac2a, can be synthesized on cellulose rapidly and inexpensively, reducing production cost [121]. It reduces the viability of S. mutans biofilms by gene suppression [122]. As an immunomodulatory peptide that has the ability to modulate human neutrophil functions [123-125], IDR-1018 significantly prevents oral multi-species plaque biofilm formation at 10 µg/mL by inducing cell lysis, without adversely being affected by saliva (Figure 4A). Combined treatment using IDR-1018 and chlorhexidine augments anti-biofilm activity, resulting in dispersion of >35% and killing of >50% of the biofilms in 3 minutes [126]. According to the ecological plaque hypothesis [127], the composition of plaque microflora is critical to the colonization of pathogenic species. Hence, treatment has been targeted at controlling problematic bacteria rather than eliminating plaque biofilm in its entirety [128]. To achieve selective elimination of a particular bacterial species from a multi-species microbial community, a novel technology known as Specifically Targeted Antimicrobial Peptides (STAMPs) has been developed [129,130]. “STAMP” is a fusion peptide with 2 main domains: a killing domain comprising a nonspecific antimicrobial peptide and a targeting domain containing a species-specific targeting peptide. The targeting domain provides specific binding to a selected pathogen and facilitates targeted delivery of the antimicrobial peptide component [131]. A precision-guided STAMP, C16G2, is capable of selectively killing cariogenic S. mutans in vitro with high efficacy in human salivaderived multispecies biofilms, whereas other natural competitors, including non-cariogenic oral streptococci, survive and become dominant within the biofilms [132-134]. Although this exciting technology may be useful in “chasitizing” plaque biofilms, it is challenging to use STAMPs for dealing with root canal infections because of the diversity of the microbiota involved [135-137], and that the difference in microbial flora between primary infections and persistent diseases [138-140]. Because only a subset of AMPs are active against biofilms, which is more relevant to prevent and 10

combat caries and root canal infections, the important database (www.BaAmps.it) provides useful resources for the study of AMPs against biofilms. 4.4

Nisin for inhibition of pathogens related to root canal infections Nisin, a natural bacteria-derived AMP (bacteriocin) isolated from Lactococcus lactis subsp.

Lactis, is highly active against Gram-positive bacteria. The antibacterial mechanism of nisin is perceived to be transmembrane pore-formation, enabling efflux of adenosine triphosphate and amino acids, or collapse of vital ionic gradients, leading to cell death (Figure 4B). Although nisin has been used to inhibit pathogens in food manufacturing [141,142] and medical infections such as staphylococcal mastitis, respiratory tract infections and atopic dermatitis [143-145], its dental application has been not fully exploited. Nisin has potent activity against caries-associated biofilms in vitro [146], remains stable at low pH, is not influenced by enzymes, proteins and other inorganic components in the saliva, and does not adversely cause apoptotic changes in human oral cells at anti-biofilm concentrations [147]. A summary of the antimicrobial activities of nisin, when employed without adjuvants, against pathogenic microorganisms involved in caries and pulpal infections is depicted in Table II. Nisin and NaF function synergistically in eradicating S. mutans biofilms; whereas the antibacterial action of fluoride occurs within the cytoplasm, nisin produces transmembrane pores that enables more fluoride ions to enter the cytoplasm [148]. When combined with D-cysteine, Daspartic acid and D-glutamic acid, nisin significantly improves killing of S. mutans biofilms, due to the ability of those amino acids in dissembling biofilms [149]. When employed as an experimental intracanal dressing, nisin inhibits pathogens in canal wall dentin, reducing S. gordonii by 49% and E. faecalis by 48% [150]. Antibacterial and anti-biofilm activities are augmented when nisin is used to replace doxycycline in a commercially available, antibiotic-containing root canal irrigant (Figure 4C) [151-154]. Although nisin is predominantly active against Gram-positive bacteria, chelating agents such as ethylenediaminetetraacetic acid (EDTA) can destabilize the outer membrane of Gram-negative bacteria [155]. This irrigation strategy potentially enhances the action of nisin on Gram-negative bacteria in root canal infections. Because nisin significantly improves the anti-biofilm activities of antibiotics such as penicillin, vancomycin or chloramphenicol that have dissimilar antibacterial mechanisms, the combined use of nisin and antibiotics is potentially useful against drug-resistant pathogens [156]. Values for evaluation of antimicrobial effectiveness are presented in Tables I and II, including IC50 (half maximal inhibitory concentration), LD50 (lethal dose that kills 50% of a sample), MBC 11

(minimal bactericidal concentration), MBIC50 (minimum biofilm inhibitory concentration), MBRC50 (minimum biofilm reduction concentration) and MIC (minimal inhibitory concentration) and MTD (maximum tolerated dose). These values should provide useful references for future clinical applications of AMPs or their peptidomimetics. However, there are limits in these contemporary research work. Although MIC testing is often considered the gold standard for antibiotic screening, in vivo antimicrobial activity may be lower than what is reported for in vitro assays. Although MBIC and MBRC performed in saliva are more useful indicators of the potential in vivo efficacy, only a limited number of peptides have been evaluated in this manner. Admittedly, much more work needs to be done to address these issues in future studies. 5.

Immunomodulatory and bioactive functions of AMPs in the oral cavity Apart from their direct antimicrobial activities, defensins stimulate chemotaxis of phagocytic

and mast cells, induce inflammatory mediators and regulate the functions of phagocytes and the complement system [46,157]. The oral commensal microbial community contributes to the overall innate immune readiness of the oral cavity components [91]; expression of hBD-2 is increased in response to the exposure of oral epithelial cells to non-pathogenic bacteria [158]. Beta-defensins play important roles in innate host defense against bacterial invasion by promoting adaptive immune responses in the human dental pulp [159,160]. In this cytokine-chemokine signal-receptor feedback mechanism, bacterial components from caries activate cytokine and chemokine release from odontoblasts, dendritic cells or macrophages via Toll-like receptors. Pro-inflammatory cytokines such as IL-1β and TNF-α secreted by these cells act as autocrine and paracrine signals to amplify AMP production by odontoblasts. The released chemokines create a gradient for migration of cells belonging to the adaptive immunity pathway to the subodontoblastic region, while AMP production by odontoblasts at the forefront of infection help reduce the microbial load [161]. Compared with healthy dental pulps, hBD-1 and hBD-4 genes are strongly expressed in inflamed pulps [162]. Expression of hBD-2 is controlled by TLR signaling in human dental pulp cells [163]. Cross-talks between hBD-2 and Toll-like receptors increases the alertness of those cells to the ingress of bacteria, with increased secretion of pro-inflammatory cytokines. Taken together, these findings are indicative of the immunomodulatory role of β-defensins in pulpal host defenses. Further elaboration of the intracellular signaling pathways involved in the immunomodulatory functions of specific AMPs or their synthetic mimics is beyond the scope of the present review. Apart from defensins, LL-37 also possess immunomodulatory properties [164]. This cathelicidin is involved in angiogenesis [165], promotion of wound-healing [166] and inhibition of osteoclastogenesis [167]. Thus, it is theoretically possible to use mimics of LL-37 in managing 12

apical periodontitis as well as internal and external root resorption. LL37 induces migration of human dental pulp stem cells by activation of epidermal growth factor receptor and C-Jun Nterminal kinase [168]. LL37 also activates the MAPK/ERK pathway to increase secretion of vascular endothelial growth factor from pulpal cells [169]. Because of the cell homing and angiogenic properties of LL-37, peptide mimics of LL-37 may find use in regenerative endodontics, as well as in pulp capping materials to promote healing of inflamed pulps and stimulate reparative dentin formation. Apart from having potent antifungal and antibacterial activities, histatins are also involved in buffering and regulation of mineralization [170,171]. In addition, when histatins are adsorbed on enamel surfaces via a phosphorylated serine moiety, the affinity of the peptide to apatite is augmented [172,173]. Presence of these peptides in the acquired pellicle prevents enamel demineralization by acidogenic bacteria [174]. In addition, binding of histatin-1 to apatite confers resistance of the peptide to proteolytic degradation, due to the relatively tight binding of histatin-1 amino acid side-chains to apatite. This prevents proteases from accessing the bound peptides. These findings open new avenues for the development of salivary protease-resistant synthetic peptides for therapeutic use against dental caries [175]. 5.1.

Microbial resistance to AMPs Although the development of resistance to AMPs is slower than that of antibiotics, it is well

established that bacteria adopt a variety of strategies to resist even antimicrobial peptides in both planktonic and sessile life-styles [176,177]. Even in the 1990s and 2000s, it was already known that microorganisms have developed many ways to limit the efficacy of AMPs [178-180]. More recent work [181,182] indicated that such bacterial evasion mechanisms may include: a) alteration bacterial cell surface net charge (e.g. modification of teicholic acids with D-alanine, modification of phospholipids with L-lysine, modification of lipid A with phosphoethanolamine and aminoarabinose); b) cell surface structural changes (e.g. acylation of lipid A, modification of lipopolysaccharides and synthesis of lipoligosaccharides, formation of extracellular cationic slime polymers and capsules for protection against host immune responses); c) entrapment and proteolytic degradation of cationic AMPs; and d) increasing the efflux of cationic AMPs by producing membrane–associated active transporters known as efflux pulps that promote extrusion of AMPs. According to LaRock and Nizet [183], the common mechanisms for the resistance of streptococcal pathogens to cationic AMPs include repulsion, sequestration, export and destruction; each pathogen possesses a different array of resistant mechanisms that may act in synergy to increase its virulence potential. Biofilm resistance to AMPs is multifactorial and may vary with the kind of microorganisms 13

present within the biofilm. Some mechanisms are common with planktonic lifestyle and include efflux pumps, secreted proteases, or alterations of bacterial surface aimed at increasing the net positive cell charge to minimize attraction of the typically cationic AMPs [176]. Until now, very little is known about the resistance of AMPs to bacteria with low metabolic activity and to persister members of biofilms.

Biofilm

resistance

appears

to be

predominantly mediated

by

exopolysaccharides and other extracellular biofilm polymeric molecules that decrease the activity of AMPs. This is likely achieved by preventing the AMPs from reaching the cytoplasmic membrane, their predominant target [177]. 6.

Conclusion and Future Perspectives The advent of novel bioinformatics and designing tools has expedited the identification of

new AMPs and development of AMP mimics [14]. Nevertheless, few synthetic peptides that demonstrated positive bench-top track records and animal study outcomes have proceeded to full Phase III clinical trials. To date, none of these molecules have been approved by the US Food and Drug Administration for therapeutic applications (note: nisin is approved for food processing only). Although AMPs exhibit significant in vitro antimicrobial activities, these activities are often abrogated under physiologic salt and serum conditions. In addition, short- and long-term AMPmediated toxicity profiles have not been established for many compounds. Another impediment to the therapeutic use of AMP is their high cost of development and commercial scale production, particularly for treatment of non-life threatening diseases. More importantly, it is only until recently that scientists discover that bacteria are capable of developing resistance to AMPs via alteration of bacterial surface properties [184,185]. Identification of new therapeutic strategies to combat biofilm-associated infections continues to be one of the major challenges in modern medicine. Despite the presence of commercialization hurdles and scientific challenges, interests in using AMPs as therapeutic alternatives and adjuvants to combat pathogenic biofilms have never been foreshortened. Not only do AMPs possess rapid killing ability, their multi-modal mechanisms of action render them advantageous in targeting different biofilm sub-populations. These cationic peptides also have the potential to intercept multiple stages of biofilm formation, including preventing cell-substrate adhesion, destroying biofilm architecture prior to its stabilization, killing of mature cell members in established consortia and facilitating biofilm detachment [186]. These factors, together with adjunctive bioactive functions such as immunomodulation and wound healing enhancement, render AMPs or their synthetic mimics exciting candidates to be considered as adjuncts in the treatment of caries, infected pulps and root canals. In this context, the most appropriate form of delivery appears to be incorporation of the 14

AMPs or their peptidomimetics into irrigants for lavage of decayed a tooth cavity prior to filling with dental materials, or as root canal irrigants for chemical debridement of the infected root canal space. This is because the cationic peptides have to be freely available in order for them to be incorporated into the cell membranes of the microbes to effectuate killing by either pore formation or intracellular targeting, and for attachment to the surface receptor of host defense cells to modulate the immune responses. More work should be directed toward the quest for combination strategies, such as the use of adjuvants (e.g. EDTA), to improve the efficacy of AMP mimics for cavity lavage and root canal debridement. In addition, cationic AMPs have been covalently immobilized on solid substrates, such as titanium dental implants to prevent peri-implant infections have also been shown to be effective against oral pathogens [65]. This highlights the possibility that immobilization of the AMPs or peptidomimetics in a resin composite filling or polymerizable root canal sealer would achieve the purpose of using cationic peptides in combating caries and pulpal infections.

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Amerongen, Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane, Biochem. J. 388 (2005) 689-695. C.R. Pusateri, E.A. Monaco, M. Edgerton, Sensitivity of Candida albicans biofilm cells grown on denture acrylic to antifungal proteins and chlorhexidine, Arch. Oral Biol. 54 (2009) 588-594. S.B. Winfred, G. Meiyazagan, J.J. Panda, V. Nagendrababu, K. Deivanayagam, V.S. Chauhan, G. Venkatraman, Antimicrobial activity of cationic peptides in endodontic procedures, Eur. J. Dent. 8 (2014) 254-260. S Pathak, VS Chauhan, Rationale-based, de novo design of dehydrophenylalanine-containing antibiotic peptides and systematic modifi cation in sequence for enhanced potency, Antimicrob. Agents Chemother. 55 (2011) 2178-2188. D.T. McLean, M.T. McCrudden, G.J. Linden, C.R. Irwin, J.M. Conlon, F.T. Lundy, Antimicrobial and immunomodulatory properties of PGLa-AM1, CPF-AM1, and magainin-AM1: potent activity against oral pathogens, Regul. Pept. 194-195 (2014) 63-68. A. Semlali, K.P. Leung, S. Curt, M. Rouabhia, Antimicrobial decapeptide KSL-W attenuates Candida albicans virulence by modulating its effects on Toll-like receptor, human β-defensin, and cytokine expression by engineered human oral mucosa, Peptides. 32 (2011) 859-867. S. Theberge, A. Semlali, A. Alamri, K.P. Leung, M. Rouabhia, C. albicans growth, transition, biofilm formation, and gene expression modulation by antimicrobial decapeptide KSL-W, BMC Microbiol. 13 (2013) 246. D. Shang, H. Liang, S. Wei, X. Yan, Q. Yang, Y. Sun, Effects of antimicrobial peptide L-K6, a temporin1CEb analog on oral pathogen growth, Streptococcus mutans biofilm formation, and anti-inflammatory activity, Appl. Microbiol. Biotechnol. 98 (2014) 8685-8695. L.L. Burrows, M. Stark, C. Chan, E. Glukhov, S. Sinnadurai, C.M. Deber, Activity of novel nonamphipathic cationic antimicrobial peptides against Candida species, J. Antimicrob. Chemother. 57 (2006) 899-907. V. Liu, S. Dashper, P. Parashos, S.W. Liu, D. Stanton, P. Shen, P. Chivatxaranukul, E.C. Reynolds, Antibacterial efficacy of casein-derived peptides against Enterococcus faecalis, Aust. Dent. J. 57 (2012) 339-343. R. Tao, Z. Tong, Y. Lin, Y. Xue, W. Wang, R. Kuang, P. Wang, Y. Tian, L. Ni, Antimicrobial and antibiofilm activity of pleurocidin against cariogenic microorganisms, Peptides 32 (2011) 1748-1754. L.H. Eckhard, A. Sol, E. Abtew, Y. Shai, A.J. Domb, G. Bachrach, N. Beyth, Biohybrid polymerantimicrobial peptide medium against Enterococcus faecalis, PLoS One 9 (2014) e109413. C. Padilla, O. Lobos, P. Brevis, P. Abaca, E. Hubert, In vitro antibacterial activity of the peptide PsVP10 against antimicrobial-resistant Enterococcus faecalis isolated from clinical samples, J. Antimicrob. Chemother. 53 (2004) 390-392. J.R. Garcia, F. Jaumann, S. Schulz, A. Krause, J. Rodríguez-Jiménez, U. Forssmann, K. Adermann, E. Klüver, C. Vogelmeier, D. Becker, R. Hedrich, W.G. Forssmann, R. Bals, Identification of a novel, multifunctional beta-defensin (human beta-defensin 3) with specific antimicrobial activity. Its interaction with plasma membranes of Xenopus oocytes and the induction of macrophage chemoattraction, Cell Tissue Res. 306 (2001) 257-264. H.Y. Wang, J.W. Cheng, H.Y. Yu, L. Lin, Y.H. Chih, Y.P. Pan, Efficacy of a novel antimicrobial peptide against periodontal pathogens in both planktonic and polymicrobial biofilm states, Acta Biomater. 25 (2015) 150-161. J. Groenink, E. Walgreen-Weterings, W. van 't Hof, E.C. Veerman, A.V. Nieuw Amerongen, Cationic amphipathic peptides, derived from bovine and human lactoferrins, with antimicrobial activity against oral pathogens, FEMS Microbiol. Lett. 179 (1999) 217-222. U.K. Gursoy, E. Kononen, N. Luukkonen, V.J. Uitto, Human neutrophil defensins and their effect on epithelial cells, J. Periodontol. 84 (2013) 126-133. P.A. Raj, K.J. Antonyraj, T. Karunakaran, Large-scale synthesis and functional elements for the antimicrobial activity of defensins, Biochem. J. 347 (2000) 633-641. S. Thennarasu, A. Tan, R. Penumatchu, C.E. Shelburne, D.L. Heyl, A. Ramamoorthy, Antimicrobial and membrane disrupting activities of a peptide derived from the human cathelicidin antimicrobial peptide LL37, Biophys. J. 98 (2010) 248-257. T. Suwandecha, T. Srichana, N. Balekar, T. Nakpheng, K. Pangsomboon, Novel antimicrobial peptide specifically active against Porphyromonas gingivalis, Arch. Microbiol. 197 (2015) 899-909.

24

217. C.G. Kelly, T. Lehner, Peptide inhibitor of Streptococcus mutans in the control of dental caries, Int. J. Pept. Res. Ther. 13 (2007) 517-523. 218. M. Drobni, T. Li, C. Krüger, V. Loimaranta, M. Kilian, L. Hammarström, H. Jörnvall, T. Bergman, N. Strömberg, Host-derived pentapeptide affecting adhesion, proliferation, and local pH in biofilm communities composed of Streptococcus and Actinomyces species, Infect. Immun. 74 (2006) 62936299. 219. S.Y. Arslan, K.P. Leung, C.D. Wu, The effect of lactoferrin on oral bacterial attachment, Oral Microbiol. Immunol. 24 (2009) 411-416. 220. A. Dobson, P.M. O'Connor, P.D. Cotter, R.P. Ross, C. Hill, Impact of the broad-spectrum antimicrobial peptide, lacticin 3147, on Streptococcus mutans growing in a biofilm and in human saliva, J. Appl. Microbiol. 111 (2011) 1515-1523. 221. Y. Porat, K. Marynka, A. Tam, D. Steinberg, A. Mor, Acyl-substituted dermaseptin S4 derivatives with improved bactericidal properties, including on oral microflora, Antimicrob. Agents Chemother. 50 (2006) 4153-4160. 222. B.R. da Silva, V.A. de Freitas, V.A. Carneiro, F.V. Arruda, E.N. Lorenzón, A.S. de Aguiar, E.M. Cilli, B.S. Cavada, E.H. Teixeira, Antimicrobial activity of the synthetic peptide Lys-a1 against oral streptococci, Peptides 42 (2013) 78-83. 223. L. Li, J. He, R. Eckert, D. Yarbrough, R. Lux, M. Anderson, W. Shi, Design and characterization of an acid-activated antimicrobial peptide, Chem. Biol. Drug Des. 75 (2010) 127-132. 224. K.P. Leung, T.D. Crowe, J.J. Abercrombie, C.M. Molina, C.J. Bradshaw, C.L. Jensen, Q. Luo, G.A. Thompson, Control of oral biofilm formation by an antimicrobial decapeptide, J. Dent. Res. 84 (2005) 1172-1177. 225. S.G. Dashper, S.W. Liu, E.C. Reynolds, Antimicrobial peptides and their potential as oral therapeutic agents, Int. J. Pep.t Res. Ther. 13 (2007) 505-516. 226. S. Taneja, P. Kumar, K. Malhotra, J. Dhillon, Antimicrobial effect of an oxazolidinone, lantibiotic and calcium hydroxide against Enterococcus faecalis biofilm: An in vitro study, Indian J. Dent. 6 (2015) 190-194. 227. G. Somanath, P.S. Samant, V. Gautam, O.J. Singh Birring, To comparatively evaluate the antimicrobial efficacy of chlorhexidine, nisin and linezolid as an intracanal medicament on Enterococcus faecalis: An in vitro study, Indian J. Dent. Res. 26 (2015) 613-618.

Figure Legends Figure 1. Mechanims of action of antimicrobial peptides (AMPs). A. Proposed models accounting for their antimicrobial activity based on membrane permeabilization and disruption [adapted from [34] with permission from Elsevier Ltd. Copyright 2012]. B. Proposed antimicrobial mechanisms of action based on intracelllular targeting [modified from [39] with permission from John Wiley & Sons Ltd. Copyright 2014]. C. Proposed models for activation of host cells in immunodulation (adapted from [7] with permission from Elsevier Ltd. Copyright 2009). Figure 2. Potential immunomodulatory and bioactive functions of AMPs or their mimics in reponse to caries and pulpal infections. Oral pathogens that degrade dentin during the early stage of dental caries release pathogen-associated molecular patterns (yellow star), diffuse through the dentinal tubules and are recognized by Toll-like receptors (red box) on the surface of the odontoblast processes. AMPs are released by odontoblasts and host phagocytes in the dental pulp. These cationic peptides induce a variety of responses in host innate immune cells such as polymorphonuclear 25

leukocytes (PMNs), monocytes, macrophages and mast cells The AMPs alter gene expression of host cells, induce chemokine and cytokine production, promote PMN and immature dendritic cell recruitment to the site of infection, influence cell differentiation and activation and block or activate TLR signaling. Immunomodulation by AMPs results in activation of innate immune responses to protection against infection, selective control of inflammation, promotion of wound healing and initiation of adaptive immune responses. LPS: lipopolysaccharide. Figure 3. AMP-mediated anti-biofilm strategies. Top panel: strategies for inhibiting biofilm formation. AMPs prevent initial adhesion of planktonic cells to a substrate by coating of the substrate surface (Left) or the bacterial surface (Middle). Prevention of biofilm maturation by killing early surface colonizers (Right). Bottom panel: strategies for eradication of biofilms. Binding of AMPs to quorum-sending molecules to prevent bacteria communication (Left). Penetration of the matrix of established biofilms to kill biofilm-associated cells (Middle). Binding and neutralizing endotocins released by members of the biofilms (Right). Figure 4. A. 3-D projections of color-adjusted confocal laser scanning microscopy images of human plaque biofilms treated with 1018 peptide. Green in merged channels: live microorganisms; red in merged channels: dead microorganisms. Bar = 100 µm. Left: 72-hour plaque biofilm treated twice with 10 µg/mL peptide (48-h treatment). Percentage of dead bacteria: 45%; Right: 72-hour plaque biofilms treated by 10 µg/mL peptide for 3 times (72-h treatment). Percentage of dead bacteria: 66% (Courtesy of Dr. Ya Shen, University of British Columbia, Vancouver, Canada; images are similar to those presented in [126]). B. Scanning electron microscopy of E. faecalis (ATCC 29212) planktonic cells (Left) after treatment for 24 hours with phosphate-buffered saline (control) or (Right) a modified root canal irrigant prepared by replacing the 3% doxycycline in the original irrigant with 3% nisin. Arrow: pore formation in bacteria in the nisin-treated group. Pores were absent in cells exposed the doxycycline-containing irrigant without nisin substitution (not shown). Bar = 100 nm. [Modified from [154] with permission from Elsevier Inc. Copyright 2011]. C. Transmission electron microscopy of the morphologic changes in E. faecalis (ATCC 29212) planktonic cells after exposure for 12 hours to phosphate-buffered saline (Left) or 2000 U/mL nisin (Right). Bar = 500 nm. Cell lysis can be seen in the high magnification image taken from the nisin group (inset). [Modified from [156] with permission from Public Library of Science. Copyright 2014].

26

Table I. Activities and cytocompatibility of AMPs against various pathogenic microorganisms involved in caries, endodontic infections and apical periodontitis

Microorganism

A. actinomycetemcomitans

AMPs

HNP-1 HNP-2 HNP-3 hBD-2 hBD-3

LL-37

mPE MUC7 20-mer MUC7 12-mer-L MUC7 12-mer-D Magainin II dhvar4a K4-S4(1-15)a KSL

A. israelii A. viscosus

hBD-2 hBD-3 mPE Bactenecin Bac2A Bac8C

A. naeslundii

Antibacterial

Anti-biofilm

No activity (＞500 µg/mL) [187] No activity (＞500 µg/mL) [187] No activity (＞500 µg/mL) [187] MIC＞250 µg/mL [76] MIC 200 µg/mL [188] MIC 9.6 - >250 µg/mL [188] MIC 45.6 µg/mL [79] MIC 37.8 µg/mL [79] MIC >100 µg/mL [189] MIC 100 µg/mL [188] MIC 0.4mµg/mL [110] MBC 1.5mµg/mL [110] MIC 125.6 – 251.3 µg/mL [191] MIC 79 – >158 µg/mL [191] MIC 39.5 – 158 µg/mL [191] MIC 3.9–98.8 mg/L [191] MIC 200 µg/mL [192] MIC 25-50 µg/mL [192] MIC 0.5 µg/mL [109] MBC 2 µg/mL [109]

MIC 9.1-10.0 µg/mL [76] MIC 9.0-10.8 µg/mL [76] MIC 0.8 µg/mL [110] MBC 1.5 µg/mL [110] MIC >256 µg/mL [122] MIC 16–32 µg/mL [122] MBC 64–25 6µg/mL [122] MIC 8 µg/mL [122] MBC 16–32 µg/mL [122]

Related niche

Ori gin

Cytotoxicity

C/E/A

N N N N N N N N

Not assessed

S

MTD 10 mg/kg in mouse models [190] Not assessed

Not assessed

50 µg/mL [189]

N N N N N N S

C/E/A

N N S N S S

dhvar4a K4-S4(1-15)a Chrysophsin 1

MIC 50 µg/mL [192] MIC µg/mL [192] MIC 8 µg/mL [193] MBC 16 µg/mL [193]

N N N

KSL

MIC 125 µg/mL [109] MBC 500 µg/mL [109]

S

Bactenecin Bac2A

MIC >256 µg/mL [122] MIC 16–32 µg/mL [122] MBC 32–64 µg/mL [122] MIC 8–16 µg/mL [122] MBC16–32 µg/mL [122]

Bac8C

Not assessed Not assessed

C/E/A

N S S

Not assessed Not assessed Not assessed <1 mg/mL No cytotoxicity to HGFs [193] No assessed No assessed MTD 10 mg/kg in mouse models [190] Not assessed Not assessed 32–128 µg/mL for 5 min. No cytotoxicity to HGFs [122] Not assessed Not assessed 8–32 µg/mL for 5 min. No cytotoxicity to HGFs [165] <1 mg/mL. No cytotoxicity to HGFs [194] Not assessed Not assessed

Chrysophsin 1

MIC 8 µg/mL [193] MBC16 µg/mL [193]

N

LL-37

MIC 31.3 µg/mL [79]

N

32–128 µg/mL for 5 min. No cytotoxicity to HGFs [122] 8–32 µg/mL for 5 min. No cytotoxicity to HGFs [193] Not assessed

hBD-2

MIC 8.2-14.0 µg/mL [76]

N

Not assessed

hBD-3

MIC 15.7 µg/mL [79]

N

Not assessed

27

C. striatum C. albicans

C16G2 KSL

MIC 4.1-7.2 µg/mL [76] IC 50 344.7 µM [133] MIC 125 µg/mL [109] MBC 250 µg/mL [109]

C16G2 HNP-1 HNP-4 HBD-2

IC 50 3.4 µM [133] MIC 52.5 µg/mL [78] MIC 0.5 µg/mL [195] MIC 4.6-59.2 µg/mL [93]

HBD-3

MIC >50 µg/mL [77]

Histatin5

MIC 2.8-7.1 µg/mL [93] MIC 25 µg/mL [196] MBC 50 µg/mL [196] LD50 1.6 µM [197]

N S

E/A E/A

25 µg/mL [196] Kill biofilm 30-60 µM [198]

E. faecalis

N

Not assessed

N

Not assessed Not assessed No toxicity to HeLa cells at MICs [200] <50 µM No toxicity to oral fibroblast [201] <1 mg/mL. No cytotoxicity to HGFs [194]

LL-37 VSL2 VS2 PGLa-AM1 CPF-AM1 Magainin-AM1 KSL

LD50 0.8µM [197] MIC 10 µM [199] MIC 10 µM [199] MIC 50 µM [201] MIC 50 µM [201] MIC 100 µM [201] MIC 250 µg/mL [109] MBC 1000 µg/mL [109]

N S S S S S S

Substance P Neuropeptide Y CGRP VIP

MIC 8.1 µg/mL [90] MIC 243.2 µg/mL [90] MIC 63.1 µg/mL[90] MIC 46.5 µg/mL [90]

S S S S

KSL-W

12.5~100 µg/mL [202]

L-K6

MIC 6.25 µM [204] MBC 25 µM [204]

Inhibit biofilm >25 µg/mL [203]

S

S

Lys

E. corrodens

N N N N

Not assessed <1 mg/mL. No cytotoxicity to HGFs [194] Not assessed Not assessed Not assessed Not assessed

Kill biofilm 320-640 µg/mL [205]

S

LL-37 HBD-3 HNP-1 HBD-1 HBD-2 HBD-3

MIC 9.9 µg/mL [79] MIC 7.8 µg/mL [79] MIC 32.0µg/ml [78] MIC 5 µM [80] MIC 5 µM [80] MIC 0.63 µM [80] MIC >50 µg/mL [77] MIC 2 µg/mL [81] MBC 15.6 µg/mL [81]

HBD-4 Chrysophsin 1

MIC 2.5 µM [80] MIC 8-16 µg⁄mL [193] MBC 16-32 µg⁄mL [193]

N N

LL-37 LL7-27 KCGP KCP Pleurocidin

MIC 12.5 µg/mL [87] MIC 12.5 µg/mL [87] MBC 0.64 mg⁄mL [206] MBC 0.67 mg⁄mL [206] MIC >256 µg/mL [207] MBC >256 µg/mL [207] MIC 10 µM [199] MIC 10 µM [199] MIC 4-5 µg/mL [208]

N

VSL2 VS2 C16-KGGK

E/A E/A

N N N N N N N N

N N N S S S

28

At MICs no toxicity to human dental pulp cells [90]

12.5~100 µg/mL No inhibitory effect on gingival epithelial cell adhesion and proliferation [202] Not assessed Little toxicity to mammalian cells [205] Not assessed Not assessed Not assessed Not assessed Not assessed Not assessed

Not assessed 8–32 µg/mL for 5 min No cytotoxicity to HGFs [193] Not assessed Not assessed Not assessed Lower hemolysis [207] No toxicity to HeLa cells at MICs [200] Not assessed

antimicrobialresistant E. faecalis F. nucleatum

C16-KKK C16-KAAK C16-KLLK PGLa-AM1

MIC 6–12.5 µg/mL [208] MIC 12.5–25 µg/mL [208] MIC 6–12.5 µg/mL [208] MIC >100 µM [201]

S S S S

CPF-AM1 Magainin-AM1

MIC >100 µM [201] MIC >100 µM [201]

S S

<50 µM No toxicity to oral fibroblast [201]

PsVP-10

MIC 0.03 µg/mL [209]

E/A

N

Not assessed

hBD-2 hBD-3

MIC 6.5->250.0 µg/mL [76] MIC 12.5 µg/mL [188] MIC 25 µg/mL [77] MIC 7.8 µg/mL [79] MIC 4.5-7.8 µg/mL [210] MIC 4.9 µg/mL [79] MIC 12.5 µg/mL [188]

E/A

N N

Not assessed Not assessed

N

Not assessed

Not assessed Not assessed

LL-37

C16G2 dhvar4a K4-S4(1-15)a PGLa-AM1 CPF-AM1 Magainin-AM1 KSL

IC50 362.3µM [133] MIC 50 µg/mL [191] MIC 5 µg/mL [191] MIC 12.5 µM [201] MIC 6.25 µM [201] MIC 12.5 µM [201] MIC 250 µg/mL [109] MBC 1000 µg/mL [109]

S N N S S S S

L-K6

MIC 4.7 µM [204] MBC 18.8 µM [204] MIC 40 µg/mL [211] MBC 160 µg/mL [211]

S

MIC 1250 µg/mL [212] MBC1250 µg/mL [212] MIC 100 µg/mL [192] MIC 10 µg/mL [192] MIC 128 µg/mL [207] MBC 256 µg/mL [207] MIC 4-8 µg/mL [193] MBC 32-128 µg/mL [193]

S

Nal-P-113 LFb 17-30 L. paracasei L. acidophilus

dhvar4a K4-S4(1-15)a Pleurocidin Chrysophsin 1

Bectenecin Bac2A Bac8C

KSL L-K6 Substance P Neuropeptide Y L. casei

hBD-3 LL-37 Pleurocidin Chrysophsin 1

S

C//E/A C/E/A

N N N N

<50 µM. No toxicity to oral fibroblasts [201] <1 mg/mL. No cytotoxicity to HGFs [194] Not assessed <320 µg/mL No toxicity to hPDLCs epi4 [211] Not assessed Not assessed lower hemolysis [207] 8–32 µg/mL for 5 min. No cytotoxicity to HGFs [193] No No

MIC >256 µg/mL [122] MIC 256 µg/mL [122] MBC >256 µg/mL [122] MIC 64-128 µg/mL [122] MBC 128-256 µg/mL [122]

N S

MIC 62.5 mg/mL [109] MBC 125 mg/mL [109] MIC 3.1 µM [204] MBC 12.5 µM [204] MIC 74.1 µg/mL [90] MIC 283.7 µg/mL [90]

S

32–128 µg/mL for 5 min. No cytotoxicity to HGFs [122] Not assessed

S

Not assessed

S

MIC 100 µg/mL [188] MIC 50 µg/mL [188] MIC 32 µg/mL [207] MBC 128 µg/mL [207] MIC 4 µg/mL [193] MBC 16-128 µg/mL [193]

C/E/A

N N N N

29

At MICs. No toxicity to human dental pulp cells [90] Not assessed Not assessed lower hemolysis [207] 8–32 µg/mL for 5 min. No cytotoxicity to HGFs [193]

Bectenecin Bac2A Bac8C

L. fermenti

Pleurocidin Chrysophsin 1

P. intermedia

HNP-1 hBD-3 LL-37

KSL LFb 17-30 P. gingivalis

hBD-1 hBD-2 hBD-3

HNP-1 HNP-2 mPE LL-37

MIC 2 µg/mL [207] MBC 8 µg/mL [207] MIC 4 µg/mL [193] MBC 4 µg/mL [193]

C/E/A

N S

Not assessed Not assessed

S

32–128 µg/mL for 5 min. No cytotoxicity to HGFs [122] lower hemolysis [207] 8–32 µg/mL for 5 min. No cytotoxicity to HGFs [193] Not assessed Not assessed

N N

MIC 5 µg/mL [213] MIC 15.7 µg/mL [79] MIC 100 µg/mL [188] MIC 15.7 µg/mL [79] MIC 100 µg/mL [188] MIC 1 mg/mL [109] MBC >2 mg/mL [109] MIC 18 µg/mL [212] MBC 18 µg/mL [212] MIC 50 µg/mL [188] MIC 34.6->250 µg/mL[76] MIC 42.1 µg/mL [79] MIC 200 µg/mL [188] MIC >250 µg/mL [76] No activity (>200 µM) [214] No activity (>200 µM) [214] MIC 2.5 µg/mL [110] MBC 2.5 µg/mL [110] MIC 12.5 µg/mL [215] MIC >125 µg/mL [79] MIC 100 µg/mL [188]

E/A

E/A

N N N N S

Not assessed

S

Not assessed

N N N

Not assessed Not assessed Not assessed

N N S

Not assessed Not assessed MTD 10 mg/kg in mouse models [190] Not assessed

N

Not assessed

LL7-27 MUC7 20-mer MUC7 12-mer-L MUC7 12-mer-D Pep-7

MIC 12.5 µg/mL [215] MIC 251.3 µg/mL [188] MIC 158 µg/mL [188] MIC 118.5 µg/mL [188] MIC 1.7µM [216]

N N N N S

KSL

MIC 1mg/mL [109] MBC 2mg/mL [109] MIC 20 µg/mL [211] MBC 160 µg/mL [211]

S

MIC 1250 µg/mL [212] MBC 1250 µg/mL [212] MIC 102.6 µg/mL [79] MIC 12.5 µg/mL [79] MIC 6.25 µg/mL [79] MIC 64 µg/mL [122] MBC 128µg/mL [122] MIC 32 µg/mL [122] MBC 128 µg/mL [122] MIC 32 µg/mL [122] MBC64-128 µg/mL [122]

S

<320 µg/mL No toxicity to hPDLCs epi4 [211] Not assessed

N

Not assessed

N S

Not assessed Not assessed

S

Not assessed

S

S

32–128 µg/mL for 5 min. No cytotoxicity to HGFs [122] Not assessed

N N N

Not assessed Not assessed Not assessed

Nal-P-113 LFb 17-30 S. gordonii

MIC >256 µg/mL [122] MIC 128 µg/mL [122] MBC >256 µg/mL [122] MIC 128 µg/mL [122] MBC 256 µg/mL [122]

LL-37 LL7-27 Bactenecin Bac2A Bac8C

C16G2 MUC7 20-mer MUC7 12-mer-L MUC7 12-merL4

S

C/E/A

IC 50 93.9 µM [133] MIC 23 µM [217] MIC 15.7-31.4 µg/mL [133] MIC 39.5 µg/mL [133] MIC 79.0 µg/mL [133]

30

Not assessed Not assessed Not assessed Not assessed <70.8 µM. Safe for HGFs [216] Not assessed

MUC7 12-mer-D Magainin II Pleurocidin P-113 hLF1–11 Chrysophsin 1

KSL Nal-P-113

MIC 39.5 µg/mL [133] MIC 61.7 µg/mL [133] MIC 8 µg/mL [207] MBC 16 µg/mL [207] MIC 40.92 µM [62] MIC 46.56 µM [62] MIC 8 µg/mL [193] MBC 16 µg/mL [193]

N N N

MIC 500 µg/mL [109] MBC 2000 µg/mL [109] MIC 80 µg/mL [211] MBC 320 µg/mL [211]

S

N N N

S

Prolone-rich peptide derivative

S. mutans

Block biofilm pH decrease 0.5-30 mM [218]

N

<320 µg/mL No toxicity to hPDLCs epi4 [211] Not assessed

N N

Not assessed Not assessed Not assessed Not assessed Not assessed Not assessed Not assessed Not assessed

HNP-1 hBD-2

MIC 4.1 µg/mL [78] MIC 4.1 µg/mL [76]

hBD-3

MIC 5.0 µg/mL [76] MIC 25 µg/mL [188] MIC 25 µg/mL [188] Adhesion inhibition 100 µg/mL [219] MIC 27.2 µM [62] MIC 4 µg/mL [188] MBC8 µg/mL [188] MIC 27.3 µM [62] MIC 8 µg/mL [207] 64 µg/mL MBC 16 µg/mL [207] [207] MIC 23 µg/mL [220] MBIC50 3.13 µM [220] MBRC50 50 µM [220] MIC 5 µg/mL [192] 50 µg/mL [192] MIC 25 µg/mL [192] Kill surface attached bacteria 50 µg/mL [192] Kill biofilm 500 µg/mL [192] Kill biofilm 140 µg/mL [221] MIC 23.6 µg/mL [192] MBIC50 15.7 µg/mL MBRC50 62.8 µg/mL [192] MIC 19.7 µg/mL [192] MBIC50 19.7 µg/mL MBRC50 >79. 5 µg/mL [192]

N

MUC7 12-merL4

MIC 39.5 µg/mL [192]

MUC7 12-mer-D

MIC 19.7 µg/mL [192]

LL-37 Lactoferrin hLF1–11 Chrysophsin-1 P-113 Pleurocidin Lacticin 3147

K4-S4(1-15)a Dermaseptin S-4 analogue

Acylated S-4 analogues MUC7 20-mer

MUC7 12-mer-L

C/E/A

MBIC50 79 µg/mL MBRC50 >79.5 µg/mL [192] MBIC50 9.9 µg/mL

31

Not assessed Not assessed lower hemolysis [207] Not assessed Not assessed 8–32 µg/mL for 5 min. No cytotoxicity to HGFs [193] Not assessed

N N N N N N N

Not assessed lower hemolysis [207] Not assessed

N

Not assessed

N

Not assessed

N

Not assessed

N

Not assessed

N

Not assessed

N

Not assessed

N

Not assessed

C16G2

MIC 3-5 µM [217] IC 50 5.9 µM [133]

mPE

MIC 0.5-1 µg/mL [110] MBC 1.25 µg/mL [110]

Bac2A

MIC 16–32 µg/mL [122] MBC 64–128 µg/mL [122] MIC 16 µg/mL [122] MBC 32 µg/mL [122]

Bac8C

S. crista S. mitis

S. pyogenes S. salivarius

MBRC50 39.5 µg/mL [192] Kill biofilm 25 µM [129]

MIC 125 µg/mL [222] MBC 250–500 µg/mL [222]

PGLa-AM1 CPF-AM1 Magainin-AM1 Magainin 2

MIC 3.1 µM [211] MIC 3.1 µM [201] MIC 50 µM [201] MIC 61.7–123.5 mg/L [201]

KSL

MIC 0.0625 mg/mL [109] MBC 0.125 mg/mL [109]

L-K6

MIC 3.13 µM [204] MBC 12.5 µM [204]

LFb 17-30

C16G2 Lys-a1 L-K6 LFb 17-30 HBD-3

No assessed

S

MTD 10 mg/kg in mouse models [190]

S

Not assessed

128 µg/mL [122]

S

63 µg/mL [222] Inhibit biofilm 7.55 µM [111]

S

32–128 µg/mL for 5 min. No cytotoxicity to HGFs [122] Not assessed

Inhibit biofilm 5 nM [110] Kill biofilm 10 nM [110]

Lys-a1

Substance P Neuropeptide Y Vasoactive intestinal peptide M8-33 M8G2 AAP2 C16G2 C16G2 HBD-3 LL-37 C16G2

S

S S N N

MBIC50 123.5 mg/L MBRC50 61.7->123.5 mg/L [201]

MBIC50 0.0625-0.125 mg/mL [109] MBRC50 0.250.5 mg/mL [109] MBIC50 3.13 µM [204] MBRC50 6.25 µM [204]

<50 µM. No toxicity to oral fibroblasts [201]

S

Not assessed

S

Not assessed

MIC 37 µg/mL [212] MBC 37 µg/mL [212] MIC 171.6 µg/mL [90] MIC 210.9 µg/mL [90] MIC 150.7 µg/mL [90]

S

Not assessed

S S S

Kill biofilm 25 µM [129] Kill biofilm 25 µM [129] Kill biofilm 40 µM [223] IC 50 257.2 µM [133] IC 50 223.6 µM [133] MIC 200 µg/mL [133] MIC 50 µg/mL [`133] MIC 28.7µM [133]

S S S S S N N S

At MICs. No toxicity to human dental pulp cells [90] Not assessed Not assessed Not assessed Not assessed Not assessed Not assessed Not assessed Not assessed

S S

Not assessed Not assessed

S

Not assessed

S

Not assessed

N

Not assessed

C/E/A

C/E/A

IC 50 5.9 µM [133] MIC 3.9–7.8 µg/mL [222] MBC 15.6–500 µg/mL [222] MIC 6.25 µM [204] MBC 25 µM [204] MIC 37 µg/ml [212] MBC 37 µg/mL [212] MIC 31.3 µg/mL [79] MIC 100 µg/mL [79]

32

C/E/A 3.9 µg/mL [222]

LL-37

MIC 37.8 µg/mL [79]

N

Not assessed

S

Not assessed

N

Not assessed

N

Not assessed

MIC 25 µg/mL [79] S. sanguinis

C16G2 Pleurocidin P-113

MIC 4-8 µg/mL [193] MBC 8-16 µg/mL [193]

N

Bactenecin

MIC 64 µg/mL [122] MBC128–256 µg/mL [122] MIC 32–64 µg/mL [122] MBC128–256 µg/mL [122] MIC 16 µg/mL [122] MBC 32-128 µg/mL [122]

N

8–32 µg/mL for 5 min. No cytotoxicity to HGFs [193] Not assessed

S

Not assessed

S

S

32–128 µg/mL for 5 min. No cytotoxicity to HGFs [122] Not assessed

S

Not assessed

S

Not assessed

Bac8C

Lys-a1 KSL L-K6

S. parasanguinis

C16G2 Lys-a1

S. oralis

C16G2 Lys-a1 dhvar4a K4-S4(1-15)a Pleurocidin Chrysophsin 1

Bac2A Bac8C

Lys-a1 KSL LFb 17-30

NoT. denticola

hBD-3 LL-37 LL-37 hBD-3 LL-37

Multi-species biofilm

hBD-3 KSL Lactoferrin

T. forsythia

C/E/A

Chrysophsin 1

Bac2A

S. sobrinus

IC 50 59.3 µM [133] MIC 19 µM [133] MIC 32 µg/mL [133] MBC 32 µg/mL [133] MIC 75 µM [133]

MIC 15.6 µg/mL [222] MBC 31.3–500 µg/mL [222] MIC 0.25 µg/mL [109] MBC 2.0 µg/mL [109] MIC 6.25 µM [204] MBC 25 µM [204] MIC 38 µg/mL [133] MIC 3.9 µg/mL [222] MBC 3.9–500 µg/mL [222] IC 50 381 µM [133] MIC 15.6 µg/mL [222] MBC 31.3-500 µg/mL [222]

15.6 µg/mL [222]

1.9 µg/mL [222]

C/E/A

S S

Not assessed Not assessed

15.6 µg /mL [222]

C/E/A

S S

Not assessed Not assessed

N N N

Not assessed Not assessed Lower hemolysis [207] 8–32 µg/mL for 5 min. No cytotoxicity to HGFs [193] No

MIC 30 µg/mL [192] MIC 5 µg/mL [192] MIC 8 µg/mL [207] MBC 16 µg/mL [207] MIC 4 µg/mL [193] MBC8 µg/mL [193]

C/E/A

N

MIC 16 µg/mL [122] MBC 64-128 µg/mL [122] MIC 16 µg/mL [122] MBC 32-64 µg/mL [122]

S

MIC 15.6 µg/mL [222] 15.6 MBC 31.3–500 µg/mL [222] µg/mL[222] MIC 0.125 µg/mL [109] MBC 0.5 µg/mL [109] MIC 37 µg/mL [212] MBC 37 µg/mL [212] MIC 50 µg/mL [212] MIC 25 µg/mL [212] MIC >125 µg/mL [79] MIC 157.5 µg/mL [79] MIC 39.4 µg/mL [79] MIC 50 µg/mL [86] MIC 15.7 µg/mL [79] Inhibition of biofilm formation 50 µg/mL [224] S. gordonii + P. gingivalis, S. gordonii + F. nucleatum Adhesion inhibition 100 µg/mL [219]

S

32–128 µg/mL for 5 min. No cytotoxicity to HGFs [122] Not assessed

S

Not assessed

S

Not assessed

N N N N N

Not assessed Not assessed Not assessed Not assessed

N S N

Not assessed Not assessed

S

33

E/A E/A

IDR-1018 Kappacin:Zn2+

10 µg/mL - prevent biofilm formation over 3 days Increase anti-biofilm activity with CHX [126] polymicrobial biofilm; immediately and continuously reduced total bacterial viability [225]

S

Not assessed

S

Not assessed

Abbreviations. AMP: antimicrobial peptide; CHX: chlorhexidine; epi4: human gingival epithelial cells; HGFs: human gingival fibroblasts; hPDLCs: human periodontal ligament stem cells; IC50: half maximal inhibitory concentration; LD50: Lethal dose that kills 50% of a sample; MBC: minimal bactericidal concentration; MBIC50: minimum biofilm inhibitory concentration; MBRC50: minimum biofilm reduction concentration; MIC: minimal inhibitory concentration; MTD: Maximum tolerated dose. Related niche. A: apical periodontitis; C: caries; E: endodontic infections Origin. N: Natural AMPs; S: Synthetic AMPs Cytotoxicity. Not assessed: cytotoxicity testing was not completed during or before the antibacterial test; lower hemolysis: lower hemolysis compared with other natural peptides in in vitro toxicity studies.

34

Table II. Antibacterial effects of nisin, without adjuvants, on bacteria commonly identified in caries and pulpal infections Microorganism S. mutans UA159 S. mutans UA159 S. sanguinis ATCC 10556 S. sobrinus ATCC 6715 S. gordonii ATCC 10558 L. acidophilus ATCC 4356 L. casei ATCC 393 L. fermenti ATCC 9338 A. viscosus ATCC 15987 A. naeslundii ATCC 12104 S. mutans UA159 S. mutans ATCC 25175 S. gordonii DL1 A. odontolyticus ATCC 17982

S. oralis SO34 F. nucleatum ATCC 25586 A. actinomycetemcomitans Y4

P. gingivalis W83 P. gingivalis ATCC 33277 P. intermedia clinical isolate T. denticola ATCC 35405

Related niche

Caries

Early and middle colonizers of oral biofilms

Late colonizers of oral biofilms

Anti-planktonic bacteria * (1 IU/mL = 1 µg/mL) MIC MBC 1000 IU/mL 2000 IU/mL 625-1250 IU/mL 1250-2500 IU/mL 156.3-312.5 IU/mL 312.5-625 IU/mL 1250-2500 IU/mL 2500-5000 IU/mL 625-1250 IU/mL 1250-2500 IU/mL 625-1250 IU/mL 1250-2500 IU/mL 312.5-625 IU/mL 625-1250 IU/mL 39-78 IU/mL 78-156 IU/mL 2500-5000 IU/mL 5000-10000 IU/mL 2500-5000 IU/mL 5000-10000 IU/mL 20 µg/mL 10 µg/mL 10 µg/mL 200 µg/mL 40 µg/mL 150 µg/mL 10 µg/mL 30 µg/mL 30 µg/mL 50 µg/mL 15 µg/mL

150 µg/mL 150 µg/mL 100 µg/ml

20µg/ml 15µg/ml 10µg/ml

100µg/ml 100µg/ml 150µg/ml

2.5µg/ml

15µg/ml

--

--

--

--

E. faecalis Pulpal infections

S. gordonii

1000 µg/mL 60 mg/mL 10 mg/mL

2000 µg/mL 70 mg/mL 20 mg/mL

Anti-biofilm --

[148]

--

[146]

≥1 µg/mL Interferes with biofilm development and reduces biofilm biomass and thickness

[147]

Antimicrobial against 2- and 7-day biofilms; effect does not change with time CFU of nisin: 10.6 at 24 h, 6.6 at 72 h, 6.3 at 1 week. CFU values < chlorhexidine or linezolid --

[156]

--

[150]

[226]

[227]

Abbreviations. CFU: colony forming units; MIC = minimum inhibitory concentrations; MBC = minimum bactericidal concentrations * MBC/MIC ratio great than 4 indicates that nisin has bacteriostatic effect. MBC/MIC ratio smaller than 4 indicates that nisin has bactericidal effect

35

Statement of significance Identification of new therapeutic strategies to combat antibiotic-resistant pathogens and biofilmassociated infections continues to be one of the major challenges in modern medicine. Despite the presence of commercialization hurdles and scientific challenges, interests in using antimicrobial peptides as therapeutic alternatives and adjuvants to combat pathogenic biofilms have never been foreshortened. Not only do these cationic peptides possess rapid killing ability, their multi-modal mechanisms of action render them advantageous in targeting different biofilm sub-populations. These factors, together with adjunctive bioactive functions such as immunomodulation and wound healing enhancement, render AMPs or their synthetic mimics exciting candidates to be considered as adjuncts in the treatment of caries, infected pulps and root canals.

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