Role of Antimicrobial Peptides in the Fight Against Infection

Dental caries is a topic frequently researched by oral health professionals in hopes of finding new treatments, as it remains one of the most prevalent infectious diseases and global public health issues.¹ Dental caries is caused by the accumulation of plaque-associated bacteria that produce demineralizing acidic by-products, which can damage tooth structures including enamel, dentin, and cementum. The development of dental caries can be attributed to continued ecological imbalance of acid-producing pathogens and exposure to their derivatives over an extended time.²

The oral cavity is one of the most diverse and populated microflora communities in the human body, with more than 700 bacterial species identified, not including fungi, viruses, and protozoa.³ The most well-studied cariogenic bacteria residing in dental plaque biofilm are Streptococcus mutans, Streptococcus sobrinus, Actinomyces, and Lactobacilli. Other pathogenic species that can contribute to dental caries include fungi, specifically Candida albicans.⁴ S. mutans is the primary bacteria species behind the development of dental caries.⁵ In addition, low pH, reduced salivary flow, and/or routine consumption of carbohydrates can exacerbate the progression of dental caries. Without protective factors, such as salivary buffers and fluoride exposure, bacterial metabolism can remain unchecked.²

Because the complete elimination of biofilm is difficult to achieve, alternative approaches to reducing harmful bacterial flora have been developed.⁶ Anticaries therapies—including various antimicrobial agents, such as fluoride, triclosan, chlorhexidine, and phenols—are used to reduce bacterial load and prevent biofilm formation.^7,8 Different delivery vehicles, such as trays and varnishes, are used for in-office treatment. More recently, silver diamine fluoride has been accepted as an off-label treatment in arresting caries lesions.⁹ Fluoride, triclosan, and phenols are commonly found in over-the-counter oral hygiene products to provide everyday caries prevention.¹⁰ 

Although current methodologies can be effective in controlling biofilm accumulation and preventing demineralization, they are not without drawbacks. Some populations are opposed to the use of fluoride and triclosan due to safety concerns. In addition, prolonged chlorhexidine use raises the risk of hypersensitivity and discoloration of hard and soft tissue in the oral cavity.¹¹ While phenolics provide antimicrobial and antigingivitis properties, they may also cause irritation and burning sensations.¹² These therapies may also disrupt microflora balance in the oral cavity when used for extended time periods.¹³ Additionally, due to increased drug resistance in medicine, conventional systemic antibiotics are not commonly prescribed to treat oral diseases.^14,15 Further, oral biofilms are already more resistant to antibiotics such as amoxicillin and metronidazole.⁶ As such, research has focused on developing alternative therapeutics, such as antimicrobial peptides (AMPs), which may offer an effective treatment for oral infections, including dental caries, periodontal diseases, oral mucosal diseases, and oral cancer.¹⁶

What are Antimicrobial Peptides?

AMPs are amino acid sequences of varying lengths that naturally occur in multicellular organisms.¹⁷ Found in the epithelial lining, blood, and lymphatic tissue, AMPs are one of the first defenses against foreign bodies. They also play an important role in the oral cavity. AMPs are produced in response to infections and subsequently bind to and neutralize invading microorganisms.¹⁸ Currently, 3,273 AMPs are known to exist, deriving from sources such as bacteria, fungi, plants, and animals, according to the Antimicrobial Peptide Database.¹⁹ This number will continue to increase as more are discovered or created. They can induce antibacterial, antibiofilm, antifungal, antiviral, anticandida, wound healing, anti-inflammatory, and/or antitumor effects.^2,18 

Due to their broad-spectrum antibacterial properties, AMPs can target specific Gram-positive and Gram-negative bacteria. They have specialized mechanisms to control bacteria, fungus, and virus populations.^20,21 Research has shown that resistance to various AMPs is rare, which makes them a promising therapeutic candidate for infectious diseases, including caries.^2,16,22 Researchers are now working on developing synthetic AMPs. Designing artificial peptides enables isolating and enhancing specific desired characteristics, providing additional benefits such as increased stability.^23,24 As AMPs can be readily applied to the oral cavity, numerous opportunities are available to develop prevention and treatment modalities for in-office and at-home oral care for caries management.²⁵

Natural Antimicrobial Peptides in the Oral Cavity

Human oral AMPs are naturally occurring and provide the following benefits:²⁶

• Defend against pathogens
• Repair cellular tissues
• Maintain the oral cavity 

Salivary proteins, such as histatins, defensins, and cathelicidins, are well-known AMPs. These salivary proteins have vastly different structures in their amino acid sequences and sizes, not only between each other but also within each type.^2,16,27 Histatins, defensins, and cathelicidins play a protective role in oral health.²⁵ Numerous histatin peptide sequences are secreted from the submandibular and parotid salivary glands. Some histatin peptides have fungicidal properties that can kill C. albicans.²⁶ Other histatins aid in enamel protection by binding to hydroxyapatite and preventing acid erosion.² Cathelicidins and defensins have displayed broad-spectrum antimicrobial activity against oral pathogens such as Gram-negative and Gram-positive bacteria, fungi, and viruses.^27,28 Cathelicidins elicit antimicrobial activity and antisepsis effects. They can trigger distinct defense systems and can repair wounds. Cathelicidins can also promote cytolysis, which is the disruption or dissolution of cells.²⁷ Research is underway on the use of defensins due to their quick ability to kill targeted microorganisms and their low risk of creating pathogenic resistance.²⁹ 

As part of the innate immune response, production of natural AMPs increases during diseased states.¹⁶ While studies have shown several human AMPs to be bactericidal to S. mutans and others to be fungicidal to C. albicans, natural AMPs are not able to completely eradicate these pathogens.^25,30 They primarily control overgrowth of harmful microorganisms and sustain the balance of oral microflora.³⁰ Naturally occurring AMPs are limited due to their instability in the ever changing oral environment.^23,31 They are susceptible to proteases—enzymes that break down proteins. As a result, they generally have short half-lives due to their rapid deterioration. Natural AMPs, therefore, have low bioavailability.^2,24 These limitations create challenges to the use of natural AMPs in the effective management of oral infections.

Synthetic Antimicrobial Peptides 

Synthetic AMPs may offer an alternative to traditional antimicrobial and antibiotic treatments. Researchers have identified several selectively targeted AMPs (STAMPS) against S. mutans. STAMPS have the potential to directly kill S. mutans within multiscience plaque biofilm.³² In one study, 43 synthetic AMPs with different mechanisms successfully inhibited the growth of S. mutans. Synthetics also demonstrated the ability to increase attraction for hydroxyapatite and others that promoted remineralizing effects on dentition.²⁵ Certain AMPs modeled from histatins have high biodegradability and demonstrate effectiveness against C. albicans.^26,33 In addition, plaque formation may be impeded by some synthetic AMPs due to their competitive blockage of bacterial adhesion.³⁴ 

Researchers are focused on developing synthetic AMPs with improved stability and modifications on the basis of natural AMPs’ structures.¹⁶ With designing synthetic peptides, researchers are aiming to produce safe and highly specified and efficient AMPs that can withstand the oral environment.^23,24 They can be created using three different methods:

1. Mimesis
2. New designs
3. Fusion of peptides

Mimesis mimics natural AMPs by using their sequence templates to create improved artificial peptides.²⁵ New design synthesis implements innovative formulations to produce original antimicrobial agents.³⁵ Fusion of peptides takes functional sequences of existing peptides and increases the specificity and/or activity of synthesized AMPs.² They can also be improved by including additional amino acids or removing sequences to strengthen peptides.³⁵ An important determination in the success of synthesized AMPs is their ability to work together to enhance each other’s effects.¹⁸ 

In order for synthetic AMPs to become more common in therapeutic applications, they must be able to withstand degradation in the oral environment by improving stability.³⁵ Without this, synthetic AMPs will be ineffective against pathogenic bacteria and biofilm. Once greater stability and effectiveness are achieved, other challenges may include the cost of production, obtaining authorization from governing bodies, and potential consumer resistance of a product labeled “synthetic.”²

Mechanisms of Action 

AMPs are diverse in their mechanism of action; they can kill cariogenic bacteria, suppress the formation of plaque biofilm, and inhibit enzymes and protein synthesis, among other activities.^5,16,36,37 Some, such as cathelicidins, can differentiate healthy host cell membranes and not act upon them.²⁷ AMPs typically disrupt their targeted microorganisms and can be selectively fatal.¹⁶ 

Many AMPs work to adhere to and anchor to cell membranes, thereby damaging and puncturing specific cells for which they are active against.²⁷ AMPs can penetrate cell walls and cytoplasmic membranes of bacteria to form channels, which lead to leakage of the cells’ contents and disintegration of membranes.^16,27 The mechanisms of antimicrobial peptides’ attachment and insertion into cell membranes are categorized by the following models:³⁸

• Barrel-stave
• Toroidal-pore
• Carpet
• Detergent-like

Barrel-stave and toroidal-pore mechanisms both create gaps, causing membranes to become more permeable, thinner, and/or leak.^27,38 Carpet and detergent-like mechanisms affect membranes by disrupting them and lowering cell resistance and density.³⁸

Alternatively, adherence to cell walls can also cause modification of bacterial structures and curvature.³⁹ After adhering to a lipid bilayer, AMPs can insert themselves into the outside layer of pathogens and destabilize cell membranes.³⁴ In Gram-negative bacteria, they can move through the outer membrane and the inner cell wall into the cytoplasm.²⁷ Once AMPs have entered a cell, if not cytolytic, they can further selectively disrupt protein synthesis and prevent cell wall formation. Some AMPs bind to intracellular organelles and inhibit enzyme activity.⁴⁰ 

A major advantage of AMPs is their specificity. They can kill pathogens without damaging surrounding host environments.¹⁶ AMPs are able to target cells and membranes, making it difficult for bacterial cells to withstand them. Studies have reported both natural and synthetic AMPs are effective against S. mutans due to their inhibitory and bactericidal qualities.²⁵ Furthermore, AMPs have synergistic abilities whereby acting together enables lower concentrations to be more effective.¹⁸

Conclusion

AMPs provide a promising approach in disease management of the oral cavity. They are being studied as alternative methods of prevention and treatment of dental caries, periodontal diseases, and oral cancer. 

In caries management, AMPs have the unique ability to specifically target and eliminate caries-causing bacteria. Naturally occurring AMPs are already found in saliva and function as part of the protective factors against disease-causing pathogens. Although natural AMPs usually have short half-lives and rapidly deteriorate in the oral cavity, synthetic AMPS may overcome these limitations. 

While numerous laboratory studies demonstrate the effectiveness of AMPs against S.mutans and other pathogenic microbes, including C. albicans, further research is required. Whether natural or synthetic, AMPs have considerable potential in the management of dental caries and other oral infections. 

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From Dimensions of Dental Hygiene. October 2021;19(10)38-41.