**Abstract**

Defensins are naturally occurring antimicrobial peptides secreted in the human body. Mammalian defensins are small, cysteine-rich, cationic peptides, generally consisting of 18–45 amino acids. The antimicrobial activity of defensins arises from their unique amino acid sequence, showing activity against both Gram-positive and Gram-negative bacteria, fungi and enveloped viruses. The use of antimicrobial peptides is rising due to their potential to control biofilm formation and kill microorganisms that are highly tolerant to antibiotics. In free-form, defensins are capable of destroying such microorganisms through numerous mechanisms mainly the *carpet*, the *toroidal* and the *Barrel-Stave* models. However, immobilization of antimicrobial peptides (AMPs) on surfaces with the help of coupling agents and spacers can improve the AMPs' lifespan and stability in the physiological environment leading to applications for medical devices and implants. Fundamental understanding of both free-form and surface-immobilized defensins is important to design more effective antimicrobial peptides and improve their performance in future developments.

**Keywords:** antimicrobial peptides, defensins, mammalian peptides, surface-immobilized antimicrobial peptides, surface-immobilized defensins

### **1. Introduction**

The innate immune system is the first line of defence in human body and vertebrates. Defensins are naturally occurring antimicrobial peptides (AMPs) that are a part of the innate immune system, protecting the body against foreign microorganisms. Defensins are produced by the interaction of antigen-presenting microbial cells with pattern recognition receptors, such as toll-like receptors that are present on the membrane of numerous immune cells (i.e., macrophages, neutrophils and leukocytes [1]. Mammalian defensins are small, cysteine-rich, cationic peptides, generally consisting of 18–45 amino acids [2]. Next to being antimicrobial, defensins also serve as immune-stimulating agents.

When synthesized *in vivo*, defensins are initially produced as inactive precursor proteins (i.e., pro-defensins), which consist of the defensin and a pro-peptide. The pro-peptides are present to ensure delivery of defensins through the body without

#### **Figure 1.**

*Schematic of a (a1) parallel and (a2) antiparallel β-sheet structure, made with the use of JSME [12]. (b) Conformation of a β-defensin (hBD3), made with the use of PEP-FOLD3 [13]. Hydrogen bonding represented by dotted lines, hydrogen atoms are white, carbon atoms are grey, oxygen atoms are red, nitrogen atoms are blue and rest-groups are pink; not exact structure.*

premature attachment of defensins to other microorganisms [3]. The pro-peptides inhibit premature attachment to other microorganisms by neutralizing the cationic charge of defensins. Also, the pro-peptides ensure subcellular localization (i.e., the location of where a protein resides in a cell) and folding of defensins into their characteristic conformation. Through proteolytic removal *in vivo* of the pro-peptides, the defensins are activated [4]. The reasoning behind the pro-peptides functioning as a folding assistant is based on the research performed on folding of defensins *in vitro* without the pro-peptide, which is found to be extremely difficult [5, 6].

α-Defensins are expressed by neutrophils and macrophages, that is, a type of white blood cell and cells that can engulf foreign particles, respectively. In general, these tend to have a broader antimicrobial activity, when compared to β-defensins, showing activity against both Gram-positive and Gram-negative bacteria, fungi and enveloped viruses [7]. Paneth cells also produce α-defensins, also known as crypticidins, which are involved in the reduction of bacteria present in the intestinal lumen. β-Defensins are primarily produced and released by epithelial cells and leukocytes, that is, a type of cell that lines the surfaces of your body and a type of blood cell that is made in the bone marrow, respectively. These are mainly active against Gram-negative bacteria and yeast; however, many also show antibacterial activity towards Gram-positive bacteria [8]. The pro-peptides of β-defensins are smaller than those of α-defensins.

Both α- and β-defensins form a triple-stranded antiparallel β-sheet structure that is stabilized by hydrogen and disulphide bonds; bond formations are schematically represented in **Figure 1**. 1 The position of the cysteines and intramolecular disulphide linkages determines the category of the defensin. The consensus of cysteine placement within the amino acid sequence for α-defensins follows C-X-C-X4-C-X9-X-X, and C-X6-C-X4-C-X9-C-X6-C-C for β-defensins [9]. When looking at the position of the disulphide linkages from cysteine in sequential order (denoted by C#), the disulphide bridges are formed between C1-C6, C2-C4 and C3-C5 for α-defensins and C1-C5, C2-C4 and C3-C6 for β-defensins [10], as shown in **Figures 2** and **3**, respectively. The disulphide bridges are important for holding the defensins in their three-dimensional structures. In addition, they contribute to the defensins chemotactic activity (i.e., movement or orientation of an organism or cell towards chemical stimulus) but when altered, only slightly affect their antimicrobial activity [11].

The adopted mechanisms of the interaction between defensins and the invading microorganism are not yet fully understood. However, disruption of

**45**

targeted cells [14, 15].

**2. Mammalian defensins**

*Defensin-Like Peptides and Their Antimicrobial Activity in Free-Form and Immobilized…*

*Schematic of HNP1 (α-defensin) showing amino acid sequence and disulphide bridges.*

the plasma membrane has been shown to be the leading cause of cell death in microbial species. The disruption caused by defensins depends on many factors, such as the polar topology, spatial separating of charges and hydrophobicity. These factors allow the attraction and subsequent interaction of defensins with the lipid bilayer of the bacterial membrane. Conversely, this interaction causes the defensins to insert themselves between the hydrophilic region of the plasma membrane and disrupt the bacterial membrane, utilizing numerous mechanisms. These include the introduction of channel-like pores and carpet-like membrane disruption, resulting in cell lysis. Simultaneously, the introduction of voltagedependent channels in the bacterial membrane allows the influx of water and results in an increase of osmotic pressure that leads to the rupture of the membrane. On the other hand, some defensins move through bacterial cell walls, bind to target cells and disrupt normal metabolism, which may lead to apoptosis of the

*Schematic of HBD1 (β-defensin) showing amino acid sequence and disulphide bridges.*

The genomic organization and evolution of defensin genes of several vertebrate species have been studied [16]. The human genome encodes, at least, 35 different defensin peptides [17]. Most of the mammalian defensin genes are divided over three chromosomes, found in four different gene clusters (**Figure 4**). All the genes

*DOI: http://dx.doi.org/10.5772/intechopen.85508*

**Figure 2.**

**Figure 3.**

<sup>1</sup> All the figures shown in this chapter have been created by the authors.

*Defensin-Like Peptides and Their Antimicrobial Activity in Free-Form and Immobilized… DOI: http://dx.doi.org/10.5772/intechopen.85508*

**Figure 2.**

*Peptide Synthesis*

**Figure 1.**

premature attachment of defensins to other microorganisms [3]. The pro-peptides inhibit premature attachment to other microorganisms by neutralizing the cationic charge of defensins. Also, the pro-peptides ensure subcellular localization (i.e., the location of where a protein resides in a cell) and folding of defensins into their characteristic conformation. Through proteolytic removal *in vivo* of the pro-peptides, the defensins are activated [4]. The reasoning behind the pro-peptides functioning as a folding assistant is based on the research performed on folding of defensins *in vitro* without the pro-peptide, which is found to be extremely difficult [5, 6]. α-Defensins are expressed by neutrophils and macrophages, that is, a type of white blood cell and cells that can engulf foreign particles, respectively. In general, these tend to have a broader antimicrobial activity, when compared to β-defensins, showing activity against both Gram-positive and Gram-negative bacteria, fungi and enveloped viruses [7]. Paneth cells also produce α-defensins, also known as crypticidins, which are involved in the reduction of bacteria present in the intestinal lumen. β-Defensins are primarily produced and released by epithelial cells and leukocytes, that is, a type of cell that lines the surfaces of your body and a type of blood cell that is made in the bone marrow, respectively. These are mainly active against Gram-negative bacteria and yeast; however, many also show antibacterial activity towards Gram-positive bacteria [8]. The pro-peptides of β-defensins are

*Schematic of a (a1) parallel and (a2) antiparallel β-sheet structure, made with the use of JSME [12]. (b) Conformation of a β-defensin (hBD3), made with the use of PEP-FOLD3 [13]. Hydrogen bonding represented by dotted lines, hydrogen atoms are white, carbon atoms are grey, oxygen atoms are red, nitrogen atoms are blue* 

Both α- and β-defensins form a triple-stranded antiparallel β-sheet structure that is stabilized by hydrogen and disulphide bonds; bond formations are schematically

linkages determines the category of the defensin. The consensus of cysteine placement within the amino acid sequence for α-defensins follows C-X-C-X4-C-X9-X-X, and C-X6-C-X4-C-X9-C-X6-C-C for β-defensins [9]. When looking at the position of the disulphide linkages from cysteine in sequential order (denoted by C#), the disulphide bridges are formed between C1-C6, C2-C4 and C3-C5 for α-defensins and C1-C5, C2-C4 and C3-C6 for β-defensins [10], as shown in **Figures 2** and **3**, respectively. The disulphide bridges are important for holding the defensins in their three-dimensional structures. In addition, they contribute to the defensins chemotactic activity (i.e., movement or orientation of an organism or cell towards chemical stimulus) but when

The adopted mechanisms of the interaction between defensins and the invading microorganism are not yet fully understood. However, disruption of

The position of the cysteines and intramolecular disulphide

**44**

smaller than those of α-defensins.

*and rest-groups are pink; not exact structure.*

1

altered, only slightly affect their antimicrobial activity [11].

<sup>1</sup> All the figures shown in this chapter have been created by the authors.

represented in **Figure 1**.

*Schematic of HNP1 (α-defensin) showing amino acid sequence and disulphide bridges.*

#### **Figure 3.**

the plasma membrane has been shown to be the leading cause of cell death in microbial species. The disruption caused by defensins depends on many factors, such as the polar topology, spatial separating of charges and hydrophobicity. These factors allow the attraction and subsequent interaction of defensins with the lipid bilayer of the bacterial membrane. Conversely, this interaction causes the defensins to insert themselves between the hydrophilic region of the plasma membrane and disrupt the bacterial membrane, utilizing numerous mechanisms. These include the introduction of channel-like pores and carpet-like membrane disruption, resulting in cell lysis. Simultaneously, the introduction of voltagedependent channels in the bacterial membrane allows the influx of water and results in an increase of osmotic pressure that leads to the rupture of the membrane. On the other hand, some defensins move through bacterial cell walls, bind to target cells and disrupt normal metabolism, which may lead to apoptosis of the targeted cells [14, 15].
