**2. Mammalian defensins**

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

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

#### **Figure 4.**

*Schematic of the genomic organization of defensins in three human chromosomes, showing chromosome 6, chromosome 8 and chromosome 20, respectively.*

expressing α-defensins and several β-defensins are found in chromosome 8 (cluster p23.1); genes that express most of the remaining β-defensins are found in chromosome 6 (cluster p21) and chromosome 20 (cluster q11.1 and p13).

Four out of the six human α-defensins are found in neutrophils and other leukocytes, specifically, human neutrophil peptides (HNPs) 1–4 (i.e., DEFA1–4). The remaining two, human α-defensin 5 and 6 (HD5 and HD6, i.e., DEFA5 and DEFA6), are expressed by Paneth cells in the intestinal lumen [18]. Numerous β-defensins are found in the respiratory system, gastrointestinal tract and urogenital system. These are expressed by epithelial cells, namely, human BD1 (hBD1, i.e., DEFB1), hBD2 (DEFB4), hBD3 (DEFB103A) and hBD4 (DEFB104) [19].

Defensins are amphipathic (i.e., having both hydrophobic and hydrophilic groups) and, it has been demonstrated that they show the ability to form dimers and oligomers with toxin molecules [20, 21]. The initial electrostatic interaction is caused by the cationic charge of the peptide and the negatively charged outer membrane of the bacterial cell wall [22]. Bacterial membranes contain many negatively charged phospholipids, lipopolysaccharides or teichoic acid, while eukaryotic membranes contain neutral phospholipids and cholesterol [23]. This explains the destructive ability of defensins towards microbes but not host cells [24].

#### **2.1 Antimicrobial mechanisms of defensins**

The activity of defensins against microorganisms is determined by the interaction of the cationic molecules with the negatively charged acidic lipopolysaccharide or teichoic acid on Gram-positive and Gram-negative bacterial membranes. The antimicrobial activity of defensins in the body depends on different factors such as salt concentration and serum components [25]. In addition, the configuration of defensins plays an important role on the activity of these molecules. For example,

**47**

bacteria [35, 36].

**2.2 Antimicrobial activity evaluation methods**

The evaluation of antimicrobial activity is widely used in the field of drug discovery as well as epidemiology and therapeutic prediction [37]. Since the low

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

helical antimicrobial peptides in solution show a typical helical amphipathic characteristic and they are either unstructured until they are in contact with the biological membrane or structured through disulphide bonds. Defensins are among the structured type of antimicrobial peptides and the hydrophilicity of the α- and β-defensins determines the extent of interaction between these molecules and the

As mentioned before, the antimicrobial mechanisms of defensins have not been fully understood. The formation of membrane pore or channel has been shown to be dependent on the membrane configuration. For example, the abundance of negatively charged phospholipids on the plasma membrane affects the concentration of peptides that are required to form a stretch or curvature on the outer layer of the bacterial membrane, and consequently lead to cell lysis [27]. Other events such as phospholipid reversal and penetration of peptides inside the cytoplasm on the inner side of the membrane leading to the loss of membrane composition and causing cellular inactivation have also been mentioned in the literature [28].

Most studies conducted on defensin mechanisms have been conducted on the α-helical structured peptides. These molecules interact with the membrane of the microorganisms. It is evident that defensins utilize membrane depolarization and permeation, against bacteria and yeast, as their most likely defence mechanisms. Defensins also aid the mobilization of T-cells and immature dendritic cells, which contribute in the activation of acquired immune responses that will trigger a longlasting cellular response to a potential pathogen [29]. Most defensins provide their antimicrobial activity through interaction with cellular membranes. The poreforming model describes the interaction between positively charged peptides and the negatively charged head of phospholipid groups of cellular membranes operates once a critical concentration is reached. This will cause the self-aggregation and perpendicular insertion of peptides inside the membrane leading to production of lined transmembrane pores, resulting in the disruption of ionic and proton gradients. The second model causes the formation of channels where the strain of peptide at the critical concentration, induces the inward curving of the membrane, creating dome-shaped channels lined with phospholipid-head groups and peptides [30]. The mechanisms in which the AMPs destroy the bacterial cells have been studied for decades. AMPs that present their secondary structure in the form of α-helix present their destructive effects on a bacterial cell based on three different documented mechanisms. First is the *Barrel-Stave model*, which is the most studied mechanism, where the peptide disrupts the membrane by exposing its hydrophobic site to the lipids in the membrane bilayer. This will force the membrane to undergo conformational changes by forming a pore on the surface of the membrane [31, 32]. The second mechanism is the *toroidal model* where the peptide and the lipid of the membrane bilayer integrate upon interaction and form torus pores leading to the death of bacterial cells. Cell death caused by these mechanisms leads to loss of compositional specificity, leakage of critical metabolites or depolarization of the membrane due to an increased rate of phospholipid reversal [33, 34]. The third mechanism is the *carpet model* where the concentration of AMP is related to the amount of interaction with the bacterial membrane. In this model, the peptides surround the cell membrane by attaching to the phosphide group of the membrane bilayer where they disrupt the curved anionic membrane and dissolve it, killing the

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

bacterial membrane [26].

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

helical antimicrobial peptides in solution show a typical helical amphipathic characteristic and they are either unstructured until they are in contact with the biological membrane or structured through disulphide bonds. Defensins are among the structured type of antimicrobial peptides and the hydrophilicity of the α- and β-defensins determines the extent of interaction between these molecules and the bacterial membrane [26].

As mentioned before, the antimicrobial mechanisms of defensins have not been fully understood. The formation of membrane pore or channel has been shown to be dependent on the membrane configuration. For example, the abundance of negatively charged phospholipids on the plasma membrane affects the concentration of peptides that are required to form a stretch or curvature on the outer layer of the bacterial membrane, and consequently lead to cell lysis [27]. Other events such as phospholipid reversal and penetration of peptides inside the cytoplasm on the inner side of the membrane leading to the loss of membrane composition and causing cellular inactivation have also been mentioned in the literature [28].

Most studies conducted on defensin mechanisms have been conducted on the α-helical structured peptides. These molecules interact with the membrane of the microorganisms. It is evident that defensins utilize membrane depolarization and permeation, against bacteria and yeast, as their most likely defence mechanisms. Defensins also aid the mobilization of T-cells and immature dendritic cells, which contribute in the activation of acquired immune responses that will trigger a longlasting cellular response to a potential pathogen [29]. Most defensins provide their antimicrobial activity through interaction with cellular membranes. The poreforming model describes the interaction between positively charged peptides and the negatively charged head of phospholipid groups of cellular membranes operates once a critical concentration is reached. This will cause the self-aggregation and perpendicular insertion of peptides inside the membrane leading to production of lined transmembrane pores, resulting in the disruption of ionic and proton gradients. The second model causes the formation of channels where the strain of peptide at the critical concentration, induces the inward curving of the membrane, creating dome-shaped channels lined with phospholipid-head groups and peptides [30].

The mechanisms in which the AMPs destroy the bacterial cells have been studied for decades. AMPs that present their secondary structure in the form of α-helix present their destructive effects on a bacterial cell based on three different documented mechanisms. First is the *Barrel-Stave model*, which is the most studied mechanism, where the peptide disrupts the membrane by exposing its hydrophobic site to the lipids in the membrane bilayer. This will force the membrane to undergo conformational changes by forming a pore on the surface of the membrane [31, 32]. The second mechanism is the *toroidal model* where the peptide and the lipid of the membrane bilayer integrate upon interaction and form torus pores leading to the death of bacterial cells. Cell death caused by these mechanisms leads to loss of compositional specificity, leakage of critical metabolites or depolarization of the membrane due to an increased rate of phospholipid reversal [33, 34]. The third mechanism is the *carpet model* where the concentration of AMP is related to the amount of interaction with the bacterial membrane. In this model, the peptides surround the cell membrane by attaching to the phosphide group of the membrane bilayer where they disrupt the curved anionic membrane and dissolve it, killing the bacteria [35, 36].

### **2.2 Antimicrobial activity evaluation methods**

The evaluation of antimicrobial activity is widely used in the field of drug discovery as well as epidemiology and therapeutic prediction [37]. Since the low

*Peptide Synthesis*

**Figure 4.**

*chromosome 8 and chromosome 20, respectively.*

expressing α-defensins and several β-defensins are found in chromosome 8 (cluster p23.1); genes that express most of the remaining β-defensins are found in chromo-

*Schematic of the genomic organization of defensins in three human chromosomes, showing chromosome 6,* 

Four out of the six human α-defensins are found in neutrophils and other leukocytes, specifically, human neutrophil peptides (HNPs) 1–4 (i.e., DEFA1–4). The remaining two, human α-defensin 5 and 6 (HD5 and HD6, i.e., DEFA5 and DEFA6), are expressed by Paneth cells in the intestinal lumen [18]. Numerous β-defensins are found in the respiratory system, gastrointestinal tract and urogenital system. These are expressed by epithelial cells, namely, human BD1 (hBD1, i.e., DEFB1), hBD2

Defensins are amphipathic (i.e., having both hydrophobic and hydrophilic groups) and, it has been demonstrated that they show the ability to form dimers and oligomers with toxin molecules [20, 21]. The initial electrostatic interaction is caused by the cationic charge of the peptide and the negatively charged outer membrane of the bacterial cell wall [22]. Bacterial membranes contain many negatively charged phospholipids, lipopolysaccharides or teichoic acid, while eukaryotic membranes contain neutral phospholipids and cholesterol [23]. This explains the

The activity of defensins against microorganisms is determined by the interaction of the cationic molecules with the negatively charged acidic lipopolysaccharide or teichoic acid on Gram-positive and Gram-negative bacterial membranes. The antimicrobial activity of defensins in the body depends on different factors such as salt concentration and serum components [25]. In addition, the configuration of defensins plays an important role on the activity of these molecules. For example,

destructive ability of defensins towards microbes but not host cells [24].

some 6 (cluster p21) and chromosome 20 (cluster q11.1 and p13).

(DEFB4), hBD3 (DEFB103A) and hBD4 (DEFB104) [19].

**2.1 Antimicrobial mechanisms of defensins**

**46**

density of peptides will ultimately result in preservation of membrane integrity and adjustment of the membrane, maintaining a higher concentration of peptides is crucial. This will result in imposed curvature strain on bacterial membrane [38]. Therefore, measurement of the minimum inhibitory concentration (MIC) is an important initial step for the confirmation of antimicrobial activity. MIC refers to the lowest concentration of compound needed for observable inhibition of bacterial cell growth. In addition, other measurements include minimal bactericidal concentration (MBC) which refers to the minimum concentration of the reagent that can cause bacterial death, the haemolytic activity (HC) that refers to the ability to break down red blood cells (i.e.,) to find the inhibitory effect of AMPs on normal mammalian cells and IC50, which refers to the half-maximal inhibitory concentration [39]. Since defensins are found in more diluted concentrations in extracellular environment than in a local environment, the measurement of the interactions between peptides and bacteria *in situ* (e.g., using a mice model) is mandatory [40].

Colorimetric assays are used to determine the concentration of peptides (MIC) where the absorbance is usually measured at 750 nm using a UV-visible spectrophotometer. The assays are performed in 96-well microtiter plates and a series of antimicrobial peptide dilutions are added to the bacterial cells immersed in growth medium. The MIC is measured based on the growth after incubation for a defined period of time (16–20 h) [41]. This methodology measures the colour of the dilutions, which is directly proportional to the number of cells in each well. The absorbance is measured with the help of a microplate reader counting the number of cells killed by the antimicrobial peptide. This methodology provides information about the susceptibility of the microorganism to the peptide [42], but it only applies to aerobic bacteria [43].

Electron microscopy is used to visualize the interaction of peptides with the phospholipid bilayer of bacterial membrane. Simultaneously, fluorescent dyes are used to observe the ability of peptides to permeate and penetrate the membrane of bacterial cells. Fluorescence spectroscopy is used to study the insertion of fluorophores into a membrane as well as provide information about the rate of peptide penetration into the membrane with the help of surface plasmon resonance. Fluorescence quenching can also be used to gain an insight into the depth of peptide penetration inside the cells [2].

In addition, both circular dichroism (CD) spectroscopy [44] and nuclear magnetic resonance (NMR) spectroscopy are used to measure the orientation and secondary structure of an antimicrobial peptide, when bound to a lipid bilayer. However, only NMR is used to measure the penetration of antimicrobial peptides into lipid bilayers in a relevant liquid-crystalline state [45]. The use of CD can distinguish between the randomly coiled, α-helical and β-sheet structures since they show wavelength-dependent differences in the absorption of the right and left circularly polarized light. Using oriented CD can also provide information about the orientation of peptide upon insertion into the membrane. This is dependent on the concentration of these peptides, the nature of the lipid and the extent of hydration [2].

In order to study the peptide configuration, both solution and solid-state NMR can be employed. For solution NMR, a mixture of peptide-detergent micelle is needed for the stabilization of the peptide in water and to overcome insufficient resolution and low (signal/noise) ratio [46]. The most recognized model membrane system used in solution NMR is the dodecylphosphocholine (DPC). This method is used for studying the interaction of peptides with the lipid bilayer since it has the ability to rotate freely in solution and mimic anisotropic environments of lipid membranes [47]. In addition, solid-state NMR is a premium technique to use for the analysis of immobile peptides that are difficult to analyse with crystallography

**49**

ends exposed [52].

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

or solution NMR. They can be used without a need for major peptide modification to determine the structure of membrane proteins in the phospholipid bilayer. This technique offers the examination of the structure and motion for the peptide-lipid interactions in physiologically relevant conditions and produces sharp resonance

In addition, to provide the dynamic interaction between the defensins and the lipid bilayer, using a monolayer mimicking the structure of bacterial membranes can also be used. To observe the interaction between the two, the sum frequency generation spectroscopy (SFG) is employed. This methodology can provide information about gas-liquid, solid-liquid and liquid-liquid interactions and is able to detect the biomolecule orientation and adsorption in sub-micron quantities. This technique uses a pulsed narrow band visible laser beam spatially and temporally overlapped with a broadband infrared laser and measures the incident beam produced from the surface [49]. The vibrational spectrum of C〓O groups from the amide backbone group can provide the secondary structure of the peptides. Consequently, vibrations from acyl chains of the lipid bilayer can provide important information about the interaction of peptide with the membrane as well as information about the molecular structure of the peptide, without the use of vesicles and

There are two types of AMPs: first AMPs that show activity towards both bacterial and mammalian cells; second, AMPs that show only activity towards the bacterial cells. Most linear cationic AMPs are unordered in aqueous solution. The balance between the positively charged and hydrophobic amino acids in cationic AMPs permits the amphipathic adaptation of these molecules in solution. This allows the interaction of AMPs with the negatively charged bacterial membrane and the subsequent penetration of these molecules inside the lipid membrane [51]. The interaction of these peptides with the bacterial membrane is increased due to their high inside-negative transmembrane potential. On the other hand, normal eukaryotic cells have a net neutral charge across their membrane bilayer, and they have reached a zwitterionic (overall neutral) point. This insight can partly explain the attraction of AMPs to prokaryotic cells and the relatively weak attraction of these molecules towards eukaryotic cells [38]. The amphipathic characteristics of defensins aid the adaptation of a folded confirmation for these molecules in both hydrophobic and hydrophilic environments [44]. These molecules are often difficult to stabilize and show poor bioavailability due to their many different cleavage points that provide susceptibility to enzyme degradation and their linear form which leaves their two

α-Defensins (DEFA1–4) are produced by endoplasmic reticulum of the bone marrow shown in **Figure 5**, and the highest concentration of defensins is found in granules (i.e., leukocyte storage organelles). Pre-pro-defensins (light purple circles in **Figure 5**), however, consist of 94 amino acids. To produce pro-defensins (dark purple circles in **Figure 5**), 19 amino acids are removed from the N-terminus of pre-pro-defensins. Further proteolysis from the N-terminal side of the sequence of amino acids results in the production of mature defensins (blue circles in **Figure 5**). The prepared defensins are encapsulated into vesicles and fused with phagocytic vacuoles. The introduction of a pathogen into cells will then result in the recognition and engulfing of such molecules by phagocytic leukocytes with the aid of defensins and the subsequent death of the pathogenic organism. Although some α-defensins are produced in the bone marrow, some (DEFA5-6) are also produced

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

lines due to macroscopic alignment [48].

labels to complicate the process of analysis [50].

**3. Antimicrobial activity of defensins in free-form**

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

or solution NMR. They can be used without a need for major peptide modification to determine the structure of membrane proteins in the phospholipid bilayer. This technique offers the examination of the structure and motion for the peptide-lipid interactions in physiologically relevant conditions and produces sharp resonance lines due to macroscopic alignment [48].

In addition, to provide the dynamic interaction between the defensins and the lipid bilayer, using a monolayer mimicking the structure of bacterial membranes can also be used. To observe the interaction between the two, the sum frequency generation spectroscopy (SFG) is employed. This methodology can provide information about gas-liquid, solid-liquid and liquid-liquid interactions and is able to detect the biomolecule orientation and adsorption in sub-micron quantities. This technique uses a pulsed narrow band visible laser beam spatially and temporally overlapped with a broadband infrared laser and measures the incident beam produced from the surface [49]. The vibrational spectrum of C〓O groups from the amide backbone group can provide the secondary structure of the peptides. Consequently, vibrations from acyl chains of the lipid bilayer can provide important information about the interaction of peptide with the membrane as well as information about the molecular structure of the peptide, without the use of vesicles and labels to complicate the process of analysis [50].

## **3. Antimicrobial activity of defensins in free-form**

There are two types of AMPs: first AMPs that show activity towards both bacterial and mammalian cells; second, AMPs that show only activity towards the bacterial cells. Most linear cationic AMPs are unordered in aqueous solution. The balance between the positively charged and hydrophobic amino acids in cationic AMPs permits the amphipathic adaptation of these molecules in solution. This allows the interaction of AMPs with the negatively charged bacterial membrane and the subsequent penetration of these molecules inside the lipid membrane [51]. The interaction of these peptides with the bacterial membrane is increased due to their high inside-negative transmembrane potential. On the other hand, normal eukaryotic cells have a net neutral charge across their membrane bilayer, and they have reached a zwitterionic (overall neutral) point. This insight can partly explain the attraction of AMPs to prokaryotic cells and the relatively weak attraction of these molecules towards eukaryotic cells [38]. The amphipathic characteristics of defensins aid the adaptation of a folded confirmation for these molecules in both hydrophobic and hydrophilic environments [44]. These molecules are often difficult to stabilize and show poor bioavailability due to their many different cleavage points that provide susceptibility to enzyme degradation and their linear form which leaves their two ends exposed [52].

α-Defensins (DEFA1–4) are produced by endoplasmic reticulum of the bone marrow shown in **Figure 5**, and the highest concentration of defensins is found in granules (i.e., leukocyte storage organelles). Pre-pro-defensins (light purple circles in **Figure 5**), however, consist of 94 amino acids. To produce pro-defensins (dark purple circles in **Figure 5**), 19 amino acids are removed from the N-terminus of pre-pro-defensins. Further proteolysis from the N-terminal side of the sequence of amino acids results in the production of mature defensins (blue circles in **Figure 5**). The prepared defensins are encapsulated into vesicles and fused with phagocytic vacuoles. The introduction of a pathogen into cells will then result in the recognition and engulfing of such molecules by phagocytic leukocytes with the aid of defensins and the subsequent death of the pathogenic organism. Although some α-defensins are produced in the bone marrow, some (DEFA5-6) are also produced

*Peptide Synthesis*

to aerobic bacteria [43].

penetration inside the cells [2].

density of peptides will ultimately result in preservation of membrane integrity and adjustment of the membrane, maintaining a higher concentration of peptides is crucial. This will result in imposed curvature strain on bacterial membrane [38]. Therefore, measurement of the minimum inhibitory concentration (MIC) is an important initial step for the confirmation of antimicrobial activity. MIC refers to the lowest concentration of compound needed for observable inhibition of bacterial cell growth. In addition, other measurements include minimal bactericidal concentration (MBC) which refers to the minimum concentration of the reagent that can cause bacterial death, the haemolytic activity (HC) that refers to the ability to break down red blood cells (i.e.,) to find the inhibitory effect of AMPs on normal mammalian cells and IC50, which refers to the half-maximal inhibitory concentration [39]. Since defensins are found in more diluted concentrations in extracellular environment than in a local environment, the measurement of the interactions between peptides and bacteria *in situ* (e.g., using a mice model) is mandatory [40]. Colorimetric assays are used to determine the concentration of peptides (MIC) where the absorbance is usually measured at 750 nm using a UV-visible spectrophotometer. The assays are performed in 96-well microtiter plates and a series of antimicrobial peptide dilutions are added to the bacterial cells immersed in growth medium. The MIC is measured based on the growth after incubation for a defined period of time (16–20 h) [41]. This methodology measures the colour of the dilutions, which is directly proportional to the number of cells in each well. The absorbance is measured with the help of a microplate reader counting the number of cells killed by the antimicrobial peptide. This methodology provides information about the susceptibility of the microorganism to the peptide [42], but it only applies

Electron microscopy is used to visualize the interaction of peptides with the phospholipid bilayer of bacterial membrane. Simultaneously, fluorescent dyes are used to observe the ability of peptides to permeate and penetrate the membrane of bacterial cells. Fluorescence spectroscopy is used to study the insertion of fluorophores into a membrane as well as provide information about the rate of peptide penetration into the membrane with the help of surface plasmon resonance. Fluorescence quenching can also be used to gain an insight into the depth of peptide

In addition, both circular dichroism (CD) spectroscopy [44] and nuclear magnetic resonance (NMR) spectroscopy are used to measure the orientation and secondary structure of an antimicrobial peptide, when bound to a lipid bilayer. However, only NMR is used to measure the penetration of antimicrobial peptides into lipid bilayers in a relevant liquid-crystalline state [45]. The use of CD can distinguish between the randomly coiled, α-helical and β-sheet structures since they show wavelength-dependent differences in the absorption of the right and left circularly polarized light. Using oriented CD can also provide information about the orientation of peptide upon insertion into the membrane. This is dependent on the concentration of these peptides, the nature of the lipid and the extent of

In order to study the peptide configuration, both solution and solid-state NMR can be employed. For solution NMR, a mixture of peptide-detergent micelle is needed for the stabilization of the peptide in water and to overcome insufficient resolution and low (signal/noise) ratio [46]. The most recognized model membrane system used in solution NMR is the dodecylphosphocholine (DPC). This method is used for studying the interaction of peptides with the lipid bilayer since it has the ability to rotate freely in solution and mimic anisotropic environments of lipid membranes [47]. In addition, solid-state NMR is a premium technique to use for the analysis of immobile peptides that are difficult to analyse with crystallography

**48**

hydration [2].

#### **Figure 5.**

*Schematic diagram of production of α-defensins and the phagocytosis of pathogens with the aid of defensins: (1) bone-marrow promyelocyte; (2) neutrophil; (3) phagocytosis.*

in the Paneth cells of the intestines [53]. β-Defensins (DEFB1–4) on the other hand are mainly produced in the epithelial cells. These regions have the highest concentration of defensins due to the higher susceptibility to a pathogenic attack, which renders them weaker and subsequently in need of the immunity provided by defensins [17].

β-Defensins mature into a secreted peptide after pre-pro-peptide state. This mature peptide has six cystine residues connected with intramolecular disulphide bonds. The connectivity of these disulphide bonds as well as the number of residues are factors differentiating α- and β-defensins [54]. The importance of a balance between the hydrophobicity of the defensins and their net positive charge is highlighted in the activity of these peptides towards the bacterial membrane [55]. The crystal formation of defensins usually results in the production of a dimeric structure. Crystallographic studies of α- and β-defensins show that free-form α-defensins have three intramolecular disulphide bonds (Paneth cell defensins and innate immunity of the small bowel) and they form a dimeric structure with six β-sheets [56]. The monomeric structure of β-defensins consists of three β-sheet folds as well as a helical N-terminus [16]. Nevertheless, defensins undergo conformational changes when introduced to a bacterial membrane. Targeted approaches on a specific protein may render the AMPs useless against the bacteria due to bacterial resistance and genetic changes to its conformation. Therefore, it is important that defensins work as non-specific agents on the membrane of the bacterial cells. Bacteria resistant to antimicrobial peptide activity usually display enzymatic covalent modification on their membrane, which reduces their negative charge.

α- and β-defensins are generally known for their cluster of positively charged amino acid residues. Although the sequence of amino acids is highly variable in defensins, their cysteine residues and their framework are highly conserved [17]. DEFA1-3 and human β-defensins are stored as mature peptides, whereas DEFA5 is stored in its pro-peptide form [57, 58]. The dimerization of β-defensins in solution is shown to be the functional structure for recognition of microorganisms [16]. However, DEFA1 takes a non-dimeric structure in solution, forming a

**51**

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

voltage-dependent channel in the planar lipid bilayer [59]. This specific example also shows that the interaction of DEFA1 with the membrane phospholipid depends on the presence of anionic phospholipids on the membrane. On the other hand, DEFA2, an α-defensin similar to DEFA1 (lack of alanine at position 1), requires the assembly of approximately two dimers for the formation of pores in uni-lamellar vesicles [60]. DEFA1 and DEFA2 are the potent forms of α-defensins and DEFA3, differing by only an additional amino acid at the N terminus, is known to be less active against *C. albicans* [61]. The dome-shaped, three-dimensional structure of amphiphilic defensins has been hypothesized to have the N- and C-termini at the two ends and the hydrophobic section having an amphiphilic structure at the lowest portion. The functional diversity of defensins and their potency mainly depend on the N- and C-terminal residues [26]. A study showed that arginine-rich cationic defensins provide a higher spectrum of antimicrobial activity due to their higher cathodal electrophoretic

The concentration of defensins in epithelial cells averages 10–100 μg ml<sup>−</sup><sup>1</sup> although the uneven distribution of these molecules results in a higher local concentration [17]. Defensins attack Gram-positive and Gram-negative bacteria with the same mechanisms; however, the attack is on the cell wall and the outer membrane of the bacteria, respectively. Accumulation of cationic peptides close to the negatively charged surface in Gram-negative bacteria may lead to binding and crossing of defensins into the cell via a charge-exchange mechanism by competing with Ca2+ and Mg2+ bound to lipopolysaccharides [63]. Although the porous surface of Gram-positive bacteria allows ease of movement for the defensins [64], safe passage of defensins through the cell wall allows via the same mechanisms to attack the membrane bilayer. Generally, defensins are known to interact with lipopolysaccharides, polysaccharides and phospholipids of Gram-negative, Gram-positive and

Defensins are active against bacteria at a concentration of 1–10 μg in optimal conditions such as low ionic strength conditions, low concentrations of proteins or other substances interfering with this activity. However, cellular conditions are harsh and the salt as well as protein concentrations inhibit defensins' antimicrobial activity depending on the sequence and bacterial target of the defensin [66]. In addition, higher concentrations of defensins have been shown to have toxic effect towards mammalian cells, specifically lung tissue [67]. Permeabilization of defensins renders the production and synthesis of DNA and their subsequent RNA and protein. The extent of interaction of defensins with the bacteria depends on the amino acid backbone and the flexibility of this chain allowing for the presence of potential spatial interactions with the head of phospholipid groups in the

Defensins provide their antimicrobial activity by the creation of pores or membrane disruption, which both lead to the release of cellular contents [69]. Other regulatory factors such as wound closures [70], fibroblast proliferation and chemotaxis of T-cells and dendritic cells can also be mentioned as the activity of defensins. Defensins also have a modulatory effect on the production of cytokines [71]. Other activities of defensins include regulation of immune and inflammatory regions by providing chemotactic activity for monocytes, T-cells and dendritic cells. In addition, nanomolar concentrations of defensins can also activate nifedipine-sensitive calcium channels of mammalian cells, reducing the electrical

Overall, defensins are important molecules for providing a cascade of antimicrobial activity in the human body in defence against pathogens and they are mainly

concentrated in regions that are more prone to pathogenic attack.

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

bacterial membrane bilayer, respectively [65].

mobility [62].

membrane [68].

conduction [72].

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

voltage-dependent channel in the planar lipid bilayer [59]. This specific example also shows that the interaction of DEFA1 with the membrane phospholipid depends on the presence of anionic phospholipids on the membrane. On the other hand, DEFA2, an α-defensin similar to DEFA1 (lack of alanine at position 1), requires the assembly of approximately two dimers for the formation of pores in uni-lamellar vesicles [60]. DEFA1 and DEFA2 are the potent forms of α-defensins and DEFA3, differing by only an additional amino acid at the N terminus, is known to be less active against *C. albicans* [61]. The dome-shaped, three-dimensional structure of amphiphilic defensins has been hypothesized to have the N- and C-termini at the two ends and the hydrophobic section having an amphiphilic structure at the lowest portion. The functional diversity of defensins and their potency mainly depend on the N- and C-terminal residues [26]. A study showed that arginine-rich cationic defensins provide a higher spectrum of antimicrobial activity due to their higher cathodal electrophoretic mobility [62].

The concentration of defensins in epithelial cells averages 10–100 μg ml<sup>−</sup><sup>1</sup> although the uneven distribution of these molecules results in a higher local concentration [17]. Defensins attack Gram-positive and Gram-negative bacteria with the same mechanisms; however, the attack is on the cell wall and the outer membrane of the bacteria, respectively. Accumulation of cationic peptides close to the negatively charged surface in Gram-negative bacteria may lead to binding and crossing of defensins into the cell via a charge-exchange mechanism by competing with Ca2+ and Mg2+ bound to lipopolysaccharides [63]. Although the porous surface of Gram-positive bacteria allows ease of movement for the defensins [64], safe passage of defensins through the cell wall allows via the same mechanisms to attack the membrane bilayer. Generally, defensins are known to interact with lipopolysaccharides, polysaccharides and phospholipids of Gram-negative, Gram-positive and bacterial membrane bilayer, respectively [65].

Defensins are active against bacteria at a concentration of 1–10 μg in optimal conditions such as low ionic strength conditions, low concentrations of proteins or other substances interfering with this activity. However, cellular conditions are harsh and the salt as well as protein concentrations inhibit defensins' antimicrobial activity depending on the sequence and bacterial target of the defensin [66]. In addition, higher concentrations of defensins have been shown to have toxic effect towards mammalian cells, specifically lung tissue [67]. Permeabilization of defensins renders the production and synthesis of DNA and their subsequent RNA and protein. The extent of interaction of defensins with the bacteria depends on the amino acid backbone and the flexibility of this chain allowing for the presence of potential spatial interactions with the head of phospholipid groups in the membrane [68].

Defensins provide their antimicrobial activity by the creation of pores or membrane disruption, which both lead to the release of cellular contents [69]. Other regulatory factors such as wound closures [70], fibroblast proliferation and chemotaxis of T-cells and dendritic cells can also be mentioned as the activity of defensins. Defensins also have a modulatory effect on the production of cytokines [71]. Other activities of defensins include regulation of immune and inflammatory regions by providing chemotactic activity for monocytes, T-cells and dendritic cells. In addition, nanomolar concentrations of defensins can also activate nifedipine-sensitive calcium channels of mammalian cells, reducing the electrical conduction [72].

Overall, defensins are important molecules for providing a cascade of antimicrobial activity in the human body in defence against pathogens and they are mainly concentrated in regions that are more prone to pathogenic attack.

*Peptide Synthesis*

defensins [17].

**Figure 5.**

reduces their negative charge.

in the Paneth cells of the intestines [53]. β-Defensins (DEFB1–4) on the other hand are mainly produced in the epithelial cells. These regions have the highest concentration of defensins due to the higher susceptibility to a pathogenic attack, which renders them weaker and subsequently in need of the immunity provided by

*(1) bone-marrow promyelocyte; (2) neutrophil; (3) phagocytosis.*

*Schematic diagram of production of α-defensins and the phagocytosis of pathogens with the aid of defensins:* 

β-Defensins mature into a secreted peptide after pre-pro-peptide state. This mature peptide has six cystine residues connected with intramolecular disulphide bonds. The connectivity of these disulphide bonds as well as the number of residues are factors differentiating α- and β-defensins [54]. The importance of a balance between the hydrophobicity of the defensins and their net positive charge is highlighted in the activity of these peptides towards the bacterial membrane [55]. The crystal formation of defensins usually results in the production of a dimeric structure. Crystallographic studies of α- and β-defensins show that free-form α-defensins have three intramolecular disulphide bonds (Paneth cell defensins and innate immunity of the small bowel) and they form a dimeric structure with six β-sheets [56]. The monomeric structure of β-defensins consists of three β-sheet folds as well as a helical N-terminus [16]. Nevertheless, defensins undergo conformational changes when introduced to a bacterial membrane. Targeted approaches on a specific protein may render the AMPs useless against the bacteria due to bacterial resistance and genetic changes to its conformation. Therefore, it is important that defensins work as non-specific agents on the membrane of the bacterial cells. Bacteria resistant to antimicrobial peptide activity usually display enzymatic covalent modification on their membrane, which

α- and β-defensins are generally known for their cluster of positively charged amino acid residues. Although the sequence of amino acids is highly variable in defensins, their cysteine residues and their framework are highly conserved [17]. DEFA1-3 and human β-defensins are stored as mature peptides, whereas DEFA5 is stored in its pro-peptide form [57, 58]. The dimerization of β-defensins in solution is shown to be the functional structure for recognition of microorganisms [16]. However, DEFA1 takes a non-dimeric structure in solution, forming a

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