**1.2 The antimicrobial peptides**

The antimicrobial peptides (AMPs) have been well conserved throughout the evolution and they ensure the organism's defense against a large number of pathogens. They serve as endogenous antibiotics that are able to rapidly kill bacteria, fungi and viruses. Interestingly, they are not toxic for the host cells. Taking into consideration the diversity of the living beings, it is presumed that a large number of specific antibiotic peptides have been developed during evolution, allowing a protection of each organism in various conditions and the last years it has clearly appeared that many of these peptides, in addition to their direct antimicrobial activity, also have a wide range of functions in modulating both innate and adaptive immunity. Most of these are small molecules (less than 40 aminoacids) but some can be proteins. To date more than 1414 antibacterial, antifungal and 107 antiviral peptides have been

<sup>\*</sup> These authors contributed equally

The Natural Antimicrobial Chromogranins/Secretogranins-Derived

Peptides – Production, Lytic Activity and Processing by Bacterial Proteases 181

*Lamina propria* is a conjunctive tissue composed of fibroblasts, immune cells and collagen. It also contains capillaries and lymphatic vessels. Epithelial cells, or enterocytes, are disposed on a single layer separating the *lumen* from the *lamina propria*. These cells are tightly bound by tight junctions forming an impermeable barrier to commensal flora and to pathogens. Brush-border *microvilli* are present on the apical surface of absorptive enterocytes, representing a large absorbing surface and allowing of microorganisms to the gut. In contrast, microfold cells are not present in *microvill*i (Figure 1); these cells express cathepsin E (a proteolytic enzyme) and Toll-like receptors able to secrete proinflammatory cytokines and chemokines. The main function of microfold cells is the transport of antigens from the

lumen to the subepithelial lymphoid tissue and thus to the adaptive immune system.

synthesize serotonin (5-HT) and numerous neuropeptides (Figure 1).

the proinflammatory bacterial molecules (Müller et al., 2005).

(Sarker, 1992).

**Lysozyme** 

**Lactoferrin** 

(Artym et al., 2005).

microbes are the same as in skin.

Several anatomical structures are present along the gastrointestinal tractus. Peyer's patches are lymphoid structures containing B and T cells, macrophages and dendritic cells (Figure 1). Lieberkühn crypts are found in the small intestine and they constitute the basis of the intestinal *villi* (Figure 1). They contain multipotent stem cells and cells, involved in gastrointestinal immunity. Two other cellular types are also present in intestine: i) goblet cells synthesize and secrete large quantities of mucin, ii) enterochromaffin cells that originate from neural crest

In addition to humoral and cellular immunity, non-immunological defense mechanisms represent an important line of intestinal defenses. Some of these protective factors have been amply documented: pancreatic and gastric juices, intestinal motility and intestinal flora

Mucosal epithelial cells and Paneth cells produce a variety of AMPs (defensins, cathelicidins, cryptdin related peptides, bactericidal/permeability increasing protein (BPI), chemokine CCL20 and bacteriolytic enzymes such as lysozyme and group IIA phospholipase A2 (Müller et al., 2005). In addition to their direct role in killing pathogenic microorganisms, AMPs are involved in attraction of leukocytes, alarming the adaptive immune system and neutralizing

Lysozyme is synthesized and secreted by Paneth cells, macrophages, neutrophils and epithelial cells (Mason & Taylor, 1975; Satoh et al., 1988). Its role and selectivity towards

Lactoferrin (LF) exhibits a wide spectrum of antimicrobial and immunotropic properties (Artym et al., 2005). In contrast to caseins, LF is particularly resistant to proteolytic degradation in alimentary tract. LF is absorbed from the intestine by means of specific receptors located on brush border cells. Orally administered LF stimulates both local and systemic immune responses. It suppresses the growth of pathogenic bacteria, while promoting the multiplication

Studies on mice showed LF to be protective against bacteremia and endotoxemia. LF inhibits the activity of proinflammatory cytokines, nitric oxide and reactive forms of oxygen. Furthermore, LF promotes the differentiation of T and B cells from their immature precursors and increases the activity of NK and LAK (lymphokine activated killer) cells

of non-pathogenic *Lactobacillus sp.* and *Bifidobacterium sp.* (Artym et al., 2005).

identified, (antimicrobial peptides database http://aps.unmc.edu/AP/main.php), including peptides from several tissues and cell types from invertebrates, plants and mammals (Wang Z. & Wang G. 2004). Among them are found cytokines and chemokines, several neuropeptides and fragments derived from proteins exhibiting antimicrobial activity. They carry an average of 40-50 percent hydrophobic residues in such a structure that the folded peptide adopts an amphipathic profile. These properties are important for their microbial killing mechanism: the cationic character of AMPs induces an electrostatic attraction to the negatively-charged phospholipids of microbial membranes and their hydrophobicity aids the integration into the microbial cell membrane, leading to membrane disruption. Furthermore, the amphipathic structure also allows the peptides to be soluble both in aqueous environments and in lipid membranes (Yeaman & Yount 2003).

In mammals, the most well studied AMPs are human defensins and cathelicidins (Zanetti, 2004; Yang et al., 2002). Furthermore, some large proteins such as lysozyme, caseins, hemoglobin, lactalbumin, secretory phospholipase A2 and lactoferrin display antimicrobial activity against numerous microorganisms. Several of them, such as lysozyme and phospholipase A2 are ubiquitous and secreted by a large number of cells (*i.e*. epithelial cells, leukocytes and Paneth cells in the small intestine) (Keshav, 2006).

Because a large number of AMPs were identified in gut and skin, in the first part of this chapter we report a review of the well-studied AMPs expressed in these tissues and in the second part we present recent data relative to the new active CGs-derived peptides in relation with pathogens involved with intestine diseases, skin infections and sepsis.

### **1.3 Gut and antimicrobial peptides**

Gastrointestinal mucosa is a large host-environmental interface, showing a remarkable organization (Figure 1) and operating several functions including the digestive absorptive processes and the nutrients peristaltism, but also a physical and immunological protection of the body against microbes and a reconnaissance between commensal and pathogenic microorganisms.

Fig. 1. Schematic representation of the gut epithelium. The different cellular actors involved in innate and/or adaptative immunity are represented. (DC: dendritic cells ; EC: enterochromaffin cells.)(According to Metz-Boutigue, M.H. et al., Curr Pharm Des.2010;16(9):1024-39).

*Lamina propria* is a conjunctive tissue composed of fibroblasts, immune cells and collagen. It also contains capillaries and lymphatic vessels. Epithelial cells, or enterocytes, are disposed on a single layer separating the *lumen* from the *lamina propria*. These cells are tightly bound by tight junctions forming an impermeable barrier to commensal flora and to pathogens. Brush-border *microvilli* are present on the apical surface of absorptive enterocytes, representing a large absorbing surface and allowing of microorganisms to the gut. In contrast, microfold cells are not present in *microvill*i (Figure 1); these cells express cathepsin E (a proteolytic enzyme) and Toll-like receptors able to secrete proinflammatory cytokines and chemokines. The main function of microfold cells is the transport of antigens from the lumen to the subepithelial lymphoid tissue and thus to the adaptive immune system.

Several anatomical structures are present along the gastrointestinal tractus. Peyer's patches are lymphoid structures containing B and T cells, macrophages and dendritic cells (Figure 1). Lieberkühn crypts are found in the small intestine and they constitute the basis of the intestinal *villi* (Figure 1). They contain multipotent stem cells and cells, involved in gastrointestinal immunity. Two other cellular types are also present in intestine: i) goblet cells synthesize and secrete large quantities of mucin, ii) enterochromaffin cells that originate from neural crest synthesize serotonin (5-HT) and numerous neuropeptides (Figure 1).

In addition to humoral and cellular immunity, non-immunological defense mechanisms represent an important line of intestinal defenses. Some of these protective factors have been amply documented: pancreatic and gastric juices, intestinal motility and intestinal flora (Sarker, 1992).

Mucosal epithelial cells and Paneth cells produce a variety of AMPs (defensins, cathelicidins, cryptdin related peptides, bactericidal/permeability increasing protein (BPI), chemokine CCL20 and bacteriolytic enzymes such as lysozyme and group IIA phospholipase A2 (Müller et al., 2005). In addition to their direct role in killing pathogenic microorganisms, AMPs are involved in attraction of leukocytes, alarming the adaptive immune system and neutralizing the proinflammatory bacterial molecules (Müller et al., 2005).

### **Lysozyme**

180 Antimicrobial Agents

identified, (antimicrobial peptides database http://aps.unmc.edu/AP/main.php), including peptides from several tissues and cell types from invertebrates, plants and mammals (Wang Z. & Wang G. 2004). Among them are found cytokines and chemokines, several neuropeptides and fragments derived from proteins exhibiting antimicrobial activity. They carry an average of 40-50 percent hydrophobic residues in such a structure that the folded peptide adopts an amphipathic profile. These properties are important for their microbial killing mechanism: the cationic character of AMPs induces an electrostatic attraction to the negatively-charged phospholipids of microbial membranes and their hydrophobicity aids the integration into the microbial cell membrane, leading to membrane disruption. Furthermore, the amphipathic structure also allows the peptides to be soluble both in aqueous environments and in lipid

In mammals, the most well studied AMPs are human defensins and cathelicidins (Zanetti, 2004; Yang et al., 2002). Furthermore, some large proteins such as lysozyme, caseins, hemoglobin, lactalbumin, secretory phospholipase A2 and lactoferrin display antimicrobial activity against numerous microorganisms. Several of them, such as lysozyme and phospholipase A2 are ubiquitous and secreted by a large number of cells (*i.e*. epithelial cells,

Because a large number of AMPs were identified in gut and skin, in the first part of this chapter we report a review of the well-studied AMPs expressed in these tissues and in the second part we present recent data relative to the new active CGs-derived peptides in

Gastrointestinal mucosa is a large host-environmental interface, showing a remarkable organization (Figure 1) and operating several functions including the digestive absorptive processes and the nutrients peristaltism, but also a physical and immunological protection of the body against microbes and a reconnaissance between commensal and pathogenic

Fig. 1. Schematic representation of the gut epithelium. The different cellular actors involved in innate and/or adaptative immunity are represented. (DC: dendritic cells ; EC: enterochromaffin

cells.)(According to Metz-Boutigue, M.H. et al., Curr Pharm Des.2010;16(9):1024-39).

relation with pathogens involved with intestine diseases, skin infections and sepsis.

leukocytes and Paneth cells in the small intestine) (Keshav, 2006).

membranes (Yeaman & Yount 2003).

**1.3 Gut and antimicrobial peptides** 

microorganisms.

Lysozyme is synthesized and secreted by Paneth cells, macrophages, neutrophils and epithelial cells (Mason & Taylor, 1975; Satoh et al., 1988). Its role and selectivity towards microbes are the same as in skin.

#### **Lactoferrin**

Lactoferrin (LF) exhibits a wide spectrum of antimicrobial and immunotropic properties (Artym et al., 2005). In contrast to caseins, LF is particularly resistant to proteolytic degradation in alimentary tract. LF is absorbed from the intestine by means of specific receptors located on brush border cells. Orally administered LF stimulates both local and systemic immune responses. It suppresses the growth of pathogenic bacteria, while promoting the multiplication of non-pathogenic *Lactobacillus sp.* and *Bifidobacterium sp.* (Artym et al., 2005).

Studies on mice showed LF to be protective against bacteremia and endotoxemia. LF inhibits the activity of proinflammatory cytokines, nitric oxide and reactive forms of oxygen. Furthermore, LF promotes the differentiation of T and B cells from their immature precursors and increases the activity of NK and LAK (lymphokine activated killer) cells (Artym et al., 2005).

The Natural Antimicrobial Chromogranins/Secretogranins-Derived

Peptides – Production, Lytic Activity and Processing by Bacterial Proteases 183

they are synthesized in germ-free conditions (Putsep et al., 2000) and/or prenatally (Mallow et al., 1996). In transfected mouse, it was shown that the alpha-defensin hBD-5 protects efficiently against *Salmonella typhimurium*, demonstrating the direct antimicrobial effect of this peptide. In mouse, alpha-defensins are named cryptdins and several families of peptides related to cryptdins are regrouped under the term CRS (Cryptdin Related Sequences). Interestingly, these CRS can form homo- or heterodimers, thus allowing a

Beta-defensins are expressed in enterocytes of the small and large intestines. 28 beta-defensin encoding genes have been identified in human genome, but only 8 were found to be expressed. hBD-1 is constitutively expressed in absence of stimulus or bacterial infection (O'Neil et al., 1999), while some nutrients can stimulate its production in cell lines (Sherman et al., 2006). In mouse, an infection by the *Cryptosporidium* parasite resulted in a down-regulation of mBD-1 (Zaalouk et al., 2004), while *in vitro*, sporozoites are killed by this defensin. Some authors conclude on an unique and important regulation of hBD-1, during small intestine infections (Dann et Eckmann, 2007). hBD-2 is not constitutively expressed, but is induced by an infection or by proinflammatory stimuli (O'Neil et al., 1999). hBD-3 and -4 are inducible and particularly expressed in crypt regions (Fahlgren et al., 2004). Defensins can also act as

Bactericidal/permeability-increasing protein (BPI), a constituent of primary neutrophil granules, is a potent natural antibiotic and anti-BPI antibodies are detected during infectious enteritis. In addition, BPI is a target antigen for anti-neutrophil cytoplasmic antoantibodies in inflammatory bowel diseases such as Crohn's disease and ulcerative colitis (Walmsley et

Enterochromaffin cells (EC) (Siddique et al., 2009) (Figure 1) are enteroendocrine cells present in the intestine, especially colon (Kuramoto et al., 2007) and containing large amounts of serotonin (5-HT). These cells can sense luminal content before its basolaterally release, and activate afferent neuron endings within *lamina propria*, allowing information exchange between gut and central nervous system (Hansen & Witte et al., 2008). Besides this important role, EC secrete also numerous other products, among which VIP (Zanner et al., 2004), Substance P (Heitz et al., 1976), CgA, CgB and secretogranin II/CgC (Cetin & Grube,

Despite the crucial role of these cells, their sparse repartition and their low number did not allowed their extensive study. However, the BON cells were proposed as a model (Kim et al., 2001), that will enhance further research. When EC were stimulated by odors, they released serotonin, showing that these cells can also be stimulated by spices and fragrances (Braun et al., 2007). Moreover, a new method was proposed allowing isolating and purifying

Mammal skin is an essential defense barrier against external aggressions, such as microbial pathogens, oxidant stress, chemical aggressions, mechanical insults, burns etc. For a long time,

combinatorial diversity to struggle against pathogens (Hornef et al., 2004).

chemotactic agents for immune cells in a similar way to that described for the skin.

**Bactericidal Permeability Increasing protein** 

1991) and melatonin (Raikhlin et Kvetnoy, 1976).

EC from biopsies (Modlin et al., 2006).

**1.4 Skin and antimicrobial peptides** 

al., 1997).

**Neuropeptides** 
