**3. Distribution and expression of cationic antimicrobial peptides**

#### **3.1. Organismal distribution**

CAPs are ubiquitous in nature and play a crucial role in first-line host defense against pathogens. Fish and marine invertebrates, because of their diversity and reliance on nonspecific innate immune defenses, have proved to be a particularly good source of novel CAPs with therapeutic potential (Otero-González, 2010; Smith and Fernandes, 2009).

#### **3.2. Expression**

groups: linear α-helical, β-sheet, loop, and extended structures enriched in certain amino acids such as Arg, Phe, Pro, Gly, Trp and His (Zasloff, 2002). Linear α-helical CAPs such as magainin, LL-37 and pleurocidin are usually unstructured in aqueous solution but adopt an α-helical conformation upon interaction with lipids, thereby acquiring antimicrobial activi‐ ty. They often contain a hinge in the middle, due to Gly or Pro residues, which enhances the ability of the peptide to enter the cell (Park et al., 2000). CAPs that form stable β-sheet struc‐ tures contain cysteine residues that are able to form disulphide bonds (usually 1-4 depend‐ ing on the type of CAP). These include the protegrins, defensins and drosomycins. Looped CAPs, thanatin and brevinin, contain a C-terminal loop carrying a strong positive charge (Fehlbaum et al., 1996). CAPs such as bactenecin, prophenin and indolicidin are enriched in specific amino acids, usually lack cysteines, and form linear or extended coils. CAPs are usu‐ ally produced as inactive pre-pro-peptides and proteolytically cleaved to release the mature

122 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

Pleurocidins comprise a family of linear α-helical CAPs consisting of a highly conserved amino-terminal signal sequence and a carboxy-terminal anionic segment flanking the more variable mature peptide of approximately 25 amino acids (Douglas et al., 2001; Patrzykat et al., 2003). This anionic portion may counteract the positively charged mature peptide, there‐ by keeping it inactive until cleaved. They show some sequence similarity to other fish CAPs, including piscidin, moronecidin and the recently discovered gaduscidin (Browne et al., 2011; Sun et al., 2007). Pleurocidins are encoded as clusters of genes comprised of four exons, and binding sites for transcription factors involved in host defense are located up‐

Pleurocidin variant NRC-04 is the best-studied of this family of peptides. Early studies showed that in 25% dodecylphosphatidylcholine (DOPC)/ dodecylphosphatidyl-ethanola‐ mine (DOPE) vesicles, it forms a structure containing between 12% and 24% α-helical con‐ tent (Yoshida et al., 2001). Supporting this, it was only weakly incorporated into neutral bilayers composed of a 7:3 mixture of DOPC and DOPE; however, it strongly associated with a slightly anionic bilayer of 7:3:1 DOPC, DOPE, and dodecylphosphatidylserine (DOPS), forming well-defined single ion channels (Saint et al., 2002). NMR studies have shown that in 0% DPC, pleurocidin has no identifiable well-defined secondary structure but in 30% TFE and in 10 mM DOPC micelles, it was 25% α-helical and this increased to 95% in 140 mM DOPC (Syvitski et al., 2005). The hydrophobic amino acids residues clustered on approximately 2/3 of the helix and hydrophilic residues on the remaining 1/3 of the helix.

**3. Distribution and expression of cationic antimicrobial peptides**

CAPs are ubiquitous in nature and play a crucial role in first-line host defense against pathogens. Fish and marine invertebrates, because of their diversity and reliance on non-

biologically active peptide.

**3.1. Organismal distribution**

stream of the promoters (Douglas et al., 2003).

**2.2. Pleurocidin**

### *3.2.1. Tissue specificity*

In mammals, CAPs are expressed in cells of the skin and mucosal surfaces, as well as blood cells such as platelets, monocytes/macropages, neutrophils, and mast cells (Guaní-Guerra et al., 2010). Tissues often express a cocktail of different CAPs with varying activities and the expression of a given CAP variant may be restricted to a specific cell type (see Wiesner and Vilcinskas, 2010). For example, of the six different α-defensins, four are produced predomi‐ nantly in neutrophils whereas two are secreted from Paneth cells of the small intestine.

Fish CAPs such as pleurocidin are expressed in a broad range of tissues and cell types in‐ cluding epithelial, immune and blood cells (Browne et al., 2011). Pleurocidin is produced by epithelial cells of the skin and gut (Cole et al., 1997; Douglas et al., 2001) as well as circulat‐ ing immune cells (Murray et al., 2007). Interestingly, different pleurocidin variants are pro‐ duced by different cell types (Douglas et al., 2003), underscoring the importance of screening multiple tissues to uncover the true diversity of CAPs.

#### *3.2.2. Developmental stage*

The presence of CAPs in neonates, who have yet to develop adaptive immunity, provides enteric protection and impacts the composition of the commensal microflora. Milk lactofer‐ rin, from which the CAP lactoferricin is produced by pepsin cleavage in the stomach, plays a major role in maternal and innate immunity (Jenssen and Hancock, 2009). Dermicidin YP-30 is important for maternal and early post-natal protection (Wiesner and Vilcinskas, 2010). Mouse cathelin-related antimicrobial peptide undergoes a developmental switch from constitutive intestinal epithelial expression in neonates to Paneth cell expression in adult in‐ testinal crypts (Menard et al., 2008).

Pleurocidin transcripts were detected early in development of winter flounder, when larvae are most susceptible to pathogen-induced mortality, and showed a progressive increase as development proceeded to adulthood (Douglas et al., 2001). Furthermore, different pleuroci‐ din variants were present at different stages of development, indicating that screening of CAP production from various developmental stages of an organism is an excellent means of discovering novel peptide variants.

#### *3.2.3. Constitutive or inducible*

CAPs may be expressed constitutively or inducibly in response to stress, poor nutrition, in‐ flammation (Wehkamp et al., 2003), injury or microbial infection (Redfern et al., 2011; Zasl‐ off, 2006), and exposure to environmental factors such as zinc (Talukder et al., 2011) or vitamin D (White, 2010). For example, hCAP-18/LL-37 protein and mRNA expression was up-regulated in neutrophils that had migrated to the inflamed gingival tissues of patients with chronic periodontitis (Turkoglu et al., 2011) and mycoplasma infection induces catheli‐ cidin expression in neutrophils of infected mice (Tani et al., 2011). Interestingly, increased endogenous glucocorticoid levels induced by psychological stress reduce CAP expression in mice, leading to increased susceptibility to infection (Aberg et al., 2007).

Expression of fish CAPs is also regulated in response to stress and disease (Douglas et al., 2003a; Sun et al., 2007), and novel variants are often induced by such stressors. Monitoring levels of CAPs in aquaculture settings provides early warning of immunosuppression due to chronic stress, and conversely induction of CAPs provides protection against anticipated stressful situations such as handling (Noga et al., 2011).
