**4. Unconventional function of AMP**

#### **4.1 Regulatory activities of AMPs.**

Besides the role of endogenous antibiotics, the antimicrobial peptides have other functions in the inflammation; wound healing and regulation of the response immune response, of which are described below.

Microbial infection of the mucosa and skin induces production of large quantities of small antimicrobial peptides, including defensins and cathelicidins, (Zasloff, 2002; Ganz, 2003a; Yang et al., 2004). They can act as chemokines, such as some β-defensins chemoattract immature iDC and other effector cells through the CCR6 receptor (Biragyn et al., 2002); (Niyonsaba et al., 2004) or human cathelicidin LL-37 recruits neutrophils, monocytes and mast cells via human formyl peptide receptor-like 1, FPRL1 (De et al., 2000) (Agerberth et

neutralize a broad range of microbes (bacteria, virus and protozoa). The ubiquitous nature of antimicrobial peptides suggests that their role in nature has been long standing and must have contributed to an organism's fitness. Many of these molecules exert mechanisms of action that appear to be unique and highly complex. However, AMPs exhibit varying, and in some cases, significant degrees of host cytotoxicity, reflecting non-selective cell targeting (Shin et al., 1999). It is likely that distinct antimicrobial peptides have evolved to function within specific physiologic and anatomic contexts to minimize their potential to concomitantly injuring the host cells. An intensive area of focus regarding antimicrobial peptide biochemistry relates to the precise mechanisms by which these molecules cause cell death. A long-held paradigm for microbicidal action has been that AMPs kill microorganisms by initiating multiple injuries in target microbial cell membranes. The principal theory suggest that peptides may create membrane pores in the organism, making a leakage of some metabolites, ensuing depolarization, loss of membrane-coupled respiration and biopolymer synthesis, and ultimately cell death. However, other authors suggest additional mechanisms, , where membrane permeabilization alone appears to be insufficient to cause cell death. These evidences come from studies documenting a clear dissociation between membrane perturbation and cell death. In these cases, cell killing may proceed in the absence of significant disruption in membrane architecture, due rather to disruptions in cellular function (Zhang et al., 2000). The functional integrity of the cytoplasmic membrane is crucial to essential functions of microbial pathogens, including gradient formation and selective permeability, cellular energetics, and synthesis of bio-

The general membrane effects of AMP are the membrane perturbation however alone may be insufficient for microbicidal effects of certain peptides. Permeabilization alone does not invariably result in staphylococcal death due antimicrobial peptides. Different peptides with varying staphylocidal potencies exhibited disparate capacities of membrane permeabilization and cell killing (Koo et al., 2001). Similar studies showed that gramicidin S rapidly depolarizes *Pseudomonas aeruginosa*, but did not kill it, suggesting that the concept of membrane perturbation and eventual cell killing may be independent (Zhang et al., 2000). Bacterial membrane energetic also appears to be involved in AMP mechanisms of action (Yeaman et al., 1998). It is now widely recognized that the AMP concept could play a promising role in fighting the presently raging microbial resistance to conventional

Besides the role of endogenous antibiotics, the antimicrobial peptides have other functions in the inflammation; wound healing and regulation of the response immune response, of

Microbial infection of the mucosa and skin induces production of large quantities of small antimicrobial peptides, including defensins and cathelicidins, (Zasloff, 2002; Ganz, 2003a; Yang et al., 2004). They can act as chemokines, such as some β-defensins chemoattract immature iDC and other effector cells through the CCR6 receptor (Biragyn et al., 2002); (Niyonsaba et al., 2004) or human cathelicidin LL-37 recruits neutrophils, monocytes and mast cells via human formyl peptide receptor-like 1, FPRL1 (De et al., 2000) (Agerberth et

molecules (Yeaman et al., 1998).

**4. Unconventional function of AMP** 

**4.1 Regulatory activities of AMPs.** 

which are described below.

antibiotics.

al., 2000; Niyonsaba et al., 2002). Defensins can also activate effector cells that can work together with the complement system to destroy microbial invaders. The α-defensins HNP1~3 have been reported to increase the production of TNFa and IL-1 while decreasing the production of IL-10 by monocytes (De et al., 2000). Some α-defensins enhance expression of adhesion molecules including ICAM-1, CD11b, and CD11c by neutrophils and facilitate the recruitment and enhance the microbicidal activity (Van Wetering et al., 1997; Chaly et al., 2000; Di Nardo et al., 2003) (FÈger et al., 2002). β-defensins induce mast cell degranulation and release of histamine and prostaglandin D2 (Yamashita and Saito, 1989; Befus et al., 1999; Niyonsaba et al., 2001) increase the expression of CXCL8 and CXCL5 (Van Wetering et al., 1997; van Wetering et al., 2002). Furthermore, murine β-defensin 2 has been shown to act directly on immature DCs as an endogeneous ligand for Toll like receptor 4 (TLR-4), inducing up regulation of co-stimulatory molecules and DC maturation, triggering robust, Th1 polarized adaptive immune responses in vivo (Biragyn et al., 2002). However, the mechanisms that regulate these functions are not well studied. Defensins attract inflammatory cells as neutrophils, B lymphocytes and macrophages. All these cells release inflammatory mediators such as IL-8, IFNγ, IL-6, IL-10 and LTB4. It is interesting that defensins may also have anti-inflammatory activity by the induction of IL-10 or SLPI.(Durr and Peschel, 2002; Zasloff, 2002).The synthesis of β-defensins by epithelial cells and the recruitment of peripheral blood granulocytes α-defensin-rich site of inflammation generates a high concentration of them. Also have direct antimicrobial effects, defensins facilitate and amplify the subsequent immune response. Indeed, spleen cells stimulated with α-defensins increase the production of human cytokines and lymphocyte proliferation. This same type of defensins, when administered to mice, produces increased serum IgG1, IgG2 and IgG2b. In addition, small amounts of HNP extend the antibody response against a singenic tumor (Tani et al., 2000).

These results indicate, without doubt, the AMPs have a role in the regulation of the immune response. On the other hand, recent studies have identified several structurally diverse endogenous mediators of innate immunity with certain features: firstly, they are rapidly released in response to infection or tissue injury; secondly, they have both chemotactic and activating effects on APCs; and thirdly, they exhibit particularly potent *in vivo* immunoenhancing activity and enhance DC differentiation from DC precursors. This subset of mediators alerts host defenses by augmenting innate and adaptive immune responses to tissue injury and/or infection. On the basis of their unique activities, they are called 'alarmins' (Oppenheim and Yang, 2005). Innate-immune mediators possessing alarmin activity include defensins, cathelicidin, eosinophil-derived neurotoxin (EDN), and high mobility group box protein 1 (HMGB1) (Oppenheim and Yang, 2005). The concept of alarmins is very interesting. This has only been observed in mammals, but is likely to exist in other groups of animals including insects. It has been observed overexpression of antimicrobial peptides during infection with various pathogens and even damage to the cuticle. Many groups of insects have been used to understand the basic characteristics of the innate immunity. However, surprisingly, the study of AMPs in insects has been limited study of their bactericidal or antiparasitic activity and virtually no information on the alternative role that could have the AMPs on the immune response in these organisms.

Differential analyses after bacterial or fungal challenge showed the regulation of more than a 100 molecules in adult *Drosophila* hemolymph (Levy et al., 2004). Using differential

Natural Antimicrobial Peptides from Eukaryotic Organisms 63

pro-inflammatory effects of the Gram-positive surface molecule lipoteichoic acid (Scott et al., 2002; Gutsmann et al., 2010) designed a new class of peptides synthetic anti-LPS peptides (SALPs). SALPs were originally based on the LPS-binding domain of the *Limulus* anti-LPS factor (LALF) but were substantially changed in length and primary sequence for optimal binding to the lipid A portion of LPS. They observed that these peptides are highly efficient in neutralization of LPS and blockage of its immunopathological consequences *in vitro* and *in vivo*. SALPs combine excellent selectivity for LPS, with high neutralizing activity *in vitro*  and potent protection to septic shock using the murine model *in vivo*. They also demonstrate the biological efficacy of rationally designed new synthetic antilipopolysaccharide peptides (SALPs) based on the *Limulus* anti-LPS factor for systemic application. Efficient inhibition of LPS-induced cytokine release and protection from lethal septic shock *in vivo* was analyzed, whereas cytotoxicity was not observed under physiologically relevant conditions and concentrations. It seems that the lipid A part of LPS is converted from its "endotoxic conformation," the cubic aggregate structure, into an inactive multilamellar structure. These

Apart from the antibacterial activity, AMPs also possess antiviral activity. For example, the α-defensins target the human immunodeficiency virus (HIV) activity by directly inactivating viral particles and affecting the ability of the virus to replicate within CD4 cells. Human α-defensins HNP-1 to -3 and HD-5 have been shown to block papillomavirus infection. Retrocyclin 2, a synthetic θ-defensin peptide that humans do not synthesize due to a mutation in the corresponding human gene, has the capacity to block influenza virus infection. Human β-defensins can also block HIV-1 replication, and interestingly, a single nucleotide polymorphism in a β-defensin gene has been associated with clinical manifestation of HIV-1 infection, suggesting that the human β-defensins play an important role in host defense against HIV. Cathelicidins, in contrast, have an inhibitory effect on lentiviral replication in vitro, and LL-37 appears capable of interfering with vaccinia virus replication in vitro and in mice. Dermaseptin S4, a 28-residue AMP isolated from frog skin, attenuates HIV infection in vitro. Other AMPs from frog skin including caerin 1.1, caerin 1.9, and maculatin 1.1 have also demonstrated inhibition of HIV in vitro(Albiol Matanic and Castilla, 2004).(Daher et al., 1986; Sinha et al., 2003; Yasin et al., 2004). Our group has worked with the peptide named scorpine from the venom of *Pandinus imperator* scorpion, where we observed a very interesting anti-virus dengue and anti-plasmodium activity (Carballar-Lejarazu et al., 2008). Scorpine is an antimicrobial peptide whose structure resembles a hybrid between a defensin and a cecropin. It exhibits antibacterial activity and inhibits the sporogonic development of parasites responsible for murine malaria. The recombinant expressed scorpine (RScp) in *Anopheles gambie* cells showed antibacterial activity against *Bacillus subtilis* and *Klebsiella pneumoniae*, at 5 and 10 µM, respectively. It also produced 98%mortality in sexual stages of *Plasmodium berghei* at 15 µM and 100% reduction in *Plasmodium falciparum* parasitemia at 5 µM. RScp also inhibited virus dengue-2 replication in C6/36 mosquito cells. In addition, we generated viable and fertile transgenic *Drosophila* that over-expresses and correctly secretes RScp into the insect hemolymph, suggesting that the generation of transgenic mosquitoes resistant to different pathogens may be viable. However, there is no knowledge of their mechanics, action. It is necessary to extend these studies with other peptides during infection

observations suggest a novel therapeutic role of AMPs.

induced with virus dengue and other pathogens.

**4.3 Anti-viral activity** 

MALDI-TOF MS, 28 peptides with a molecular mass below 15 kDa and belonging to different structural families were identified and could be classified into two groups. The first group contains the AMPs and their different isoforms. DIMs belonging to this group are likely to be effectors molecules of the immune response through their antimicrobial activity. The second group contains molecules for which the lack of similarity to any peptide prevents the proposition of any precise function. These peptides are suspected to serve as chemokines during the *Drosophila* immune response but the different approaches for investigating their role have so far been unsuccessful (Levy et al., 2004). On the other hand, our group has analyzed the peptides in the hemolymph of mosquitoes *An. albimanus* infected with malaria parasites. We found a complex pattern of peptides, including cecropin, which are released into the hemolymph. Similarly, gambicin, cecropin, and defensin are over-expressed in the intestinal epithelium and fat body of mosquitoes infected with *Plasmodium*. However, it is unknown whether these peptides participate in the elimination of the parasite. Cecropin has been consider an important AMP against Plasmodium, but in vitro assays with synthetic cecropin did not affect *Plasmodium* viability (unpublished results), but this peptide is over-expressed in mosquitoes infected with the parasite (Herrera-Ortiz, A. et al., 2010.). It would be interesting to analyze the peptides released into the hemolymph of these insects and their role in regulating the immune response.
