**2. Antimicrobial mechanisms of AMP**

The activity of AMPs must start at the cytoplasmic membrane since most AMPs permeabilize microbial membranes. Several models have been proposed on how AMPs insert into the membrane leading to the formation of ion channels, transmembrane pores or extensive membrane rupture. These models are: 1) transmembrane pore models and 2) nonpore models activity. Here we will also review other antimicrobial mechanisms that have been found. For example, the antimicrobial mechanisms of apidaecin do not rely on pore forming activity, this peptide does have antimicrobial activity at a concentration at least four order of magnitude below the concentration that disturbs the bacterial membrane. Peptides from each structural family have been reported to rely on antimicrobial mechanism that would not imply membrane depolarization of the target microorganism, suggesting internal molecular determinant. Certain peptides are unable to cause membrane depolarization at the minimal inhibitory concentration, while other cause maximal depolarization well below the MIC (Minimal Inhibitory Concentration) value. Evidences are mounting that involve particular macromolecules as well as intracellular functions as the final target for antimicrobial activity of the AMP. Even though, bacterial membranes are a necessary entry path for the AMP, therefore determining part of the AMP selectivity as well as efficiency.

#### **2.1 Transmembrane pore models of AMPs**

There are more than 1,000 known AMPs (Brahmachary et al., 2004; Wang and Wang, 2004; Fjell et al., 2007), and for the majority of them, there is little or no evidence for

Natural Antimicrobial Peptides from Eukaryotic Organisms 59

carpet and detergent models into a single idea in which the catastrophic collapse of membrane integrity includes membrane fragmentation. Others distinguish between the two models based on whether or not the peptide-induced leakage efficiency depends on the size of the entrapped solutes (Ostolaza et al., 1993). Bechinger and Lohner (Bechinger and Lohner, 2006; Salnikov et al., 2009) recently discussed molecular shape models in which AMP lipid interactions could be depicted with phase diagrams to describe the propensity of an AMP to permeabilize a membrane by disrupting the lipid packing. Epand and colleagues have proposed a lipid clustering model in which AMPs induce clustering or phase separation of lipids, with leakage occurring due to boundary defects (Epand et al., 2009; Epand et al., 2010). Almeida and colleagues have described AMP activity in terms of binding, insertion and perturbation using the sinking raft model, which they recently augmented by adding a formal thermodynamic analysis to predict activity (Pokorny and Almeida, 2004; Gregory et al., 2008; Almeida and Pokorny, 2009; Gregory et al., 2009; Almeida and Pokorny, 2010). Interfacial activity is defined as the propensity of an imperfectly amphipathic peptide to partition into the bilayer interface and drive the vertical rearrangement of the lipid polar and non polar groups. The disruption of the normally strict

segregation of polar and nonpolar groups causes membrane permeabilization.

**2.2 Non membrane mediated models of AMP activity** 

design AMPs.

**3. AMP classical functions** 

While these models, and others, have been useful in discussing AMPs, the molecular organization of the lipid bilayer undergoing solutes leakage in the presence of AMPs is still very much unknown. Without this knowledge, it remains challenging to predict structure– sequence activity relationships; thus, it also remains challenging to engineer or *de novo*

In addition to the pore models described above, AMP activity has been described using antimicrobial mechanism that do not involve bacterial membrane permeability impairement. For instance PR-39, a porcine proline-arginine-rich antibacterial peptide was found to lyse the microbes through a non pore forming mechanism, altering the microbe division and septum formation (Shi et al., 1996). Small Anionic antimicrobial peptides have been found in ovine lungs, that appear to function without the need for the initial electrostatic interaction with the bacterial membranes, and kill the bacteria through intracellular content flocculation (Brogden et al., 1998). Mersacindin, a lantibiotic, has been shown to inhibits bacterial cell wall formation (Brotz et al., 1997). Some antimicrobial peptides like the pleurocidin and dermaseptin inhibit bacterial DNA synthesis while buforin II and tachyplesin bind to nucleic acid in general (Yonezawa et al., 1992). The proline rich AMP family target proteicious or nucleic acid intracellular molecules (Park et al., 2008), indeed members of the proline-rich AMP family like Drosocin, pyrrhocoricin, and apidaecin

have been shown to act on bacterial GRO EL and Dna K proteins(Otvos et al., 2000).

The classical function of AMP has been their role as major effectors of the innate immune system; AMPs complement the highly specific but relatively slow adaptive immune system. Unlike the acquired immune mechanisms, endogenous AMPs, which are constitutively expressed or induced, provide fast and effective means of defense. Most of these geneencoded peptides are mobilized shortly after microbial infection and act rapidly to

transmembrane pores (Wimley, 2010). Instead, there is compelling evidence that many AMPs function by binding to membrane surfaces and disrupting the packing and organization of the lipids in a nonspecific way. The simplest models of membrane permeation by peptides involve the formation of membrane-spanning pores. These pores have been studied in lipid bilayers and have been proposed to be the major cause of bacterial membrane depolarization. As bacteria ATP synthesis is linked to the transmembranal potential of the cell, any perturbation of the ion partition may potentially lead to the cell death. The structure of these peptides induced pores have been analyzed and several pore structure were proposed.

The barrel stave pore model (Rapaport and Shai, 1991) involves a mechanism where the peptides interact laterally with one another to form a specific structure enclosing a waterfilled channel, much like a protein ion channel. Bioinformatic analysis of protegrin 1 insertion in membranes concluded that this model was most consistant with the observed energy of insertion of the peptide in artificial membranes (Langham et al., 2008). Furthermore the electrophysiology record analysis of Ceratotoxin and pleuricidin peptides inserted in lipid bilayers show that they form a peptide filled pore isolated from the lipids from the membranes. The pore formation dynamics correlates with their antimicrobial activity (Bessin, 2004).

In the toroidal pore model (Ludtke et al., 1996), specific peptide–peptide interactions are not present Instead, the peptides affect cooperatively the local curvature of the membrane, forming a peptide lipid toroid pore in the membrane. The cathelicidin peptide LL 37 (an amphipathic, alpha-helical, antimicrobial peptide) appears to form such kind of pore in the microbial membranes. NMR spectra studies of LL 37 shows that its pore channel is filled with the membrane lipids phosphate heads. The resulting ionic leakage ultimately leads to bacterial membrane depolarization (Henzler Wildman et al., 2003). The pore formation of cateslytin, a beta sheet conformation peptide, has been studied through patch clamp and NMR experiments, and has been found to fit a pore formation model involving transient dissymmetry between the phospholipid leafs of the membrane as a key ingredient to explain the formation of the pore (Jean-Francois et al., 2008)

The carpet model for AMP antimicrobial activity , originally described by Shai (Gazit et al., 1996), is the most commonly cited model of membrane destabilization by AMPs. The carpet/detergent model proposes that the accumulation of the peptides imbedded in the microbial membrane provoques perturbation in the membrane integrity. Antimicrobial peptides accumulate on the membrane surface with an orientation that is parallel to the membrane untill peptide concentration has reached a critical level (i.e., a peptide-rich ''carpet'' has formed on the membrane surface). Then permeabilization occurs, via global bilayer destabilization. PMAP-23, a cationic peptide member of the cathelicidin family, is considered to induce membrane permeability according to the Shai-Matsuzaki-Huang "carpet" model (Bocchinfuso et al., 2009). Cecropin P1, another alpha helical AMP, imbedded in reconstituted phospholipid bilayer is preferentially oriented nearly parallel to the surface of the lipid membranes, a position that is incompatible with the proteinaceous pore model, as demonstrated by polarized ATR-FTIR spectroscopy analysis(Gazit et al., 1996) The detergent model is also often cited to explain the catastrophic collapse of membrane integrity, observed with some AMPs at high peptide concentration (Ostolaza et al., 1993; Hristova et al., 1997; Bechinger and Lohner, 2006). Some authors combine the

transmembrane pores (Wimley, 2010). Instead, there is compelling evidence that many AMPs function by binding to membrane surfaces and disrupting the packing and organization of the lipids in a nonspecific way. The simplest models of membrane permeation by peptides involve the formation of membrane-spanning pores. These pores have been studied in lipid bilayers and have been proposed to be the major cause of bacterial membrane depolarization. As bacteria ATP synthesis is linked to the transmembranal potential of the cell, any perturbation of the ion partition may potentially lead to the cell death. The structure of these peptides induced pores have been analyzed and several

The barrel stave pore model (Rapaport and Shai, 1991) involves a mechanism where the peptides interact laterally with one another to form a specific structure enclosing a waterfilled channel, much like a protein ion channel. Bioinformatic analysis of protegrin 1 insertion in membranes concluded that this model was most consistant with the observed energy of insertion of the peptide in artificial membranes (Langham et al., 2008). Furthermore the electrophysiology record analysis of Ceratotoxin and pleuricidin peptides inserted in lipid bilayers show that they form a peptide filled pore isolated from the lipids from the membranes. The pore formation dynamics correlates with their antimicrobial

In the toroidal pore model (Ludtke et al., 1996), specific peptide–peptide interactions are not present Instead, the peptides affect cooperatively the local curvature of the membrane, forming a peptide lipid toroid pore in the membrane. The cathelicidin peptide LL 37 (an amphipathic, alpha-helical, antimicrobial peptide) appears to form such kind of pore in the microbial membranes. NMR spectra studies of LL 37 shows that its pore channel is filled with the membrane lipids phosphate heads. The resulting ionic leakage ultimately leads to bacterial membrane depolarization (Henzler Wildman et al., 2003). The pore formation of cateslytin, a beta sheet conformation peptide, has been studied through patch clamp and NMR experiments, and has been found to fit a pore formation model involving transient dissymmetry between the phospholipid leafs of the membrane as a key ingredient to

The carpet model for AMP antimicrobial activity , originally described by Shai (Gazit et al., 1996), is the most commonly cited model of membrane destabilization by AMPs. The carpet/detergent model proposes that the accumulation of the peptides imbedded in the microbial membrane provoques perturbation in the membrane integrity. Antimicrobial peptides accumulate on the membrane surface with an orientation that is parallel to the membrane untill peptide concentration has reached a critical level (i.e., a peptide-rich ''carpet'' has formed on the membrane surface). Then permeabilization occurs, via global bilayer destabilization. PMAP-23, a cationic peptide member of the cathelicidin family, is considered to induce membrane permeability according to the Shai-Matsuzaki-Huang "carpet" model (Bocchinfuso et al., 2009). Cecropin P1, another alpha helical AMP, imbedded in reconstituted phospholipid bilayer is preferentially oriented nearly parallel to the surface of the lipid membranes, a position that is incompatible with the proteinaceous pore model, as demonstrated by polarized ATR-FTIR spectroscopy analysis(Gazit et al., 1996) The detergent model is also often cited to explain the catastrophic collapse of membrane integrity, observed with some AMPs at high peptide concentration (Ostolaza et al., 1993; Hristova et al., 1997; Bechinger and Lohner, 2006). Some authors combine the

explain the formation of the pore (Jean-Francois et al., 2008)

pore structure were proposed.

activity (Bessin, 2004).

carpet and detergent models into a single idea in which the catastrophic collapse of membrane integrity includes membrane fragmentation. Others distinguish between the two models based on whether or not the peptide-induced leakage efficiency depends on the size of the entrapped solutes (Ostolaza et al., 1993). Bechinger and Lohner (Bechinger and Lohner, 2006; Salnikov et al., 2009) recently discussed molecular shape models in which AMP lipid interactions could be depicted with phase diagrams to describe the propensity of an AMP to permeabilize a membrane by disrupting the lipid packing. Epand and colleagues have proposed a lipid clustering model in which AMPs induce clustering or phase separation of lipids, with leakage occurring due to boundary defects (Epand et al., 2009; Epand et al., 2010). Almeida and colleagues have described AMP activity in terms of binding, insertion and perturbation using the sinking raft model, which they recently augmented by adding a formal thermodynamic analysis to predict activity (Pokorny and Almeida, 2004; Gregory et al., 2008; Almeida and Pokorny, 2009; Gregory et al., 2009; Almeida and Pokorny, 2010). Interfacial activity is defined as the propensity of an imperfectly amphipathic peptide to partition into the bilayer interface and drive the vertical rearrangement of the lipid polar and non polar groups. The disruption of the normally strict segregation of polar and nonpolar groups causes membrane permeabilization.

While these models, and others, have been useful in discussing AMPs, the molecular organization of the lipid bilayer undergoing solutes leakage in the presence of AMPs is still very much unknown. Without this knowledge, it remains challenging to predict structure– sequence activity relationships; thus, it also remains challenging to engineer or *de novo* design AMPs.

#### **2.2 Non membrane mediated models of AMP activity**

In addition to the pore models described above, AMP activity has been described using antimicrobial mechanism that do not involve bacterial membrane permeability impairement. For instance PR-39, a porcine proline-arginine-rich antibacterial peptide was found to lyse the microbes through a non pore forming mechanism, altering the microbe division and septum formation (Shi et al., 1996). Small Anionic antimicrobial peptides have been found in ovine lungs, that appear to function without the need for the initial electrostatic interaction with the bacterial membranes, and kill the bacteria through intracellular content flocculation (Brogden et al., 1998). Mersacindin, a lantibiotic, has been shown to inhibits bacterial cell wall formation (Brotz et al., 1997). Some antimicrobial peptides like the pleurocidin and dermaseptin inhibit bacterial DNA synthesis while buforin II and tachyplesin bind to nucleic acid in general (Yonezawa et al., 1992). The proline rich AMP family target proteicious or nucleic acid intracellular molecules (Park et al., 2008), indeed members of the proline-rich AMP family like Drosocin, pyrrhocoricin, and apidaecin have been shown to act on bacterial GRO EL and Dna K proteins(Otvos et al., 2000).
