Review Article Antimicrobial Peptides: Their Role As Infection .

BioMed Research International3Table 1: Representative antimicrobial peptides of different classifications (modified from anatinTachyplesinsProtegrinsPlant defensin VrD2PlectasinInsect defensin nTritrpticinHistatinsPR-39HostMammal: humanInsect: mothInsect: honey beeAmphibian: frogAve: chickenInsect: soldier bugArthropod: horseshoe crabMammal: pigPlant: mung beanFungus: ebony cupInsect: northern blow flyMammal: humanMammal: humanMammal: rhesus monkeyMammal: cowMammal: pigMammal: humanMammal: pigof two to three antiparallel 𝛽-sheets stabilized by three tofour intramolecular disulfide bonds; however in some casesan 𝛼-helical or unstructured segment is found at the Nor C-terminus. Unlike the 𝛼-helical antimicrobial peptides,which are unstructured in aqueous solutions, the defensinsmaintain a compact globular structure under such conditions [12, 13]. Apart from overall similarity in secondarystructure, most mammalian-derived 𝛼-defensins possess twoadditional common features, namely, a protruding loopresulting from a conserved arginine/glutamate salt bridgeand a 𝛽-bulge caused by a conserved glycine-X-cysteine (X:any amino acid) motif between the first and second cysteineresidues [13].2.3. Flexible Antimicrobial Peptides Rich in Specific AminoAcids. A minority of antimicrobial peptides contain a highproportion of certain amino acids such as proline, tryptophan, histidine, arginine, and glycine. Representative members of this class include the tryptophan rich bovine indolicidin and porcine tritrpticin, histidine rich human histatins,and the arginine and proline-rich porcine PR-39. Due totheir unusual amino acid compositions, these peptides havehighly variable secondary structures. The 13-amino acidindolicidin (ILPWKWPWWPWRR), for example, adopts alargely extended conformation in the presence of zwitterionic micelles composed of substances such as dodecylphosphocholine or anionic sodium-dodecyl sulfate [13].3. Mechanisms of Cell Specificity andSelectivity of Antimicrobial PeptidesInherent differences in the microbial versus the host cellmembrane composition and architecture aid selectivity of theantimicrobial peptides. Regulation of expression or localisation of the peptides is also thought to prevent unwantedinteractions with vulnerable host cells.3.1. Target Specificity and Selective Cell Toxicity. A biologicalmembrane can be thought of as simply a fluid mosaicconsisting of phospholipids interspersed with proteins. Indifferent organisms glycerides and sterols may also contributeto the biochemical architecture and surface topology of suchmembranes. There are, however, fundamental differences thatexist between microbial and animal cell membranes thatallow the antimicrobial peptides to distinguish between thesecells and selectively target one over the other as sketched inFigure 1 [9].3.2. Membrane Composition, Charge, and Hydrophobicity.The core component of almost all natural biomembranesis the phospholipid bilayer. These bilayers are amphipathic,meaning they have both hydrophobic and hydrophilicregions. However, eukaryotic and prokaryotic cell membranes differ significantly in terms of exact compositionand cell energetics (Figure 2). Phosphatidylcholine (PC) andits analogue sphingomyelin (SM) as well as phosphatidylethanolamine (PE) have no charge under physiological conditions [9]. Cholesterol and other sterols such as ergosterolwhich are abundantly found in eukaryotic membranes, butvery seldom in prokaryotic membranes, are also generallyneutrally charged (Figure 2) [15]. Hydroxylated phospholipids such as phosphatidylglycerol (PG), cardiolipin (CL),and phosphatidylserine (PS) possess a net negative chargeunder physiological conditions. It can be seen how the chargeof the membrane is mainly due to the ratio and location ofthe various phospholipids, with cell membranes comprisingmostly PG, CL, and PS, as is the case in most pathogenic bacteria, being very electronegative, whereas those membranesthat are rich in PC, PE, or SP tend to have a net neutral charge,as is the case in mammalian cell membranes [15, 16].3.3. Membrane Asymmetry. Although cellular membranesare neither symmetric nor static, differences between mammalian and microbial phospholipid bilayers can serve aspotential targets for antimicrobial peptides. In some cellssuch as the bovine erythrocyte, only 2% of the total PE content is located on the outer membrane leaflet [13]. Differencesin membrane symmetry, saturation of phospholipid bilayers,and compositional stoichiometry will influence the membrane’s fluidity and phase transition. In a similar manner, thecharge of the inner and outer leaflets of the cellular bilayermay also be different [15].3.4. Microbial Ligands and Receptors as Targets for Antimicrobial Peptides. Experiments have shown that D-and Lamino acid versions of antimicrobial peptides exhibit similarbinding affinities to targets cells, suggesting that stereospecific receptors are not involved in targeting pathogeniccells [9]. However, several studies appear to refute this andsuggest that certain proteins located in the microbial cellmembrane may serve as binding targets for certain classes of

4BioMed Research InternationalPrototypic plasmamembrane of a multicellularanimal letElectrostatic andhydrophobic interactionsHydrophobic interactionsBacterial holesterolAntimicrobial peptideAcidic lasmic5040302010CytoplasmicRelative composition (%)Figure 1: Membrane targeting of antimicrobial peptides and basis of their selectivity (adapted from [45]).Membrane constituentFigure 2: Comparative lipid architecture of microbial and humancytoplasmic membranes. Cytoplasmic membranes of bacterial(Escherichia coli, Staphylococcus aureus, or Bacillus subtilis) andfungal (Candida albicans) pathogens are compared with that ofthe human erythrocyte in relative composition and distributionbetween inner and outer membrane leaflets. Membrane constituentsranging from anionic (left) to neutral (right) are CL, PG, PE, PC,SM, and sterols (cholesterol or ergosterol, ST). Note the markeddifference among microbial pathogens and human erythrocytesresides in the phospholipid composition and asymmetry. Thesedifferences are believed to account for the selective antimicrobialpeptide affinity for microbial versus host cells to the extent thatit exists for a given antimicrobial peptide. Keys: open, E. coli;horizontal hatching, S. aureus; shaded, B. subtilis; checkered, C.albicans; solid, human erythrocyte (adapted from [9]).antimicrobial peptides such as histatins. This would supportthe findings why histadins are involved in local defencemechanisms with particular type of pathogens and have beenrecovered in dental or skin wounds. Some researchers alsopostulate that anionic components of cell membranes, forexample, CL, PG, or lipopolysaccharide (LPS), may serveas pseudoreceptors, enabling the initial interaction betweenthe antimicrobial peptide and the microbial cell target [13].Hence, antimicrobial-binding receptors may be an alternativepathway of AMP interaction with the bacterial cell envelop.3.5. Transmembrane Potential. The transmembrane potentialis yet another way in which microbial and mammalian cellsvary and it is in the charge separation that exists betweenthe inner and outer layers of the cytoplasmic membrane. Anelectrochemical gradient, resulting from the differing ratesor proton exchange across the cell membrane, is referred toas the transmembrane potential (Δ𝜓). A normal mammaliancell has a Δ𝜓 between 90 and 110 mV in range. Pathogenicbacteria, however, generally exhibit Δ𝜓 in the 130 to 150 mV range. This significant difference in electrochemicalpotential may be another factor that allows antimicrobialpeptides to distinguish between host and target cells [9].4. Selective Toxicity Based onAntimicrobial Peptide DesignIn the aqueous intercellular environment, many antimicrobial peptides are believed to adopt extended or unstructuredconformations, although this may not be the case if there areintramolecular bonds present, which will ensure a specificconformation in a variety of environments due to inducedrigidity. Once the antimicrobial peptide binds to the cellmembrane of a pathogenic microbe, it may undergo significant conformational change and adopt a specific conformation, such as a 𝛼-helix. Studies suggest that dynamic and/orinherent conformations of antimicrobial peptides have aneffect on their selective cytotoxicity [9, 17, 18]. Additionally,antimicrobial peptides may undergo conformational transition, self-association, or oligomerization within the target

BioMed Research Internationalpathogen membrane, but not the host cell membrane toincrease cell-specific toxicity [13]. Zhang and coworkers [16]employed synthetic test peptides that were uniformly cationicbut varied in conformation and included extended, cyclic,𝛼-helical, and 𝛽-sheet structures. It was determined thatall test peptides were able to interact with and penetratelipid monolayers composed of PG, a negatively chargedphospholipid. However, only the 𝛼-helical and extendedpeptides were able to interact with the more neutrally chargedPC membrane. In the same study it was also found that 𝛽sheet peptides were able to translocate phospholipids fromthe inner to the outer leaflet at concentrations that were lowerthan those that were required to permeabilize the membrane.Similarly, Kol and coworkers [19] showed that peptides withcomparable conformation, but rich in histidine and lysineand lacking in tryptophan, were also able to induce significantlevels of phospholipid translocation. It can be concluded fromthese studies that not only do antimicrobial peptides interactwith phospholipid membranes of only specific compositionand symmetry, but they are also able to affect remodelling ofthe membranes in specific cells.4.1. In Vivo Preferential Affinity for Microbial versus Mammalian Cells. Welling and colleagues [20] conducted an invivo experiment where they tested the binding affinity of aradiolabeled fragment of the cationic ubiquicidin antimicrobial peptide 99m Tc-UBI 29–41 for microbial cells as comparedto host cells. In the study, animals were infected with Candidaalbicans, Klebsiella pneumonia, or Staphylococcus aureus.Sterile inflammations were also induced in the thigh musclesof animals through injection of heat-killed microorganismsor purified LPS, to serve as controls. The radiolabeled peptides accumulated to a significant extent in the infected sitesrelative to sterile or noninfectiously inflamed parts of thebody. This in vivo experiment demonstrated that the peptidescould distinguish between host and microbial cells and alsoaccumulate at the infected sites. Through scintigraphic measurements it was determined that the radiolabeled peptidesaccumulated in infected tissues at a rapid rate and that therewas up to a fivefold increase in rates of accumulation ininfected tissues relative to noninfected tissues. This rapidlocalization was interpreted as the peptides having a higher orpreferential affinity for the target cell surface relative to thatof the host cell surface.4.2. Localisation of Cytotoxic Antimicrobial Peptides LimitsExposure of Vulnerable Host Tissues. It is possible that hostcell cytotoxicity is reduced in many multicellular organismsdue to their localization to tissues that are not vulnerableto their cytotoxic effects. In most animals these peptides aresecreted by cells onto relatively inert and robust surfaces suchas the epithelia of the intestines or lung, or in amphibians,onto the skin. These localities are most likely to interactwith potentially harmful microbes most frequently, and theexpression of most of the antimicrobial peptides is eitherconstitutive or rapidly inducible, to allow them to form partof the first defences against pathogens [9]. Another means ofprotecting sensitive host tissues from antimicrobial peptides5is by containing them within granules in the phagocytingleukocytes, which engulf pathogens and expose them tolethal concentrations of antimicrobial peptides and oxidizing agents. The defensin class of antimicrobial peptides isdeployed in this way, since they are some of the most toxic andleast selective of the host produced antimicrobial peptides.The slightly acidic microenvironment within the maturephagolysosome is also the most effective environment for thedefensins, as they exhibit maximum cytotoxicity under theseconditions [12].5. Mechanisms of AntimicrobialPeptide ActionThe generally conserved structures of antimicrobial peptides,across a wide variety of organisms, lend some clues as to theirmechanisms of action. They are almost exclusively amphipathic and cationic under physiological conditions, and this isbelieved to aid their target cell selectivity. The ideal antimicrobial peptide should have low host cell cytotoxicity but be toxicto a wide range of pathogenic microbes. The antimicrobialdeterminants should be easily accessible and should not beprone to change or alteration. In general, antimicrobial peptides have amphipathic structures that allow them to interactwith phospholipid membranes, structures that are essentialto all pathogens [17]. Parameters such as conformation (𝑋),hydrophobicity (𝐻), hydrophobic moment (𝑀𝐻), charge (𝑄),polar angle (𝜃), and amphipathicity (𝐴) are all importantto the functioning of antimicrobial peptides. Furthermore,all these determinants are interrelated and modification ofone of these features will lead to alteration of the others[9].5.1. Conformation (𝑋). Although antimicrobial peptides maybe found in a wide range of host organisms and have differingamino acid sequences, they can be classified into a fewdiscrete groups based on their secondary structure. The twolargest groups include peptides that possess a 𝛽-sheet or 𝛼helical secondary structure. The majority of the remainingantimicrobial peptides are those that have an unusually highproportion of one or more amino acids such as tryptophanor proline and arginine. The 𝛼-helical peptides are frequentlyfound in the intercellular fluid of insects and amphibians andgenerally adopt an unstructured or extended conformation inaqueous solution, only adopting their helical structure uponinteraction with a phospholipid membrane [21]. The reasonfor this is that the intramolecular hydrogen bonding requiredfor an 𝛼-helic conformation is disrupted in a polar solventsuch as water. In a membrane, the polar hydrogen bondinggroups are shielded from lipophilic (apolar) membrane environment through 𝛼-helic formation. The helix conformationalso exposes the apolar side chains to the neutral lipidenvironment inside the membrane. Although the primarystructure of the 𝛽-sheet class of antimicrobial peptides showsa level of dissimilarity in amino acid sequence, they allshare common features with regard to amphipathic structure,possessing distinct hydrophilic and hydrophobic domains[9].

65.2. Charge (𝑄). Most of the antimicrobial peptides areoverall cationic and have charges ranging from 2 to 9, withmany possessing highly defined negatively charged domains.This positive charge is important for the initial attractionto and interaction with the anionic cellular membranes ofbacteria and other pathogenic microorganisms. Likewisethe relatively less anionic membranes of the host do notelectrostatically attract the antimicrobial peptides and mayconfer some target cell selectivity to the peptides. Pathogenicbacteria are generally rich in acidic phospholipids such asCL, PG, and PS. Additionally the teichoic and teichuronicacids of the cell walls of Gram-positive bacteria and the LPSof Gram-negative bacteria confer additional electronegativecharge to the bacterial cell surface. It has been determinedthat the Δ𝜓 of bacteria is typically 50% higher than that ofmammalian cells and it has been proposed that antimicrobialpeptides may be concentrated onto the surface of pathogenicmicrobes in an electrophoretic manner [22]. Although manystudies were able to correlate the cationicity of antimicrobialpeptides with their antimicrobial activity, a strictly linearrelationship does not exist. Dathe and coworkers [23] demonstrated in studies with analogues of magainin that increasingthe cationicity from 3 to 5 resulted in an increase inantibacterial activity against both Gram-positive and Gramnegative species. They did, however, note that there wasa limit to cationicity, after which any increases in positivecharge no longer increase antibacterial activity. It is believedthat this decrease in antibacterial activity may have been dueto the peptides binding so strongly to the negatively chargedphospholipid head group that translocation of the peptideinto the cell was impossible [9].5.3. Amphipathicity (𝐴) and Hydrophobic Moment (𝑀𝐻).Amphipathicity is a nearly universal feature amongst antimicrobial peptides and is achieved through a number of different peptide structures. The amphipathic 𝛼-helix is oneof the most common and simplest of these features. Byalternating anionic and cationic amino acid residues at everythree to four positions the peptide is able to adopt a secondarystructure that allows for optimal electrostatic interactionwith amphipathic phospholipid membranes (Figure 3). Thisfeature allows the peptide to exert cytotoxic activity towardsnot only negatively charged cell membranes but also thosewith a neutral charge or amphipathic nature [14].Amphipathicity of a peptide can be described by itshydrophobic moment (𝑀𝐻) which can be calculated as thevectorial sum of individual amino acid hydrophobicities,normalized to an ideal helix. An increase in hydrophobicmoment correlates to increased permeabilization of the targetcell membrane. This is especially significant in interactionswith lipid membranes that are neutrally charged, wherecharge factors are unlikely to bring about the requiredattraction to and interaction with the target cell membrane[17]. Like the 𝛼-helical antimicrobial peptides, the 𝛽-sheethost defence peptides also exhibit amphipathicity. This ismanifested as varying numbers of 𝛽-strands organised toform hydrophobic and hydrophilic surfaces. The 𝛽-strands,which are often antiparallel, are stabilised by regularly spacedBioMed Research Internationaldisulphide bonds or by cyclisation of the peptide backbone.This intramolecular bonding allows 𝛽-sheet antimicrobialpeptides to maintain a rigid conformation even in aqueousextracellular fluid and also facilitates multimerization, as thehydrophobic surfaces will cluster together to avoid exposureto the aqueous environment. Although the exact mechanismsby which amphipathic antimicrobial peptides bring aboutmembrane disruption in the target cell membrane is undetermined at present, largely because the exact conformationof the peptides in the membranes is not known, studieshave shown that segregated amphipathicity in both 𝛼-helicaland 𝛽-sheet antimicrobial peptides has a profound effect onpeptide disruption of natural biomembranes [9].5.4. Hydrophobicity (𝐻). The hydrophobicity of a peptidemay be defined as the percentage of hydrophobic aminoacid residues making up its primary structure. For mostantimicrobial peptides the hydrophobicity is around 50% andis essential for the functioning of the peptide as it allowsthe peptide to interact with and penetrate into the phosp

-Helical Antimicrobial Peptides. Approximately to % of all antimicrobial peptides identi ed and studied to date contain predominant -helical structures. is may be due the relative ease with which these peptides are chemically synthesised, which allows for extensive charac-terisation in the laboratory. e se peptides usually consist