review article antimicrobial peptides: their role as...

Review ArticleAntimicrobial Peptides Their Role as Infection-SelectiveTracers for Molecular Imaging

Thomas Ebenhan12 Olivier Gheysens3 Hendrick Gert Kruger2

Jan Rijn Zeevaart4 and Mike Machaba Sathekge1

1 Department of Nuclear Medicine University of Pretoria amp Steve Biko Academic Hospital Corner of Malherbe and Steve Biko RoadPretoria 0001 South Africa

2 Catalysis and Peptide Research Unit School of Health Sciences University of KwaZulu Natal E-Block 6th FloorWestville Campus University Road Westville Durban 3630 South Africa

3 Department of Nuclear Medicine University Hospitals Leuven Katholieke Universiteit Leuven Campus GasthuisbergHerestraat 49 3000 Leuven Belgium

4Department of Science and Technology Preclinical Drug Development Platform North West University11 Hoffman Street Potchefstroom 2520 South Africa

Correspondence should be addressed to Mike Machaba Sathekge mikesathekgeupacza

Received 7 June 2014 Accepted 29 July 2014 Published 27 August 2014

Academic Editor Filippo Galli

Copyright copy 2014 Thomas Ebenhan et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Antimicrobial peptides (AMPs) are a heterogeneous class of compounds found in a variety of organisms including humans andso far hundreds of these structures have been isolated and characterised They can be described as natural microbicide selectivelycytotoxic to bacteria whilst showing minimal cytotoxicity towards the mammalian cells of the host organism They act by theirrelatively strong electrostatic attraction to the negatively charged bacterial cells and a relatively weak interaction to the eukaryotehost cells The ability of these peptides to accumulate at sites of infection combined with the minimal hostrsquos cytotoxicity motivatedfor this review to highlight the role and the usefulness of AMPs for PET with emphasis on their mechanism of action and thedifferent interactions with the bacterial cell These details are key information for their selective properties We also describe thestrategy design and utilization of these peptides as potential radiopharmaceuticals as their combination with nuclear medicinemodalities such as SPECT or PET would allow noninvasive whole-body examination for detection of occult infection causing forexample fever of unknown origin

1 Introduction

Compared with other conventional technologies tomo-graphic imaging can evaluate disease processes deep withinthe body noninvasively and relatively rapidly It is thereforenot surprising that molecular imaging has powerfully aug-mented the investigation of various disease processes and hasbecome an essential tool in the field of oncology for bothresearch and patient care [1] Another major advantage ofimaging is its ability to provide a holistic three-dimensionalassessment of the whole organ or body less likely to belimited by sampling errors and therefore corelating well withthe overall disease process While continued advances in

molecular imaging have provided unparalleled opportunitiesfor more refined methods to monitor diseases tools forevaluating infection and inflammation remain limited Twoimagingmethods widely used in the clinics currently includehigh resolution computed tomography (CT) that measuresanatomic (and therefore late) changes or 18F-labeled 2-fluoro-deoxy-D-glucose (18F-FDG)-positron emission tomography(PET) which is a general marker of metabolic activityAs 18F-FDG is also accumulating in sites of infection andinflammation due to the elevated glucose metabolism inthese loci [2] thus it is nonspecific for infection Therefore itbecame increasingly important to develop more specific and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 867381 15 pageshttpdxdoiorg1011552014867381

2 BioMed Research International

selective infection imaging agents Direct ex vivo labelingof leukocytes is considered the ldquogold standardrdquo for infectionimaging by PET Unfortunately this process is very laboriousand time-consuming and requires the handling of bloodproducts [3ndash5] Alternatively indirectly labeled leukocytescan be achieved using radiolabeled molecules such aschemotactic peptides or cytokines that bind to receptorson the leukocytes [3] Unfortunately the biological effectsof some of the leukocyte receptor-targeting compoundshave limited their clinical use as infection-specific molecularimaging agents [5] Although the most commonly labeledleukocytes neutrophils and lymphocytes are quite selectivefor infection there are cases when they may fail to detect aninfection or accumulate in noninfected sites If the infectionfails to elicit an immune response labeled leukocytes will notaccumulate at the infected loci which may be the case ina severely immune-compromised individual or in the caseof infection by certain pathogens such as Mycobacteriumtuberculosis or Pneumocystis carinii Some noninfectiousimmune conditions such as rheumatoid arthritis may alsoprovoke an immune response and accumulate the tracer[3] Through the use of different tracers different targetingstrategies are possible to image infection using PET

Tracers that interact directly with the pathogenicmicrobes responsible for infection are by nature highlyspecific for infection and unlike labeled leukocytes should notaccumulate in sterile inflammations These types of tracersinclude radiolabeled antibiotics and antimicrobial peptidesTechnetium-99m labeled ciprofloxacin (99mTc-ciprofloxacin)has been the most widely studied antibiotic-based tracerfor SPECT infection imaging [6] targeting DNA Gyrase anenzyme present in all dividing bacteria and is not thoughtto accumulate in dead bacteria or sterile inflammationsSome problems associated with its use as a tracer in SPECTinfection imaging have occurred with regards to poorradiochemical purity and stability [3] More recently it hasbeen reported that localisation at infected foci takes placeprimarily through increased extravasation and stasis Thisprocess also occurs at uninfected sites with increased vascularpermeability and 99mTc-ciprofloxacin may accumulate atsites of sterile inflammation thereby reducing its specificityfor infection [7]

Antimicrobial peptides (AMP) have attracted interest aspotential targeting vectors for the development of PET tracersdesigned for the detection of infection These peptides arefound in a variety of organisms including humans andso far hundreds have been isolated and characterised Itis believed that these peptides function as broad-spectrummicrobicides and form part of the innate immune systemof many eukaryotes including humans Regardless of theirorigin they share many common properties such as havinga net positive charge being amphipathic and in most casesare membrane active [8] Due to their role in the bodyas a natural microbicide these antimicrobial peptides areselectively cytotoxic to bacteria whilst showing minimalcytotoxicity towards cells of the host organism It is thoughtthat the net cationic nature of the peptides results in arelatively strong electrostatic attraction to negatively chargedbacterial cells and a relativelyweak attraction to the eukaryote

host cells which are usually less negatively charged thanprokaryotes and is believed to form the basis of this cell-typediscrimination [9]The ability of these peptides to accumulateat sites of infection combined with their almost negligiblecytotoxicity or attraction to host cells makes these peptidesattractive as targeting vectors for PET imaging of infection[10]

2 Overview of Antimicrobial Peptides

Antimicrobial peptides are evolutionarily conserved biom-olecules that form part of the defence mechanisms in manyorganisms [11] ranging from prokaryotes to multicellularanimals such as humans [9] They form part of the first lineof defence against pathogenic microbes in higher animalsand in many lower forms of life they are the only form ofdefence against pathogenic and saprophytic microbes [12]The selective cytotoxicity of these peptides where they attackthe pathogenic microbes and leave the host cells unharmedis due to the fundamental differences in composition andstructure of the host cells to those of the pathogenic bacteriaand yeasts Despite some AMPs showing immunomodula-tory effects andor chemotactic behaviour a common featureof these antimicrobial peptides is that they are amphipathicbut possess an overall positive charge [9] Approximately1500 antimicrobial peptides have been characterised in awiderange of organisms and classification of these peptides canbe complicated due to the high degree of sequence dissim-ilarity between the various peptides However classificationhas been attempted based on amino acid composition andsecondary structures

Three large groups (Table 1) have been identified namely120572-helical peptides cysteine-containing 120573-sheet peptides andflexible peptides rich in specific amino acids such as prolinetryptophan histidine arginine and glycine [13]

21 120572-Helical Antimicrobial Peptides Approximately 30 to50 of all antimicrobial peptides identified and studied todate contain predominant 120572-helical structures This maybe due the relative ease with which these peptides arechemically synthesised which allows for extensive charac-terisation in the laboratory These peptides usually consistof 12ndash40 amino acid residues and contain an abundance ofhelix stabilising residues such as alanine leucine and lysinebut never cysteine In aqueous solutions these peptides areoften unstructured but assume their amphipathic 120572-helicalconformations when associated with a cell membrane or ina membrane mimetic environment Often these peptides arenot strictly 120572-helices and may contain an internal kink [14]

22 120573-Sheet Antimicrobial Peptides Theothermajor group ofantimicrobial peptides are those that typically contain two toten cysteine residues that form one to five interchain disulfidebonds This bonding interaction allows these peptides toadopt the 120573-sheet conformation Most 120573-sheet antimicrobialpeptides are part of the defensin family and these peptidesare evolutionarily conserved across plants fungi insectsmolluscs and vertebrate animals Defensins typically consist

BioMed Research International 3

Table 1 Representative antimicrobial peptides of different classifi-cations (modified from [13])

Class Representatives Host

120572-helical

LL-37 Mammal humanCecropins Insect mothMelittin Insect honey beeMagainins Amphibian frogFowlicidins Ave chicken

120573-sheet

Thanatin Insect soldier bugTachyplesins Arthropod horseshoe crabProtegrins Mammal pig

Plant defensin VrD2 Plant mung beanPlectasin Fungus ebony cup

Insect defensin A Insect northern blow fly120572-defensin Mammal human120573-defensin Mammal human120579-defensin Mammal rhesus monkey

Flexible

Indolicidin Mammal cowTritrpticin Mammal pigHistatins Mammal humanPR-39 Mammal pig

of two to three antiparallel 120573-sheets stabilized by three tofour intramolecular disulfide bonds however in some casesan 120572-helical or unstructured segment is found at the N-or C-terminus Unlike the 120572-helical antimicrobial peptideswhich are unstructured in aqueous solutions the defensinsmaintain a compact globular structure under such condi-tions [12 13] Apart from overall similarity in secondarystructure most mammalian-derived 120572-defensins possess twoadditional common features namely a protruding loopresulting from a conserved arginineglutamate salt bridgeand a 120573-bulge caused by a conserved glycine-X-cysteine (Xany amino acid) motif between the first and second cysteineresidues [13]

23 Flexible Antimicrobial Peptides Rich in Specific AminoAcids A minority of antimicrobial peptides contain a highproportion of certain amino acids such as proline trypto-phan histidine arginine and glycine Representative mem-bers of this class include the tryptophan rich bovine indoli-cidin and porcine tritrpticin histidine rich human histatinsand 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 zwitte-rionic micelles composed of substances such as dodecyl-phosphocholine or anionic sodium-dodecyl sulfate [13]

3 Mechanisms of Cell Specificity andSelectivity of Antimicrobial Peptides

Inherent differences in the microbial versus the host cellmembrane composition and architecture aid selectivity of the

antimicrobial peptides Regulation of expression or locali-sation of the peptides is also thought to prevent unwantedinteractions with vulnerable host cells

31 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 sterolsmay also contributeto the biochemical architecture and surface topology of suchmembranesThere 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]

32 Membrane Composition Charge and HydrophobicityThe core component of almost all natural biomembranesis the phospholipid bilayer These bilayers are amphipathicmeaning they have both hydrophobic and hydrophilicregions However eukaryotic and prokaryotic cell mem-branes differ significantly in terms of exact compositionand cell energetics (Figure 2) Phosphatidylcholine (PC) andits analogue sphingomyelin (SM) as well as phosphatid-ylethanolamine (PE) have no charge under physiological con-ditions [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 phospho-lipids 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 bac-teria being very electronegative whereas those membranesthat are rich in PC PE or SP tend to have a net neutral chargeas is the case in mammalian cell membranes [15 16]

33 Membrane Asymmetry Although cellular membranesare neither symmetric nor static differences between mam-malian and microbial phospholipid bilayers can serve aspotential targets for antimicrobial peptides In some cellssuch as the bovine erythrocyte only 2 of the total PE con-tent is located on the outer membrane leaflet [13] Differencesin membrane symmetry saturation of phospholipid bilayersand compositional stoichiometry will influence the mem-branersquos fluidity and phase transition In a similar manner thecharge of the inner and outer leaflets of the cellular bilayermay also be different [15]

34 Microbial Ligands and Receptors as Targets for Antimi-crobial Peptides Experiments have shown that D-and L-amino acid versions of antimicrobial peptides exhibit similarbinding affinities to targets cells suggesting that stereospe-cific 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


Externalleaflet

Internalleaflet

Internalleaflet

Externalleaflet

Weak

Acidic phospholipidZwitterionic

CholesterolAntimicrobial peptide

Bacterial cytoplasmicmembrane

Prototypic plasmamembrane of a multicellular

animal (erythrocyte)

Hyd

roph

obic

inte

ract

ions

Elec

tros

tatic

and

hydr

opho

bic i

nter

actio

ns

Strong

Figure 1 Membrane targeting of antimicrobial peptides and basis of their selectivity (adapted from [45])

Rela

tive c

ompo

sitio

n (

)

CL PG PE PC SM ST

Exop

lasm

icCy

topl

asm

ic

Membrane constituent

40302010

50

20304050

10

Figure 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 outermembrane leafletsMembrane constituentsranging from anionic (left) to neutral (right) are CL PG PE PCSM 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 colihorizontal hatching S aureus shaded B subtilis checkered Calbicans 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 serve

as pseudoreceptors enabling the initial interaction betweenthe antimicrobial peptide and the microbial cell target [13]Hence antimicrobial-binding receptorsmay be an alternativepathway of AMP interaction with the bacterial cell envelop

35 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 (Δ120595) A normal mammaliancell has a Δ120595 between minus90 and minus110mV in range Pathogenicbacteria however generally exhibit Δ120595 in the minus130 tominus150mV rangeThis 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 Design

In the aqueous intercellular environment many antimicro-bial 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 signif-icant conformational change and adopt a specific conforma-tion such as a 120572-helix Studies suggest that dynamic andorinherent conformations of antimicrobial peptides have aneffect on their selective cytotoxicity [9 17 18] Additionallyantimicrobial peptides may undergo conformational tran-sition self-association or oligomerization within the target


pathogen 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 cyclic120572-helical and 120573-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 120572-helical and extendedpeptides were able to interact with themore neutrally chargedPC membrane In the same study it was also found that 120573-sheet peptides were able to translocate phospholipids fromthe inner to the outer leaflet at concentrations that were lowerthan those that were required to permeabilize the membraneSimilarly 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

41 In Vivo Preferential Affinity for Microbial versus Mam-malian Cells Welling and colleagues [20] conducted an invivo experiment where they tested the binding affinity of aradiolabeled fragment of the cationic ubiquicidin antimicro-bial peptide 99mTc-UBI 29ndash41 for microbial cells as comparedto host cells In the study animals were infected withCandidaalbicans Klebsiella pneumonia or Staphylococcus aureusSterile inflammations were also induced in the thigh musclesof animals through injection of heat-killed microorganismsor purified LPS to serve as controls The radiolabeled pep-tides accumulated to a significant extent in the infected sitesrelative to sterile or noninfectiously inflamed parts of thebodyThis in vivo experiment demonstrated that the peptidescould distinguish between host and microbial cells and alsoaccumulate at the infected sites Through scintigraphic mea-surements 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 rapidlocalizationwas interpreted as the peptides having a higher orpreferential affinity for the target cell surface relative to thatof the host cell surface

42 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 amphibiansonto 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 peptides

is by containing them within granules in the phagocytingleukocytes which engulf pathogens and expose them tolethal concentrations of antimicrobial peptides and oxidiz-ing agents The defensin class of antimicrobial peptides isdeployed in thisway since they are someof themost toxic andleast selective of the host produced antimicrobial peptidesThe 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 Action

The generally conserved structures of antimicrobial peptidesacross a wide variety of organisms lend some clues as to theirmechanisms of action They are almost exclusively amphi-pathic and cationic under physiological conditions and this isbelieved to aid their target cell selectivityThe ideal antimicro-bial 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 pep-tides have amphipathic structures that allow them to interactwith phospholipid membranes structures that are essentialto all pathogens [17] Parameters such as conformation (119883)hydrophobicity (119867) hydrophobicmoment (119872

119867) charge (119876)

polar angle (120579) and amphipathicity (119860) are all importantto the functioning of antimicrobial peptides Furthermoreall these determinants are interrelated and modification ofone of these features will lead to alteration of the others[9]

51 Conformation (119883) Although antimicrobial peptidesmaybe 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 120573-sheet or 120572-helical 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 arginineThe 120572-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 120572-helic conformation is disrupted in a polar solventsuch as water In a membrane the polar hydrogen bondinggroups are shielded from lipophilic (apolar) membrane envi-ronment through 120572-helic formation The helix conformationalso exposes the apolar side chains to the neutral lipidenvironment inside the membrane Although the primarystructure of the 120573-sheet class of antimicrobial peptides showsa level of dissimilarity in amino acid sequence they allshare common features with regard to amphipathic structurepossessing distinct hydrophilic and hydrophobic domains[9]


52 Charge (119876) Most of the antimicrobial peptides areoverall cationic and have charges ranging from +2 to +9 withmany possessing highly defined negatively charged domainsThis 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 Δ120595 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] demon-strated in studies with analogues of magainin that increasingthe cationicity from +3 to +5 resulted in an increase inantibacterial activity against both Gram-positive and Gram-negative 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]

53 Amphipathicity (119860) and Hydrophobic Moment (119872119867)

Amphipathicity is a nearly universal feature amongst antimi-crobial peptides and is achieved through a number of dif-ferent peptide structures The amphipathic 120572-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 (119872

119867) which can be calculated as the

vectorial sum of individual amino acid hydrophobicitiesnormalized 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 120572-helical antimicrobial peptides the 120573-sheethost defence peptides also exhibit amphipathicity This ismanifested as varying numbers of 120573-strands organised toform hydrophobic and hydrophilic surfaces The 120573-strandswhich are often antiparallel are stabilised by regularly spaced

disulphide bonds or by cyclisation of the peptide backboneThis intramolecular bonding allows 120573-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 exactmechanismsby which amphipathic antimicrobial peptides bring aboutmembrane disruption in the target cell membrane is unde-termined at present largely because the exact conformationof the peptides in the membranes is not known studieshave shown that segregated amphipathicity in both 120572-helicaland 120573-sheet antimicrobial peptides has a profound effect onpeptide disruption of natural biomembranes [9]

54 Hydrophobicity (119867) 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 phos-pholipid bilayer Although a certain amount of hydropho-bicity is essential for the functioning of the antimicrobialpeptide excessive hydrophobicity will increase its likelihoodof destroying the hostrsquos cells and reduce its specificity formicrobial cells [24] Wieprecht and coworkers [25] studiedthe relationship between the hydrophobicity of peptides andtheir ability to permeabilize biomembranes Using maga-inin analogues as model antimicrobial peptides they wereable to keep factors such as hydrophobic moment helicityand charge nearly constant whilst producing analoguesof variable hydrophobicity Their experiments showed thathydrophobicity had little or no effect on the peptidersquos abilityto bind to or permeabilize the membrane when it consistedexclusively of PG However inmembranes consisting of a 3 1ratio of PC PG the peptides with the highest hydrophobicityhad an approximately 60-fold higher permeabilizing abilitythan the least hydrophobic peptide and in membranescomposed of only PC there was a 300-fold difference

55 Polar Angle (120579) A peptidersquos polar angle refers to therelative proportion of polar to nonpolar facets of the peptideconformed to an amphipathic helix A helical peptide withone facet composed entirely of polar amino acid residuesand the other facet composed entirely of nonpolar residueswould have a polar angle of 180∘ Less segregation betweenthe domains or an overabundance of hydrophobic residueswould lead to a lower polar angle Studies conducted byUematsu andMatsuzaki [26] on both synthetic and naturallyoccurring peptides have shown that a lower polar angleand therefore a more hydrophobic facet is more conduciveto membrane permeabilization Polar angle has also beencorrelated with the stability of peptide induced pores inbiomembranes They also demonstrated that antimicrobialpeptides with smaller polar angles were able to induce higherdegrees of membrane permeabilization and translocation athigher rates than peptideswith greater polar angles Howeverthe pores formed by the peptides with smaller polar angleswere less stable than those formed by peptides with greater


1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Charged polar

Neutral polar

Hydrophobic

Other (gly)

Figure 3 Statistical analysis of residue distribution in the 20-residue N-terminal stretch 120572-helical AMPs from natural sources A graphicalrepresentation of the frequency of different types of residue at each position on a helical wheel projection is shown The uneven distributionof hydrophobic and charged peptides contributes to the amphipathic nature of the peptide (adapted from Tossi et al [17])

polar angles Hydrophobic and hydrophilic properties ofantimicrobial peptides can be seen to play vital roles in theinteractions with and permeabilization of phospholipid cellmembranes [18]

56 Common Structural Features of Antimicrobial PeptidesWhilst a wide variety of antimicrobial peptides do exist innature conservation of key features and secondary structureshas been noted Extremes of features such as amphipathicitycharge hydrophobic moment or polar angle are not benefi-cial since they tend to compromise either antimicrobial activ-ity or lead to increased host cell cytotoxicity The minimumcharge that peptides may possess in order to exert any kindof antimicrobial activity appears to be +2 This minimumcationicity is important because it allows for the initialelectrostatic attraction to the bacterial membrane which isnegatively charged It also allows for the displacement ofany other cations that may already be bound to the targetcell membrane and for the translocation into the interiorof the membrane bilayer Similarly the hydrophobicity ofthe peptide should be moderate since very hydrophobicantimicrobial peptides would target membranes with a net-neutral charge such as the host cells leading to a reductionin target selectivity and damage to the host organism Itcan be seen that selective targeting of pathogenic microbesis largely due to a balance between electronegativity andhydrophobicity of the antimicrobial peptides [9]

6 Initial Interactions with the TargetedCellular Membrane

The initial interaction between the antimicrobial peptideand the cellrsquos phospholipid membrane is important as itdetermines target cell selectivity and also influences anysubsequent interactions with the target cell The initialinteractions are largely determined by physical and chemicalfeatures of both the antimicrobial peptide and the target cellmembrane [12]

61 Electrostatic Interactions Electrostatic interactions arewidely believed to be responsible for the initial targetingof the microbial cell A study by Matsuzaki [27] correlatedantimicrobial peptide cationicity with membrane bindingability and the fact that cationicity is a conserved feature ofalmost all antimicrobial peptides in awide range of organismsfurther supports this argument Electrostatic forces act overa long range and the abundance of lysine and arginineresidues in antimicrobial peptides which are attracted tothe negatively charged phosphate groups of biomembraneslends further credibility to the theory that these interactionsare responsible for the initial attraction to the target cellmembrane [12] In Gram-negative bacteria it is believed thatthe antimicrobial peptides displace cations that are normallyassociated with the LPS since antimicrobial peptides possessa binding affinity for the LPS that is approximately threeorders of magnitude greater than the divalent cations usually


associated with this moiety Strains of bacteria where the LPSis highly substituted with 4-amino-4-deoxy-L-arabinose or ishighly acylated show greater resistance to positively chargedantimicrobial peptides lending further credibility to thetheory that electrostatic charge is important for interactionwith the target cell membrane [28] Gram-positive bacterialack an LPS or outer cell membrane but they do havea thick cell wall made up of teichuronic or teichoic acidpolymersThese highly anionic structures are ideal targets forthe cationic antimicrobial peptides Strains of Staphylococcusaureuswhere the teichoic acids have beenmodified resultingin increased anionic charge are more susceptible to cationicantimicrobial peptides [29ndash31] The fact that most bacteriahave a strong electrochemical gradient (Δ120595) relative tomammalian cells is also thought to increase target selectivityof antimicrobial peptides [9]

62 Receptor-Ligand Interactions with the Membrane Somestudies have shown that both naturally occurring and syn-thetic peptides interact with the membrane equally wellregardless of whetherD-amino or L-amino acids are used [3233] This would suggest that interactions with biomembranesare not dependant on receptor-ligand mechanisms howeverother studies have shown that this may not be the case withall antimicrobial peptides Nisin a naturally occurring cyclicpeptide with powerful antimicrobial action has been foundto bind specifically to bacterial membrane bound lipid II[34 35] Similarly tachyplesin has been shown to have aspecific affinity for LPS The data from these studies suggeststhat receptor mediated binding is important for cell targetingin a small number of antimicrobial peptides [34]

7 Events following Initial Membrane Binding

Experimental determination of initial attraction of peptidesto and interaction with cellular membranes is usually sim-pler than determination of interactions that follow this Avariety of methodologies such as circular dichroism [2236] X-ray crystallography nuclear magnetic resonance [37]reverse phase-high performance liquid chromatography andsurface plasmon resonance [22] amongst other techniqueshave been used to elucidate peptide-membrane interactionsHowever it is suggested that the antimicrobial efficacy andmechanisms are extremely sensitive to conditions such aspH osmotic strength solution viscosity and temperature soany data obtained by the above mentioned techniques mustbe viewed with regard to these conditions [9] Subsequentto the initial membrane binding antimicrobial peptidespenetrate the outer phospholipidmembrane a phase referredto as threshold concentration and in doing so is able toexert their cytotoxic effects in the interior of the cell Theentry into the cell by the peptides requires a minimumnumber or threshold concentration of antimicrobial peptidesto accumulate on the surface of the lipid membrane Thisevent can be affected by factors other than concentrationsuch as the ability of the peptides to multimerize andalso features of the phospholipid membrane itself such asits lipid composition head group size and fluidity [38]

The transmembrane potential of the bilayer may also influ-ence theway inwhich the peptide enters themembrane sincea highly negative transmembrane potential will facilitate poreformation by drawing the positively charged peptide into themembrane [39]

8 Changes in Peptide Conformation uponInteraction with the Membrane

Many antimicrobial peptides especially those with 120572-helicalsecondary structures undergo significant conformationalrearrangement upon entering the nonpolar environment ofthe inner membrane The 120572-helical antimicrobial peptidesare normally disordered in the extracellular environmentexhibiting random coil or extended structures but rapidlyconform to a structured 120572-helix when associated with thebiomembrane [18] Some antimicrobial peptides can onlyundergo this conformational change in association with anegatively charged bilayer membrane This may be due tothe way the lipids are arranged in such membranes withthe phospholipid head groups inducing optimum periodicityof the cationic amino acid residues in the peptide whichin turn promotes correct conformation into the helicalsecondary structure [40 41] It has been suggested that thisfeature ensures that the antimicrobial peptides will onlybe ldquoactivatedrdquo into the cytotoxic form in the presence ofthe target cell membrane in this case a negatively chargedbacterium and will not indiscriminately damage nontargethost cells [17] The intramolecular disulphide bonds found in120573-sheet peptides ensure that they maintain their secondarystructure even in aqueous environments and so they do notundergo the drastic conformational rearrangements seen in120572-helical peptides although quaternary peptide structuresmay disassociate upon entering the membrane and thiscould facilitate selective toxicity [9] Following the initialinteraction with the cell membrane many peptides mayundergo self-association which when combined with lipid-peptide interactions may lead to the creation of complexstructures that contribute to the cytotoxic effects of thepeptide The antimicrobial peptidersquos amino acid sequenceand conformation in the monomer form will dictate itsability to form these structures In amphipathic peptides thehydrophobic domains are able to interact with the nonpolarhydrophobic core region of the lipid bilayer thereby drivingthe peptide deeper into the membrane Alternatively theycould also interact with the hydrophobic facets of otherpeptides promoting multimerization in an attempt to avoidexposure of these facets to the aqueous environment Thistype of multimerization and interaction with the interiorof the lipid bilayer may result in peptide lined pores orchannels being formed in the biomembrane resulting inloss of integrity and permeabilization Since biomembranesare highly variable in composition and structure it is pos-sible that a peptide may behave in a number of differentways when associated with different cellular membranes [9]Several models have been proposed to describe the poreformation observed in membranes that have been exposed toantimicrobial peptides


81 The Barrel-Stave Model This mechanism of membranepore formation is so named because the transmembranepeptides or peptide complexes lining the channel arepositioned in a barrel-like ring with the peptides formingtransmembrane staves Amphipathic peptides are orientedso that the hydrophobic domains interact with the nonpolarhydrocarbon tails located in the interior of the lipid mem-brane whereas the hydrophilic domains are oriented so thatthey face the aqueous channel of the pore and form its lining[24] Initially the monomer peptides accumulate at the cellsurface and undergo conformational rearrangement whenthey contact themembrane (Figure 4)This is thought to forcethe phospholipid head groups aside and induce thinningof the membrane This allows the hydrophobic part of thepeptide to enter into the nonpolar interior of the membranewhilst the cationic amino acids of the antimicrobial peptideinteract with the negatively charged head groups When thethreshold concentration of the peptides is reached the peptidemonomers are able to aggregate to form multimers whichfurther forces the peptides into the hydrophobic centre ofthe membrane as the aggregation prevents the hydrophilicparts of the peptide from being exposed to the hydrophobicparts of the innermembrane (Figure 4(a)) As ever increasingnumbers of peptide monomers aggregate the pore in themembrane is expanded [9 24]

82The Toroidal Pore orWorm-HoleMechanism Thismech-anism of pore formation has been well studied using the120572-helical magainin peptides Upon contacting the chargedcellular membrane the disorganised peptides take on the120572-helical structure Initially the helices orientate themselvesso that they are parallel with the surface of the membraneThe polar phospholipid head groups are displaced and thesurface of the membrane is weakened resulting in a positivecurvature strain in the membrane As a result of this strainand thinning the membrane is destabilised and becomesmore susceptible to further peptide interactions Once athreshold concentration of peptides is reached the peptidesreorientate so that they are perpendicular to the membraneand begin to multimerize so that the hydrophilic parts ofthe peptides are not in contact with the hydrophobic partsof the membrane (Figure 4(b)) The newly formed toroidalpore is unstable and upon disintegration some of the peptidesare forced into the inner leaflet of the cell membrane Itis therefore believed that the disintegration step of thesetransient pores is important as it allows the peptides totranslocate into the intracellular space where they may acton other targets [42]

83 The Carpet Model The carpet model of membrane per-meabilization is based on diffuse action by many monomerpeptides on the cellular membrane When sufficiently highconcentrations of certain antimicrobial peptides are presenton the cell membrane some of the phospholipids of themembrane are displaced which results in changes to themembranes fluidity or brings about weaknesses in the barrierproperties of the membrane The cumulative effect of thesedisplacements is that the membrane is weakened and loses

its integrity As suggested previously the initial attractionof the antimicrobial peptides to the membrane is throughelectrostatic attraction forces No specific channels or poresare formed and it is believed that permeabilization and loss ofmembrane integrity are through the unfavourable energeticproperties that dispersion of the phospholipids brings about(Figure 4(c)) [22]

9 Impact of Bacterial Infections to HumanHealth and Traditional Methods ofInfection Diagnosis

It is estimated that up to 85 of patients that are criticallyill in hospital have a fever but display no other outward signof infection Since prolonged episodes of fever can be fatalit is essential that any underlying infection is detected assoon as possible so that the correct treatment regime can beinitiated [43] Traditional methods of diagnosis may includeexamination of tissue biopsies and attempting to culturepathogens an often inaccurate and time-consuming taskwhich can delay the onset of treatment Diagnostic imagingprocedures are also employed and may include computedtomography (CT) scanning or magnetic resonance imaging(MRI) However these techniques are generally not able todetect early stage infections as they require morphologicalchanges in the tissues to take place a feature usually asso-ciated with advanced infections [44] Furthermore they aregenerally focussed on specific parts of the body meaningthat it is possible that the infection may be missed or thetrue extent of the infection may not be detected Gallium-radiolabeled antibodies or -immunoglobulins or complexessuch as 6768Ga-citrate may be employed to highlight regionswhere leukocyte trafficking is occurring using SPECT orPET scanning However these technologies are unable todefinitively distinguish between infected tissues and thosethat are inflamed but sterile since leukocyte traffickingoccurs in both cases [44] Given the high specific affinityof naturally occurring antimicrobial peptides for pathogenicbacteria or fungi as opposed to cells of the host organism itwas envisaged that theymay be employed to aid the resolutionof diagnostic imaging processes [45]

91 The Use of Antimicrobial Peptides as Radiopharmaceuti-cals Ideally a radiopharmaceutical employed for infectionimaging should allow for rapid detection of bacteria andrapid clearance from the noninfected sites It should alsoexhibit high and specific uptake at the infected site withminimal amounts accumulating in sterile or nontarget tissueThe compound should also have low toxicity and not inducean immune response Very importantly it should be able todistinguish between a sterile and an infected inflammation[45] Since antimicrobial peptides generally show a broadspectrum of activity against a wide range of pathogenicyeasts and bacteria they are ideal targeting molecules forinfections where the suspected pathogen has not been iden-tified Additionally their mode of action requires them tophysically associate with the pathogen and so they would beable to bring a gamma or positron emitting source such as


(a) (b) (c)

Figure 4 Overview of possible interactionmechanism following peptide interaction with the bacterial cell membrane [53] that is (a) barrel-stave model (pore formation) (b) toroidal model (pore formation) and (c) carpet model (membrane disruption) Red coloured peptideregions hydrophilic blue coloured peptide regions hydrophobic

Tc

Reducing agents

99mTcO minus

4

(a)

OH

O

Tc

NNH

OH

HN

O

N

OO

OHOHHO

H

N

O

OC Peptide

(b)

Figure 5 Approaches in radiolabeling of peptides The direct method (a) where radionuclides are covalently attached to the peptide and theindirect method (b) where radionuclides are attached to targeting peptides by means of bifunctional chelators [8]

technetium-99m (99mTc) or gallium-67 (67Ga) to the exactlocation of the infection Their lack of affinity for the hostorganismrsquos cells alsomeans that they would not accumulate insterile inflamed tissues Radiolabeled antimicrobial peptidesare also attractive because they are cleared rapidly from thecirculatory system and excreted by the body In addition theyare also able to penetrate the extravascular tissues and therebyaccumulate at infected sites in a very short space of time [46]Ideally the radiolabeling procedure of a targeting moleculeshould allow for the firm attachment of a radionuclide to themolecule without it adversely affecting its targeting ability orthe pharmacokinetics of the molecule Labelling approachescan either be direct or indirect as follows

(i) A direct labelling (Figure 5(a)) approach involvesincorporation of the radionuclide onto the targetingmolecule via a covalent bond In the case of peptidetargeting molecules a covalent bond may be formedbetween the radionuclide and a suitable free amide

residue of Lys andArg [47] Using the tyrosine residuemay cause problems associated with labelling includ-ing nonspecific or poor binding in vivo instability ofthe complex and unwanted alterations to the peptidestructure such as the cleaving of internal disulphidebonds which can alter its functioning [8]

(ii) An indirect labelling strategy can be used throughaddition of chelating agents to the targeting molecule(Figure 5(b)) [8] Bifunctional chelates have beenused to label peptide carrier molecules with radionu-clides The chelating agent may be preloaded withthe radionuclide prior to being bonded to the carriermoiety or it could be firstly attached to the carriermolecule and then exposed to the nuclide for chela-tion in a process known as postlabelling Postlabellinghas the advantage that the carrier molecule canbe stored for a long period of time until neededand the radionuclide which undergoes decay can


KVHGSLARAG KVRGQTPKVA KQEKKKKKTG RAKRRMQYNR RFVNVVPTFG KKKGPNANS

101 20 30 40 50 59

Figure 6 Primary structure of ubiquicidin as originally reported by Hiemstra and coworkers [49]

Figure 7 Anterior whole-body image taken at 30min after tracerinjection showing kidneys (dotted arrow) liver (solid arrow) andurinary bladder (ball arrow) (adapted from [52])

be added shortly before the radiopharmaceutical isadministered This benefits commercialisation of thecarrier molecule and makes the technology easier touse in hospitals or clinics [48]

92 Ubiquicidin Exemplifies an Approach for AntimicrobialPeptide Derived Radiopharmaceuticals The 59-amino acidresidue antimicrobial peptide ubiquicidin (UBI) is a 67 kDapeptide that was first discovered in cytosolic extracts of themurine macrophage (Figure 6) This peptide was shown toexhibit antimicrobial effects against Salmonella typhimuriumand Listeria monocytogenes It was subsequently found in awide range of other organisms including humans [49] Sinceit occurs naturally in man ubiquicidin is not an immuno-genic entity which makes it suitable for administration asa diagnostic tool It also has high affinity for bacterial cellsbut does not target mammalian cells rendering it nontoxicto the patient and selective in that it is unlikely to accumulateat sterile inflammation sites [50] Several studies have beenperformed on fragments of ubiquicidin both in vitro and invivo to assess its ability to bind to bacterial cells

Welling and coworkers [51] evaluated the whole 99mTclabeled ubiquicidin and various radiolabeled fragments of thepeptide including UBI1-18 (KVHGSLARAGKVRGQTPK)UBI29-41 (TGRAKRRMQYNRR) UBI18-29 (KVAKQEKKKKKT) UBI 18ndash35 (KVAKQEKKKKKTGRAKRR) UBI31-38 (RAKRRMQY) and UBI22-35 (QEKKKKKTGRAKRR)for their ability to bind to bacterial cells andor humanleukocytes in vitro They found that the ubiquicidin peptidefragments UBI 18ndash35 UBI 31ndash38 UBI 22ndash35 and UBI 29ndash41 showed considerably higher binding affinities for the

bacterial cells than they did for the human leukocytes Thein vivo results obtained by scintigraphy of experimentallyinfected mice following intravenous administration of thevarious radiolabeled peptides showed that the UBI18-35and UBI29-41 peptides appeared to be the most promisingcandidates After a postadministration period of 2 h and 24 hthe leukocyte to bacteria binding ratios were 1 36 1 166and 1 73 1 220 for UBI18-35 andUBI29-41 respectivelyTheresearchers concluded that UBI29-41 and UBI18-35 were theoptimal peptides for distinguishing infections from sterileinflammations

93 Human Clinical Trials of 99mTc-Ubiquicidin 29ndash41 as anInfection Imaging Agent Akhtar and coworkers [52] studiedthe efficacy of 99mTc-UBI 29ndash41 as an infection imaging agentin eighteen patients with suspected prosthetic or soft tissueinfections Using scintigraphy to monitor the radiolabeledpeptide the researchers were able to monitor the target tonontarget (TNT) ratios of the imaging agent Infection in thepatients was confirmed through culture of bacteria from theinfected site or where this was not possible through completeblood examinationThe study found that all patients toleratedthe radiolabeled peptide well no significant changes to theirvital signs were noted and no related side effects were seenfollowing the administration of the 99mTc-UBI 29ndash41 TheTNT ratio was determined at 30 60 and 120 minutes withthe 30-minute scan showing the highest mean TNT valueThe anterior whole-body scan (Figure 7) gave informationabout the biodistribution of the tracer and its routes ofelimination by the body It can be seen that the tracer ismostlyeliminated by the urinary system and also some perfusion-dependant liver activity was noted The imaging agent wasfound to have a sensitivity of 100 and a specificity of 80The researchers concluded that the 99mTc-UBI 29ndash41 hada positive predictive value of 929 a negative predictivevalue of 100 and an overall diagnostic accuracy of 944The radiolabeled peptide displayed efficacy against a rangeof different bacteria including Pseudomonas aeruginosaStaphylococcus aureus and Streptococcus pyogenes It was theopinion of the researchers that 99mTc-UBI 29ndash41 is a highlysensitive and specific imaging agent for detecting soft tissueand bone infections in humans

10 Discussion and Perspective

The usage of nuclear medicine modalities such as SPECTor PET allows clinicians for noninvasive whole-body exam-ination of physiological processes such as occult infectionat cellular level and apart from being a useful tool forphysiological and medical research these highly sensitivetechnologies are capable of detection of diseases without or


prior to anatomical change (fever of unknown origin) Todate radiolabeled leukocytes monoclonal antibodies againstcytokinesleukocytes and tracers associated with specificmolecular targets or metabolic processes are utilized [54]Radiolabeled leukocytes have a spectrum of limitations(alteration of leukocyte function due to radiation damage)that is they have a cumbersome pharmakokinetic and arealso relatively nonspecific Moreover labeled leukocytes andhigh molecular weight tracers such as antibodies may alsohave limited penetration into infected or diseased tissuesThe latter presented overview which clarifies the widespreadpotential of AMPs to be evaluated as imaging probes giventheir unique selective involvement with bacteria A sim-ple literature query searching for ldquoantimicrobial peptidesrdquoresulted in ca 6000 publications However as soon as thequery is combined with the term ldquoimagingrdquo it resulted inonly 63 publications only 17 of those have clinical relevance(99mTc-UBI-29-41 related studiestrials)This is an importantobservation as this ubiquicidin fragment may representsa near-perfect carrier for targeting molecules for infectiondetection The human clinical trials conducted by Akhtarand coworkers [52] with 99mTc-UBI 29ndash41 did not find anyevidence of cytotoxicity in the patients which supports thefindings of the current study Even though it was statedthat the signal to noise ratio is low [46] it has been usedsuccessfully for 10 years now In 2010 the clinical trialsto date were justified by de Murphy et al towards theirdiagnostic value over the initial 7-year period 99mTc-UBI 29ndash41 meta-analysis returned high values for sensitivity (963)specificity (941) and accuracy (953) with high positivepredictive (951) and negative predictive values (955)[54] From 2011 onwards seven additional clinical studies(enrolling together over 160 patients) have been successfullycarried out and all demonstrated 99mTc-UBI29-41-SPECTas a highly accurate and selective diagnostic tool for boneinfection in diabetic foot [55] hip prostheses [56] or otherimplant related infections [57 58] moreover it also detectsosteomyelitis [59 60] and infective endocarditis [61] Itcan be ascertained that this field of applications for 99mTc-UBI29-41 imaging will keep growing also because researchwith alternative radioisotopes other than 99mTc may yielda new group of radiopharmaceutical agents for medicaldiagnostic imaging using clinical PETCT or PETMRI infuture Novel radioisotopes such as 68Ga 82Rb or 62Cucan be produced on-demand from a radioisotope generatorwithout the need for an on-site cyclotron and may serveas radionuclides for PET 68Ga has garnered interest as apositron emitter for molecular imaging due to some of theadvantages it offers in a tracer It has a radioactive half-lifeof 6771 minutes which makes it compatible with biokineticsof most low molecular weight radiopharmaceuticals such aspeptides oligonucleotides aptamers or antibody fragmentsThe nuclear decay of the isotope is mainly through positronemission (89) with average positron energy of 740 keVAdditionally the coordination chemistry of Ga3+ is wellunderstood which is helpful in designing chelating agentswhich can be used to link this radionuclide to a targeting

vector [62] Recently UBI29-41 was conjugated to themacro-cycle 147-triazacyclononane-147-triacetic acid (NOTA)and subsequently labeled with 68Ga [63] This approachwas initially utilized with 14710-tetraazacyclododecane-N1015840N10158401015840N101584010158401015840N1015840101584010158401015840-tetraacetic acid (DOTA) to yield the peptidederivatives such as DOTA-TOC or DOTA-TATE for 68Ga-complexation which subsequently allowed tumor-receptor-based PET imaging In a preclinical study using 68Ga-NOTAUBI29-41-PET it was shown that the macrocycleconjugation did not compromise the peptidersquos ability toselectively bind to bacteria in vivo [64] Aside from UBIthere are other compounds evaluated for infection andinflammation imaging [54] but the majority of antimicro-bial peptides available remain underinvestigated in termsof infection imaging In 2000 the human neutrophil pep-tides (HNP1-3) were considered amongst other peptides asuseful agent for targeting infection as part of the defencemechanism in monocytelymphocyte cultures HNPs playsa chemotactic role as mediating molecules This ambiguousrole may be a drawback in developing HNPs for imaginghence the usage of particular peptides as targeting vec-tors may have some secondary limitations despite of theirfavourable cellular properties [65] As radiopharmaceuticalsare mostly administered by iv injection the peptides canbe prone to enzymatic degradation or the destabilisationof the radioisotope as reported for 18F-UBI29-41 [66] Thelactoferrin-derived peptide hLF(1-11) showed great sensitivityas infection agent targetingmultidrug-resistantAcinetobacterbaumannii strains however binding to Candida albicans afungus and the hepatobiliar excretionmade it less favourablefor imaging [51] Moreover hLF showed immune-activatingor bactericidal effects depending on the dose administeredthat is encountered with a negative feedback mechanism byinterleukin-10 modulation [67 68] Another example theAMP Latarcin-2a extracted from the venom of the CentralAsian spider Lachesana tarabaevi has undesirable lytic activ-ity against Gram-positive and Gram-negative bacteria ery-throcytes and yeast at micromolar concentrations and thusmakes it less considered for bacterial detectionwith PET [69]Moreover most bacteria are able to produce both surface-bound andor secretory proteases a defence strategy thatcan degrade or inactivate AMPs Consequently using AMP-derived compounds as imaging agents would result in false-negative diagnostics where a persistent infectionmight easilybe misjudged or overseen entirely Through understandingthese specific bacteria-intrinsic defence mechanisms it canbe avoided to use vulnerable AMP-derived structures asinfection imaging agents It should also be noted that exceptfor a few structures research did not reveal a bacteria-specific receptor-like target that complements the potentialpeptides as a ligands or allosteric modulators Tumor cellsin contrast express specific receptors integrin- bombesin-or somatostatin ligands or antagonists which are targetedby SPECT or PET tracers [10 70] Moreover the hostrsquosimmune system when reacting on infections has patho-logic pathways that can be imaged using PET Activatedmacrophages may act as an equivalent host-dependent targetwhich can be visualized by 18F-FDG nonspecifically [3] but


the actual bacterial burden remains overseen In contrastAMP-derived peptides are acting in a host-independentmechanism radiolabeled peptides will bind to free andto cell-adherent but not phagocytised bacteria and hencebacteria become invisible for 99mTc-UBI29ndash41-SPECT oncethey are incorporated by macrophages [71] The use of thismodality potentially allows for the early detection of infectionprior to any morphological changes in the body taking place[7] It also allows for the discrimination of infection from asterile inflammation which may appear superficially similaras both may present as reddened swollen and unusuallywarm areasThis is due to the increased blood flow enhancedvascular permeability and influx of white blood cells whichare common in both situations [4]The latter approachwouldemphasize a dual tracer imaging regimen in future clinicalstudies or even dual tracer administration (if the respectiveradioisotope properties and pharmacokinetic properties arecomplementing the approach) In summary the ideal tracerfor clinical PET imaging of infection should meet severalcriteria (1) It should sustain substantial blood degradationand have a reasonable degree of lipophilicity (2) it shouldaccumulate and be retained at the site of infection (ideally byinternalization and subsequent amplification) with minimalaccumulation in noninfected sites (3) it should have rapidclearance of nonspecific activity uptake from the surroundingregions for high signal-to-noise-ratios and (4) it shouldhave minimal side effects and should be easy to prepareat low cost UBI29-41 has proven its usefulness towardsgeneric infection imaging and other suitable AMPs basedradiopharmaceuticals will follow undoubtedly

Abbreviations

AMP Antimicrobial peptidesB subtilis Bacillus subtilisC albicans Candida albicansCL CardiolipinCT Computed tomographyDNA Deoxyribonucleic acidE coli Escherichia coliFDG FluorodeoxyglucoseLPS LipopolysaccharideMRI Magnetic resonance imagingPC PhosphatidylcholinePE Phosphatidyl-ethanolaminePET Positron emission tomographyPG Phosphatidyl-glycerolPS Phosphatidyl-serineS aureus Staphylococcus aureusSM SphingomyelinSPECT Single photon emission computed tomographyST SterolsTNT Target to nontarget ratioTATE (Tyrosine3)octreotateTOC (Phenylalanin1-Tyrosine3)octreotideUBI Ubiquicidin (fragment)

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

The work related to this review was funded and kindlysupported by National Research Foundation (NRF) theInstitute of Cellular andMolecular Medicine and the NuclearTechnologies in Medicine and the Biosciences Initiative(NTeMBI) a national technology platform developed andmanaged by the South African Nuclear Energy Corporation(Necsa) and funded by the Department of Science andTechnology (DST)

References

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[2] S Basu T Chryssikos S Moghadam-Kia H Zhuang D ATorigian and A Alavi ldquoPositron emission tomography as adiagnostic tool in infection present role and future possibil-itiesrdquo Seminars in Nuclear Medicine vol 39 no 1 pp 36ndash512009

[3] C J Palestro ldquoRadionuclide imaging of infection In search ofthe grailrdquo Journal of NuclearMedicine vol 50 no 5 pp 671ndash6732009

[4] H J J M Rennen O C Boerman W J G Oyen and FH M Corstens ldquoImaging infectioninflammation in the newmillenniumrdquo European Journal of Nuclear Medicine vol 28 no2 pp 241ndash252 2001

[5] O C Boerman E TM DamsW J G Oyen F HM CorstensandG Storm ldquoRadiopharmaceuticals for scintigraphic imagingof infection and inflammationrdquo Inflammation Research vol 50no 2 pp 55ndash64 2001

[6] A Signore M Chianelli C DAlessandria and A AnnovazzildquoReceptor targeting agents for imaging infammatininfectionwhere are we nowrdquo Quarterly Journal of Nuclear Medicine andMolecular Imaging vol 50 no 3 pp 236ndash242 2006

[7] S J Goldsmith and S Vallabhajosula ldquoClinically provenradiopharmaceuticals for infection imaging mechanisms andapplicationsrdquo Seminars in Nuclear Medicine vol 39 no 1 pp2ndash10 2009

[8] C P J M Brouwer M Wulferink and M M Welling ldquoThepharmacology of radiolabeled cationic antimicrobial peptidesrdquoJournal of Pharmaceutical Sciences vol 97 no 5 pp 1633ndash16512008

[9] M R Yeaman and N Y Yount ldquoMechanisms of antimicrobialpeptide action and resistancerdquoPharmacological Reviews vol 55no 1 pp 27ndash55 2003

[10] S M Okarvi ldquoPeptide-based radiopharmaceuticals futuretools for diagnostic imaging of cancers and other diseasesrdquoMedicinal Research Reviews vol 24 no 3 pp 357ndash397 2004

[11] E Guanı-Guerra T Santos-Mendoza S O Lugo-Reyes andL M Teran ldquoAntimicrobial peptides general overview andclinical implications in human health and diseaserdquo ClinicalImmunology vol 135 no 1 pp 1ndash11 2010

[12] M Zasloff ldquoAntimicrobial peptides of multicellular organismsrdquoNature vol 415 no 6870 pp 389ndash395 2002


[13] D Takahashi S K Shukla O Prakash and G Zhang ldquoStruc-tural determinants of host defense peptides for antimicrobialactivity and target cell selectivityrdquo Biochimie vol 92 no 9 pp1236ndash1241 2010

[14] I Zelezetsky and A Tossi ldquoAlpha-helical antimicrobial pept-ides-using a sequence template to guide structure-activityrelationship studiesrdquo Biochimica et Biophysica Acta vol 1758no 9 pp 1436ndash1449 2006

[15] R M Epand and H J Vogel ldquoDiversity of antimicrobial pep-tides and their mechanisms of actionrdquo Biochimica et BiophysicaActa vol 1462 no 1-2 pp 11ndash28 1999

[16] L Zhang A Rozek and R E W Hancock ldquoInteraction ofcationic antimicrobial peptides with model membranesrdquo TheJournal of Biological Chemistry vol 276 no 38 pp 35714ndash35722 2001

[17] A Tossi L Sandri and A Giangaspero ldquoAmphipathic alpha-helical antimicrobial peptidesrdquo Biopolymers vol 55 no 1 pp4ndash30 2000

[18] M Dathe and T Wieprecht ldquoStructural features of helicalantimicrobial peptides their potential to modulate activity onmodel membranes and biological cellsrdquo Biochimica et Biophys-ica ActamdashBiomembranes vol 1462 no 1-2 pp 71ndash87 1999

[19] M A Kol A I P M de Kroon D T S Rijkers J A Killianand B De Kruijff ldquoMembrane-spanning peptides induce phos-pholipid flop amodel for phospholipid translocation across theinner membrane of E colirdquo Biochemistry vol 40 no 35 pp10500ndash10506 2001

[20] M M Welling A Lupetti H S Balter et al ldquo99mTc-labeledantimicrobial peptides for detection of bacterial and Candidaalbicans infectionsrdquo Journal of Nuclear Medicine vol 42 no 5pp 788ndash794 2001

[21] N Y Yount and M R Yeaman ldquoMultidimensional signaturesin antimicrobial peptidesrdquo Proceedings of the National Academyof Sciences of the United States of America vol 101 no 19 pp7363ndash7368 2004

[22] N Sitaram and R Nagaraj ldquoInteraction of antimicrobial pep-tides with biological and model membranes structural andcharge requirements for activityrdquo Biochimica et Biophysica ActaBiomembranes vol 1462 no 1-2 pp 29ndash54 1999

[23] M Dathe H Nikolenko J Meyer M Beyermann and MBienert ldquoOptimization of the antimicrobial activity ofmagaininpeptides by modification of chargerdquo FEBS Letters vol 501 no1ndash3 pp 146ndash150 2001

[24] Y Shai ldquoMode of action of membrane active antimicrobialpeptidesrdquo Biopolymers vol 66 no 4 pp 236ndash248 2002

[25] T Wieprecht M Dathe M Beyermann et al ldquoPeptidehydrophobicity controls the activity and selectivity of magainin2 amide in interaction with membranesrdquo Biochemistry vol 36no 20 pp 6124ndash6132 1997

[26] N Uematsu and K Matsuzaki ldquoPolar angle as a determinant ofamphipathic 120572-helix-lipid interactions a model peptide studyrdquoBiophysical Journal vol 79 no 4 pp 2075ndash2083 2000

[27] K Matsuzaki ldquoWhy and how are peptide-lipid interac-tions utilized for self-defense Magainins and tachyplesins asarchetypesrdquo Biochimica et Biophysica ActamdashBiomembranes vol1462 no 1-2 pp 1ndash10 1999

[28] T Ganz ldquoDefensins antimicrobial peptides of innate immu-nityrdquo Nature Reviews Immunology vol 3 no 9 pp 710ndash7202003

[29] LV Collins S A Kristian CWeidenmaier et al ldquoStaphylococ-cus aureus strains lacking D-alanine modifications of teichoic

acids are highly susceptible to human neutrophil killing and arevirulence attenuated in micerdquoThe Journal of Infectious Diseasesvol 186 no 2 pp 214ndash219 2002

[30] M Gross S E Cramton F Gotz and A Peschel ldquoKey role ofteichoic acid net charge in Staphylococcus aureus colonizationof artificial surfacesrdquo Infection and Immunity vol 69 no 5 pp3423ndash3426 2001

[31] A Peschel ldquoHow do bacteria resist human antimicrobial pep-tidesrdquo Trends in Microbiology vol 10 no 4 pp 179ndash186 2002

[32] K Matsuzaki ldquoControl of cell selectivity of antimicrobialpeptidesrdquo Biochimica et Biophysica Acta vol 1788 no 8 pp1687ndash1692 2009

[33] W L Maloy and U P Kari ldquoStructure-activity studies onmagainins and other host defense peptidesrdquo Biopolymers vol37 no 2 pp 105ndash122 1995

[34] E Breukink I Wiedemann C van Kraaij O P Kuipers H-Sahl and B de Kruijff ldquoUse of the cell wail precursor lipid II bya pore-forming peptide antibioticrdquo Science vol 286 no 5448pp 2361ndash2364 1999

[35] I Wiedemann E Breukink C van Kraaij et al ldquoSpecific bind-ing of nisin to the peptidoglycan precursor lipid II combinespore formation and inhibition of cell wall biosynthesis forpotent antibiotic activityrdquo The Journal of Biological Chemistryvol 276 no 3 pp 1772ndash1779 2001

[36] S E Blondelle K Lohner and M Aguilar ldquoLipid-inducedconformation and lipid-binding properties of cytolytic andantimicrobial peptides determination and biological speci-ficityrdquo Biochimica et Biophysica Acta Biomembranes vol 1462no 1-2 pp 89ndash108 1999

[37] L H Kondejewski M Jelokhani-Niaraki S W Farmer etal ldquoDissociation of antimicrobial and hemolytic activitiesin cyclic peptide diastereomers by systematic alterations inamphipathicityrdquo The Journal of Biological Chemistry vol 274no 19 pp 13181ndash13192 1999

[38] L Yang T M Weiss R I Lehrer and H W Huang ldquoCrys-tallization of antimicrobial pores in membranes magainin andprotegrinrdquo Biophysical Journal vol 79 no 4 pp 2002ndash20092000

[39] H W Huang ldquoAction of antimicrobial peptides two-statemodelrdquo Biochemistry vol 39 no 29 pp 8347ndash8352 2000

[40] K A Henzler Wildman D Lee and A Ramamoorthy ldquoMech-anism of lipid bilayer disruption by the human antimicrobialpeptide LL-37rdquo Biochemistry vol 42 no 21 pp 6545ndash65582003

[41] J M Sanderson ldquoPeptide-lipid interactions insights and per-spectivesrdquo Organic amp Biomolecular Chemistry vol 3 no 2 pp201ndash212 2005

[42] L Yang T A Harroun T MWeiss L Ding and HW HuangldquoBarrel-stavemodel or toroidal model A case study onmelittinporesrdquo Biophysical Journal vol 81 no 3 pp 1475ndash1485 2001

[43] B Circiumaru G Baldock and J A Cohen ldquoA prospectivestudy of fever in the intensive care unitrdquo IntensiveCareMedicinevol 25 no 7 pp 668ndash673 1999

[44] W Becker and J Meller ldquoThe role of nuclear medicine ininfection and inflammationrdquoTheLancet Infectious Diseases vol1 no 5 pp 326ndash333 2001

[45] A Lupetti P H Nibbering MMWelling and E K J PauwelsldquoRadiopharmaceuticals new antimicrobial agentsrdquo Trends inBiotechnology vol 21 no 2 pp 70ndash73 2003

[46] LMelendez-Alafort J Rodrıguez-Cortes G Ferro-Flores et alldquoBiokinetics of 99mTc-UBI 29-41 in humansrdquoNuclear Medicineand Biology vol 31 no 3 pp 373ndash379 2004


[47] LMelendez-Alafort F DM Ramırez G Ferro-Flores C A deMurphy M Pedraza-Lopez and D J Hnatowich ldquoLys and Argin UBI a specific site for a stable Tc-99m complexrdquo NuclearMedicine and Biology vol 30 no 6 pp 605ndash615 2003

[48] D Blok R I J Feitsma P Vermeij and E J K Pauwels ldquoPeptideradiopharmaceuticals in nuclearmedicinerdquoEuropean Journal ofNuclear Medicine vol 26 no 11 pp 1511ndash1519 1999

[49] P S Hiemstra M T van den Barselaar M Roest P H Nibber-ing and R van Furth ldquoUbiquicidin a novel murine microbi-cidal protein present in the cytosolic fraction of macrophagesrdquoJournal of Leukocyte Biology vol 66 no 3 pp 423ndash428 1999

[50] G Ferro-Flores C Arteaga De Murphy M Pedraza-Lopez etal ldquoIn vitro and in vivo assessment of 99mTc-UBI specificityfor bacteriardquo Nuclear Medicine and Biology vol 30 no 6 pp597ndash603 2003

[51] M M Welling A Paulusma-Annema H S Balter E KJ Pauwels and P H Nibbering ldquoTechnetium-99m labelledantimicrobial peptides discriminate between bacterial infec-tions and sterile inflammationsrdquo European Journal of NuclearMedicine vol 27 no 3 pp 292ndash301 2000

[52] M S Akhtar A Qaisar J Irfanullah et al ldquoAntimicrobialpeptide 99mTc-Ubiquicidin 29-41 as human infection-imagingagent clinical trialrdquo Journal of Nuclear Medicine vol 46 no 4pp 567ndash573 2005

[53] K A Brogden ldquoAntimicrobial peptides pore formers ormetabolic inhibitors in bacteriardquoNature ReviewsMicrobiologyvol 3 no 3 pp 238ndash250 2005

[54] C A deMurphy F Gemmel and J Balter ldquoClinical trial of spe-cific imaging of infectionsrdquo Nuclear Medicine Communicationsvol 31 no 8 pp 726ndash733 2010

[55] S Saeed J Zafar B Khan et al ldquoUtility of 99119898Tc-labelledantimicrobial peptide ubiquicidin (29ndash41) in the diagnosis ofdiabetic foot infectionrdquo European Journal of Nuclear Medicineand Molecular Imaging vol 40 no 5 pp 737ndash743 2013

[56] K Aryana A Hootkani R Sadeghi et al ldquo99mTc-labeledUbiquicidin scintigraphy a promisingmethod in hip prosthesisinfection diagnosisrdquoNuklearMedizin vol 51 no 4 pp 133ndash1392012

[57] D Beiki G Yousefi B Fallahi et al ldquo99mtc-Ubiquicidin [29-41] a promising radiopharmaceutical to differentiate ortho-pedic implant infections from sterile inflammationrdquo IranianJournal of Pharmaceutical Research vol 12 no 2 pp 347ndash3532013

[58] B Nazari Z Azizmohammadi M Rajaei et al ldquoRole of99mTc-ubiquicidin 29ndash41 scintigraphy to monitor antibiotic therapyin patients with orthopedic infection a preliminary studyrdquoNuclear Medicine Communications vol 32 no 8 pp 745ndash7512011

[59] M Assadi K Vahdat I Nabipour et al ldquoDiagnostic value of99mTc-ubiquicidin scintigraphy for osteomyelitis and compar-isons with 99mTc-methylene diphosphonate scintigraphy andmagnetic resonance imagingrdquo Nuclear Medicine Communica-tions vol 32 no 8 pp 716ndash723 2011

[60] C Dillmann-Arroyo R Cantu-Leal H Campa-Nunez CLopez-Cavazos M Bermudez-Arguelles and J C Mejıa-Herrera ldquoApplication of the ubiquicidin 29-41 scan in thediagnosis of pyogenic vertebral osteomyelitisrdquo Acta OrtopedicaMexicana vol 25 no 1 pp 27ndash31 2011

[61] A M Taghizadeh M H Mandegar and M AssadildquoTechnetium-99m-ubiquicidin scintigraphy in the detectionof infective endocarditisrdquo Hellenic Journal of Nuclear Medicinevol 17 no 1 pp 47ndash48 2014

[62] M Fani J P Andre and H R Maecke ldquo68Ga-PET a powerfulgenerator-based alternative to cyclotron-based PET radiophar-maceuticalsrdquo Contrast Media ampMolecular Imaging vol 3 no 2pp 67ndash77 2008

[63] T Ebenhan N Chadwick M M Sathekge et al ldquoPeptidesynthesis characterization and 68Ga-radiolabeling of NOTA-conjugated ubiquicidin fragments for prospective infectionimaging with PETCTrdquo Nuclear Medicine and Biology vol 41no 5 pp 390ndash400 2014

[64] T Ebenhan J R Zeevaart J D Venter et al ldquoPreclinical eval-uation of 68Ga-labeled 147-triazacyclononane-147-triaceticacid-ubiquicidin as a radioligand for PET infection imagingrdquoJournal of Nuclear Medicine vol 55 no 2 pp 308ndash314 2014

[65] B Agerberth J Charo J Werr et al ldquoThe human antimicrobialand chemotactic peptides LL-37 and 120572-defensins are expressedby specific lymphocyte and monocyte populationsrdquo Blood vol96 no 9 pp 3086ndash3093 2000

[66] D Salber J Gunawan K J Langen et al ldquoComparisonof 99mTc- and 18F-ubiquicidin autoradiography to anti-Staphylococcus aureus immunofluorescence in rat muscleabscessesrdquo Journal of Nuclear Medicine vol 49 no 6 pp 995ndash999 2008

[67] L Dijkshoorn C P J M Brouwer S J P Bogaards A NemecP J Van Den Broek and P H Nibbering ldquoThe syntheticn-terminal peptide of human lactoferrin hLF(1-11) is highlyeffective against experimental infection caused by multidrug-resistant Acinetobacter baumanniirdquo Antimicrobial Agents andChemotherapy vol 48 no 12 pp 4919ndash4921 2004

[68] C P J M BrouwerM Rahman andMMWelling ldquoDiscoveryand development of a synthetic peptide derived from lactoferrinfor clinical userdquo Peptides vol 32 no 9 pp 1953ndash1963 2011

[69] A Won A Ruscito and A Ianoul ldquoImaging the membranelytic activity of bioactive peptide latarcin 2ardquo Biochimica etBiophysica ActamdashBiomembranes vol 1818 no 12 pp 3072ndash3080 2012

[70] X Chen E Sievers Y Hou et al ldquoIntegrin 120572v1205733-targetedimaging of lung cancerrdquo Neoplasia vol 7 no 3 pp 271ndash2792005

[71] P H Nibbering M M Welling A Paulusma-Annema C P JM Brouwer A Lupetti and E K J Pauwels ldquo99mTc-labeledUBI 29-41 peptide for monitoring the efficacy of antibacterialagents in mice infected with Staphylococcus aureusrdquo Journal ofNuclear Medicine vol 45 no 2 pp 321ndash326 2004

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


selective infection imaging agents Direct ex vivo labelingof leukocytes is considered the ldquogold standardrdquo for infectionimaging by PET Unfortunately this process is very laboriousand time-consuming and requires the handling of bloodproducts [3ndash5] Alternatively indirectly labeled leukocytescan be achieved using radiolabeled molecules such aschemotactic peptides or cytokines that bind to receptorson the leukocytes [3] Unfortunately the biological effectsof some of the leukocyte receptor-targeting compoundshave limited their clinical use as infection-specific molecularimaging agents [5] Although the most commonly labeledleukocytes neutrophils and lymphocytes are quite selectivefor infection there are cases when they may fail to detect aninfection or accumulate in noninfected sites If the infectionfails to elicit an immune response labeled leukocytes will notaccumulate at the infected loci which may be the case ina severely immune-compromised individual or in the caseof infection by certain pathogens such as Mycobacteriumtuberculosis or Pneumocystis carinii Some noninfectiousimmune conditions such as rheumatoid arthritis may alsoprovoke an immune response and accumulate the tracer[3] Through the use of different tracers different targetingstrategies are possible to image infection using PET

Tracers that interact directly with the pathogenicmicrobes responsible for infection are by nature highlyspecific for infection and unlike labeled leukocytes should notaccumulate in sterile inflammations These types of tracersinclude radiolabeled antibiotics and antimicrobial peptidesTechnetium-99m labeled ciprofloxacin (99mTc-ciprofloxacin)has been the most widely studied antibiotic-based tracerfor SPECT infection imaging [6] targeting DNA Gyrase anenzyme present in all dividing bacteria and is not thoughtto accumulate in dead bacteria or sterile inflammationsSome problems associated with its use as a tracer in SPECTinfection imaging have occurred with regards to poorradiochemical purity and stability [3] More recently it hasbeen reported that localisation at infected foci takes placeprimarily through increased extravasation and stasis Thisprocess also occurs at uninfected sites with increased vascularpermeability and 99mTc-ciprofloxacin may accumulate atsites of sterile inflammation thereby reducing its specificityfor infection [7]

Antimicrobial peptides (AMP) have attracted interest aspotential targeting vectors for the development of PET tracersdesigned for the detection of infection These peptides arefound in a variety of organisms including humans andso far hundreds have been isolated and characterised Itis believed that these peptides function as broad-spectrummicrobicides and form part of the innate immune systemof many eukaryotes including humans Regardless of theirorigin they share many common properties such as havinga net positive charge being amphipathic and in most casesare membrane active [8] Due to their role in the bodyas a natural microbicide these antimicrobial peptides areselectively cytotoxic to bacteria whilst showing minimalcytotoxicity towards cells of the host organism It is thoughtthat the net cationic nature of the peptides results in arelatively strong electrostatic attraction to negatively chargedbacterial cells and a relativelyweak attraction to the eukaryote

host cells which are usually less negatively charged thanprokaryotes and is believed to form the basis of this cell-typediscrimination [9]The ability of these peptides to accumulateat sites of infection combined with their almost negligiblecytotoxicity or attraction to host cells makes these peptidesattractive as targeting vectors for PET imaging of infection[10]

2 Overview of Antimicrobial Peptides

Antimicrobial peptides are evolutionarily conserved biom-olecules that form part of the defence mechanisms in manyorganisms [11] ranging from prokaryotes to multicellularanimals such as humans [9] They form part of the first lineof defence against pathogenic microbes in higher animalsand in many lower forms of life they are the only form ofdefence against pathogenic and saprophytic microbes [12]The selective cytotoxicity of these peptides where they attackthe pathogenic microbes and leave the host cells unharmedis due to the fundamental differences in composition andstructure of the host cells to those of the pathogenic bacteriaand yeasts Despite some AMPs showing immunomodula-tory effects andor chemotactic behaviour a common featureof these antimicrobial peptides is that they are amphipathicbut possess an overall positive charge [9] Approximately1500 antimicrobial peptides have been characterised in awiderange of organisms and classification of these peptides canbe complicated due to the high degree of sequence dissim-ilarity between the various peptides However classificationhas been attempted based on amino acid composition andsecondary structures

Three large groups (Table 1) have been identified namely120572-helical peptides cysteine-containing 120573-sheet peptides andflexible peptides rich in specific amino acids such as prolinetryptophan histidine arginine and glycine [13]

21 120572-Helical Antimicrobial Peptides Approximately 30 to50 of all antimicrobial peptides identified and studied todate contain predominant 120572-helical structures This maybe due the relative ease with which these peptides arechemically synthesised which allows for extensive charac-terisation in the laboratory These peptides usually consistof 12ndash40 amino acid residues and contain an abundance ofhelix stabilising residues such as alanine leucine and lysinebut never cysteine In aqueous solutions these peptides areoften unstructured but assume their amphipathic 120572-helicalconformations when associated with a cell membrane or ina membrane mimetic environment Often these peptides arenot strictly 120572-helices and may contain an internal kink [14]

22 120573-Sheet Antimicrobial Peptides Theothermajor group ofantimicrobial peptides are those that typically contain two toten cysteine residues that form one to five interchain disulfidebonds This bonding interaction allows these peptides toadopt the 120573-sheet conformation Most 120573-sheet antimicrobialpeptides are part of the defensin family and these peptidesare evolutionarily conserved across plants fungi insectsmolluscs and vertebrate animals Defensins typically consist




120572-helical


120573-sheet




Flexible












Externalleaflet

Internalleaflet

Internalleaflet

Externalleaflet

Weak






Hyd

roph

obic

inte

ract

ions

Elec

tros

tatic

and

hydr

opho

bic i

nter

actio

ns

Strong


Rela

tive c

ompo

sitio

n (

)

CL PG PE PC SM ST

Exop

lasm

icCy

topl

asm

ic


40302010

50

20304050

10














119867) charge (119876)














1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Charged polar

Neutral polar

Hydrophobic

Other (gly)
























(a) (b) (c)


Tc

Reducing agents

99mTcO minus

4

(a)

OH

O

Tc

NNH

OH

HN

O

N

OO

OHOHHO

H

N

O

OC Peptide

(b)








101 20 30 40 50 59















Abbreviations




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















120572-helical


120573-sheet




Flexible












Externalleaflet

Internalleaflet

Internalleaflet

Externalleaflet

Weak






Hyd

roph

obic

inte

ract

ions

Elec

tros

tatic

and

hydr

opho

bic i

nter

actio

ns

Strong


Rela

tive c

ompo

sitio

n (

)

CL PG PE PC SM ST

Exop

lasm

icCy

topl

asm

ic


40302010

50

20304050

10














119867) charge (119876)














1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Charged polar

Neutral polar

Hydrophobic

Other (gly)
























(a) (b) (c)


Tc

Reducing agents

99mTcO minus

4

(a)

OH

O

Tc

NNH

OH

HN

O

N

OO

OHOHHO

H

N

O

OC Peptide

(b)








101 20 30 40 50 59















Abbreviations




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Externalleaflet

Internalleaflet

Internalleaflet

Externalleaflet

Weak






Hyd

roph

obic

inte

ract

ions

Elec

tros

tatic

and

hydr

opho

bic i

nter

actio

ns

Strong


Rela

tive c

ompo

sitio

n (

)

CL PG PE PC SM ST

Exop

lasm

icCy

topl

asm

ic


40302010

50

20304050

10














119867) charge (119876)














1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Charged polar

Neutral polar

Hydrophobic

Other (gly)
























(a) (b) (c)


Tc

Reducing agents

99mTcO minus

4

(a)

OH

O

Tc

NNH

OH

HN

O

N

OO

OHOHHO

H

N

O

OC Peptide

(b)








101 20 30 40 50 59















Abbreviations




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























119867) charge (119876)














1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Charged polar

Neutral polar

Hydrophobic

Other (gly)
























(a) (b) (c)


Tc

Reducing agents

99mTcO minus

4

(a)

OH

O

Tc

NNH

OH

HN

O

N

OO

OHOHHO

H

N

O

OC Peptide

(b)








101 20 30 40 50 59















Abbreviations




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Charged polar

Neutral polar

Hydrophobic

Other (gly)
























(a) (b) (c)


Tc

Reducing agents

99mTcO minus

4

(a)

OH

O

Tc

NNH

OH

HN

O

N

OO

OHOHHO

H

N

O

OC Peptide

(b)








101 20 30 40 50 59















Abbreviations




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































(a) (b) (c)


Tc

Reducing agents

99mTcO minus

4

(a)

OH

O

Tc

NNH

OH

HN

O

N

OO

OHOHHO

H

N

O

OC Peptide

(b)








101 20 30 40 50 59















Abbreviations




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















101 20 30 40 50 59















Abbreviations




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Abbreviations




Acknowledgments


References
















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















review article antimicrobial peptides: their role as...

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