Antibacterial free fatty acids: activities, mechan

50 Pages·2010·0.89 MB·English

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ANTIBACTERIAL FREE FATTY ACIDS: ACTIVITIES,
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MECHANISMS OF ACTION AND
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BIOTECHNOLOGICAL POTENTIAL
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Andrew P. Desbois1 and Valerie J. Smith2*
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12 1Biomedical Sciences Research Complex,School of Biology,Universityof St
13 Andrews, Fife,KY169ST,UK
14 2Scottish Oceans Institute(formerlyGattyMarineLaboratory),Universityof St
15 Andrews, Fife,KY168LB,UK
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18 *Authorforcorrespondence: email [email protected]; phone +44(1334)
19 463474; fax [phone].
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21 Runningtitle:ANTIBACTERIALFREEFATTYACIDS
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1 Abstract
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3 Amongst thediverseand potent biological activities offreefattyacids (FFAs)is the
4 abilitytokill orinhibit thegrowthofbacteria. The antibacterial properties ofFFAs
5 areusedbymanyorganisms todefendagainst parasiticorpathogenicbacteria.Whilst
6 theirantibacterial mode ofactionis still poorlyunderstood,theprimetarget ofFFA
7 actionis thecell membrane.Here,FFAs disrupt theelectrontransport chain and
8 oxidativephosphorylation.Besides interferingwithcellularenergyproduction,FFA
9 actionmayalso result from theinhibitionofenzymeactivity,impairment ofnutrient
10 uptake, generationoftoxicperoxidationandauto-oxidationdegradationproducts or
11 direct lysis ofbacterial cells. Theirbroadspectrum ofactivity,non-specificmodeof
12 actionandsafetymakes them attractive as antibacterial agents forvarious applications
13 inmedicine,agriculture andfoodpreservation,especiallywheretheuseof
14 conventional antibiotics is undesirableorprohibited.Moreover,theevolutionof
15 inducibleFFA-resistant phenotypes is less problematicthanwithconventional
16 antibiotics. Thepotential forcommercial orbiomedical exploitationofantibacterial
17 FFAs, especiallyforthosefrom natural sources, is discussed.
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19 Keywords:antibiotic; antimicrobial; drugresistance; lipid; natural products.
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21 1.Introduction
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23 Fattyacids (FAs) areubiquitous molecules typicallyfoundboundtoother compounds
24 such as glycerol,sugars orphosphateheadgroups toform lipids. Lipids are integral
25 components ofcell structures, e.g.membranes, whicharemadeupofphospholipids,
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1 andenergystores that are oftencomposedoftriglycerides. FAs canbereleasedfrom
2 lipids, typicallybyenzymeaction,tobecome freefattyacids (FFAs),whichhave
3 diverseandpotent biological activities (Table1).
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5 FFAs consist ofachainofcarbon atoms attachedtohydrogenatoms (Figure1).The
6 numberofcarbonatoms varies but thoseinbiological systems usuallyhave aneven
7 numberbetween10and 28andthis reviewmainlyconcentrates onthese. At oneend
8 ofthecarbonchainis a carboxyl group(–COOH) andat theother endis a methyl
9 group (–CH )(Figure1). Thecarboxyl groupis hydrophilicandionised when
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10 solubilisedinwaterwhereas thecarbonchainis hydrophobic,makingtheentire
11 moleculeamphipathic.FAs with<8carbon atoms areconsideredshort-chainwhereas
12 thosewith>16carbon atoms areregardedas long-chain.Unsaturated FAs haveoneor
13 moreC=C doublebonds inthecarbonchainwhile thecarbon atoms insaturatedFAs
14 areall joinedbyC–C singlebonds (Figure1). Lipases (the groupoflipolyticenzymes
15 that cleave FAs from lipidheadgroups byhydrolysis togiveFFAs) canbe specificfor
16 certaintypes oflipidbut theymayalso discriminatetheFAs that theycleaveontheir
17 positiononthelipidheadgroup andthelengthand unsaturationoftheFA’s carbon
18 chain.
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20 Thebiological activities ofFFAs have roles inhost defences against potential
21 pathogenicoropportunisticmicro-organisms. An important aspect ofthis is growth
22 inhibitionorthedirect killingofbacteria.Thereis nowanextensiveliterature
23 concerningthe antibacterial effects ofvarious FFAs from awide rangeof biological
24 sources, includingalgae, animals andplants (McGawet al.2002; Willeand
25 Kydonieus 2003; Desbois et al.2008,2009). Indeed,FFAs areoftenidentifiedas the
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1 activeingredients inethnicandherbal medicines (Yffet al.2002; McGaw et al.
2 2002).This review aims tosummarisesomeofthis workandtodiscuss the various
3 mechanisms andstructural features of FFAs that causes them toprevent bacterial
4 growthorsurvival.Furthermore,thepotential for commercial orbiomedical
5 exploitationofantibacterial FFAs is discussed.
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7 2.Freefattyacids in antibacterial defence
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9 Theantibacterial effects ofFFAs arefrequentlyobservedduringbioassay-guided
10 fractionationof extracts from avarietyoforganisms (HemsworthandKochan1978;
11 McGaw et al.2002; WilleandKydonieus 2003; Desbois et al.2009).The
12 antibacterial actions ofFFAs aretypicallybroadspectrum andofpotencies
13 comparabletonatural antimicrobial peptides (AMPs)invitro (Georgel et al.2005).
14 FFAs functionintheantimicrobial defences ofmanymulticellularorganisms,
15 includingmammals (HemsworthandKochan1978; Georgel et al.2005), plants
16 (Weber2002),molluscs (Benkendorff et al.2005), seaweeds (Küpperet al. 2006)and
17 amphibians (Rickrode1986).Whilst FFAs arenot as structurallydiverseas themore
18 widelystudiedAMPs theirimportanceinthehumaninnateimmunesystem is well
19 established,particularlyinthedefenceofskinand mucosal surfaces (Thormarand
20 Hilmarsson2007; Drake et al.2008). Indeed,FFAs arethemost active antimicrobial
21 agents present inhuman skinlipidsamples (WilleandKydonieus 2003).Thereis 10-
22 15µgof FFAs persquarecentimetreonhumanskin,ofwhichlauric acid (C12:0),
23 myristicacid(C14:0),palmiticacid(C16:0),sapienicacid(C16:1n-10) and cis-8-
24 octadecenoicacid(C18:1n-10)arethemost abundant (WilleandKydonieus 2003;
25 Takigawa et al.2005). FFAs areproducedonthe skinbylipolyticcleavageoflipids
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1 secretedfrom thesebaceous glands (Shalita1974; Fluhret al.2001; Drake et al.2008)
2 andtheirpresenceonthe skinis sufficient tocontrol thebacterial microbiota
3 (Takigawaet al.2005; Georgel et al.2005; Kennyet al.2009).Themost important
4 antibacterial FFAinhumanskinexudateis C16:1n-10,along-chainmonounsaturated
5 FFA, andskindeficient inthis andotherFFAs tends tobemoresusceptible to
6 colonisationbytheopportunisticpathogen, Staphylococcus aureus (Takigawaet al
7 2005; Georgel et al.2005).However,iftheskinis treatedwithC16:1n-10protection
8 against colonisationis bolstered(Takigawa et al 2005; Georgel et al.2005).Besides
9 inhibitingorkillingbacteriadirectly, FFAs also makeconditions unfavourableforthe
10 growthofcertainbacteriaontheskinsurfacebymaintainingan acidicpH (Fluhret al.
11 2001; Takigawaet al.2005). FFAs mayfurtheraffect the expressionofbacterial
12 virulencefactors (Table 1),whichareimportant oressential forthe establishment of
13 aninfection,probablybydisruptingcell-to-cell signalling.Thus, saturated and
14 unsaturated FFAs canprevent initial bacterial adhesionandsubsequent biofilm
15 formation(Kuriharaet al 1999; Osawaet al 2001; Kankaanpää et al.2004; Wonet al
16 2007; Stenz et al.2008;Davies andMarques 2009).Moreover,theswarming
17 behaviouroftheurinarytract pathogen, Proteus mirabilis,isinhibitedbymedium-
18 andlong-chainsaturated FFAs (Liaw et al.2004). Theexpressionofcertain toxins,
19 haemolysins and enzymes conferringdrugresistanceareall down-regulated inthe
20 presenceofvarious saturatedandunsaturated FFAs (RuzinandNovick2000; Liaw et
21 al.2004; Clarkeet al 2007)while genes responsibleforironuptakeandextracellular
22 proteases canbesimilarlyreduced (Kennyet al 2009).Theabilityofvarious species
23 ofbacteriatoresist theactionofFFAs andsubvert theseepithelial defences certainly
24 explains, at least inpart,thesuccess of certainskinandmucosal pathogens (Clarkeet
25 al.2007; Drake et al.2008).
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2 Perhaps less well known is therolethat FFAs playinthedefenceofsingle-celled
3 eukaryoticorganisms against bacterial threats. In microbial eukaryotes, suchas
4 microalgae, FAs arefoundprimarilyinthelipids that constitutethecell membranes
5 andenergystoragestructures but duringcellulardisintegrationlargequantities of
6 FFAs are releasedfrom cellularlipids byhost lipolyticenzymes (Jüttner2001;
7 Wichardet al.2007).Ahighproportionofthe FFAs that arefreedfrom thecell
8 membranes, includingthosearoundthephotosyntheticplastid,aremono- and
9 polyunsaturatedvarieties (Cutignanoet al.2006). TheseFFAs aretoxicto
10 invertebrate grazers, whichmayhave causedthe microalgal cell toloseits integrityin
11 thefirst instance(Jüttner2001; Wichardet al.2007).Therefore,thetoxicFFAs act to
12 reduce grazernumbers andultimatelygrazingpressure(Jüttner2001).At first it might
13 seem counter-intuitivethat this defencestrategyrequires thehost cell toundergo
14 mechanical damage and deathbut inevolutionaryterms it has benefits because
15 neighbouringmicroalgal cells, particularlyinbiofilms, wouldbeexpectedtobe
16 clones orverycloselyrelated.That thesesameFFAs arepotentlyantimicrobial means
17 similarprotectionmaybeaffordedtomicroalgae underthreat from pathogenic
18 bacteriaorviruses. Whilst theinitial host will not survive,FFAs released from a
19 microalgal cell that has beendamagedbyapathogenwill act onpathogens inthelocal
20 vicinityreducingtheirnumbers, therefore conferringsomeprotectionofits
21 neighbouringrelatives from onwardtransmission.This ‘populationlevel’defence
22 maybeconsideredmetabolicallyinexpensiveas theFFAs form essential cellular
23 components withthelipases alreadysynthesisedandpresent withinthecell tocarry
24 out vital processes.
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1 3.Antibacterial activity and FFAstructure
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3 Theantibacterial activityofeach FFAis influencedbyits structure andshape.This, in
4 turn,is afunctionofthelengthofthecarbonchainandthepresence,number,position
5 andorientationofdouble bonds (Figure2).Theliteraturecontains contrastingreports
6 concerningthe relationshipbetweena FFA’s structureandits antibacterial activitybut
7 somegeneral trends doemerge.The–OH groupofthecarboxyl groupseems tobe
8 important fortheantibacterial activityof FFAs as methylated FFAs (Figure 1)often
9 havereducedornoactivity(KodicekandWorden 1945; Zhenget al.2005).
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11 Medium-andlong-chain unsaturated FFAs tendtobemoreactiveagainst Gram-
12 positivebacteriathan Gram-negatives (Kodicek andWorden1945; Galbraithet al.
13 1971). In general,unsaturatedFFAs tendtohave greaterpotencythansaturatedFFAs
14 withthesamelengthcarbonchain(Kabaraet al. 1972; GreenwayandDyke1979;
15 Feldlauferet al.1993; Zhenget al.2005; Desbois et al.2008).Withinseries of
16 monounsaturatedFFAs, themost potent usuallyhave14or16carbonatoms (Kabara
17 et al.1972; Feldlaufer et al.1993).Often adirect correlationexists betweenthe
18 numberofdoublebonds inanunsaturated FFA’s carbonchainandits antibacterial
19 efficacy(Saitoet al.1984; KnappandMelly1986; Feldlauferet al.1993). Thedouble
20 bonds innaturallyoccurringFFAs typicallyhave cis orientationandthesetendto
21 have greater antibacterial activitythan FFAs withdoublebonds in trans orientation
22 (Galbraithet al.1971; Kabara et al.1972; Feldlauferet al.1993),probablybecause
23 thestructures oftrans-bondedunsaturated FFAs resemblesaturatedFFAs (Figure2).
24 Whilst onlyafewstudies haveinvestigatedthe effect ofbondpositioninthecarbon
25 chainof FFAs thereis someevidencethat thepositionofdoublebonds can affect
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1 potencyandspectrum of antibacterial action(Kabaraet al.1977; Feldlauferet al.
2 1993; WilleandKydonieus 2003).
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4 Forsaturated FFAs, the most activehave10or12carbons inthechainand
5 antibacterial efficacytends todecrease,as the chainlength gets longerorshorter
6 (Galbraithet al.1971; Kabara et al.1972; Bergssonet al.2001; Sunet al.2003; Wille
7 andKydonieus 2003).However,others workers havereportedthat FFAs with14,16
8 or18carbon atoms canbemorepotent than FFAs with10or12carbons against
9 certainspecies ofbacteria(Willett andMorse1966; GalbraithandMiller1973a;
10 Milleret al.1977).Comparisons betweenstudies arecomplicatedbecausedifferent
11 authors haveusedavarietyofmethodological approaches todetermineand measure
12 potencywithconsiderablevariationintheinoculum andincubationconditions.
13 Moreover,the relative activityof FFAs maydependonwhether acompletegrowth
14 inhibitionassayoran IC determinationis used(Willett andMorse1966).Toenable
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15 simplecomparison, ideallyall determinations ofminimum inhibitoryconcentration
16 (MIC)andminimum bactericidal concentration(MBC)for FFAs needto adhereto
17 standardiseddefinitions andprotocols suchas thosepublishedbytheClinical and
18 LaboratoryStandards Institute(CLSI)(CLSI,2000).
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20 4.Mechanisms of antibacterial activity
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22 It remains unclearexactlyhow FFAs exert theirantibacterial activities but theprime
23 target seems tobethebacterial cell membrane andthevarious essential processes that
24 occurwithinandat themembrane (Figure3).Someofthedetrimental effects on
25 bacterial cells canbeattributedtothedetergent properties of FFAs on account oftheir
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1 amphipathicstructures. This allows them tointeract withthecell membranetocreate
2 transient orpermanent pores ofvariablesize.At higher concentrations detergents,
3 such as FFAs, cansolubilisethemembranetosuchanextent that various membrane
4 proteins orlargersections ofthelipidbilayer arereleased.Thekeymembrane-located
5 process affectedbyFFAs is theproductionofenergycausedbyinterferencewiththe
6 electrontransport chain andthedisruptionofoxidativephosphorylation(Sheuand
7 Freese1972; GalbraithandMiller1973b; Milleret al.1977; Boyaval et al.1995;
8 WojtczakandWięckowski 1999).Otherprocesses that maycontributetobacterial
9 growthinhibitionordeathinclude: cell lysis, inhibitionofenzymeactivity,
10 impairment ofnutrient uptakeandthe generationoftoxicperoxidationandauto-
11 oxidationproducts (Figure3).FFAs cankill abacterium outright (bactericidal action)
12 orinhibit its growth(bacteriostaticaction),whichis reversible andmeans that the
13 bacterium remains viable but cannot undergocell divisioninthepresenceoftheFFA
14 (KodicekandWorden1945; SheuandFreese1972).Assays usedtoinvestigatethe
15 antibacterial activities of FFAs donot always discriminatebetweenbactericidal and
16 bacteriostaticactions but it is reasonabletoassume that growthinhibitioncannot
17 continueindefinitelyand eventuallya growth-inhibitedbacterium will die. In
18 describingtheprocesses ofantibacterial activitybelow,littledistinctionismadeas to
19 whethertheoutcomeis bactericidal orbacteriostatic.
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21 4.1Disruptionof electron transport chain
22 Theinnermembraneof Gram-positiveandGram-negativebacteriais animportant
23 siteforenergyproductionandit is wherethe electrontransport chainis located.The
24 various carriers inthe electrontransport chain,whichare embeddedwithin the
25 membrane,pass electrons from onecarriertoanotheruntil twoelectrons combine
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1 withthefinal acceptor,usuallyoxygen, andtwoprotons toform water (Mitchell
2 1961).Duringthis process protons areexportedfrom theinsideofthecell whilst the
3 concentrationofelectrons inthecytosol increases. This generates aproton gradient
4 andmembranepotential whichare crucial forthe productionofATP bytheenzyme,
5 ATP synthase (Mitchell 1961).Medium-andlong-chainsaturatedandunsaturated
6 FFAs that gainaccess throughthe cell wall oroutermembraneofabacterium,can
7 perhaps bindtothecarriers oftheelectrontransport chaindirectlyorinsert intothe
8 innermembranecausingtheelectron carriers tomoveapart orbedisplaced from the
9 membraneentirely(GalbraithandMiller1973b; Peters andChin2003). In each case
10 theabilityofthe electron transport chaintotransferelectrons is impairedso that the
11 protongradient andmembranepotential arereduced.This results inareductionin
12 ATP productionandthe bacterium becomes deprivedofanessential sourceofenergy.
13 Bothunsaturated andsaturatedFFAs couldtranslocateorbindthe electron carriers
14 directlybut completedisplacement from themembraneis likelyachievedby
15 unsaturated FFAs only,probablybecausetheyincreasemembrane fluidity(Greenway
16 andDyke1979; Chamberlainet al.1991; Stulniget al.2001).This is becausethecis-
17 bonds inunsaturatedFAs causeakinkinthe carbonchain(Figure2)that prevents
18 theseFAs from packingtightlyinthemembrane. Thus, whenmedium-and long-
19 chainunsaturatedFFAs areincorporatedintothe membranethereis anincreasein
20 fluiditythat candevelop intomembraneinstability(Chamberlainet al.1991; Stulnig
21 et al.2001).Conversely, medium-andlong-chain saturated FFAs (and trans-bonded
22 unsaturated FFAs)that lackakinkedstructurecan bepackedmoretightly(Figure2).
23 Hence,medium-andlong-chainsaturatedFFAs canreducemembranefluidityand
24 disrupt electrontransport perhaps byrestrictingthemovement ofcarriers withinthe
25 membrane(Sheu andFreese1972).Moreover, as explainedabove,solubilisationof
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