Antimicrobial peptides (AMPs)1960s: John Spitznagel observed proteins in guinea-pig &rabbit granulocytes that could kill bacteria1970s: Robert Lehrer isolated 2antibacterial peptides fromrabbitsPeptides christened ‘defensins’
Tried to develop therapeutic usefor magaininsCurrently researching humanpeptidesMichael Zasloff: 1986Observed antibacterial resistance inXenopus laevis• Identified 1 st amphibian peptide –magainin• Magainins stored in neuroepithelial cells• Form bacteria-resistant ‘mesh’• Amphipathic α helixXenopus laevis
ANTIMICROBIAL DATABASES ON THE WEBThis laboratory has created and maintains theAntimicrobial Sequences DatabaseANTIMIC: adatabase ofantimicrobialsequencesM. BrahmacharyInstitute of InfocommResearch, Singaporehttp://nar.oxfordjournals.org/cgi/content/full/32/suppl_1/D586#SEC3Welcome to the Antimicrobial Peptide Database
Why the interest?• Increasing interest in innateimmunity• Need for new antibioticsAnimals:Feedadditives15%Animaltherapeuticuse 33%Human use:G.P./hospitals52%Increase in AntibioticResistanceAntibiotic use in the E.U. in 1997% resistant bacterial strains isolated from infected patients (U.K.)
Data from theWellcome Trust, U.K.
Emerging Vancomycin-resistant EnterococcalInfections*252015105ICUNon-ICU01989 1990 1991 1992 1993 1994 1995 1996 1997 199828% of the bacteria that most frequently cause hospitalacquiredinfections are resistant to the preferred antibiotic fortreatment Data from CDC, U.S.A.
A GLOBAL PROBLEM:
PREDICTED VALUE OF NEW ANTIBIOTICS(“new” - introduced since 2000)
Where are AMPs found ?ProcaryotesMicroalgaeHigher plantsAnimalsNumerous studies, mainly eubacteriaNo clear reportsFew; mostly reports for seedsMajority of studiesProportions oforganisms studiedfor AMPsAmphibia39%Mammalia27%Amphibia – recent interestin AMP activity againstfungal pathogensOther11%Insecta23%
• Small, 3-4 kDa peptidesMAMMALIAN AMPSDEFENSINS• Active against Gram + & Gram – bacteria• Structural motif – 3 disulphide bonds• Bonds essential for maintaining hydrophobic β sheet• All evolved from a common ancestral gene• Not homologous to invertebrate defensins• Active against Gram +ve & Gram -ve bacteria,fungi, some enveloped viruses and eukaryotic cells
α - Defensinssignal propiece active peptide19 aa 40-45 aa30 aa• First reported 1960s from rabbit neutrophils• Usually found in neutrophils, macrophages and Paneth cells• Constitutive expression; mature defensins released onmicrobial invasion• 6-cysteine motif, forming 3 disulphide bonds
β-DEFENSINSprepropiece20 - 30 aaactive peptide29 - 35 aa• First discovered 1990s in bovine granulocytes &tracheal epithelia• Predominantly found in epithelial tissues• Expression constitutive or inducedRecently (1999), circular θ minidefensins identified inRhesus monkeys
signal20-30 aaconserved prepro regionCATHELICIDINSpropiece100-120 aapeptide19-94 aavariable active domainFamily found only in mammals, ca. 40 membersN-terminal highly conserved, identifies cathelicidinsExpressed in bone marrow, stored in peroxidase-negativeneutrophil granules, then exocytosed from activated cellsMost are without antibacterial activity until C-terminalproteolytically removed
Structural diversity of AMPs1-disulphide bondeg bactenecinα-helicaleg cecropin2-disulphide bondseg protegrin3-disulphide bondseg α-defensinlinear non α-helicaleg indolicidinlactoferricin- the smallest known
Antibacterial fragments of larger proteinsHistonesRibosomal proteinsApolipoproteins
How do AMPs work?eg protegrin
Clustering of cationic and hydrophobic domainsBasic (+ ve) amino acids Hydrophobic (-ve) a-a
Hydrophobic interactionsANTIMICROBIAL PEPTIDE++Electrostatic &hydrophobic interactionsWeakStrongOuter leaflet+-+ +- --+Inner leaflet ------Plasma membrane of amulticellular animalBacterial cytoplasmicmembrane
α−helicalpeptideThe Shai-Matsuzaki-Huang ModelPhysicaldisruption ofmembrane &fragmentationCarpeting ofouter leaf.Peptidemoves intoinner leaf.Integration.Outerleaf stretchesrelative to inner.Stress results.Diffusion frommembrane tointracellulartargets.Phase transition & ‘wormhole’formation. Transient pores form.
SYNERGISM• Mor et al., 1994 demonstrated potent synergy between the 5dermaseptins (DSs1 to s5) from Phyllomedusa sauvagii (frog)• Occurs despite similar structure and proposed mode of action?• Proposed that significance of 5 closely related peptides is toprovide potent, broad-spectrum protection from invading microorganisms• Also known that antimicrobial synergy occurs betweendifferent peptides from Xenopus laevis eg., magainins• Multi-site mode of action means lower concentrations ofindividual active peptides are required
RegulationAMPs may be expressed constitutively andstored in cells before release at site or time ofneed, eg carcinin in crab haemocytesIn some cells/tissues AMP expression is inducedby micro-organisms, eg most insects, humandefensinsHow is AMP expression induced ?
Toll receptors• Originally discovered in Drosophila by Nüsselein-Volhard & Wieschaus as part of the pathway thatdetermines the dorso-ventral axis in the embryo•Named Toll as it is slang for ‘far out / wild’ in German• Only later found to be associated with innateimmunity - Toll deficient mutant flies more susceptibleto fungal infections•Acitivation of Toll leads ultimately to up-regulation ofimune effector genes, including AMPs
OTHER BIOLOGICAL FUNCTIONSOF ANTIMICROBIAL PEPTIDESAMPS: CHEMOTAXISAMP Target Refα- defensinsβ-defensinscathlicicidinsmonocytes + dendritic cellsT cells + dendritic cellsneutrophils,monocytes + T cellsTerrito et al1989Yang et al 1999Yang et al 2000
AMPS: ACTIVATION & STIMULATIONDEFENSINSUpregulate TNFα & IL-1β in monocytes (Van Wetering 1997)Bind C1q and activate classical pathway (Prohaska et al 1997)Enhance adaptive system via CD4 + T cells (Lillard et al 1999)CATHELICIDINSPR-39 assists in wound repair by inducing expression ofsyndecans (Gallo et al 1994)PR-39 regulates angiogenesis (Li et al 2000)
AMPS IN OTHER ANIMALSMOLLUSCSDefensins(mussels)Myticin(mussels)Mytilin(mussels)severaluncharacterisedpeptides (oysters)
AMPs in MytilusAMPs produced in haemocytes, stored in haemocytegranulesSmall peptides, characterised by high cysteine contentDefensins and myticins principally active against Grampositivebacteria, mytilins have much wider spectrumMajority of mytilin isoforms active against Vibrio speciesLack of expression of a mytilin isoform in early stage larvaemay contribute to susceptibility of larval bivalves toVibriosis(Mitta et al, 2000 a and b)
AMPs in crustaceansPenaeidins(shrimps)Crustins(shrimps)severaluncharacterisedpeptides variousspecies‘Carcinin’(shore crab)‘Bac-C’(shore crab)KILLING
Antibacterial peptides in C. maenas3 unidentifiedantibacterialproteins70 kDa45 kDa14 kDaGRANULARHAEMOCYTES‘Bac-C’‘Carcinin’ Kills G + & G - bacteria11.5 kDa 6.5 kDa mol massCationic, proline-rich60% similarity withbovine cathelicidins(Schapp et al, 1996)
CarcininSDS-PAGERelf et al, 199911.5 kDa14.5 kDa6.5 kDaCharacteristics• molecular mass: 11.5 kDa• cationic• constitutive• kills Gram +ve bacteria• salt requiring• heat stable to 98ºC
Penaeidins(shrimps)Destoumieux et al, 2000Family of antimicrobial peptides, 5.5-6.5 kDa,from Pennaeus vannameiPredominantly active against Gram-+ bacteria & fungiSynthesized and stored in granular haemocytesCationic, constitutive
Crustins(shrimps)Bartlett et al, 2002Family of antimicrobial peptides, also identified fromfrom Pennaeus vannameiSequence similarities to Carcinin, but higher molecularmass (16.5 kDa)Not so far as well investigated with respect to expressionand function
Summary• Diverse group of low molecular mass (< 10 kDa)proteins with broad-spectrum microbicidal activity• Produced by many organisms• Often amphipathic and cationic• May act synergistically with lysozyme or otherAMPs• Expression induced through TLRs• Important effectors of innate immunity
References:Hans Boman (1981) Structure and immune function of cecropins Nature 292: 246-48Robert Hancock, Daniel Chapple (1999) Peptide Antibiotics. Antimicrobial Agents &Chemotherapy, 46, 1317-1323Robert I Lehrer, Tomas Ganz (2002) Defensins of vertebrate animals Current Opinionin Immunology 14:96–102Louise Rollins-Smith et al (2002) Antimicrobial peptide defences against pathogensassociated with global amphibian declines. Developmental & ComparativeImmunology, 26, 63-72.Michael Zasloff (1987) Magainins, a class of antimicrobial peptides from Xenopusskin: isolation, characterization of two active forms, and partial cDNA sequence of aprecursor. Proceedings of the National Academy of Sciences of the USA 84: 5549–5453.Michael Zasloff (2002) Antimicrobial peptides of multicellular organisms. Nature, 415,389-395