It has been shown that all types of living organismsare able to produce a variety of ribosomally synthe-sized antibacterial peptides or proteins.1 This kind ofmolecule has in recent years attracted a great deal ofattention because of the potential to fight pathogenicmicroorganisms, and because bacteria are becomingmore and more threatening to human health due to theincrease in multiple antibiotic resistances. Althoughantimicrobial peptides obtained either from highereukaryotes, lower eukaryotes, or bacteria are differentwith respect to both their activity and structure, mostshare some common properties. They normally con-sist of 20–60 amino acid residues, their net charge ispositive, they are hydrophobic and/or amphiphilic,and they are usually membrane active.
During the past 10 years the ribosomally synthe-sized antimicrobial peptides of gram positive bacteria,
and in particular of lactic acid bacteria (LAB), haveattracted considerable interest and numerous peptideshave been characterized. These peptides are com-monly referred to as bacteriocins, partly so as toclearly separate them from traditional antibiotics, towhich they are in most respects different. The bacte-riocins from lactic acid bacteria are commonly di-vided into three groups: class I—the lantibiotics (seearticle by Guder et al. in this issue); class II—the heatstable unmodified bacteriocins; and class III—thelarger heat labile bacteriocins (Table I).
The bacteriocins that will be discussed in thisreview should not be confused with the colicins, pro-duced by gram-negative bacteria, or the bacteriocinsthat are larger proteins (.20 kD) and that interferewith sensitive bacteria in many different ways, notonly by permeabilization of the membrane but also byinhibiting protein, RNA, and DNA synthesis. How-ever, it has been recognized that gram-negative bac-
teria can also produce class II like bacteriocins such ascolicin V, microcin 24, and haemocin.2–5
In recent years several review articles have beenpublished on the bacteriocins from gram-positive bac-teria, providing detailed information on different as-pects of these peptides . The present review will dealmainly with the diversity, structure, and antimicrobialactivity of class II bacteriocins from lactic acid bac-teria without dealing with their genetics, includingregulation of synthesis.
THE DIVERSITY OF CLASS IIBACTERIOCINS IN LAB
Many LAB such asLactococcus lactis, Enterococcus,Pediococcus, Leuconostoc, Streptococcus thermophi-lus, and Lactobacillus species are actively used intraditional fermented food produced from milk, meatand vegetables. In addition, other LAB includingCar-nobacteriumand Streptococcusisolates have beenshown to produce such bacteriocins. More than 50
class II bacteriocins from LAB have been isolated andcharacterized, and this has led to subgrouping accord-ing to various criteria (Table I),1,10,13 although withincreased knowledge it has become apparent that thepresent classification needs revision. In this respect, itshould also be strongly emphasized that many small,nonmodified LAB bacteriocins, such as lactococcin Aand B, are not included in any of the class II sub-groups.14,15 This review is not intended to cover thelarge field of class II bacteriocins in detail, but ratherto give a conceptual view of what is known aboutthese peptides.
The largest subgroup of class II bacteriocins is that of theso-called pediocin-like bacteriocins that are effective inkilling Listeria. Fourteen such peptides have been iden-tified, and their amino acid sequences consist of between
Table I Classification of Bacteriocins from Lactic Acid Bacteria
Class I: Lantibiotics Type A: Elongated shaped moleculesType B: Globular molecules
Class II: Nonmodified heat stable bacteriocins Subclass IIa: Pediocin-like bacteriocins (listeria active)Subclass IIb: Two-peptide bacteriocinsSubclass IIc: Other bacteriocins
Class III: Large heat-labile bacteriocins Proteins: Helveticin J,72 enterolysin A73
FIGURE 1 Alignment of pediocin-like bacteriocins. (a) Enterocin P (EntP) is the only class IIabacteriocin known to have asec-dependent leader. (b) Acidocin A (AcdA) does apparently belongto the class IIa bacteriocins but does not possess the conserved N-terminal disulfide bridge.
Class II Antimicrobial Peptides 51
37 and 58 residues. The pediocin family constitutes adistinct group of homologous peptides as shown in Fig-ure 1. They share an overall sequence identity of be-tween 40 to over 70%, and the most striking structuralfeature is the conserved sequence motif identified in theN-terminal part of the molecule—namely, YYGNGVX-CXKXX CXVD/NWG/A. This group of bacteriocins isby far the most investigated with respect to mode ofaction, structure, and structure–function relationshipstudies.
Structure–Function Relationship Studies
Due to the considerable number of natural variantswith both conserved N-terminal sequences and con-served biological activity, the pediocin-like bacterio-cins are a common object for structure-function rela-tionship and genetic engineering studies within classII bacteriocins. With few exceptions, members of thisgroup have a similar spectrum of bacterial inhibition,although the MIC values toward target organismsvary among them. In general, those peptides that havefour cysteines (two disulfide bridges, see Figure 1)show higher activity than those with only two cys-teines. Several papers have discussed possible struc-tural models for class IIa bacteriocins,17–23and it hasbeen suggested that the C-terminal half forms a hy-drophobic or amphiphilic transmembranea-helix, al-lowing the formation of a so-called barrel-stave po-ration complex.24 This structural model is, however,apparently incompatible with the structure of bacte-riocins having two disulfide bonds.25–27
Several studies addressed the question on whichparticular structural element or amino acid residuesare required for, or are involved in, the killing ofbacteria by pediocin-like peptides. Analysis of theamino acid sequence clearly shows that the conservedN-terminal part of these molecules is the most hydro-philic and cationic while the less conserved C-termi-nal half is more hydrophobic. It has been suggestedthat the two domains take a different role in the killingprocess, the N-terminal half mediating the binding ofthe bacteriocin to the target organism via the chargedresidues, while the hydrophobic/amphiphilic C-termi-nal part penetrates into the target membrane, therebypermeabilizing the cell.19,20,28,29
It has been shown that the N-terminal portion wasindeed important for the high bacteriocin activity ofmesenterocin Y105 (MesY105). By removal of onlythe three residues KYY from the highly conservedN-terminal region, the activity of MesY105 was re-duced to 0.04%.21 A similar reduction of activity wasalso observed when the cysteines involved in disulfide
bridging were chemically modified or replaced byserine.
Various analogues of MesY105 were chemicallysynthesized and when the C-terminal Trp residue wasabsent the antimicrobial activity of the molecule wasreduced by 4 logs compared with the native peptide.28,30,31 It is striking in this respect to observe thedifference between MesY105 and broad-spectruma-helical antimicrobial peptides from vertebrate ani-mals (see article by Tossi et al. in this issue), whichcan, in some cases, be shortened to 14–18 residueswithout deleterious effect on the antimicrobial po-tency. The work with MesY105 has suggested that theamphiphilic-helical domain interacts with the mem-brane lipid bilayer whereas residues 1–14 form part ofa recognition structure for a receptor; however, dataobtained with other bacteriocins are not in agreementwith this model.
As already mentioned, some class IIa peptides,such as divergicin DV41 (Div41) contain two disul-fide bridges (Figure 1). To establish the structure–activity relationships of this bacteriocin, chemicalmodification of charged amino acids and enzymatichydrolysis of the peptide were performed.26 Alter-ation of the net charge of this cationic peptide, withincertain limits, surprisingly did not affect its activity.Endoproteinase Asp-N cleavage of Div41 releasedtwo fragments, N1 (amino acid residues 1–17) and N2(amino acid residues 18–43), respectively corre-sponding to the conserved hydrophilic N-terminal andthe variable hydrophobic C-terminal regions. Micro-bial inhibition assays showed that the C-terminal frag-ment remained active but that it lost its inhibitoryactivity when the C-terminal Lys 42 was cleaved bytrypsin, or when the disulfide bridge was reduced.These results suggested that both the hydrophilicityand the folding imposed by the Cys25—Cys43 disul-fide bond were essential for antilisterial activity. Asfor MesY105, the Trp residue of Div41 was alsocrucial for antimicrobial activity, while conversely,chemical modification of the three N-terminal ty-rosine residues had less of an effect and only simul-taneous modification of all three Tyr residues causeda complete loss of antilisterial activity. This work ledto the conclusion that the YGNGV consensus se-quence is probably not involved in binding to a spe-cific site on listerial target cells.26 Thus, manipulationof the conserved N-terminal part of Div41 apparentlyresulted in different effects compared with that of theN-terminal part of MesY105, although the results aredifficult to compare because different experimentalprocedures were used.
Fimland et al.28 looked at the antimicrobial activ-ity of hybrid bacteriocins derived from three different
52 Nes and Holo
pediocin-like peptides (sakacin P, curvacin A, andpediocin PA-1). Four such hybrids were constructedby solid-phase peptide synthesis by combining thehydrophilic N-terminal half of one peptide with theless conserved hydrophobic C-terminal half of an-other. All of these hybrid molecules had an antimi-crobial toward different target strains that was moresimilar to that of the bacteriocins from which theC-terminal half was derived, although some were lesspotent than the natural counterparts. Thus, this regionis apparently an important determinant for the speci-ficity of pediocin-like bacteriocins and must conse-quently interact with some entity of the target cell.However, it cannot be conclusively determined fromthese experiments whether the target specificity en-compasses a receptor like molecule or not. Theseresults, in agreement with those reported for Div41,26
support the notion that the specificity of pediocin-likebacteriocins does not directly involve the conservedN-terminal half of the molecule. Another relevantfinding was that a 15-mer fragment of pediocin PA-1that spans the region from residue 20 to 34 inhibits theantimicrobial activity of native pediocin PA-1, andalso of enterocin A, sakacin P, leucocin UAL-187 andcurvacin A (although to a much lesser extent).31 Theresults clearly suggest that the C-terminal locatedfragment interacts specifically and competitively withsome target cell entity either in the cell wall or in themembrane, strengthening the notion that class IIabacteriocins interact with some kind of a receptorentity.
The significance of the nonconserved C-terminaldisulfide bridge found in some of the pediocin-likebacteriocins derives from the fact that it seems tostabilize their structure and increase their potencywith respect to those lacking this additional bridge.16
By site-directed mutagenesis it has been possible tointroduce a C-terminal disulfide bridge into sakacinA, which is otherwise devoid of this structure. Thispeptide is 10–20-fold more potent than its naturalcounterpart against some target cells while otherswere unaffected.30 Furthermore, while the antimicro-bial activity of the wild-type peptide is known to bereduced at increased temperature, the disulfide-con-taining mutant retained it, as observed with naturalpediocin-like bacteriocins that contain both N-termi-nal and C-terminal disulfide bridges.28 Thus, the C-terminal bridge found in some pediocin-like bacterio-cins apparently stabilizes a structure that is particu-larly important for the target cell specificity.
Detailed information on the structures of two classIIa bacteriocins, leucocin AUL 186 (LeuA) and car-nobacteriocin B2 (CbnB2) (Figure 1), respectively of37 and 48 residues, has been obtained by two dimen-
sional 1H-nmr spectroscopy.17,22 These peptides are66% identical and contain only one disulfide bridge inthe highly conserved N-terminal sequence. LeuA wasshown to adopt a well-defined tertiary structure in theregion stretching from residue 2 to residue 31, theregion between residues 2 and 16 adopting a three-stranded antiparallelb-sheet conformation, followedby an amphiphilic helix covering residues 17–31. Onthe other hand, structural analysis of CbnB2 revealeda well-defined helical sequence in the region fromresidue 18 to 38 but the N-terminal region was foundto be highly disordered. This surprising structuraldifference between two quite homologous peptideswas not explained, but several possibilities were ruledout, such as that the observed N-terminal disorder inCbnB2 was a consequence of thermal denaturation orlow thermal stability, or of poor/incomplete structur-al/conformational sampling by the nmr technique. Itwas furthermore concluded that a three-strandedb-sheet as found in LeuA is incompatible with theinterpretation of the nmr data of CbnB2. While theN-terminal part of LeuA is amphipatic, with fourhydrophobic residues (Y2, H8, T10, and A24) on oneside of theb-sheet and four hydrophilic residues (L1,N5, C9, and C14) on the other side, the N-terminalregion of CbnB2 cannot adapt to a similar amphipaticfold. A key question that remains to be answered iswhether the solvent used in these nmr studies, triflu-oroethanol, which is known to stabilize amphiphilicstructures, actually simulates the interaction betweenthe bacteriocins and the target membrane, and conse-quently if the reported structures are actually of bio-logical significance. Nevertheless, these results arevery interesting, and it will be useful to bear them inmind for future structural studies, studies of interac-tions with membranes, mode of action studies, etc.
Mode of Action
A number of mode of action studies have been con-ducted on the bacteriocins from lactic acid bacteria(for a review, see Ref. 7). All bacteriocins, includingthe pediocin-like peptides, are positively charged atphysiological pH and their pI varies between 8 and11. It has been shown that the cytoplasmic membraneis the principal target for bacteriocins, and not sur-prisingly, that the antibacterial activity of many bac-teriocins is decreased by reduced positive charge. It isthus likely that the conserved, cationic N-terminalregion is involved in the initial interaction with thenegatively charged phospholipid headgroups in thecell membrane. Results demonstrate that it is thiselectrostatic interaction, and not the presence of thehighly conserved YGNGV motif, that governs pedi-
Class II Antimicrobial Peptides 53
ocin binding to the target membrane,19 although con-flicting data have been obtained, as already men-tioned. The initial electrostatic interaction can be ad-versely affected by the presence of positive andnegative ions or pH values that change the net chargeof bacteriocin molecules, thereby also affecting theantimicrobial activity.
The first study on the mode of action of class IIabacteriocins was performed on pediocin PA-1 and itwas concluded that this peptide promoted the perme-abilization of the target cell membrane, resulting inthe leakage of various types of cations.33 It was pos-tulated that pediocin-PA1 forms pores and that theirformation is not dependent on a membrane potential,as observed with some other bacteriocins. However,other authors demonstrated that the activity of pedi-ocin PA-1 is enhanced by the presence of a membranepotential when tested on liposomes. Increasedamounts of the peptide produced leakage of largermolecules from the target cells, so it was proposedthat larger pores are produced at increased peptideconcentrations.33
Another open and important question concerningthe microbial killing mechanism that has not yet beenanswered is whether a bacteriocin receptor is in-volved. No conclusive experimental evidence hasbeen presented, and published results so far are con-tradictory. It has been shown that pediocin PA-1 isable to permeabilize vesicles made fromListeriamonocytogeneslipids, which may support a nonrecep-tor model.20,34 On the other hand, it has been recog-nized that antibacterial peptides from eukaryotes,which do not have a receptor, are equally active intheir D and L forms. TheD-enantiomer of LeuA hasbeen synthesized and has been shown to be inactive,indicating a stereospecific interaction with a targetmolecule that strongly suggests the presence of areceptor or docking molecule. In addition, it has nowbeen proved that both the class I bacteriocins mer-cacidin and nisin require lipid II as receptor or dock-ing molecule in order to exert their antimicrobialactivity (see article by Guder et al. in this issue).35,36
Also, the receptor-dependent peptide-pheromoneplantaricin A possesses its pheromone activity only byits natural L configuration. However, plantaricin Aalso possesses a low but significant antimicrobial ac-tivity also in its D configuration. As discussed in theprevious section, it appears that the C-terminal part ofthe class IIa bacteriocins is of major importance forthe target specificity and is thus most likely to beinvolved in specific interactions. In balance, the ma-jority of findings support the notion that these bacte-riocins need a receptor-like molecule on susceptible
bacteria in order to exert a maximum antimicrobialactivity, but a receptor has yet to be identified.
Immunity
All bacteriocin producers are insensitive to their ownbacteriocin. For most of the nonlantibiotics, the im-munity gene codes for a single polypeptide and islocated in the vicinity of, and in the same operon as,the strutural bacteriocin gene.10 Cloning in sensitivestrains has proved the functionality of the immunitygenes as the transformants have been shown to be lesssensitive to the bacteriocin than the parental strain.The proteins involved in immunity range in size from51 to 254 amino acids, and they are all cationic, likethe bacteriocins themselves. To understand how theyprotect the producer strain against its own bacteriocinmay help to understand how the bacteriocins mecha-nistically kill sensitive bacteria.
It is likely that the bacteriocin operons of thepediocin family have evolved from a common ances-tor. The finding that the more similar bacteriocins alsohave the more similar immunity proteins supports thisidea. MesY105 and LeuA differ by only two aminoacids, and their immunity proteins show 74% identity.Similarly, sakacin A and CbnBM1 are 67% identical,and this relationship is reflected in similar immunityproteins (47% identity). However, in general a muchlower overall sequence similarity is apparent betweenthe immunity proteins than between the bacteriocinsthemselves, and no conserved regions are found. Oneremarkable exception are the immunity proteinsagainst sakacin A and curvacin A, which are quitedifferent, although the bacteriocins themselves arehighly similar.37,38 On the other hand, the putativecurvacin A immunity protein is similar to that ofacidocin A (overall identity of 41.2% at the aminoacid level), although these two bacteriocins have nomore than an average similarity (34% identity). In thiscontext it is worthwhile mentioning that acidocin A,which has rather poorly conserved N- and C terminalsequences with respect to the other pediocin-like pep-tides (Figure 1), is included in this group also on thebasis of the similarity between its immunity proteinand that of curvacin A. These two proteins are muchsmaller (55 and 51 residues respectively) than thoseprotecting against the rest of the class IIa bacteriocins.Structural prediction by the method of Argos andRao39 indicates that both these proteins can form amembrane spanning helix in their N-terminal region,respectively encompassing amino acids 11–27 and2–28.40
The amino acid sequences of many of the otherimmunity proteins of the bacteriocins in this family do
54 Nes and Holo
not indicate that they are membrane associated. Thegenes necessary for carnobacteriocin B2 (CbnB2)production are located on the 61 kb plasmid pCP40.41,42 When the producer strain LV17B is cured ofthis plasmid, it becomes sensitive to the bacteriocin.However, when the gene downstream to the bacteri-ocin gene was cloned in the cured strain, it was shownto confer protection against CbnB2,43 indicating thatit codes for the immunity protein (CbniB2). By usingantibodies against CbniB2, the authors showed that ina bacteriocin-producing strain only about 1% wasfound in the cytoplasmic membrane, while the restwas in the cytoplasm. Furthermore, no in vitro inter-action was observed between the CbniB2 and thebacteriocin, and sensitive cells did not become im-mune to the bacteriocin if the immunity protein wasadded from the outside, or if bacteriocin and immu-nity protein were combined prior to exposure to sen-sitive cells.43 The authors thus suggested that theimmunity protein may interfere with bacteriocin-in-duced pore formation from the inside of the cells.Similar results also were previously obtained with theimmunity protein of lactococcin A.44
Since the bacteriocins of the pediocin family are sosimilar in sequence, it is conceivable that they shouldhave a common mode of action, although, as dis-cussed above, NMR analysis suggested major struc-tural differences in two peptides.17,22 It has beendemonstrated by Chikindas et al. that pediocin PA-1acts on the cytoplasmic membrane of a sensitive cell,causes pore formation and leakage of low molecularweight compounds.33 Other work cited by Quadri etal. indicates that carnobacteriocin B2 has the samemode of action.43 These bacteriocins show only minordifferences with respect to inhibitory spectrum, butthe corresponding immunity proteins are very specificand cannot provide cross-immunity to other bacterio-cins of the same family. When membrane vesiclesprepared from pediococci transformed with the pedi-ocin PA-1 immunity gene were compared with vesi-cles from wild type cells,33 the latter vesicles wereshown to leak intracellular components when exposedto the bacteriocin, while this effect was much reducedin membrane vesicles from cells that contained theimmunity gene. This suggests that the immunity pro-tein interacts with the cytoplasmic membrane, thuspreventing pore formation.
A coherent model of how an immunity proteinprotects the bacteriocin-producing cell has thus as yetto be provided, but it is important to note these pro-teins can be functionally expressed and thereby pro-tect a sensitive bacterium independently from bacte-riocin production. Furthermore, in spite of an appar-ently very loose interaction with the cytplasmic
membrane, it is likely that interaction with the mem-brane itself or some entity of the membrane is re-quired for the immunity protein to protect the pro-ducer against its bacteriocin.
Antimicrobial Activity Spectrum
Surprisingly, few studies greatly concern themselveswith determining the antimicrobial spectra of activityfor these commercially interesting bacteriocins withany detail, although most papers describing the isola-tion and characterization of novel peptides providesome information on the antimicrobial spectrum. Thisis however usually of low significance due to thelimited number of target strains tested. In addition, theantimicrobial assays used are often different so thatthe activity of the various bacteriocins is difficult tocompare. In this respect, Blom et al.45 describe howvarious external factors can actually interfere withantimicrobial activity determination in the well-diffu-sion assay. The antimicrobial effect can also be con-siderably affected by numerous known or unknownphysical, chemical, and nutritional environmental fac-tors.46,47
In one study, the antimicrobial activity of fourpurified pediocin-like bacteriocins was determinedand compared.16 The bacteriocins included in thisstudy were pediocin PA-1 and enterocin A, both con-taining two cysteine bridges, and sakacin P and cur-vacin A, both containing one cysteine bridge (seeFigure 1). The four-cysteine containing bacteriocinsgenerally had a higher specific antimicrobial activity,and this was directed against more strains than thetwo-cysteine bacteriocins. They were, however, alsomore affected by reduction of disulfide bonds, con-firming that the second cysteine bridge found in theC-terminal half of pediocin PA-1 and enterocin Acontributes to their enhanced antimicrobial activity.
In general, the class IIa bacteriocins are very ef-fective againstListeria. Studies including severalhundredListeria monocytogensstrains have shownthat close to 100% of the strains are killed effectivelyby such bacteriocins or LAB producing such bacte-riocins. However, it is important to note that thebacteriocin concentrations required to kill the mostand least sensitiveL. monocytogenesvary by 100-foldor more. The most sensitive strains are killed at con-centrations as low as less than 0.1 ng/mL in liquidculture.16
Among the pediocin-like bacteriocins, enterocin P,which unlike the others is secreted by thesec-depen-dent pathway, has also been shown to inhibit thegrowth or kill other gram-positive pathogens such asClostridium perfringens, Clostridium botulinum,and
Class II Antimicrobial Peptides 55
Staphylococcus aureus.48 This is also partly true forthe other pediocins-like bacteriocins, although mostremain to be tested systematically against such bac-teria.
SUBCLASS IIB: TWO-PEPTIDEBACTERIOCINS
An increasing number of bacteriocins have been char-acterized (Table II) whose activities are distinguishedby the fact that they depend on the complementaryaction of two peptides. These two peptides are clearlydifferent in sequence, suggesting that they originatefrom two independent and probably different bacte-riocins. One can speculate that these binary peptidesystems may have developed as a consequence of thecomponent peptides being more efficient when actingtogether, a notion supported by the observation thatthe individual peptides sometimes have a modest an-timicrobial activity of their own. For example, theindividual peptides in the two-peptide bacteriocinsPlnEF and PlnJK do posses a low but significantantibacterial activity that may suggest such a devel-opment 49 Furthermore, one of the partners in theclass IIb bacteriocin lactacin F shows homology to an
equivalent peptide of another two-peptide bacteriocin,plantaricin S, which suggests a common ancestor,while the other partners do not share any significanthomology and are probably unrelated.50,51An impor-tant criterion defining a two-peptide bacteriocin is thatthe two peptides act synergistically and have onlyonededicated immunity protein whose gene is linked tothose of the two structural genes for the peptides,usually in an operon structure.10 Interestingly, of theclass IIB bacteriocins characterized to date, only twohave been shown to have a significant sequence ho-mology. The first two-peptide bacteriocin to be re-ported, lactococcin G, was isolated fromLactococcuslactis, and hasa andb chains consisting respectivelyof 39 and 35 amino acid residues.52,53 Recently, en-terocin 1071A and enterocin1071B have been char-acterized fromEnterococcus faecalisand were provento constitute a two-peptide bacteriocin.54 These twopeptides were respectively shown to be 64 and 61%homologous to the corresponding lactococcala andbpeptides.
Mode of Action
Class IIb bacteriocins usually act by killing targetbacteria, although bacteriostatic activity has also beenobserved, depending on the target organisms and/orthe concentration of bacteriocin used. In detailed stud-ies on the mode of action of several two-peptidebacteriocins, it has been shown that the membrane isthe target for the antimicrobial compound, and theinteraction always leads to its permeabilization. Theclass IIb bacteriocins seem to form relatively specificpores that are all found to dissipate the transmem-brane potential (DC).7 However, some differenceshave been observed between the various antimicrobialsystems. Thea andb peptides composing lactococcinG do not individually have an antimicrobial activityand an optimal activity is obtained when the twopeptides are present in a 1:1 ratio.53,55Addition of thisbacteriocin to sensitive cells results in a collapse oftransmembrane potential but not in the dissipation ofthe pH gradient. Cellular ATP is rapidly depleted andintracellular potassium ions are released as measuredby 86Rb1 efflux that is pH dependent (occurring onlyabove pH 5).
The class IIb bacteriocins plantaricin EF and plan-taricin JK do not form a complex at a strictly definedratio of 1:1 as observed for lactococcin G.56 More-over, in contrast to the latter bacteriocin, three (PlnJ,K, and F) out of the four of the plantaricin peptidesshowed a small but significant individual antimicro-bial effect at concentrations of 20 nM, 50 nM, and 4mM, respectively, while the fourth (PlnE) did not
Table II Class IIb Bacteriocins—The Two-PeptideBacteriocins
Bacteriocin PeptidesAminoAcids Ref.
Lactococcin G Peptidea 39 52Peptideb 35
Lactacin F Laf F 57 50Laf X 48
Lactococcin MN Lcn M 482,68
Lcn N 47Plantaricin EF PlnE 33 87
PlnF 34Plantaricin JK Pln J 25 87
Pln K 32Plantaricin S Plc A 27 51
Plc B 25Enterocin 1071 Ent1071A 39 54
Ent1071B 34Thermophilin 13 ThmA 43 18
ThmB 62Acidocin J1132a a 6220 Da 59
b 5 a 1 Gly 6280 Da
a Acidocin J1132 should probably be defined as a one-peptidebacteriocin because the two peptides appear to originate from thesame gene.
56 Nes and Holo
show any activity up to the highest concentrationtested (13mM).57 It should be noted that these studiesused synthetic peptides, so that it can be excluded thatany unintentional cross-contamination with comple-mentary peptides occurred. When the complementarypeptides were tested together, at least a 1000-foldincrease in antimicrobial activity was achieved withoptimal activity observed at a ratio of approximately1:1, while it was not possible to achieve this increasedantimicrobial activity by interchanging the peptidesfrom the two plantaricins. That the lactococcin Gsystem requires a strict 1:1 operational ratio while thisis not necessary for the two plantaricin systems ismost probably due to the low but significant antimi-crobial activity of individual peptides, so that a defi-ciency in one partner may partly be made up for by anexcess of the complementary peptide.
The class IIb bacteriocins lactacin F50 and planta-ricin S58 also have at least one of their componentpeptides with some individual antimicrobial activity;however, these have not been so rigorously testedusing highly purified or synthetic peptides, so thatcross-contamination with the complementary peptidescannot be excluded. In addition, the antibacterial ac-tivity of the individual peptides has not been tested athigh peptide concentration. Consequently, one cannotdefinitely conclude if the lactocin F and plantaricin Ssystems resemble either the lactococcin G or planta-ricin EF and JK bacteriocins.
A detailed mechanism of action for plantaricinsEF and JK has been studied withLactobacillusplantarum965, one of the most susceptible bacteriato the bacteriocins produced byL. plantarumC11.56 Both bacteriocins permeabilized the targetcells and dissipated the transmembrane electricalpotential and pH gradient, and both resulted in theefflux of small cations (Rb1 and choline ions), withplantaricin EF appearing to be the most effective inthis respect. Furthermore, both bacteriocins weremore effective in permeabilizing the bacterial mem-branes than any of their individual peptides. Con-versely, neither of the bacteriocins caused any ef-flux of phosphate ions, while efflux of glutamatewas most efficiently observed with plantaricin JK.It has also been shown that acidocin J1132 permitsglutamate efflux.59 One may conclude that planta-ricin JK permeabilization results in a higher con-ductivity for specific anions while that by plantari-cin EF is for cations. It is also interesting to notethat plantaricin JK is much more effective thanplantaricin EF in killing L. plantarum 965 (MICvalues of 0.1 and about 7 nM, respectively).
Structure– Function Studies
Computer analyses have shown that the individual pep-tides of the two-peptide bacteriocins generally containputative amphiphilic regions that may penetrate themembrane of sensitive bacteria. Although no nmr anal-ysis has been performed in order to obtain experimentalevidence of such structural motifs, some conformationalstudies have been performed using circular dichroism(CD).57,60All bacteriocin peptides that have been stud-ied are unstructured in aqueous solution, but adopt somea-helical structure in the presence of trifluoroethanol,micelles of dodecylphosphocholine or negativelycharged dioleoylphosphoglycerol (DOPG) liposomes.Far less structure was obtained when liposomes of zwit-terionic dioleoylglycerophosphocholine (DOGPC) wereused. This observation suggests that a net negativecharge on the phosopholipid bilayer is important for theinduction of structure in the positively charged bacteri-ocin peptides. Furthermore, studies with the two-peptidelactococcin G showed that most of the induced helicalstructure was actually situated in the region of the pep-tides that had been predicted by computer analysis.52,60
Of particular interest was the observation that when bothpeptides of lactococcin G were addedsimultaneouslytoDOPG micelles, more helical structure was induced ascompared to the summed helical structure of the indi-vidual peptides. However,consecutiveaddition of thetwo peptides to DOPG liposomes did not induce thisadditional helical structuring. These results suggest thatthe two peptides interact in solution in a manner thatenhances helical formation when they interact with themembrane, but that this interaction becomes inaccessibleafter the peptides interact with the membrane. These CDstudies suggest that the two peptides form a complexwith the target membrane at a ratio of 1:1 and that thecomplex consists of approximately 52% amphiphilica-helices when measured in DOPG. They support thenotion that these bacteriocins interact with the mem-branes of target bacteria via formation of a “barrelstave” pore, leading to an irreversible permeabiliza-tion and consequently to death of the target bacteria.Furthermore, the active complex requires the syner-gistic interaction of thea andb peptides at a 1:1 ratiobut cannot be formed by individual peptides alone,even though these can adopt helical structures (44 and33%, respectively), in a membrane-mimicking envi-ronment (DOPG).
Similar studies have also been performed on plan-taricins EF and JK, encoded in the plantaricin regulonof Lactobacillus plantarumC1.57 The individual pep-tides adopt helical structure in the presence of tri-flouroethanol (TFE) and dodecylphosphocholine mi-celles, and in negatively charged DOPG liposomes,
Class II Antimicrobial Peptides 57
while less structure was formed in the presence of thezwitterionic DOGPC liposomes. When the comple-mentary peptides were added simultaneously toDOPG liposomes, additional helical structure was in-duced as compared to the sum of helical structure forindividual peptides, although this effect was not ob-served with TFE and dodecylphosphocholine micelles.
OTHER CLASS II BACTERIOCINS
Enterocin B and Carnobacteriocin A
Another group of LAB bacteriocins, showing a dis-tinct amino acid sequence homology, is representedby enterocin B (EntB) and carnobacteriocin A (CbnA;Figure 2), which are respectively produced by strainsof E. faeciumand C. piscicola.61,62The same twobacterial strains also produce the class IIa bacterio-cins, enterocin A and carnobacteriocin B2. EntB andCbnA are both 53 amino acid residues long and share47% identity. Although of a different subclass, EntBshows an inhibitory spectrum close to that of EntA,being able to kill lactobacilli, enterococci, propionicacid bacteria, listeria, and staphylococci, but it isgenerally less potent than the pediocin-like peptide.An important feature of this pair is that while eitherpeptide is individually capable of causing several logsdecrease of sensitive bacteria, a certain number ofthese survive or are able to recover and will eventu-ally start growing. However, when the two bacterio-cins are combined, the same target organisms seem to
be 100% killed, and no survivors or growth are de-tected within the next 24 h.62 This synergistic effect atthe moment appears to be quite unique to this pair, butmay be more common than previously supposed. Theincreased efficacy of killing by the combined action ofmultiple bacteriocins, even those belonging to differ-ent subclasses, may suggest why LAB often producemore than one such peptide.10 This effect may also beof considerable commercial interest as it could makebacteriocins more reliable as agents for killing patho-gens and other undesirable bacteria.
Enterocin L50
Enterocin L50 represents a new subgroup of LABbacteriocins, which seems to have more in commonwith members of a small group of antimicrobialand/or hemolytic peptides secreted by staphylococcithan with other class II bacteriocins (Figure 3). Theselatter peptides are referred to as the SLUSH A, B, andC , produced byStaphylococcus lugdunensis,andAGS-1, -2, and –3, produced byStaphylococcus hae-molyticus.63–65 These peptides are also synthesizedribosomally, but they lack a leader sequenc, and fur-thermore, no immunity gene is cotranscribed. In con-trast to the SLUSH and AGS systems, enterocin L50is only bactericidal and not hemolytic. It consists oftwo peptides, A and B (44 and 43 amino acid residues,respectively), and in order to achieve maximum ac-tivity both are needed. Contrary to the well-charac-terized two-peptide bacteriocins (class IIb), these two
FIGURE 2 Homology between the prebacteriocins of enterocin B and carnobacteriocin A. (Thearrow indicates the cleavage site of the double-
FIGURE 3 Alignment of enterocin L50 peptides and hemolytic/bacteriocin peptides.
58 Nes and Holo
L50 peptides are 72% identical and while the individ-ual peptides have significant antimicrobial activity,when they are combined this increases by approxi-mately 5–100-fold, depending on the indicator straintested and which of the peptides the synergistic activ-ity is compared to. As to the spectrum of activity,enterocin L50 can kill a variety of gram-positivebacteria.
Lactococcin A
Lactococcin A, which is produced by selected strainsof Lactococcus lactisssp. cremori, was the first bac-teriocin that was shown to possess the unique double-glycine leader presequence.14 The importance of thedouble-glycine leader was demonstrated in the secre-tion process that requires a dedicated ABC transport-er.66 It was shown that this transporter is actuallyrecognizing the leader sequence and in the process ofsecreting the peptide will specifically cleave it off.67
Lactococcin A seems to be produced only byL. lactisstrains and several independent lactococcin A produc-ers have been isolated from milk sources from differ-ent parts of the world. The mature peptide is 54 aminoacid residues long and hydrophobic. Its genetic deter-minant was initially found on a 60 kb plasmid, whichalso encodes two additional bacteriocins, lactococcinB and the two-peptide lactococcin MN (Table II).68
One of the unique features of lactococcin A is itsvery narrow spectrum of action. It has been shownthat it kills only other lactococci, while bacteria ofother species have so far been found to be unsuscep-tible. For 120 different lactococci that have beentested, the minimal inhibitory concentrations (50%growth inhibition) vary 2500-fold between the mostsensitive and least sensitive strain, but only one strainappeared to be completely insensitive to this pep-tide.14 The most susceptible strains were inhibited byas low as 7 pM lactococcin A, which corresponds toapproximately 400 lactococcin A molecules per targetbacteria (calculated from colony forming units).14
Mode of action studies performed with lactococcinA have shown that it permeabilizes both whole cellsand cytoplasmic membrane vesicles ofL. lactis, andthat this permeabilization takes place in the absence ofa proton motive force, thus being voltage indepen-dent. On the other hand, liposomes derived fromphospholipids of sensitive lactococcin were not af-fected by lactococcin A. Cytoplasmic membrane ves-icles derived from resistant bacteria were also unaf-fected by this peptide. These observations suggest thata specific molecular entity, such as a receptor, isinvolved in recognition by susceptible cells. As nopermeabilization was observed in lactococcin A im-
mune cells, or in membrane vesicles derived fromimmune lactococci, this was taken to indicate that theimmunity protein also acts through the membrane.69
This protein has in fact been purified and is a majorcell protein component—one cell may contain (to anorder of magnitude) 105 molecules—and it is in partassociated with the cell membrane, as judged by im-munoblot analysis of membrane vesicle-associatedproteins. Exposing lactococcin A sensitive cells to anexcess of the immunity protein did not affect thelactococcin-A induced killing of the cells, indicatingthat the immunity protein does not protect cells bysimply binding to lactococcin A, nor to externallyexposed domains on the cell surface.44 Exposing im-mune-positive cells to antiserum against the immuneprotein also did not sensitize the cells to lactococcinA, confirming that the immunity protein in fact doesnot act extracellularly.44 It has been postulated that itis associated with the membrane via a transmembraneanchoring through its C-terminal end, as it contains aputative amphiphilica-helix from residue 29 to 47. Amodel has been proposed in which this helix isthought to traverse the membrane in such a way thatthe C-terminal part of the protein is on the outside ofthe cell.70, 71
CONCLUDING REMARKS
Presently, the nonmodified class II bacteriocins fromlactic acid bacteria have found limited industrial ap-plication. Most of the LAB they originate from areconsidered “food grade” bacteria, being the naturalflora of a number of fermented food products includ-ing dairy products, fermented sausage, and vegeta-bles. In addition, LAB are common bacteria in variouscavities of the human body and constitute part of thehealthy bacterial flora.
Most of the commercial use of bacteriocins isthrough the nondeliberate use of bacteriocin-produc-ing starter cultures intended for fermenting variousfoods. In addition, fermentates containing bacterio-cins have been commercialized as food ingredientsintended to increase shelf life and to provide a micro-bially safer food product. Presently, class II bacterio-cins have not been tried out in personal hygienicproducts (such as tooth paste, skin lotions, etc.) or asantimicrobial compounds for veterinary or humanmedical applications. However, it must be said thatthese peptides are still relatively “young” as antimi-crobial compounds, having been known for just alittle over ten years. Although their spectra of inhib-itory activity is fairly narrow and to some extentunpredictable, and their potency can be severely af-
Class II Antimicrobial Peptides 59
fected by environmental conditions, they are probablyamong the most potent antimicrobial peptides knowntoward their target microorganisms. They are alsonontoxic to eukaryotic cells, at least to the extent thatthey are not hemolytic. The emergence of increasedbacterial resistance to traditional antibiotics suggeststhat class II bacteriocins be seriously considered as agroup of antimicrobial compounds worth investigat-ing for future medical use.
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