Published online before print December 6, 2004
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* Institute for Medical Microbiology and Immunology, University of Bonn, Germany; and
Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Italy
1 Correspondence: Institute for Medical Microbiology and Immunology, University of Bonn, Sigmund-Freud-Str. 25, 53105, Bonn, Germany. E-mail: sahl{at}mibi03.meb.uni-bonn.de
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Key Words: human ß-defensin 3 • membrane depolarization • staphylococci
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The body of in vivo evidence for a substantial role of these peptides in host defense is growing rapidly; Mentioned is a brief selection of recent, prominent examples.
Paneth cells in mouse small intestinal crypts secrete granules rich in microbicidal cryptdins when exposed to bacteria or bacterial antigens. The dose-dependent secretion occurs within minutes, and these 
-defensins account for 
70% of the released bactericidal peptide activity. Gram-negative bacteria, Gram-positive bacteria, lipopolysaccharide (LPS), lipoteichoic acid (LTA), lipid A, and muramyl dipeptide elicit cryptdin secretion [9
].
There is direct evidence of a correlation between defensin expression and incidence of infection in man. Human ß-defensins (hBDs) 1 and 2 are widely expressed in oral-inflamed tissue samples and primary oral keratinocytes [10 ]. In patients affected by psoriasis, epithelial BDs are overexpressed, and skin lesions are relatively free from infection, and in atopic dermatitis, their expression is suppressed, and lesions are infection-prone [11 , 12 ].
Inhibition of the proteolytic activation of cathelicidins in neutrophils had a drastic effect on the clearance of bacteria from wounds in an experimental porcine skin-wound model [13 ], and in mice, cathelicidins were shown to protect against necrotic skin infection caused by Group A streptococci [14 ]. An absence of cathelicidin, as well as defensins, has been demonstrated in patients suffering from Morbus Kostmann [15 ].
The relevance of AMPs to host defense is also illustrated by counter-strategies developed by the microorganisms to escape their activities.
During Shigella infections, expression of the antibacterial peptides LL-37 and hBD-1 is reduced or turned off. The down-regulation was detected in patients with bacillary dysenteries and in Shigella-infected cell cultures of epithelial and monocyte origin. The down-regulation of immediate defense effectors might promote bacterial adherence and invasion into host epithelium and could be an important virulence parameter. Shigella plasmid DNA was identified as a putative mediator causing the down-regulation [16 ].
The reduction of the overall negative charge of surface components appears to be the most effective strategy of Gram-positive and Gram-negative microbes to reduce the accumulation of AMPs in the cell envelope and thus, to escape from the direct killing activity [17 , 18 ]. For example, MprF is a membrane protein of Staphylococcus aureus, which is involved in modification of phosphatidylglycerol with L-lysine; the modified phospholipid resulting from its activity is a major constituent of the staphylococcal membrane, and the negative surface charge of such a membrane is significantly reduced [19 ]. An mprF– mutant strain was killed considerably faster by human neutrophils and exhibited attenuated virulence in mice, indicating a key role for defensin resistance in the pathogenicity of S. aureus. MprF homologues were identified in various other human pathogens [17 ]. Similarly, the alanylation of teichoic acid and LTA reduces the surface charge [20 ] but also interferes with Toll-like receptor 2-mediated defense responses [21 ].
As mentioned above, the role of HDPs in innate immunity is more complex than just a capacity to antagonize microbes directly, and an increasing number of other functions, in addition to their antibiotic activity, including chemotactic or regulatory functions, are being discovered (e.g., refs. [22 23 24 ]). In fact, the peculiar sensitivity of the antibiotic activity of some HDPs, in particular, most mammalian defensins and some cathelicidins, to environmental factors such as salt concentration and serum components, may cast some doubt on this being their principal role. However, although it is reasonable to suppose that the defensive role of HDPs derives from a combination of direct and defense-stimulatory activities, there is sufficient support for the significance of the direct antibiotic effect (e.g., refs. [17 , 19 ]).
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-helical structure (e.g., cecropins, magainins, and some cathelicidins) [25
]; and linear-extended peptides, which are Gly-, Pro-, or Trp-rich.
The crystal structures of several defensins have been determined and indicate surprising similarity, based on a central, three-stranded ß-sheet core, despite different sizes, residue distributions, and cysteine-pairing schemes. Regarding the mammalian peptides, the structures of various -defensins and BDs have been determined, and it appears that each group represents a conserved scaffold, where the folding is apparently determined principally by the formation of the disulfides. As the sequences vary considerably, this conserved, tertiary-structure scaffold allows the molecules to display considerably different surfaces and a remarkable variation of cationicity, which ranges from almost neutral in mouse BD-2 to 11 in human BD-3. This reflects on their quaternary structure; some defensins form dimeric or oligomeric structures; others apparently remain monomeric. This likely represents evolved characteristics used to modulate the mode of action [26
]. In contrast, the helical peptides are usually unstructured in solution and adopt the typical helical, amphipathic structures in appropriate media (e.g., solvents such as trifluoroethanol or detergent micelles) and in physiological settings, in contact with biological membranes.
Although our knowledge about a number of aspects regarding the defense peptides (including biological roles, genetics of production, and structures) is currently, rapidly expanding, our understanding of the bactericidal modes of action is based mainly on pioneering studies of the last decade [27 ] but is now rather stagnant. The fact that small, cationic, amphiphilic peptides are so widespread led to the postulation of a general principle of antibiotic activity, which has supposedly been conserved throughout the evolution of these molecules; i.e., these antibiotics should have a common molecular target, which should not be subject to substantial, evolutionary changes. From the defensive point of view, it is preferable to have a moderate activity against a broad range of target organisms with a common target rather than potent activities but restricted to a particular group of microorganisms having a specific target. In fact, AMPs often tend to act on several groups of micoorganisms but have relatively moderate potency [minimal inhibitory concentrations (MICs) ranging from 100 nM to 10 µM and higher], and there is considerable evidence that in most cases, the microbial membrane is involved as a target in their antibiotic action. The destructive effects on membranes with a negative surface charge by amphiphilic compounds have been known for a long time, and there is a wealth of information about the biophysical behavior of synthetic and natural defense peptides in and on model membranes (reviews are in, e.g., two special issues of Biopolymers [28 , 29 ]).
The best-studied amongst AMPs, regarding the mode of action, are the -helical peptides, and various models have been elaborated to explain their membrane-compromising activity. The most prominent models are summarized in Figure 1
. These range from discrete, "barrel-stave" pore formation, which, however, seems limited to peptides such as alamethicin, to a more dynamic type of pore or channel suggested for several peptides (e.g., including magainin and melittin), diversely described as "toroidal," "wormhole," or dynamic peptide-lipid supramolecular pores [30
, 31
] or from less well-defined apertures, termed "aggregate channels" [32
]. A more generalized carpeting and subsequent disruption of the membrane also seem possible, especially at high peptide concentrations [33
].
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Figure 1. Models for pore formation by helical, amphiphilic antibiotic peptides (adapted from ref. [25
]).  Outer membrane in Gram-positive bacteria, thick peptidoglycan in Gram-negative bacteria;  electrostatic attraction to anionic bacterial membrane surface;  formation of an amphipathic structure capable of membrane insertion;  possible permeabilization mechanisms, which depend on peptide sequence/concentration and membrane characteristics;  translocation and binding to internal molecular targets, including proteins and nucleic acids:  e.g., release of autolytic enzymes.
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A more complex treatment of the interaction of AMPs with the microbial surface may much better explain the observed biological phenomena, involving components other than the membrane itself. For example, the Gram-positive bacterial cell wall has been implicated as a potential target involved in the action of BDs [10 ]. Lehrer suggests [37 ] for the porcine protegrin peptides a mechanism based on hydro-osmotic transtesseral extrusion and rupture mechanism; i.e., the peptides affect membrane integrity, which promotes K+ efflux and water influx, thus increasing the tugor and inducing cell rupture, at particular, yet-unidentified sites in the cell wall, which are for yet-unknown reasons, less osmotically stable. An important aspect of these considerations, however, is that although the membrane remains a principal factor, most likely the modes of action of peptides from the five structural groups mentioned above may differ and possibly even within these groups. It would thus be necessary to study different HDPs case-by-case.
A further complication for the elucidation of the modes of action is that the killing activities may not only vary or even differ for the individual groups of peptides but also for individual groups of target organisms. For example, when passing through the cell wall of a Gram-positive bacterium, defensins accumulate on anionic polymers and can activate autolysins (e.g., ref. [19 ]; see below and ref. [38 ]), which by dismantling the cell wall in an uncontrolled manner, contribute to the overall killing activity. In Gram-negative bacteria, they must instead pass through the LPS layer, in which its physical destabilization is an antibiotic activity by itself. In this respect, it cannot escape notice that ever since AMPs were discovered, much effort has been dedicated to developing such molecules as a new generation of antibiotics [28 , 29 ] but so far, with little success. One reason may be that rational design strategies were based too narrowly on optimization of membrane interactions; i.e., they did not include all the relevant targets. These strategies were effective in increasing the membranolytic efficiency but eventually resulted in an increased, general toxicity, which reduced their applicability considerably.
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Figure 2. Lipid II-mediated activities of lantibiotics. Lipid II carries the completed subunit of the bacterial cell wall, which is shuttled across the cytoplasmic membrane and polymerized on the outside into the murein network. Many lantibiotics can form a complex with lipid II and thereby block the polymerization reactions (A). After binding to lipid II, some lantibiotics, e.g., nisin, can insert into the membrane and form a well-defined pore (B).
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However, this is not the complete picture. We had previously shown that nisin and other cationic peptides interfere with the separation of daughter cells, which normally occurs through site-specific hydrolysis of wall material after completion of the septum cell wall. Cationic peptides can displace the cell-wall lytic enzymes involved in these processes from the teichoic acid and LTA, where the enzymes are compartmentalized and thus, kept inactive when not required. This results in premature and uncontrolled lytic activity and subsequent cell lysis (Fig. 3 ; ref. [47 ]). Such an activity of nisin and other lantibiotics is independent from lipid II binding and contributes significantly to the overall bactericidal activity. There is every reason to suppose that such an activity could be a common feature of amphiphilic, cationic AMPs [48 ].
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Figure 3. Induction of autolysis in Staphylococcus simulans 22 by cationic peptides. Cells had been treated for 10 min with the lantibiotic Pep5 [38
]. Premature separation of the daughter cells as a result of activation of cell-separating enzymes is visible.
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-helical segment of differing length and stability present in all but the bovine peptide [49
50
51
52
53
]. As this conserved structural scaffold supports a considerable sequence variation, these molecules present quite different surfaces and dimerization or oligomerization patterns, which are likely important for their function. In the hBD-1, dimerization is observed in the crystal structure but not in solution and occurs longitudinally, involving a symmetric pair of intermolecular salt bridges so that the dimer may form a sickle-shaped structure with a hydrophobic convex surface, which could lead to string-like oligomers. It has been suggested but not yet demonstrated that dimerization could have a biological significance [52 ]. In contrast, hBD-2 dimerizes side-on via the formation of an extended, six-stranded ß-sheet in the crystal structure [52 ]. It is surprising that the highly cationic hBD-3 forms stable dimers or oligomers in solution [53 , 54 ], which appear to involve symmetric pairs of intermolecular and intramolecular salt bridges. It had been suggested that these structural characteristics could explain why of all the tested BDs, only hBD-3 shows a measurable antimicrobial activity in vitro at physiological salt concentration [10 ].
It is reasonable to suppose that the variability in charge, surface properties and dimerization capacities and patterns, could play an important role in modulating the antimicrobial activity of these molecules. During the course of studies aimed at determining the effect of evolutionary variations in primate BDs, the human and macaque (Macaca fascicularis, mfa), congeners of BD-2, and human and gibbon (Hylobates concolor, hc), congeners of BD-3 (Fig. 4
), have been chemically synthesized, and structural characteristics and in vitro antimicrobial activity were determined [54
, 55
]. A completely artificial defensin, tBD, was used as a reference molecule. Its sequence was designed using a template obtained by the positional frequency analysis of 60 mammalian and avian defensins (N. Antcheva et al., unpublished results) in a manner similar to that used successfully with -helical peptides [25
].
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Figure 4. Sequences of described BDs. Natural peptides are BD-2 variants from man and mfa and BD-3 variants from man and from the gibbon hc. Template BD (tBD) is an artificial defensin designed on the basis of a sequence template derived from the positional frequency analysis of 60 BDs from various mammalian and avian species. *E, Pyroglutamic acid.
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Circular dichroism spectroscopy of the synthesized and folded peptides indicated that hBD-2 is well-structured in aqueous buffer [55
] and is consistent with the presence of the stable -helical, N-terminal stretch seen in the crystal structure [50
]. Spectra are largely unaffected by interaction with membrane-mimicking sodium dodecyl sulfate (SDS) micelles. The macaque congener shows a looser structure, in particular, with respect to -helical content. This was also the case of hBD-3 [54
] with the only ß-sheet core evident in aqueous buffer, and some -helical content became evident in the presence of SDS micelles. Furthermore, the structural stability appeared to decrease in the gibbon congener so that the intramolecular salt-bridging may also be relevant in stabilizing the tertiary structure. The synthetic tBD showed a stable ß-sheet structure in the presence of buffer and SDS with no evidence of helical content, likely a result of the N-terminal stretch being too short. In this respect, it could resemble the structure of the bovine neutrophil BD-12 [49
].
The peptides were tested for in vitro antimicrobial activity using different conditions, including low (5% v/v) and high (50% v/v) medium concentrations, showing somewhat differing potency patterns. The human and macaque BD-2 congeners were active only in special, low-concentration media [5% v/v Mueller-Hinton (MH) broth or Sabouraud (SAB) medium, see Table 1 ], in line with the reported salt-sensitivity of defensins in general. hBD-2 was more active toward Gram-negative species and mfaBD-2 toward Gram-positive bacteria and the yeast C. albicans [55 ]. Sequence and structure variations in these congeners thus seem to have a significant, functional effect with respect to antimicrobial activity. This is evidenced also by the behavior of the synthetic tBD, which has a similar cationicity but lacks the N-terminal stretch and has a somewhat different residue arrangement (Fig. 4) ; tBD showed a broad spectrum activity on all tested microorganisms, at least in terms of MIC values.
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Table 1. Comparison of Antimicrobial Activity (MIC, µM) of Human and Primate BDs
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Figure 5. Bacterial inactivation and membrane permeabilization kinetics. (a) Time-killing plots for E. coli ML-35 (107 CFU/ml, 8 µM peptide in 10 mM SPB); (b) time-killing plots for S. aureus 710A (107 CFU/ml, 8 µM peptide in 10 mM SPB); hBD-2 ( ), mfaBD-2 (•), hBD-3 ( ), and hcBD-3 ( ); (c) bacterial killing in terms of percent survival with respect to untreated bacteria, after exposure of 105 CFU/ml S. aureus (squares), E. coli (circles), and P. aeruginosa (diamonds) to 8 µM human (open symbols) and gibbon (filled symbols) BD-3 at increasing concentrations of NaCl; (d, e) Membrane permeabilization kinetics determined spectrophotometrically by following the unmasking of cytoplasmic ß-galactosidase or 6-phospho-ß-galactosidase, which were constitutively produced in specific strains of E. coli (ML-35 pYC, d) and S. aureus (710A, e), to the extracellular chromogenic substrates o-nitrophenyl-ß-D-galactopyranoside (ONPG) or ONPG-6P, respectively, as described previously (107 CFU/ml bacteria and 5 µM peptide in 10 mM SPB for both) [56
]; hBD-2 (—), mfaBD-2 (– –), hBD-3 (••••), hcBD-3 (····), and tBD (_.._; N. B., hBD-3, hcBD-3, and tBD transiently bind ONPG-6P, resulting in a signal that prevents an accurate assessment of S. aureus permeabilization kinetics. It was, however, estimated to be slow).
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Membrane permeabilization studies were carried out by following the kinetics of hydrolysis of extracellular chromogenic substrate to cytoplasmic galactosidases constitutively produced by special strains of E. coli and S. aureus (Fig. 5 d and e) [54 , 55 ]. These experiments confirmed a membrane-poration capacity for the BDs in general, although they displayed different permeabilization kinetics (Fig. 5 d and e) [54 , 55 ]. In line with the killing experiments, hBD-2 was the most efficient at permeabilizing the E. coli cytoplasmic membrane after an initial lag period, and mfaBD-2 was by far the most active on the S. aureus membrane. hBD-3 and hcBD-3 showed slower and quite different permeabilization curves so that their permeabilization mechanisms may differ in a manner reflecting their dimerization capacities, although the end effect in terms of MIC values and bacterial killing is similar. It was not possible to accurately assess their behavior with respect to the S. aureus membrane as a result of an artifactual signal deriving from their binding with the chromogenic substrate, but it was assessed to be slow. The synthetic tBD, with a minimized structure, was also less effective than the natural molecules in permeabilizing the bacterial membrane.
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Table 2. MICs of Defensins (µM/µg/ml)
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Figure 6. Bactericidal activity of tBD at 5x MIC () or 10x MIC () and hcBD-3 at 5x MIC (•) for S. simulans 22 (a) and S. aureus LT1334 (b); control ( ) without addition of peptide. (a, inset) Optical density at 600 nm (OD600) taken in parallel with the colony count.
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Figure 7. Influence of tBD on the membrane potential of S. simulans 22, S. haemolyticus I-10925, and S. aureus LT1334. tBD was applied at a concentration of 5x MIC. The potential was calculated from the distribution of the lipophilic cation triphenyphosphonium+ (TPP+) inside and outside the cells. TPP+ concentration was inserted into the Nernst equation. tBD ( ); hcBD-3 ().
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Figure 8. Incorporation of glucosamine (A), uridine (B), thymidine (C), and proline (D) into macromolecules in S. simulans. Radiolabeled precursurs were added to the culture medium, culture aliquots were treated with 10% trichloroacetic acid and incubated for 30 min on ice, and the precipitate was washed and counted for incorporation. Controls (); treated cells ().
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Figure 9. Accumulation of [3H]-L-glutamate into chloramphenicol-treated cells () and efflux of the labeled amino acid after addition of tBD (5x MIC; ); uptake of the radioactive marker by cells pretreated with tBD ().
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The comparative experiments conducted with the staphylococcal strain also demonstrate that defensins have an impact on microbial membranes. However, the effects observed were smaller than anticipated when compared with agents forming defined pores. Complete disruption of the barrier function did not occur, at least in a major proportion of the treated cells and under the conditions tested, i.e., at 5x MIC. Possibly, membrane disruption may be more dramatic at higher peptide concentrations [58 ]; however, to elucidate the complexity of defensin activities, it seems more relevant to avoid concentrations that overwhelm subtle effects that clearly contribute to killing or growth inhibition. Indeed, it will be interesting to study the physiology of cells surviving such conditions.
The staphylococcal strains tested here also differed to some extent in their response to the peptides. Particularly S. simulans 22 showed biphasic killing kinetics, indicative of two possibly independent mechanisms, which may partially overlap in time, e.g., membrane pertubation and cell-wall lysis. The lytic processes occurring with this strain could have several molecular reasons; e.g., premature activity of cell wall-degrading enzymes involved in separation of daughther cells produces rapid lysis, which agrees well with the killing kinetics observed here; however, inhibition of the polymerization steps in cell-wall biosynthesis cannot be excluded as a mechanism.
Our results indicate that although perturbation of membrane-barrier functions contributes significantly to killing, HDPs exhibit additional antibiotic activities. Their amphiphilic and cationic nature provides high reactivity and enables a maximum of interactions in biological systems. Thus, HDPs may produce, through interaction with membranes and a number of other low-affinity targets, inhibitory effects, which may result in additive or synergistic combinations during the killing process. Moreover, which of the inhibitory effects contributes most to killing may largely depend on the structure of the individual peptide, on its concentration, as well as on the target microbe species and physiological conditions of the target cells.
Received August 13, 2004; revised October 29, 2004; accepted November 3, 2004.
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-defensins by intestinal Paneth cells in response to bacteria Nat. Immunol. 1,113-118[CrossRef][Medline]-helical antimicrobial peptides Biopolymers 55,4-30[CrossRef][Medline]-helical antimicrobial peptides Biopolymers 47,451-463[CrossRef][Medline]This article has been cited by other articles:
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