Plectasin, a fungal defensin, targets the bacterial cell wall precursor Lipid II.

Host defense peptides such as defensins are components of innate immunity and have retained antibiotic activity throughout evolution. Their activity is thought to be due to amphipathic structures, which enable binding and disruption of microbial cytoplasmic membranes. Contrary to this, we show that plectasin, a fungal defensin, acts by directly binding the bacterial cell-wall precursor Lipid II. A wide range of genetic and biochemical approaches identify cell-wall biosynthesis as the pathway targeted by plectasin. In vitro assays for cell-wall synthesis identified Lipid II as the specific cellular target. Consistently, binding studies confirmed the formation of an equimolar stoichiometric complex between Lipid II and plectasin. Furthermore, key residues in plectasin involved in complex formation were identified using nuclear magnetic resonance spectroscopy and computational modeling.

Artificial beta-defensin based on a minimal defensin template.

We have designed and chemically synthesized an artificial beta-defensin based on a minimal template derived from the comparative analysis of over 80 naturally occurring sequences. This molecule has the disulfide-bridged beta-sheet core structure of natural beta-defensins and shows a robust salt-sensitive antimicrobial activity against bacteria and yeast, as well as a chemotactic activity against immature dendritic cells. An SAR (structure-activity relationship) study using two truncated fragments or a Cys-->Ser point-mutated analogue, from which one or two of the three disulfide bridges were absent, indicated that altering the structure resulted in a different type of membrane interaction and a switch to different modes of action towards both microbial and host cells, and that covalent dimerization could favour antimicrobial activity. Comparison of the structural, aggregational and biological activities of the artificial defensin with those of three human beta-defensins and their primate orthologues provided useful information on how their mode of action may relate to specific structural features.

Mode of action of human beta-defensin 3 against Staphylococcus aureus and transcriptional analysis of responses to defensin challenge.

Human beta-defensin 3 (hBD3) is a highly charged (+11) cationic defence peptide, which retains activity against staphylococci even at elevated salt concentrations. We studied the antibiotic mode of action of hBD3 against Staphylococcus aureus SG511, using whole-cell assays and analysing the transcriptional response to hBD3 treatment. hBD3 caused rapid killing and simultaneously blocked all biosynthetic pathways, however, significant depolarisation was not observed and permeabilisation of the membrane was incomplete. The transcriptional response pattern was in part similar to those of strongly cationic amphiphiles, e.g. in that anaerobic energy production was downregulated. Significantly, part of the staphylococcal cell wall stress stimulon were upregulated. The most prominent microbial counterstrategy appears to be based on the upregulation of ABC transporters possibly functioning in detoxification of the membrane environment; the transporter genes were highly upregulated in both, a short-term response and a long-term adaptation experiment. Knockout of the most highly induced transporter VraDE significantly enhanced hBD3 susceptibility. We propose that the antibiotic activity of hBD3 is based on interference with the organisation over space and time of membrane-bound multienzyme machineries such as the electron transport chain and, in particular, the cell wall biosynthesis complex rather than on formation of defined transmembrane pores.

Tuning the biological properties of amphipathic alpha-helical antimicrobial peptides: rational use of minimal amino acid substitutions.

In nature, alpha-helical antimicrobial peptides present the small and flexible residue glycine at positions 7 or 14 with a significant frequency. Based on the sequence of the non-proteinogenic alpha-helical model peptide P1(Aib7), with a potent, broad spectrum antimicrobial activity, six peptides were designed by effecting a single amino acid substitution to investigate how tuning the structural characteristics at position 7 could lead to optimization of selectivity without affecting antimicrobial activity against a broad panel of multidrug resistant bacterial and yeast indicator strains. The relationship between structural features (size/hydrophobicity of the side chain as well as conformation and flexibility) and biological activity, in terms of minimum inhibitory concentration, membrane permeabilization kinetics and lysis of red blood cells are discussed. On conversion of the peptide to proteinogenic residues, these principles allowed development of a potent antimicrobial peptide with a reduced cytotoxicity. However, while results suggest that both hydrophobicity of residue 7 and chain flexibility at this position can be modulated to improve selectivity, position 14 is less tolerant of substitutions.

Controlled alteration of the shape and conformational stability of alpha-helical cell-lytic peptides: effect on mode of action and cell specificity.

A novel method, based on the rational and systematic modulation of macroscopic structural characteristics on a template originating from a large number of natural, cell-lytic, amphipathic alpha-helical peptides, was used to probe how the depths and shapes of hydrophobic and polar faces and the conformational stability affect antimicrobial activity and selectivity with respect to eukaryotic cells. A plausible mode of action explaining the peptides' behaviour in model membranes, bacteria and host cells is proposed. Cytotoxic activity, in general, correlated strongly with the hydrophobic sector depth, and required a majority of aliphatic residue side chains having more than two carbon atoms. It also correlated significantly with the size of polar sector residues, which determines the penetration depth of the peptide via the so-called snorkel effect. Both an oblique gradient of long to short aliphatic residues along the hydrophobic face and a stabilized helical structure increased activity against host cells but not against bacteria, as revealed by haemolysis, flow cytofluorimetric studies on lymphocytes and surface plasmon resonance studies with model phosphatidylcholine/cholesterol membranes. The mode of interaction changes radically for a peptide with a stable, preformed helical conformation compared with others that form a structure only on membrane binding. The close correlation between effects observed in biological and model systems suggests that the 'carpet model' correctly represents the type of peptides that are bacteria-selective, whereas the behaviour of those that lyse host cells is more complex.

Identification and optimization of an antimicrobial peptide from the ant venom toxin pilosulin.

A template based on positional residue frequencies in the N-terminal stretch of natural alpha-helical antimicrobial peptides was used to prepare sequence patterns and to scan the Swiss-Prot Database, using the ScanProsite tool. This search identified a segment in pilosulin 1, a cytotoxic peptide from the venom of the jumper ant Myrmecia pilosula, as a potential novel antimicrobial peptide sequence. This segment, corresponding to the 20 N-terminal residues, was synthesized and its structural properties and biological activities were investigated. It showed a potent and broad spectrum antimicrobial activity including standard and multi-drug resistant gram-positive and gram-negative bacteria and Candida albicans, confirming the validity of the search method. A rational redesign approach resulting in four amino acid substitutions yielded a variant with improved antibacterial and significantly reduced hemolytic activity.

Mammalian defensins: structures and mechanism of antibiotic activity.

Antibiotic peptides are important effector molecules in host-parasite interactions throughout the living world. In vertebrates, they function in first-line host defense by antagonizing a wide range of microbes including bacteria, fungi, and enveloped viruses. The antibiotic activity is thought to be based on their cationic, amphipathic nature, which enables the peptides to impair vital membrane functions. Molecular details for such activities have been elaborated with model membranes; however, there is increasing evidence that these models may not reflect the complex processes involved in the killing of microbes. For example, the overall killing activity of the bacterial peptide antibiotic nisin is composed of independent activities such as the formation of target-mediated pores, inhibition of cell-wall biosynthesis, formation of nontargeted pores, and induction of autolysis. We studied the molecular modes of action of human defense peptides and tried to determine whether they impair membrane functions primarily and whether additional antibiotic activities may be found. We compared killing kinetics, solute efflux kinetics, membrane-depolarization assays, and macromolecular biosynthesis assays and used several strains of Gram-positive cocci as test strains. We found that membrane depolarization contributes to rapid killing of a significant fraction of target cells within a bacterial culture. However, substantial subpopulations appear to survive the primary effects on the membrane. Depending on individual strains and species and peptide concentrations, such subpopulations may resume growth or be killed through additional activities of the peptides. Such activities can include the activation of cell-wall lytic enzymes, which appears of particular importance for killing of staphylococcal strains.

Mersacidin eradicates methicillin-resistant Staphylococcus aureus (MRSA) in a mouse rhinitis model.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) often colonize the anterior nares, and nasal carriage remains the main source of bacterial dissemination. The aim of this study was to assess the in vivo activity of the lantibiotic mersacidin against MRSA colonizing nasal epithelia. METHODS: The efficiency of mersacidin in the eradication of MRSA was tested employing mice pre-treated with hydrocortisone and inoculated intranasally either three or six times with a bacterial suspension. RESULTS: In mersacidin-treated animals, pre-colonized with MRSA, bacteria could not be detected in blood, lungs, liver, kidney, spleen or nasal scrapings and there were no lesions manifested after intraperitoneal drug application. Blood samples from infected mice obtained 2 h after mersacidin therapy revealed anti-MRSA activity in a serum bactericidal test. Moreover, elevated interleukin-1beta and tumour necrosis factor-alpha titres were noticed in the pre-infected but not in cured animals. In contrast, mersacidin did not induce differences in the cytokine profiles of treated uninfected control mice. CONCLUSIONS: In the mouse rhinitis model, mersacidin was able to eradicate MRSA colonization. The site of action (epithelium versus blood) of mersacidin needs to be further explored.

Localization and functional analysis of PepI, the immunity peptide of Pep5-producing Staphylococcus epidermidis strain 5.

Pep5 is a cationic pore-forming lantibiotic produced by Staphylococcus epidermidis strain 5. The producer strain protects itself from the lethal action of its own bacteriocin through the 69-amino-acid immunity peptide PepI. The N-terminal segment of PepI contains a 20-amino-acid stretch of apolar residues, whereas the C terminus is very hydrophilic, with a net positive charge. We used green fluorescent protein (GFP)-PepI fusions to obtain information on its localization in vivo. PepI was found to occur outside the cytoplasm and to accumulate at the membrane-cell wall interface. The extracellular localization appeared essential for conferring immunity. We analyzed the functional role of the specific segments by constructing various mutant peptides, which were also fused to GFP. When the hydrophobic N-terminal segment of PepI was disrupted by introducing charged amino acids, the export of PepI was blocked and clones expressing such mutant peptides were Pep5 sensitive. When PepI was successively shortened at the C terminus, in contrast, its export properties remained unchanged whereas its ability to confer immunity was gradually reduced. The results show that the N-terminal part is required for the transport of PepI and that the C-terminal part is important for conferring the immunity phenotype. A concept based on target shielding is proposed for the PepI immunity mechanism.

Combination of antibiotic mechanisms in lantibiotics.

Recent studies on the mode of action have revealed exciting features of multiple activities of nisin and related lantibiotics making these peptides interesting model systems for the design of new antibiotics (Molec. Microbiol. 30 (1998) 317; Science 286 (1999) 2361; J. Biol. Chem. 276 (2001) 1772.). In contrast to other groups of antibiotic peptides, the lantibiotics display a substantial degree of specificity for particular components of bacterial membranes. Mersacidin and actagardine were shown to bind with high affinity to the lipid coupled peptidoglycan precursor, the so-called lipid II, which prevents the polymerisation of the cell wall monomers into a functional murein sacculus. The lantibiotics nisin and epidermin also bind tightly to this cell wall precursor; however, for these lantibiotics the binding of lipid II has two consequences. Like with mersacidin blocking of lipid II inhibits peptidoglycan biosynthesis; in addition, lipid II is used as a specific docking molecule for the formation of pores. This combination of lethal effects explains the potency of these peptides, which are active in nanomolar concentration. Other type-A lantibiotics are believed to also use docking molecules for pore formation, although identification of such membrane components has not yet been achieved.

The consequence of sequence alteration of an amphipathic alpha-helical antimicrobial peptide and its diastereomers.

The search for antibiotics with a new mode of action led to numerous studies on antibacterial peptides. Most of the studies were carried out with l-amino acid peptides possessing amphipathic alpha-helix or beta-sheet structures, which are known to be important for biological activities. Here we compared the effect of significantly altering the sequence of an amphipathic alpha-helical peptide (15 amino acids long) and its diastereomer (composed of both l- and d-amino acids) regarding their structure, function, and interaction with model membranes and intact bacteria. Interestingly, the effect of sequence alteration on biological function was similar for the l-amino acid peptides and the diastereomers, despite some differences in their structure in the membrane as revealed by attenuated total reflectance Fourier-transform infrared spectroscopy. However, whereas the all l-amino acid peptides were highly hemolytic, had low solubility, lost their activity in serum, and were fully cleaved by trypsin and proteinase K, the diastereomers were nonhemolytic and maintained full activity in serum. Furthermore, sequence alteration allowed making the diastereomers either fully, partially, or totally protected from degradation by the enzymes. Transmembrane potential depolarization experiments in model membranes and intact bacteria indicate that although the killing mechanism of the diastereomers is via membrane perturbation, it is also dependent on their ability to diffuse into the inner bacterial membrane. These data demonstrate the advantage of the diastereomers over their all l-amino acid counterparts as candidates for developing a repertoire of new target antibiotics with a potential for systemic use.