a Bayer AG, PH-Research Antiinfectives I, D-42096 Wuppertal; b Institute of Medical Microbiology and Immunology, University of Bonn, D-53105 Bonn, Germany
The spread of bacterial resistance leads to a growing demand for novel antibiotics. However, despite significant efforts in academia and the pharmaceutical industry, no genuinely new class of antibacterial compounds has reached the market for almost 20 years.
Industrial strategies for searching for antibiotics with novel mechanisms of action include the screening of large libraries' of synthetic compounds for inhibitors of targets that have not yet been exploited in antibacterial chemotherapy. The advantage of this approach is that it leads to compounds of low molecular mass which are relatively easy to modify by medicinal chemistry. On the other hand, such hits' obtained from in vitro assays still have to overcome major obstacles, e.g. penetration into the intact bacterial cell, an appropriate antibacterial spectrum, favourable pharmacokinetics and low toxicity, before they can be marketed. Therefore, other approaches, such as a detailed evaluation of the potential of existing classes of antimicrobial compounds, are still valuable. Although many of these substances have been known for years, the molecular mechanisms by which they kill bacteria are sometimes poorly characterized. In some cases, a better understanding of this process may help to improve the utility of the class of agents. One example is the lantibiotics, where recent investigations into the molecular mechanism of action have led to unexpected results. It became evident that representatives of different classes of lantibiotics share the same molecular target although this interaction leads to different antibacterial effects. In one particular case, two different mechanisms of action appear to reside in the same molecule and these combine to produce high-potency antimicrobial activity.
Lantibiotics are a group of antimicrobial peptides that are produced by and primarily act upon Gram-positive bacteria.1 Their inactivity against Gram-negative bacteria results from their relatively large size (approximately 18004600 Da) which prevents them from penetrating the outer membrane of the Gram-negative cell wall. The term lantibiotics (lanthionine-containing antibiotics) refers to their unique intramolecular ring structures formed by the thioether amino acids lanthionine or methyllanthionine. These and further post-translational modifications strongly influence the structure of the peptides as well as their stability against protease degradation.
The most well known of the lantibiotics is nisin, which was first described in 1928,2 although its structure was not elucidated until 1971.3 Nisin has been widely used as a natural and safe food preservative in the dairy industry for almost 30 years4,5 and it is chiefly the success of nisin that has stimulated the interest in other lantibiotics. Production of lantibiotics is widespread among Gram-positive bacteria. To date about 30 lantibiotics have been described.1 Lantibiotics are grouped into two major categories based on their structural features and differences in their modes of action (Figure 1). Type A lantibiotics (e.g. nisin, epidermin and Pep5) are flexible, elongated, amphipathic molecules which act mainly by forming pores in the bacterial cytoplasmic membrane.6 In contrast, type B lantibiotics (e.g. mersacidin, actagardine and cinnamycin) have a rigid globular shape and inhibit particular enzymes by forming a complex with their membrane-bound substrates.7,8
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Although this model takes into account many of the results obtained in experiments with model membrane systems, a number of phenomena that are observed with intact bacteria remain unexplained. If a negative surface charge and a sufficiently high level of energization were the sole requirements for pore formation, why is the antibacterial spectrum of many of the lantibiotics rather narrow and not inclusive of all Gram-positive bacteria? Why does nisin effectively kill bacteria in nanomolar concentrations, whereas numerous non-lantibiotic pore-forming peptide antibiotics act in the micromolar range? Why can nisin be used as a non-toxic food preservative and why are eukaryotic membranes affected by nisin and Pep5 only in the millimolar range of concentrations?13,14 Furthermore, there is the interesting observation that incubation of susceptible bacteria with a proteolytic amino-terminal fragment of nisin (nisin 112) prevents pore formation by intact nisin.21 Although it had no antibacterial activity of its own, nisin 112 seemed to compete with intact nisin for defined but limited binding sites in the membrane.
The early observations of Linnet & Strominger22 were instrumental in identifying specific nisin-binding sites. They reported that, in an in vitro system based on isolated bacterial membranes, nisin interferes with peptidoglycan biosynthesis. Later it was demonstrated that the lantibiotic caused this inhibition by binding to the lipid-bound peptidoglycan precursors23 and the same was shown for epidermin.24 Thus, nisin and epidermin from the type A category of lantibiotics, as well as the structurally unrelated type B peptides mersacidin and actagardine, form complexes with the membrane-bound peptidoglycan precursors. Once purified lipid II was available from the studies on mersacidin,8 it was possible to investigate whether the peptidoglycan precursors were the particular membrane components involved in pore formation. Lipid II is the ultimate monomeric peptidoglycan precursor and the first that is available for interaction with lantibiotics on the outside of the cytoplasmic membrane. Incorporation of lipid II into artificial phospholipid vesicles increased the activity of nisin by three orders of magnitude (from micromolar to nanomolar concentrations), with approximately two lipid II molecules per 105 phospholipid molecules being sufficient to yield this effect.2425 In addition, vesicles made of the cytoplasmic membrane of Micrococcus flavus were more sensitive to nisin when their lipid II content was increased.25
Studies with whole cells indicated that these results are relevant for the pore-forming process in intact bacteria. When Micrococcus luteus or Staphylococcus simulans was preincubated with ramoplanin, a non-lantibiotic lipopeptide that forms a complex with the membrane-bound peptidoglycan precursors itself and thus blocks the binding sites for the lantibiotics,24,26 pore formation by nisin or epidermin was prevented. The protective effect of ramoplanin was only observed when nisin or epidermin was applied in concentrations up to five to ten times the MIC but disappeared when larger amounts of the lantibiotics were added.24
In the light of these recent results, the model of pore formation has to be refined (Figure 2b). The lantibiotics nisin and epidermin do not merely interact with the phospholipids but target a specific integral component of the eubacterial cytoplasmic membrane. The lipid-bound peptidoglycan precursor significantly facilitates pore formation. Lipid II may serve as a docking molecule for the peptides to bind to the membrane surface and to adopt the correct position for pore opening, or might be part of the pore itself since it has an influence on pore architecture: in the absence of lipid II pores are more permeable for anions,20 but no ion selectivity is observed in the presence of the peptidoglycan precursor (I. Wiedemann et al., unpublished results). Whether in vivo the peptides also interact with lipid I (undecaprenyl-diphosphoryl-N-acetylmuramic acid-pentapeptide), which is only present on the cytoplasmic side of the membrane, remains an open question. In a cell-free system with a mixed population of isolated right-side out and inside-out vesicles, nisin and epidermin have affinity for both lipids.23,24
Thus, binding of lantibiotics from two different subgroups to the same target molecule has completely different consequences for the bacterial cell. Bacteria treated with mersacidin or actagardine lyse as a consequence of cell wall thinning, whereas in the case of nisin and epidermin the effect on peptidoglycan biosynthesis is overtaken by the much faster killing by de-energization and loss of vital metabolites. It is interesting to speculate which structural features of the peptides may cause their different modes of action. Nisin and epidermin share a homologous amino-terminal ring pattern (Figure 1), whereas the structurally different Pep5 has no affinity for lipid II and may use a different docking molecule.24 This is in agreement with the observation that the fragment nisin 112 prevents the adsorption of intact nisin to target cells.21 In addition, site-directed mutagenesis experiments with nisin indicate that relatively subtle variations in the amino-terminal ring system have strong effects on the interaction with lipid II. The mere exchange of the lanthionine in the first ring for ß-methyllanthionine reduced the binding constant for the peptidoglycan precursor 50-fold, which correlated with a parallel decrease in pore-forming activity in lipid II-containing liposomes (I. Wiedemann et al., unpublished results). A flexible hinge region in the central part of the type A lantibiotics is indispensable for the pore-forming process and although this portion of nisin may also contribute to the interaction with lipid II, its primary function seems to be to provide the peptide with enough conformational freedom to adopt a conducting position in the membrane. Variations in the carboxyl-terminal region of nisin had relatively minor influence on the binding to lipid II (I. Wiedemann et al., unpublished results).
The type B lantibiotics mersacidin and actagardine are rigid, globular molecules and have no flexible hinge region. Their structure allows tight binding to lipid II but lacks the portion necessary for pore formation. Mersacidin and actagardine contain a conserved sequence that extends over more than one-third of the two peptides and includes one complete ring system (Figure 1). It is tempting to suggest that this conserved central core of the otherwise dissimilar peptides is the structural basis for their activity.
With respect to the putative target site at the lipid II molecule, it is noteworthy that nisin and epidermin have affinity for both lipid I and lipid II,24 while mersacidin and actagardine are remarkably selective in their recognition of lipid II.8 As lipid I and lipid II differ only in the N-acetylglucosamine residue, it is likely that the disaccharide moiety is involved in the interaction with mersacidin and actagardine. Furthermore, the peptide side chain could be excluded as the target side of the two type B lantibiotics.11
Elucidation of the molecular mode of action of nisin has revealed some remarkable features of this peptide. Firstly, nisin is the first amphiphilic cationic peptide shown to form pores in a targeted rather than in a non-specific way. The identification of a specific target now enables rational design strategies since lantibiotics can be modified by site-directed mutagenesis of their structural genes.1 By analogy, it may be possible to find some interesting lead structures for drug development among the numerous antibiotics found decades ago for which little, if any, knowledge of the molecular activities is available. Secondly, the remarkable difference in potency displayed by nisin as compared with other amphiphilic peptide antibiotics25 may result not only from targeted pore formation but may be based on a combination of two killing mechanisms. Although nisin derivatives with a mutated hinge region are defective in pore formation and have reduced antibacterial activity (I. Wiedemann et al., unpublished results), their MICs are still in the same range as those of mersacidin which acts solely by blocking peptidoglycan biosynthesis.10,11 While this effect is still under investigation, it seems possible that mutant peptides in which structural features essential for pore formation are destroyed may not necessarily lose their affinity for lipid II, thus leaving unaltered the ability to kill via inhibition of cell wall synthesis. Therefore, it appears that nisin intrinsically has two antibiotic functions for which defined structural features can be identified. It may be unusual but stimulating to consider such dual functions when designing the next generation of antibiotics.
Notes
* Tel: +49-202-364561; Fax: +49-202-364116; E-mail: heike.broetz.hb{at}bayer-ag.de
References
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