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Multiple Activities in Natural Antimicrobials Multiple activities of natural agents such as defensins and bacteriocins suggest a change in strategy when developing new antimicrobials Hans-Georg Sahl and Gabriele Bierbaum any antibiotics, such as -lactams, tetracyclines, and chloramphenicol, target specific molecules and act in a single, welldefined manner. However, other larger antibiotic molecules typically have more complex inhibitory patterns and multiple activities. This rule of thumb applies to host-defense peptides such as the defensins from multicellular organisms and also bacteriocins from bacteria, and may also hold true for lipopeptide and glycopeptide antibiotics. This multiplicity of activities provides an attractive strategy when designing antiinfective drugs. M Summary • Nature’s most abundant antibiotics, cationic amphiphilic host defense peptides, cause pleiotropic effects on membrane processes suggesting that they act like what medicinal chemists call “dirty” or “promiscuous” drugs; unlike with targeted, small molecule antibiotics there is less in the way of resistance development • Bacterial defense peptides, called bacteriocins, combine pleiotropic membrane activities with targeting of specific molecules such as the cell wall precursor Lipid II, strongly increasing their potency • Clinically used and newly developed large natural antimicrobials such as lipopeptide and glycopeptide antibiotics may also have multiple activities • Designing truly effective multiply-active antibiotics appears an attractive strategy, but is likely to require a good deal of basic research Host-Defense Peptides Act Like Promiscuous Drugs Host-defense peptides (HDPs) are amphiphilic cationic peptides of 20 –50 amino acids and include the ␣- and -defensins, cathelicidins, cecropins, magainins, bactenecins, and protegrins. HDPs are part of the innate immune systems of almost every life form, with highly conserved and very broad activities against bacteria, viruses, and fungi in the micromolar range of concentration. The designation “host-defense peptide” was coined when researchers realized that these antimicrobial peptides have not only direct antibiotic activities but also immune modulatory functions. For example, some of them recruit and activate immune cells, enhance bacterial clearance, alter gene expression, and neutralize lipopolysacharides (LPS). A crucial physicochemical feature for the antibiotic activity of HDPs is their amphiphilic character, which enables them to adopt conformations in which polar and charged amino acid side chains orient to one side and apolar residues to the other (Fig. 1). Thus, these peptides can bind to negatively charged microbial surfaces, and then integrate into and disrupt the underlying cytoplasmic membranes. Despite substantial evidence that charge-mediated binding of HDPs is critical for their antibiotic activity, most knowledge about their lipid bilayerperturbing activities is based on analysis of model membranes, leaving many questions as to how they kill microbes. Further, the disturbance of bilayer functions may be only one of several ways that Hans-Georg Sahl is Professor of Medical and Pharmaceutical Microbiology and Gabriele Bierbaum is Professor of Medical Microbiology at the Institute of Medical Microbiology, Immunology and Parasitology, University of Bonn, Germany. This article is based on an invited presentation given at the 47th ICAAC in Chicago, September 2007. Volume 3, Number 10, 2008 / Microbe Y 467 Sahl Believes Science Requires “Quite a Bit of ‘Creative Playing’” Hans-Georg Sahl says that doing science satisfies him more than do the results. “Too many people are pushing science toward applications,” he says. “It would have been a nightmare for me to put a scientifically interesting project aside just because business people had a different opinion. We need curiosity-driven basic research in the first place, and then we will always have something to apply— the other way around simply will not work.” Nevertheless, Sahl hopes that his research will benefit public health. He studies antibiotic mechanisms—what happens at the molecular level when an antibiotic kills bacteria—a focus that could lead to new therapeutics when resistance to available drugs is increasing. “There is an urgent need for new antibiotics, particularly compounds with unprecedented mechanisms,” he says. “Multiresistant bacteria are really a major health threat, and the medical need for new antibiotics is not adequately met by the pharmaceutical industries. Identifying new antibiotic paradigms is a very rewarding business that can certainly contribute to overcoming resistance problems.” Sahl is professor of pharmaceutical and medical microbiology at the University of Bonn in Bonn, Germany. “How are complex biosynthetic pathways organized in space?” he asks. “How do multiple enzymes function in a coordinated fashion so that a complex macromolecule such as the bacterial cell wall keeps growing from one cell to the next generation of cells without losing functionality? Antibiotics are wonderful tools to 468 Y Microbe / Volume 3, Number 10, 2008 interfere with such processes, and, from disturbing a process, you can learn much about the process itself.” Sahl, 58, was not always pointed toward a career in biology. He grew up in Engers, a small town near Koblenz in the central Rhine valley, where the river Mosel enters the Rhine. As a child, he had an aquarium and a terrarium, and received his first microscope when he was 15. But he was also interested in geography, listening to rock and pop music and playing the guitar, sports, and studying German, English, and American literature. Tom Sawyer and Huckleberry Finn are among his favorite books. “I definitely cannot say that I wanted to become an academic researcher from childhood on,” he says. Nevertheless, growing up, Sahl nurtured a love for nature and the outdoors, a fondness inspired by his childhood environment. “It was not too far from a city, but far enough to have fields, a small forest, small lakes and the river Rhine,” he says. “What I remember most is that we had endless time for playing outside and never got tired; that certainly implanted my love for outdoor activities. “I definitely kept that love for nature and activities, and playing outdoors,” he adds. “Building a small dam in a creek is still a fascinating thing to do. I guess I have kept a lot of the homo ludens [man at play] of my childhood. Science actually allows you to do that—it requires quite a bit of ‘creative playing’—it is just that the toys are getting so much more expensive.” During his final year of high school, his biology and chemistry teacher led him to think about a career in biology. “He taught us exciting new things which just had appeared in textbooks,” Sahl says. “I remember very well when we learned about the chemistry behind converting sugars into energy. This was really hot stuff in those days for high school teaching. It gave me, for the first time, a concrete molecular idea for an essential process of life, energy generation. Those experiences actually made me enroll in biology and chemistry—rather than geography, languages, or other things.” Sahl received his Diploma in Biology from the University of Bonn in 1975, and his Ph.D. in microbiology, also from the University of Bonn, in 1978. Between 1979 and 1983, he held a postdoctoral fellowship in medical microbiology, which included a year at Purdue University. “I spent almost all of my scientific life at the University of Bonn, which is a bit unusual, but it was perfect for me and for the University, so why not?” he says. “Bonn is a beautiful place to live.” After deciding to study biology, “I just did what I found most interesting, and I was lucky enough to find support, positions, and the right mentors,” Sahl says. “After undergraduate studies, I specialized in microbiology because I thought that a bacterium would be biggest organism that I could possibly understand on the molecular level. I had tried virology before, because a virus was much simpler and seemed to be more easy to understand, but I returned to bacteria when I found out that you cannot study a virus without studying eukaryotic cell biology.” Sahl, who is unmarried, enjoys travel and takes long trips whenever he can spare the time. “I love to be in other places of the world, although getting there is more and more of a nuisance,” he says. “My love for nature and outdoor activities is one of the reasons why I have to get away every once in a while— on a three-week canoe trip into the wilderness, or to the outback and the desert in Australia, or to go hiking in the mountains.” When he is not traveling or engaged in his research, he works on his house. “I have quite a bit of land around it, which I turned into a little park,” he says. “It always keeps me busy over the weekend, in winter even more than in summer, trimming bushes or cutting a tree, planting new trees, chopping wood, cutting the grass. It is the right exercise, physically and mentally, for somebody who sits at a desk most of the week.” Marlene Cimons Marlene Cimons is a freelance writer in Bethesda, Md. HDPs affect target cells. For example, when we Bacteriocins Combine Targeted and treated staphylococcal cells with human -deNontargeted Activities fensin 3 (hBD3, Fig. 1), the cell wall stress reguBacterial peptides with antibacterial activity, lon including the two-component regulatory called bacteriocins—whether posttranslationsystem vraSR was upregulated at the transcripally modified (lantibiotics) or not—typically tional level. The cell wall stress regulon consists share features with other HDPs, including relaof a group of genes that typically is upregulated tive size, cationicity, and amphiphilicity. Addiin response to vancomycin, oxacillin, and other tionally, some bacteriocins contain structural agents that inhibit cell wall biosynthesis in motifs that also are found among eukaryotic Staphylococcus aureus. defensins, such as the ␥-core motif that appears This response indicates that the peptides do not simply disrupt cytoplasmic membranes. Instead, they also interfere with FIGURE 1 highly coordinated, membrane-bound processes, of which cell wall biosynthesis appears the most vulnerable. Electron transport chains may also be impaired, particularly by helical peptides such as human cathelicidin LL37 that have strong amphiphilic features. In general, the antibiotic activity of host defense peptides appears not to be based on targeting specific molecules. Instead, they produce pleiotropic effects at various sites—acting as if they were what some medicinal chemists and other experts call “dirty drugs.” Nonetheless, conventional drug-design strategies aim not at developing promiscuous drugs but at developing agents that have high affinity for well-defined targets. However, nature apparently Primary sequence of the human beta defensin 3 (hBD3) and the human cathelicidin LL 37 (amino acids in the one-letter code); hBD3 has an overall positive charge of ⫹11 due to works differently in the case of so many a surplus of lysine (K) and arginine (R) residues over negatively charged residues (D, E); antimicrobial peptides. Moreover, units structure is stabilized by 3 disulfide bridges. LL37 is a linear, ␣-helical peptide with a like antibiotics which act on a single net charge of ⫹ 6; its pronounced amphiphilicty is demonstrated in the spatial structure with hydrophobic residues in green, negatively charged side chains in red and positively target, there is little in the way of resischarged residues in blue. tance developing for these antimicrobial peptides. Volume 3, Number 10, 2008 / Microbe Y 469 onine-containing antibiotics) are about the same molecular weight as defensins but contain thioether amino acids instead of disulfide bonds. The thioether amino acids in these bacteriocins, namely lanthionine and methyllanthionine, are produced by posttranslational changes from cysteine and serine and from cysteine and threonine residues, respectively (Fig. 2A). Bacteriocins were thought to kill exclusively by disrupting membranes until researchers realized that they also act on other targets. Indeed, many of them act like HDPs by disrupting lipid bilayer functions at micromolar concentrations. However, against a narrow spectrum of closely related strains, lantibiotics have minimal inhibitory concentrations (MICs) in the nanomolar range; in such cases, specific target-mediated activities typically are involved. For about 40 of the lantibiotics described so far, the molecular target is the membrane-bound cell wall precursor Lipid II. Researchers assign lantibiotics that bind Lipid II to two groups (Fig. 2B). The first, typified by the food preservative nisin, consists of elongated molecules, while lantibiotics in the second group, such as mersacidin, have a more globular shape. After these lantibiotics have formed stoichiometric complexes with Lipid II, they sequester the cell wall precursor, blocking its further incorporation into the cell envelope. Thus, these lantibiotics inhibit cell wall biosynthesis in a way that resembles (A) Primary structure of the lantibiotics nisin and mersacidin. Lantibiotics contain numerous unusual amino acids which are produced through posttranslational modificaglycopeptide antibiotics. However, the tion of precursor peptides; such amino acids include, e.g., didehydroalanine (Dha, from lantibiotics bind the highly conserved serine), didehydrobutyrine (Dhb, from threonine), lanthionine (Lan, from Dha and cyssugar-pyrophosphate moiety of Lipid II teine), methyllanthionine (MeLan, from Dhb and cysteine). The thioether containing residues Lan and MeLan introduce intramolecular ring structures; the two sequence rather than the D-Ala-D-Ala peptide motifs involved in Lipid II binding which are characteristic for each group of nisin- and side chain of the building block for the mersacidin-related lantibiotics are boxed. (B) Structure of the membrane-bound cell wall cell wall (Fig. 2B). precursor Lipid II, consisting of the disaccharide-pentapeptide cell wall subunit and the bactoprenol-pyrophosphate membrane anchor; the interpeptide bridge attached to the Binding to Lipid II may yet provoke lysine residues of the stem peptide is found in most gram-positive bacteria and may additional activities for some lantibiotconsist of five glycine residues in many staphylococci. The binding sites on Lipid II for ics. For example, when nisin binds to lantibiotics (light blue) and glycopeptide antibiotics (dark blue) are indicated. Lipid II, the cell wall precursor acts like an anchor molecule for the lantibiotic, which subsequently inserts into the memamong class IIa bacteriocins from gram-positive brane to form a defined and stable pore consisting bacteria. Meanwhile, the lantibiotics (lanthiof Lipid II and nisin molecules (Fig. 3). FIGURE 2 470 Y Microbe / Volume 3, Number 10, 2008 FIGURE 3 Dual mode of action of the lantibiotic nisin. Nisin forms a stoichiometric complex with Lipid II which triggers two fatal events (i) the cell wall precursor is abducted and no longer available for incorporation into the growing peptidoglycan network (left); binding of Lipid II via its N-terminal binding motif and the carbohydrate-pyrophosphate moiety on Lipid II enables the C-terminal segment of nisin to insert into the membrane (right); several nisin: Lipid II complexes assemble to form a stable pore with an overall diameter of 2 nanometers. Besides these Lipid II-mediated activities, nisin can induce cell wall lytic processes, particularly in staphylococci, and, at micromolar concentrations, impair vital lipid bilayer functions. Several other lantibiotics, including nisinrelated epidermin and gallidermin as well as plantaricin C, which has a mersacidin-like Lipid II binding motif, also form Lipid IIdependent pores. However, these peptides form pores in membranes of only some bacterial strains, such as the micrococci, but not the lactococci. The overall length of the individual lantibiotic molecule as well as the lipid composition of the target-strain membranes determine whether pores can form. When Lipid II-lantibiotic complexes insert into membranes, they may dislocate from the sites where they are needed for cell wall synthesis and may lead to septum formation at inappropriate sites. In addition to these Lipid II-mediated activities, lantibiotics have other effects. For instance, when treated with nisin, dividing cells of Staphylococcus simulans prematurely split the new cell wall between the two daughter cells, leading to rapid lysis when the septum is in- complete. Other cationic peptides, including defensins, also rapidly lyse staphylococci— possibly triggering the same cell wall lytic mechanism. The complexity of the lantibiotics apparently is matched by that of another prominent group of bacteriocins—namely, the class II peptides from gram-positive bacteria. For instance, the Listeria-active class IIa peptides combine with protein components of the mannose phosphotranferase sugar uptake system to form pores through target-cell membranes. More generally, many naturally occurring antimicrobial peptides appear to combine broad toxicity with the targeting of specific molecules. This property of dual or multiple activities also helps to explain how peptide bacteriocins have such remarkably high efficacies, with MICs typically in the nanomolar concentration range, against some targeted bacteria; but low-level background activity against the rest of the grampositive bacteria. Volume 3, Number 10, 2008 / Microbe Y 471 FIGURE 4 Structures of the glycopeptide antibiotic vamcomycin and of the lipodepsipeptide antibiotic daptomycin; both are clinically important natural product antibiotics for treatment of multiresistant Gram-positive bacterial infections. Glycopeptide antibiotics other than vancomycin may have increased amphiphilic properties through attached strongly hydrophobic moieties such as chlorophenyl-benzyl rings (oritavancin) or acyl chains (e.g., teicoplanin) linked to the disaccharide units. Some Lipopeptide and Glycopeptide Antibiotics Show Multiple Activities Some experts argue that such complex activity patterns are found mainly among highermolecular-weight, natural compounds that are unsuitable for commercial development. However, there are noteworthy exceptions to this rule. For instance, established low-molecularweight drugs such as isoniazide cause pleiotropic effects. Meanwhile, several commercially successful antibiotics that overcome drug resistance among some gram-positive pathogens also appear to have multiple activities. For instance, glycopeptide antibiotics such as vancomycin (Fig. 4) interfere with cell wall synthesis by attaching to the Lipid II precursor at its D-AlaD-Ala peptide side chain (Fig. 2B), thereby inhibiting transglycosylation of cell wall subunits. However, some other glycopeptide antibiotics have pronounced amphiphilic properties, particularly those containing lipid side chains such 472 Y Microbe / Volume 3, Number 10, 2008 as teicoplanin as well as telavancin and ramoplanin, both of which are under development. Thus, these drugs may impair membrane barrier functions in addition to inhibiting cell wall biosynthesis. Another glycopeptide, oritavancin, which is also in clinical development, appears to have an additional binding site for the interpeptide bridge, that occurs in a modified form of Lipid II in gram-positive cells. The lipodepsipeptide daptomycin (Fig. 4), which was approved for use in the United States in 2003, supposedly forms pores in gram-positive bacteria. This cyclic molecule has a net negative charge and requires calcium ions for activity. When it forms complexes with calcium ions, the antibiotic has an overall positive charge and is assumed to behave like other cationic peptides with antimicrobial activity—that is, to oligomerize within membranes, forming pores that disrupt vital ion gradients within target bacterial cells. However, according to some investigators, daptomycin also may inhibit either peptidoglycan or lipoteichoic acid biosynthesis. Daptomycin, in contrast to cationic HDPs, is a very potent compound. Designing Anti-Infective Drugs with Multiple Modes of Action During the past few decades, hybrid antibiotics were designed with dual modes of action by combining various beta-lactam, macrolide, quinolone, oxazolidinone, or glycopeptide antibiotics. The goal was to have drugs with extended activity spectrums while reducing the risk that pathogens would develop resistance to them. However, inferior physicochemical and pharmacological properties tended to counterbalance those advantages—reducing potency and introducing other adverse features. Whether more recently developed rifamycin-quinolone hybrids overcome those problems and fulfill their promise remains to be seen. Natural peptides with multiple antimicrobial activities, of course, differ from synthetic hybrid antibiotics because the former combine innate, pleiotropic antibiotic activity, which is based on cationic amphiphilicity, with specific target- mediated activities, mainly involving interference with cell wall biosynthesis. Cationic amphiphilicity is also an indispensable feature for the immunomodulatory activity of HDPs. Some synthetic short-chain amphiphilic HDP derivatives have been designed that are devoid of direct antibiotic activity, but fully retain immunoregulatory functions and effectively control bacterial infec- tions in mice. Thus, combining those three functions—immunomodulation, basic, and targeted antibiotic activities—into one molecule appears a particularly promising approach. However, succeeding with that approach is likely to require a good deal of basic research. The increasingly dramatic buildup of antibiotic resistance among human pathogens warrants such efforts. ACKNOWLEDGMENTS We gratefully acknowledge the many excellent contributions by members of our research groups and collaborators. Financial support was obtained from the German Research Foundation (DFG, various projects to both of us), the Federal Ministry of Education and Research (BMBF, Pathogenomics network to G.B. and SkinStaph network to H.G.S.) and the BONFOR programme of the Medical Faculty, University of Bonn. SUGGESTED READING Baltz, R. H., V. Miao, and S. K. Wrigley. 2005. Natural products to drugs: daptomycin and related lipopeptide antibiotics. Nat. Prod. Rep. 22:717–741. Diep, D. B., M. Skaugen, Z. Salehian, H. Holo, and I. F. Nes. 2007. Common mechanisms of target cell recognition and immunity for class II bacteriocins. Proc. Natl. Acad. Sci. USA 104:2384 –2389. Hancock, R. E., and H. G. Sahl. 2006. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nature Biotechnol. 24:1551–1557. Lange, R. P., H. H. Locher, P. C. Wyss, and R. L. Then. 2007. The targets of currently used antibacterial agents: lessons for drug discovery. Curr. Pharm. Des. 13:3140 –3154. Peschel, A., and H. G. Sahl. 2006. The co-evolution of host cationic antimicrobial peptides and microbial resistance. Nature Rev. Microbiol. 4:529 –536. Sass, V. U. P., A. Tossi, G. Bierbaum, and H. G. Sahl. 2008. Mode of action of human beta-defensin 3 against Staphylococcus aureus and transcriptional analysis of responses to defensin challenge. Int. J. Med. Microbiol., in press. Scott, M.G., E. Dullaghan, N. Mookherjee, N. Glavas, M. Waldbrook, A. Thompson, A. Wang, K. Lee, S. Doria, P. Hamill, J. J. Yu, Y. Li, O. Donini, M. M. Guarna, B. B. Finlay, J. R. North and R. E. Hancock. 2007. An anti-infective peptide that selectively modulates the innate immune response. Nature Biotechnol. 25:465– 472. Wiedemann, I., E. Breukink, C. van Kraaij, O.P. Kuipers, G. Bierbaum, B. de Kruijff, and H. G. Sahl. 2001. Specific binding of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity. J. Biol. Chem. 276:1772–1779. Willey, J. M., and W. A. van der Donk. 2007. Lantibiotics: peptides of diverse structure and function. Annu. Rev. Microbiol. 61: 477–501. Yeaman, M. R. and N. Y. Yount. 2007. Unifying themes in host defence effector polypeptides. Nature Rev. Microbiol. 5:727–740. Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415:389 –395. Volume 3, Number 10, 2008 / Microbe Y 473