Cell, Vol. 65, 205-207, April 19, 1991, Copyright 0 1991 by Cell Press
Antibacterial Peptides: Key Components Needed in Immunity Hans G. Boman Department of Microbiology Stockholm University S-106 91 Stockholm Sweden
Twenty years ago, biochemistry, immunology, and entomology represented rather different intellectual traditions. In biochemistry, a strong belief in model systems (yeast extracts, rat liver homogenates, and of course E. coli) generated a large body of data with only modest theoretical speculations and consequently rather few disagreements. This was quite contrary to immunology, where an impressive building of speculative theories used to result in some rather outspoken disagreements. In fact, such arguments may sometimes have delayed the acceptance of novel experimental findings. In the third corner, the large number of insect species (l-3 million) adapted to very different ecological niches convinced entomologists that no generality was within reach. Thus, there was an outspoken disbelief in model systems and perhaps as a consequence, a surprising respect for contradictory facts. However, immunology and entomology have drifted toward the traditions of biochemistry, largely because of classical and molecular genetics. During the last decade, the field of antibacterial peptides, which is rooted in the distant corners of immunology and entomology, has developed into biochemistry and molecular biology. Granular Proteins and Defensins tram Phagocytes Phagocytosis was discovered more than 100 years ago, but only after 80 years were the mechanisms by which engulfed bacteria are killed divided into two different groups, dependent on the absence or presence of oxygen. The oxygen-independent killing depends on several large proteins, such as bactericidal-permeability increasing protein and cathepsin G, as well as a group of small peptides called defensins (Lehrer et al., 1991). The defensins were isolated in the early 80’s by Lehrer’s group, and the first sequences were reported a few years later (more than 15 sequences have been published to date). All defensins contain 29-34 amino acid residues, have three intramolecular disulfide bonds, and are quite basic. The threedimensional structure is known both from X-ray and NMR studies. Defensins act on a wide variety of bacteria but usually better on gram-positive than on gram-negative bacteria. They also work on fungi and are somewhat cytotoxic for normal eukaryotic cells. However, this does not seem to cause any problems, because defensins are not normally released but are sequestered in granules of neutrophils. Defensins are made as preproproteins of 93-95 residues containing one defensin copy. A smaller peptide with only 12 residues and one intramolecular disulfide bond was found in bovine neutrophils (Romeo et al., 1988) but so far nowhere else.
Minireview
Frog Skins and the Magainins Frog skins contain glands that produce a large number of biologically active peptides, many of which have counterparts in neural and intestinal tissues of mammals (reviewed by Bevins and Zasloff, 1990). The secretion of these peptides can be induced by epinephrine or norepinephrine. Some are general toxins-crude toxin from the frog Bombina was early reported to act on bacteria. Twenty years later, Xenopus had become a widely used laboratory animal from which oocytes were removed surgically. These frogs could thrive in waters quite dense with bacteria, yet the wounds from their operation never became infected. This observation led Zasloff to the finding of the magainins (Zasloff, 1987), two closely related antibacterial peptides produced in the skin of Xenopus. Another frog skin peptide, PGLa, has also been shown to be antibacterial. Magainins are only 23 residues, devoid of cysteine, and form an amphipathic a helix. They act on both grampositive and gram-negative bacteria, fungi, and protozoa (Bevins and Zasloff, 1990). The gene structure for the magainins is not yet known, but cDNA cloning has shown that these peptides are made as large precursor molecules containing six copies interspaced by segments from which a short acidic hexapeptide is liberated (Bevins and Zasloff, 1990). A surprising finding is the diversity of the frog antibiotic peptide family: magainins may be unique to Xenopus. In Bombina, the major antibacterial peptide, bombinin, is hardly related in sequence to magainin (Simmaco et al., 1991). insect immunity and the Cecropins The cecropins were originally identified as highly potent antibacterial peptides in immune hemolymph from the cecropia moth. All cecropins are 31-39 residues, are devoid of cysteine, and have a strongly basic N-terminal region and a long hydrophobic stretch in the C-terminal half. The structure of cecropins deduced from NMR studies is two a helices joined by a hinge region containing Gly-Pro (Holak et al., 1988). Although cecropins were thought to be unique to insects, a cecropin has been found in pig intestine (Lee et al., 1989), which implies that cecropins are widespread in the animal kingdom. In two dipterans, Sarcophaga and Drosophila, the amino acid sequences of cecropins A and IA are identical, although there is only 73% homology at the nucleotide level. Thus, there appears to be strong selective pressure to conserve the cecropin protein sequence in different insects. Insect cecropins are induced by bacterial infection and are synthesized as preproproteins of 62-64 residues. Two IO Human HNP-1 Rabbit NP-1 Rat RatNP-1
ACYCRIPAC VVCACRRALC VTCYCRRTRC
20 IAGERRYGTC LPRERRAGFC GFRERLSGAC
30 IYQGRLWAFC C RIRGRIHPLCCRR GYRGRIYRLC CR
Figure 1. Defensin Sequences from Three Mammals Disulfide bridges link residues 3 and 31, 5 and 20, and
10
and 30.
Cdl 206
10
Xenopus
Bombina Xenopus Figure
Magainin
Bombinin pGLa
2. Antibacterial
*, amidated
i
i-IGKFLHSAK GIGGALLSAA GMASKAGAIA
Peptides
from
Zll
KFGKAFVGEI KVGLKGLAKG GKIAKVALKA
MNs LAEHFN I,"
Two Frogs
C-terminus.
preprocecropins and some truncated analogs have been synthesized by the solid phase method, and these polypeptides were used in processing studies with partially purified enzymes (Boman et al., 1989). The pro part, which contains one or two dipeptides, was removed stepwise by a dipeptidyl aminopeptidase that was found to be highly specific. The C-terminal amide present in insect cecropins (Boman and H&mark, 1987) is presumably derived from a glycine residue that follows the last residue in the mature protein. Many other active peptides are processed in the same way as the cecropins, and a glycine-dependent amidation reaction has been demonstrated for brain peptides. In Cecropia (Xanthopoulos et al., 1988), Sarcophaga (Kania and Natori, 1989) and Drosophila (Kylsten et al., 1990) each cecropin is encoded by a single-copy gene that has a single intron with a conserved splice site. Mechanism of Action Cecropins A and B are highly active against several grampositive and gram-negative bacteria, while the porcine form shows high activity only against gram-negatives. The original observation that cecropins lysed E. coli formed the basis of an assay used to isolate cecropins. Subsequently, cecropins were found to lyse bacteria but not eukaryotic cells. Cecropins will also lyse artificial liposomes composed of phospholipids having zwitterionic or negatively charged head groups. The rate and extent of lysis are dependent on the cecropin concentration. Thus, the cecropins act stoichiometrically and not catalytically. There is every reason to believe that this would also apply to defensins and magainins. While defensins and magainins are about equally potent, cecropins are an order of magnitude more active on a molar basis against gram-negative bacteria (Wade et al., 1990). The primary targets of defensins, magainins, and cecro pins are the inner and outer bacterial membranes. All three groups of peptides have also been shown to form channels in artificial membranes, but it is not yet clear if channel formation is the mechanism by which these peptides kill microorganisms. Both the natural L- and the o-enantiomers of a cecropin, a magainin, and some hybrid peptides form voltage-dependent anion channels in artificial membranes. The D-peptide exhibits a circular dichroism spectrum that is the mirror image of the natural cecropin, which was interpreted
II,
JO
xl
BOIllbYX
A
RW--KIFKKI
Antheraea
B
KW--KIFKKI
EKMGRNIRDG EKVGRNIRNG
Hyalophora
B
KW--KVFKKI
EKMGRNIRNG
Drosophila Porcine
A
GWLKKIGKKI
Pl
SWLSKTAKKL
ERVGQBTRDA ENSAK-KR--
to mean that the o-enantiomer forms a left-handed helix and the natural L-enantiomer forms a right-handed one (Wade et al., 1990). Because the antibacterial activity of both enantio mers is the same, the mechanism of antibiotic activity does not appear to depend on an interaction of the peptide with chiral centers on bacterial membranes. Other Antibacterial Peptides For some time, cecropins and defensins were the only antibacterial peptides known; at that time, they were considered to be unique to that group of animals in which they were discovered. However, the isolation of magainins four years ago was soon followed by the discovery of defensinlike peptides in insects (see Lehrer et al., 1991) and a cecropin in pig intestine (Lee et al., 1989). Simultaneously, other peptides were found in other insects, barley, horseshoe crab, and in bovine tracheal mucosa (Diamond et al., 1991, and references therein). A male-specific peptide protects the male reproductive tract of Drosophila against infections (Samakovlis et al., 1991). Of special structural interest are peptides that are either proline rich or arginine-proline rich (Frank et al., 1990, and references therein). Antibacterial peptides with new properties have also been obtained by making hybrid molecules with one domain from a cecropin and another from the bee venom toxin melittin (Wade et al., 1990). Work on the larger antibacterial proteins from phagocytes has yielded the cDNA sequences for both bactericidalpermeability increasing protein and cathepsin G. Interestingly, two degradation products from cathepsin G, a pentapeptide and a heptapeptide, have antibacterial activity (Bangalore et al., 1990). Taken together, these data strongly indicate that antibacterial peptides are widespread in nature. Since the potential role of antibacterial peptides in immunity is not generally appreciated, a brief explanation of their role in host defense may be justified. In Defense of Peptides Let us first assume that a prime function of any immune system is to offer protection against the natural flora of microorganisms. The defense against pathogens probably differs between fast-growing insects and long-lived mammals with few offspring. When the immune system is impaired by AIDS or immunosuppressive agents, it is the natural flora and opportunistic pathogens that take over. Also, a fixed repertoire of defensive agents makes sense against the natural flora, less so against occasional pathogens. If this is the case, then bacteria should be a prime target of the immune system, both now and during the evolution of immunity. Secondly, the rate of an immune response in any animal must be faster than the rate of multiplication of the invading organism-if not, the animal will not survive. During favorable growth conditions, the generation time of bacteria is
IVKAGPAIEV IIKAGPAVAV IVKA GPAIAV TI-QGLGIAQ -1SEGIAIAI
LG~AKAI LGEAKAL* LGEAKAL* QAANVAATAR* QGGPR
Figure 3. Cecropin Sequences sects and One Mammal *, amidated
C-terminus.
from
Four
In-
Minireview 207
about 50 times faster than that of B cells, which produce immunoglobulins. Finally, the prepro form of an antibacterial peptide is made almost 180 times faster than IgM (assuming a constant rate of peptide bond synthesis). Small peptides also diffuse faster than large defensive cells and antibodies, a property which is also likely to be beneficial when combating an infection. Antibacterial peptides lyse both gram-positive and gram-negative bacteria (and liposomes) but cause little or no harm to eukaryotic cells (defensins and magainins are slightly cytotoxic). Thus, self destruction is largely avoided by membrane specificity, presumably without the recognition of any bacterial proteins (because peptides with only D amino acids are equally active as the natural ones). In general, the survival advantages of antibacterial peptides are their potency (almost like broad-spectrum antibiotics) and their low cost (in terms of time, information, and energy) of production. Thus, they can form an important part of the primary defense against bacterial infections. Evolutionary Aspects Lymphocyte activation is generally accepted to require two signals, as originally proposed by Bretscher and Cohn 20 years ago. It is reasonable to assume that one signal, a “pattern recognition” event (Janeway, 1989), evolved initially, while a second, highly specific signal was added much later, perhaps in response to the increasing complexity of the organism. By several criteria, insect immune systems represent a defense against bacterial infections that arose early in evolution; a single signal, such as a degradation product of murein or free lipopolysaccharide, is sufficient to induce cecropins and other antibacterial proteins in insects. Insects may have diverged from vertebrates before the need for a second signal developed. Immune systems as such may have evolved in concert with the eukaryotic cell, because even the earliest eukaryotes had to compete with bacteria. Since antibacterial peptides probably do not require specialized cells for their synthesis, it is likely that this type of defense predates the separation of vertebrates and invertebrates, which may have occurred some 800 million years ago. Finally, the evolution of immunity within a group of species must be regarded not only in relation to the size of an animal (see Janeway, 1989) but also in relation to its life span and its reproductive rate (Boman and Hultmark, 1987). Small and rather short-lived animals with a high reproductive rate such as Drosophila can afford to lose 90% of the population in an occasional virus infection (and a good virus should not kill its host completely). In contrast, “classical” immune mechanisms (i.e., the major histocompatibility loci and the lymphatic system) are needed in slow-growing populations of large mammals. However, a defense against the natural flora of bacteria is a must for all types of animals; that is why they have antibacterial peptides.
Bangalore, N., Travis, (1990). J. Biol. Chem. Bevins, 41 4.
J., Onunka, V. C., Pohl, J., and Shafer, 265, 13564-13588.
C. L., and Zasloff.
M. (1990).
Annu.
Rev. Biochem.
W. M.
59, 395-
Boman, 126.
H.G.,and
H&mark.
D. (1987). Annu. Rev. Microbial.
47,103-
Boman, H. G., Soman, I. A., Andre& D., Li, Z-q., Merrifield, R. B, Schlenstedt. G., and Zimmermann, R. (1969). J. Biol. Chem. 264, 5652-5860. Diamond, G.. Zasloff. M., Eck, H., Bressaur, M., Malay, W. L., and Bevins, C. L. (1991). Proc. Natl. Acad. Sci. USA 88, in press. Frank, R. W., Gennaro, R., Schneider, K., Przybylski, D. (1990). J. Biol. Chem. 265, 16671-16674.
M., and Romeo,
Holak, T. A., Engstrom, A., Kraulis, P. J., Lindeberg, G., Bennich, H., Jones, T. A., Gronenborn, A. M., andclore, G. M. (1966). Biochemistry 27, 7620-7629. Janeway. 1-13.
C. A. (1969).
Kania. A., and Natori, Kylsten, 224.
Cold
Spring
S. (1969).
P., Samakovlis,
Harbor
FEBS
Symp.
Quant.
Biol. 54,
Lett. 258, 199-202.
C.. and Hultmark,
D. (1990). EMBO J. 9,217-
Lee. J.-Y., Boman, A., Chuanxin, S.. Andersson, M.. Jornvall, H., Mutt, V., and Boman, H. G. (1969). Proc. Natl. Acad. Sci. USA 88, 91599162. Lehrer,
R. I., Ganz,
T.. and Selsted,
Romeo, D., Skerlavaj, B., Bolognesi, Biol. Chem. 283, 9573-9575.
M. E. (1991).
Cell 84, 229-230.
M.. and Gennaro,
Samakovlis, C., Kylsten. P., Kimbrell, D. A., Engstrom, mark, D. (1991). EMBO J. 70, 163-169. Simmaco, M., Barra, D., Chiarini, F., Noviello, G., and Richter, K. (1991). Eur. J. Biochem.,
R. (1966).
J.
A., and Hult-
L., Melchiorri, in press.
P., Kreil,
Wade, D.. Boman, A., Wihlin. B., Drain, C. M., Andreu, D., Boman, H. G., and Merrifield, R. 8. (1990). Proc. Natl. Acad. Sci. USA 87, 4761-4765. Xanthopoulos, K. G., Lee, J. Y.. Gan, R., Kockum, K., Faye, Boman, H. G. (1966). Eur. J. Biochem. 772, 371376. Zasloff,
M. (1987).
Proc.
Natl. Acad.
Sci. USA 84, 5449-5453.
I., and
Antibacterial Peptides: Key Components Needed in Immunity Hans G. Boman Department of Microbiology Stockholm University S-106 91 Stockholm Sweden
Twenty years ago, biochemistry, immunology, and entomology represented rather different intellectual traditions. In biochemistry, a strong belief in model systems (yeast extracts, rat liver homogenates, and of course E. coli) generated a large body of data with only modest theoretical speculations and consequently rather few disagreements. This was quite contrary to immunology, where an impressive building of speculative theories used to result in some rather outspoken disagreements. In fact, such arguments may sometimes have delayed the acceptance of novel experimental findings. In the third corner, the large number of insect species (l-3 million) adapted to very different ecological niches convinced entomologists that no generality was within reach. Thus, there was an outspoken disbelief in model systems and perhaps as a consequence, a surprising respect for contradictory facts. However, immunology and entomology have drifted toward the traditions of biochemistry, largely because of classical and molecular genetics. During the last decade, the field of antibacterial peptides, which is rooted in the distant corners of immunology and entomology, has developed into biochemistry and molecular biology. Granular Proteins and Defensins tram Phagocytes Phagocytosis was discovered more than 100 years ago, but only after 80 years were the mechanisms by which engulfed bacteria are killed divided into two different groups, dependent on the absence or presence of oxygen. The oxygen-independent killing depends on several large proteins, such as bactericidal-permeability increasing protein and cathepsin G, as well as a group of small peptides called defensins (Lehrer et al., 1991). The defensins were isolated in the early 80’s by Lehrer’s group, and the first sequences were reported a few years later (more than 15 sequences have been published to date). All defensins contain 29-34 amino acid residues, have three intramolecular disulfide bonds, and are quite basic. The threedimensional structure is known both from X-ray and NMR studies. Defensins act on a wide variety of bacteria but usually better on gram-positive than on gram-negative bacteria. They also work on fungi and are somewhat cytotoxic for normal eukaryotic cells. However, this does not seem to cause any problems, because defensins are not normally released but are sequestered in granules of neutrophils. Defensins are made as preproproteins of 93-95 residues containing one defensin copy. A smaller peptide with only 12 residues and one intramolecular disulfide bond was found in bovine neutrophils (Romeo et al., 1988) but so far nowhere else.
Minireview
Frog Skins and the Magainins Frog skins contain glands that produce a large number of biologically active peptides, many of which have counterparts in neural and intestinal tissues of mammals (reviewed by Bevins and Zasloff, 1990). The secretion of these peptides can be induced by epinephrine or norepinephrine. Some are general toxins-crude toxin from the frog Bombina was early reported to act on bacteria. Twenty years later, Xenopus had become a widely used laboratory animal from which oocytes were removed surgically. These frogs could thrive in waters quite dense with bacteria, yet the wounds from their operation never became infected. This observation led Zasloff to the finding of the magainins (Zasloff, 1987), two closely related antibacterial peptides produced in the skin of Xenopus. Another frog skin peptide, PGLa, has also been shown to be antibacterial. Magainins are only 23 residues, devoid of cysteine, and form an amphipathic a helix. They act on both grampositive and gram-negative bacteria, fungi, and protozoa (Bevins and Zasloff, 1990). The gene structure for the magainins is not yet known, but cDNA cloning has shown that these peptides are made as large precursor molecules containing six copies interspaced by segments from which a short acidic hexapeptide is liberated (Bevins and Zasloff, 1990). A surprising finding is the diversity of the frog antibiotic peptide family: magainins may be unique to Xenopus. In Bombina, the major antibacterial peptide, bombinin, is hardly related in sequence to magainin (Simmaco et al., 1991). insect immunity and the Cecropins The cecropins were originally identified as highly potent antibacterial peptides in immune hemolymph from the cecropia moth. All cecropins are 31-39 residues, are devoid of cysteine, and have a strongly basic N-terminal region and a long hydrophobic stretch in the C-terminal half. The structure of cecropins deduced from NMR studies is two a helices joined by a hinge region containing Gly-Pro (Holak et al., 1988). Although cecropins were thought to be unique to insects, a cecropin has been found in pig intestine (Lee et al., 1989), which implies that cecropins are widespread in the animal kingdom. In two dipterans, Sarcophaga and Drosophila, the amino acid sequences of cecropins A and IA are identical, although there is only 73% homology at the nucleotide level. Thus, there appears to be strong selective pressure to conserve the cecropin protein sequence in different insects. Insect cecropins are induced by bacterial infection and are synthesized as preproproteins of 62-64 residues. Two IO Human HNP-1 Rabbit NP-1 Rat RatNP-1
ACYCRIPAC VVCACRRALC VTCYCRRTRC
20 IAGERRYGTC LPRERRAGFC GFRERLSGAC
30 IYQGRLWAFC C RIRGRIHPLCCRR GYRGRIYRLC CR
Figure 1. Defensin Sequences from Three Mammals Disulfide bridges link residues 3 and 31, 5 and 20, and
10
and 30.
Cdl 206
10
Xenopus
Bombina Xenopus Figure
Magainin
Bombinin pGLa
2. Antibacterial
*, amidated
i
i-IGKFLHSAK GIGGALLSAA GMASKAGAIA
Peptides
from
Zll
KFGKAFVGEI KVGLKGLAKG GKIAKVALKA
MNs LAEHFN I,"
Two Frogs
C-terminus.
preprocecropins and some truncated analogs have been synthesized by the solid phase method, and these polypeptides were used in processing studies with partially purified enzymes (Boman et al., 1989). The pro part, which contains one or two dipeptides, was removed stepwise by a dipeptidyl aminopeptidase that was found to be highly specific. The C-terminal amide present in insect cecropins (Boman and H&mark, 1987) is presumably derived from a glycine residue that follows the last residue in the mature protein. Many other active peptides are processed in the same way as the cecropins, and a glycine-dependent amidation reaction has been demonstrated for brain peptides. In Cecropia (Xanthopoulos et al., 1988), Sarcophaga (Kania and Natori, 1989) and Drosophila (Kylsten et al., 1990) each cecropin is encoded by a single-copy gene that has a single intron with a conserved splice site. Mechanism of Action Cecropins A and B are highly active against several grampositive and gram-negative bacteria, while the porcine form shows high activity only against gram-negatives. The original observation that cecropins lysed E. coli formed the basis of an assay used to isolate cecropins. Subsequently, cecropins were found to lyse bacteria but not eukaryotic cells. Cecropins will also lyse artificial liposomes composed of phospholipids having zwitterionic or negatively charged head groups. The rate and extent of lysis are dependent on the cecropin concentration. Thus, the cecropins act stoichiometrically and not catalytically. There is every reason to believe that this would also apply to defensins and magainins. While defensins and magainins are about equally potent, cecropins are an order of magnitude more active on a molar basis against gram-negative bacteria (Wade et al., 1990). The primary targets of defensins, magainins, and cecro pins are the inner and outer bacterial membranes. All three groups of peptides have also been shown to form channels in artificial membranes, but it is not yet clear if channel formation is the mechanism by which these peptides kill microorganisms. Both the natural L- and the o-enantiomers of a cecropin, a magainin, and some hybrid peptides form voltage-dependent anion channels in artificial membranes. The D-peptide exhibits a circular dichroism spectrum that is the mirror image of the natural cecropin, which was interpreted
II,
JO
xl
BOIllbYX
A
RW--KIFKKI
Antheraea
B
KW--KIFKKI
EKMGRNIRDG EKVGRNIRNG
Hyalophora
B
KW--KVFKKI
EKMGRNIRNG
Drosophila Porcine
A
GWLKKIGKKI
Pl
SWLSKTAKKL
ERVGQBTRDA ENSAK-KR--
to mean that the o-enantiomer forms a left-handed helix and the natural L-enantiomer forms a right-handed one (Wade et al., 1990). Because the antibacterial activity of both enantio mers is the same, the mechanism of antibiotic activity does not appear to depend on an interaction of the peptide with chiral centers on bacterial membranes. Other Antibacterial Peptides For some time, cecropins and defensins were the only antibacterial peptides known; at that time, they were considered to be unique to that group of animals in which they were discovered. However, the isolation of magainins four years ago was soon followed by the discovery of defensinlike peptides in insects (see Lehrer et al., 1991) and a cecropin in pig intestine (Lee et al., 1989). Simultaneously, other peptides were found in other insects, barley, horseshoe crab, and in bovine tracheal mucosa (Diamond et al., 1991, and references therein). A male-specific peptide protects the male reproductive tract of Drosophila against infections (Samakovlis et al., 1991). Of special structural interest are peptides that are either proline rich or arginine-proline rich (Frank et al., 1990, and references therein). Antibacterial peptides with new properties have also been obtained by making hybrid molecules with one domain from a cecropin and another from the bee venom toxin melittin (Wade et al., 1990). Work on the larger antibacterial proteins from phagocytes has yielded the cDNA sequences for both bactericidalpermeability increasing protein and cathepsin G. Interestingly, two degradation products from cathepsin G, a pentapeptide and a heptapeptide, have antibacterial activity (Bangalore et al., 1990). Taken together, these data strongly indicate that antibacterial peptides are widespread in nature. Since the potential role of antibacterial peptides in immunity is not generally appreciated, a brief explanation of their role in host defense may be justified. In Defense of Peptides Let us first assume that a prime function of any immune system is to offer protection against the natural flora of microorganisms. The defense against pathogens probably differs between fast-growing insects and long-lived mammals with few offspring. When the immune system is impaired by AIDS or immunosuppressive agents, it is the natural flora and opportunistic pathogens that take over. Also, a fixed repertoire of defensive agents makes sense against the natural flora, less so against occasional pathogens. If this is the case, then bacteria should be a prime target of the immune system, both now and during the evolution of immunity. Secondly, the rate of an immune response in any animal must be faster than the rate of multiplication of the invading organism-if not, the animal will not survive. During favorable growth conditions, the generation time of bacteria is
IVKAGPAIEV IIKAGPAVAV IVKA GPAIAV TI-QGLGIAQ -1SEGIAIAI
LG~AKAI LGEAKAL* LGEAKAL* QAANVAATAR* QGGPR
Figure 3. Cecropin Sequences sects and One Mammal *, amidated
C-terminus.
from
Four
In-
Minireview 207
about 50 times faster than that of B cells, which produce immunoglobulins. Finally, the prepro form of an antibacterial peptide is made almost 180 times faster than IgM (assuming a constant rate of peptide bond synthesis). Small peptides also diffuse faster than large defensive cells and antibodies, a property which is also likely to be beneficial when combating an infection. Antibacterial peptides lyse both gram-positive and gram-negative bacteria (and liposomes) but cause little or no harm to eukaryotic cells (defensins and magainins are slightly cytotoxic). Thus, self destruction is largely avoided by membrane specificity, presumably without the recognition of any bacterial proteins (because peptides with only D amino acids are equally active as the natural ones). In general, the survival advantages of antibacterial peptides are their potency (almost like broad-spectrum antibiotics) and their low cost (in terms of time, information, and energy) of production. Thus, they can form an important part of the primary defense against bacterial infections. Evolutionary Aspects Lymphocyte activation is generally accepted to require two signals, as originally proposed by Bretscher and Cohn 20 years ago. It is reasonable to assume that one signal, a “pattern recognition” event (Janeway, 1989), evolved initially, while a second, highly specific signal was added much later, perhaps in response to the increasing complexity of the organism. By several criteria, insect immune systems represent a defense against bacterial infections that arose early in evolution; a single signal, such as a degradation product of murein or free lipopolysaccharide, is sufficient to induce cecropins and other antibacterial proteins in insects. Insects may have diverged from vertebrates before the need for a second signal developed. Immune systems as such may have evolved in concert with the eukaryotic cell, because even the earliest eukaryotes had to compete with bacteria. Since antibacterial peptides probably do not require specialized cells for their synthesis, it is likely that this type of defense predates the separation of vertebrates and invertebrates, which may have occurred some 800 million years ago. Finally, the evolution of immunity within a group of species must be regarded not only in relation to the size of an animal (see Janeway, 1989) but also in relation to its life span and its reproductive rate (Boman and Hultmark, 1987). Small and rather short-lived animals with a high reproductive rate such as Drosophila can afford to lose 90% of the population in an occasional virus infection (and a good virus should not kill its host completely). In contrast, “classical” immune mechanisms (i.e., the major histocompatibility loci and the lymphatic system) are needed in slow-growing populations of large mammals. However, a defense against the natural flora of bacteria is a must for all types of animals; that is why they have antibacterial peptides.
Bangalore, N., Travis, (1990). J. Biol. Chem. Bevins, 41 4.
J., Onunka, V. C., Pohl, J., and Shafer, 265, 13564-13588.
C. L., and Zasloff.
M. (1990).
Annu.
Rev. Biochem.
W. M.
59, 395-
Boman, 126.
H.G.,and
H&mark.
D. (1987). Annu. Rev. Microbial.
47,103-
Boman, H. G., Soman, I. A., Andre& D., Li, Z-q., Merrifield, R. B, Schlenstedt. G., and Zimmermann, R. (1969). J. Biol. Chem. 264, 5652-5860. Diamond, G.. Zasloff. M., Eck, H., Bressaur, M., Malay, W. L., and Bevins, C. L. (1991). Proc. Natl. Acad. Sci. USA 88, in press. Frank, R. W., Gennaro, R., Schneider, K., Przybylski, D. (1990). J. Biol. Chem. 265, 16671-16674.
M., and Romeo,
Holak, T. A., Engstrom, A., Kraulis, P. J., Lindeberg, G., Bennich, H., Jones, T. A., Gronenborn, A. M., andclore, G. M. (1966). Biochemistry 27, 7620-7629. Janeway. 1-13.
C. A. (1969).
Kania. A., and Natori, Kylsten, 224.
Cold
Spring
S. (1969).
P., Samakovlis,
Harbor
FEBS
Symp.
Quant.
Biol. 54,
Lett. 258, 199-202.
C.. and Hultmark,
D. (1990). EMBO J. 9,217-
Lee. J.-Y., Boman, A., Chuanxin, S.. Andersson, M.. Jornvall, H., Mutt, V., and Boman, H. G. (1969). Proc. Natl. Acad. Sci. USA 88, 91599162. Lehrer,
R. I., Ganz,
T.. and Selsted,
Romeo, D., Skerlavaj, B., Bolognesi, Biol. Chem. 283, 9573-9575.
M. E. (1991).
Cell 84, 229-230.
M.. and Gennaro,
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