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Mode of action of diptericin A a bactericidal peptide induced in the hemolymph of Phormia terranovae larvae.

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Archives of Insect Biochemistry and Physiology 10:229- 239(1989)
Mode of Action of Diptericin A, a Bactericidal
Peptide Induced in the Hemolymph of
Phormia terranovae Larvae
Elisabeth Keppi, Anthony P. Pugsley, Jean Lambert, Claude Wicker,
Jean-Luc Dimarcq, Jules A. Hoffmann, and Daniele Hoffmann
Unite' Associe'e au Centre National de la Recherche Scientifique 672, Endocrinologie et
Immunologie des Insectes, Laborafoire de Biologie Ge'ne'rale de I'Universite' Louis Pasteur,
Strasbourg ( E .K., J.L., C .W., 1.-L.D., J.A.H., D. H.) and Unit6 de Gtnttique Mole'culaire,
Institut Pasteur, Paris (E.K., A.P.P.), France
Diptericin A is a member o f a multigenic family of antibacterial peptides that
are synthesized by larvae of fhormia terranovae (Diptera) in response to a bacterial injection o r to injury. The 82-residue peptide is active only against a
limited range o f Gram-negative bacteria. Data presented suggest that the primary action of diptericin A is on the cytoplasmic membrane of growing bacteria.
Key words: insect immunity, antibacterial protein, bacterial cytoplasmic membrane
Larvae of the dipteran insect Phorrnia terranovae synthesize several peptides
with potent antibacterial activity [l]in response to bacterial challenge or injury.
Three of these peptides have recently been isolated, and their amino acid
sequences have been fully or partially determined [2,3]. They show significant sequence homologies and represent members of a family of new inducible antibacterial peptides that we have termed diptericins. They are basic (pl
7.8-8.5) heat-stable molecules with a molecular weight of 8,600 Da and containing high levels of Asx, Pro, and Gly. They are distinct from other inducible antibacterialpeptides isolated from Lepidopterans (lysozymes [4], attacins
[5], and cecropins [6]) and from Dipterans (the cecropin-like sarcotoxins [7];
for reviews, see [8] and [9]).
Received October 29,1988; accepted February 15,1989.
Acknowledgments: We express our gratitude to R. Barker, A. Klier, C. Schnaitrnan, C. Elmerich,
W. Lubitz, H.G. Bornan, C. Campelli, Y. Piernont, J.Millet, and L. Le Minor who kindly supplied the bacterial strains used in this study. We are indebted to R. Klock, A. Meunier, and C.
Heyer for skillful technical assistance. We also wish to acknowledge the many helpful discussions with Prof. J. Fothergill (University of Aberdeen).
Address reprint requests to Dr. D. Hoffrnann, Laboratoire de Biologie Generale-UA CNRS 672-12,
rue de I'Universite 67000 Strasbourg, France.
0 1989 Alan R. Liss, Inc.
Keppi et al.
Preliminary data on the mode of action of diptericins against the Gramnegative bacterium Escherichia coli [3] indicated that they are probably
bacteriolytic and active only on growing cells. The present paper provides a
more detailed analysis of the antibacterial spectrum of diptericins and of their
mode of action.
Preparation of Semipure Diptericin A (SP-Diptericin A)
Diptericin A was semipurified from the hemolymph of immunized larvae
of Phorrnia terranovae by heat treatment, cation exchange chromatography and
gel permeation as previously described [3]. A HPLC profile of an aliquot of
SP*-diptericin showed that it consisted of 20% pure diptericin and contained
no other antibacterialpeptide. The concentration of pure diptericin A was estimated to be roughly equal to 1pg per ml of immune hemolymph.
Bacterial Strains and Media
The strains used for the study of the antibacterial spectrum were obtained
from several laboratories (see Text and "Acknowledgments"). The mode of
action of diptericin A was primarily investigated on E. coli K12 strains BZBlOll
(gyrA) and D31 (a mutant with a defective lipopolysaccharide). DAP uptake
assays and peptidoglycan degradation assays were performed with E. coli K12
strain W7 (dap A, Lys).
Bacteria were grown in L broth or minimal medium M63 containing 0.2%
glucose as described by Miller [lo]. Xenorhabdus nernatophizus was grown at 30°C
in 0.4% Oxoid bacteriological peptone containing 0.5% NaCl and 0.4% glucose (pH 7.4).
Antibacterial Spectrum
Assay conditions were essentially those described by Pugsley and Oudega
[ll]. Soft agar (3 ml) was seeded with approximately lo6 indicator cells from
an exponential phase culture and poured onto the surface of normal-strength
nutrient agar in Petri dishes. Samples of SP-diptericin A (5 p1 containing the
equivalent of approximately 1.5 pg of pure diptericin A) were deposited onto
the indicator lawn. The plates were incubated for 8 h at 37"C, and the diameters of the clear zones were recorded.
P-Galactosidase Assay
E. coli D31 cells were grown to an absorbance of 0.1 at 600 nm in L broth
medium containing 1mM IPTG (Sigma, St. Louis). SP-diptericinA was added
at a final concentration equivalent to 3 pg/ml of pure substance, and samples
(200 p1) were removed periodically and centrifuged at 3,500g for 5 min. The
culture supernatants were kept on ice until further use. The pellets were resuspended in 200 p1 of L broth, and the cells were sonicated to release
P-galactosidase. P-galactosidase in the supernatants and in the lysed cells was
assayed according to Miller [101.
*Abbreviations used: DAP = Diaminopimelic acid; 3H-DAP = (DL + meso) - 2,6-diamino[3,4,5-3H]
pimelicacid; IPTC = isopropyl p-D-thiogalactopyranoside;LPS = lipopolysaccharide;
SDS = sodium dodecyl sulfate; SP = semipurified.
Mode of Action of Diptericin A
Degradation of 3H-DAP-LabeledPeptidoglycan
Exponential phase E . coli W7 cells (5 x lo7) were prelabeled with 20 pl of
3H-DAP(36 Ci/mmol; 1mCi/ml) (CEA, Paris) and then grown in L broth containing unlabeled DAP (20 pg/ml) and SP-diptericinA at a final concentration equivalent to 24 pg/ml of pure substance. Samples (100 p1) were removed periodically, mixed with hot 10% SDS, and further heated at 100°Cfor 10 min. SDSinsoluble material was collected by filtration through Millipore membrane filters
(HAWP, 0.45 pm pore diameter), washed with fresh medium, air-dried, and
the radioactivity on the filter was determined by liquid scintillation counting.
Measurement of Lysine and DAP Uptake
Exponential-phase E. coli BZB 1011were suspended at a density of 5 x lo7
cells/ml in minimal M63 glucose medium at 37"C, and SP-diptericin A was
added at a final concentration equivalent to 15 pg/ml of pure substance. Samples (400 pl) were removed at intervals and incubated at 37°C with 25 pg/ml
chloramphenicol and 0.3 pCi of [14C]-lysine(336 mCi/mmol; Amersham) at a
final concentration of 2 pM- Aliquots (100 pl) were removed at intervals and
filtered through Millipore membrane filters. The radioactivity in the cells was
measured on the filter by liquid scintillation counting. In a parallel experiment, the bacteria were killed by adding toluene, which permeabilized the
bacterial envelope.
The procedure used to measure 3H-DAPuptake was essentially as described
above except that strain W7 was used.
Antibacterial Spectrum of Diptericin A
The activity of SP diptericin A was tested against a battery of Gram-negative
and Gram-positive bacteria. Among the Gram-negative strains, E . coli K12,
Erwinia herbicola T, E . carotovora 113, Shigella dispar P15, Klebsiella pneumoniae
UNF 5023, and Xenorhabdus nematophilus Xn21 all exhibited varying degrees of
sensitivity. All other Gram-negative bacteria tested (two strains each of E . coli,
Salmonella fyphimurium, and Enterobacter cloacae, and one each of Salmonella wien,
S.enferitidis, S.derby, Serratia marcescens, Citrobacterfreundii, Aeromonas hydrophila,
Pseudomonas aeruginosa, and Acinetobacter calcoaceticus) were fully resistant to
diptericin A. Other than for the specific case of E. coli K 12 discussed below,
there was no obvious feature (presence of capsule or long oligosaccharideson
outer membrane lipopolysaccharides) that distinguished between diptericin
A-resistant and -sensitive strains. Likewise, all Gram-positive bacteria tested
(five strains of Bacillus subtilis, three strains of B. thuringiensis and one each of
B. megaterium, Staphylococcus aureus, and Micrococcus luteus), were also fully
diptericin A-resistant.
E. coli K12 strain D31 is routinely used by us 111and by others [12] to study
the action of antibacterial agents of insect origin because of its high level of
sensitivity. This strain carries a mutation, which makes it highly sensitive to
other agents that cannot normally cross the E . coli outer membrane or that do
so only inefficiently [13]. This phenotype is often caused by the production of
truncated outer membrane LPS core sugars and consequent insertion of phos-
Keppi et al.
pholipids into the outer leaflet of the outer membrane. We therefore tested
two pairs of isogenic E . coli K12 strains carrying, respectively, transposon TnlO
insertions in rfu genes involved in LPS core oligosaccharide biosynthesis and
a A prophage carrying the wild-type allele (strains CS1716WCS1717X and
CS1716/CS1717, respectively; strains generously supplied by C. Schnaitman).
All four strains were sensitive to diptericin A, but CS1716X and CS1717X (lacking the A prophage) exhibited considerably greater sensitivity than the corresponding strains with the A prophage (i.e., rfa+). The same strains were also
more sensitive to the detergents sodium desoxycholate and SDS, to EDTA,
and to the antibiotics rifampicin and chloramphenicol. This result confirms
that the outer membrane is a barrier to the penetration of diptericin A.
No mutants of E. coli K12 survived diptericin A treatment either on plates
or in liquid culture, and a battery of colicin and bacteriophage-resistantmutants
of E . coli K12 [ l l ] were all diptericin sensitive. It therefore seems unlikely that
diptericin uses any of the known outer membrane transport systems to penetrate the cell envelope.
Diptericin A Causes Lysis of E. coli K12
SP-diptericin A was added at concentrations ranging from 0.75 pg/ml to 24
pg/ml to growing cells of E. coli strain D31, and the effects were monitored by
measuring culture absorbance at 600 nm. As shown in Figure 1, growth was
affected by concentrations as low as 0.75 pg/ml, and the cells lysed markedly
at a concentration of 6 pg/ml. Interestingly, the lowest active concentrations of
diptericin in these tests are inferior to the actual concentration of diptericin in
Time (h)
Fig. 1. Dose-dependent effect of diptericin Aon the absorbance of E. coli D31cultures: 5 X 10’
bacteridml in exponential growth phase were incubated in the presence of SP-diptericin A at
the concentrations of 0.75 (I), 1.5 (2), 3 (3), 6 (4),12 (5), and 24 pg/ml (6). The absorbance of
the cultures at 600 nm was measured at different time intervals and compared to that of a control culture (C)where diptericin A was replaced by an equivalent volume of distilled water.
Mode of Action of Diptericin A
the immune hemolymph (estimated to be 1 pg of pure diptericin per ml).
Increasing concentrations of diptericin A had a stronger effect; not only did
the culture absorbance decrease more dramatically, but the lag period between
the addition of diptericin A and the decrease of absorbance was also reduced.
These observations indicate that diptericin A causes lysis of E . coZi cells. Viability (ability to form colonies on L broth agar) declined after 30 min of treatment [3]. The addition of SP-diptericin A to €. coli also caused a sharp decrease
in the level of cytoplasmic P-galactosidaseafter 60 min, coincident with a sharp
drop in culture absorbance (Fig. 2).
These results indicate that diptericin A causes complete lysis of E . coli K12.
To see whether lysis was caused by peptidoglycan breakdown, cells of E . coli
strain W7 were prelabeled with 3H-DAP,which is incorporated exclusively in
peptidoglycan. Subsequent addition of diptericin A caused the release of the
label as hot SDS-soluble material after 2 h (Fig. 3).
While this result confirms that peptidoglycan breakdown does occur, it does so
only 30-60 min after the first detectable signs of lysis, as indicated by the release
of P-galactosidase and the decline in culture absorbance. To see whether there
were any detectableeffects on membrane function prior to this, we measured the
rates of 3H-DAP and [14C]-lysineuptake in treated and control cells. Accumulation of [14C]-Lysineby the strain BZBlOll was almost immediately reduced upon
addition of diptericin A (Fig. 4). Similar results were obtained with the strain W7
for the accumulation of 3H-DAP (data not shown). Thus, inhibition of active
transport is the earliest detectable effect of diptericin A, preceding by 30 min
any effect on viability and by 60 min any detectable signs of lysis.
Time (h)
Fig. 2. Diptericin A-induced release of P-galactosidase from F. coli. E. coli D31 was grown in L broth
supplemented with 1 mM IPTG to induce p-galactosidase synthesis; 3 p@nl SP-diptericin A was
added to the culture, and 200 JLI
aliquots were removed at time intervals and assayed for P-galactosidase activity. The figure represents the cell-associated activity expressed as percentage of the total
activity present in the culture (cellsand medium). It also shows the evolution of the cell culture
(increase up to 1 h, followed by a marked decrease) monitored by absorbance measurements.
Keppi et at.
Time (h)
Fig. 3. Effect of diptericin A on 3H-DAP-labeledpeptidoglycan in E. coli. Cells of E. coli W7
prelabeled with 3H-DAPwere grown to an absorbance of 0.1 at 600 nm in L broth containing 20
pg/ml of DAP SP-diptericinA (24 pg/ml) was added to the culture at time0 (-.-). A parallel experiment was run in which 20 m M MgS04were added together with diptericin A to the bacteria
Samples (100 pl) were removed at hires indicated and processed as described in “Materials
and Methods.” The cell-associated radioactivity is expressed as a percentageof the radioactivity in the control without diptericin A.
Mg+ Reduces the Lytic Effect of Diptericin A
The above results suggested that lysis was not the primary cause of death
of the diptericin A-treated cells but occurred as a consequence of an earlier,
less dramatic perturbation of membrane functions. We have previously observed
that M g + + can reduce or eliminate lysis in such a situation [15]. We therefore compared the effects of diptericin A in the presence or absence of varying
concentrations of MgS04. Figure 5 shows that the addition of MgS04 to cultures of E. coli D31 reduced the lytic effect of diptericin A. This reduction was
observed with concentrations of Mg++ ranging from 5 to 20 mM. The lytic
effect of diptericin A was completely abolished when the concentration of
Mg+ was higher than 20 mM. Mg+ did not prevent killing by diptericin,
as determined by the plate count assay, nor did it affect the action of diptericin
on amino acid uptake. The way in which Mg++ exerts its effect remains
unkown, but it presumably stabilizes the outer membrane rather than acting
as an osmoprotectant.
Potentiation of the Lytic Effect of Diptericin A by Triton X-100
When E. coli cells were pretreated with the equivalent of 0.6 pg/ml pure
diptericin A, they became sensitized to 0.1% Triton X-100. The treated cells
lysed more rapidly and more extensively upon the addition of the detergent
than in its absence. When the detergent was added 15min after the diptericin,
there was a considerable delay before lysis occurred, whereas there was no
delay when Triton was added 45-60 min after the diptericin. Detergent alone
Mode of Action of Diptericin A
- 0
Time (min)
Fig. 4. Effects of diptericin A on [14C]-lysinetransport in E. coli. E. coli BZB 1011 was grown in
M63 medium containing 0.4% glucose to an absorbance of 0.1 at 600 nm. SP-diptericin A (15
pg/ml) was added, and 400 pI samples were removed at times indicated, supplemented with
chloramphenicol (25 pg/ml) and 2 pM [14C]-lysine(0.3 pCi/sample). After 1 min, the samples
were filtered through nitrocellulose filters, which were dried and counted. Hatched columns:
control culture without diptericin: dotted columns: diptericin treated culture; open columns:
killed bacteria.
did not cause lysis. This result indicates that diptericin and Triton X-100 act
synergistically (Fig. 6).
Diptericin A is an antibacterial peptide that is effective only against a limited range of Gram-negative bacteria. Gram-positive bacteria and eukaryotic
cells (e.g., sheep red blood cells) are not affected by diptericin A. Mutants of
E. coZi with modified LPS exhibit the highest sensitivity, while two "smooth"
strains with long LPS oligosaccharide side chains (E. coZi serotypes 06 and 010)
were completely resistant, possibly because diptericin A could not penetrate
the cell surface (see [14]). These data indicate that diptericin A alone would
not protect insect larvae against general microbial attack. However, Phormia larvae produce other agents that might act together with diptericin A to provide
fuller protection. it is interesting to note that other antibacterial agents isolated from the hemolymph of immunized P. terranova larvae have different activity spectra and that one of them is related to cecropin [3,16].
Figure 7 shows the kinetics of the various effects observed in this study.
Amino acid transport is affected within a very short time. The viability decreases
after 30 min (the effects of diptericin A on membrane functions are presumably reversible up to this time), and lysis and the release of cytoplasmic
P-galactosidase follow after a further 30 min. Eventually, the cell wall
Keppi et al.
Time (h)
Fig. 5. Effect of diptericin A on €. coli in the presence of varying concentrations of M g + + .
E. coli D31 in exponential growth phase was incubated with SP-diptericin A (3 @ml) in L broth
medium at 37°C; the following concentrations of MgS04were added: 2.5 m M (1); 5 m M (2);
10 m M (3); 20 m M (4); diptericin Alone (A); control (C); incubationwith 20 m M MgS04(C').
disintegrates as indicated by the release of DAP. Diptericin A therefore
appears to cause a succession of events resulting most probably from a
primary effect on the functioning of the cytoplasmic membrane. This would
be consistent with the synergistic effects of diptericin A and Triton X-100.
Previous studies showed that Triton X-100 potentiated the effect of another
antimicrobial agent, microcin E492, which also seems to affect the E . coli
cytoplasmic membrane 1171.
Among the antibacterial peptides isolated so far from insects, some information on the mode of action is available for cecropins and the cecropin-like
sarcotoxins I, two groups of related molecules isolated, respectively, from
Hyulophoru cecropia [6] and Surcophugu peregrinu [18,19], the latter species being
relatively close to P. terrunovue. Cecropins and sarcotoxins act on both Grampositive and Gram-negative bacteria, which is in contrast to the narrow activity spectrum of diptericin A. Moreover, diptericin A acts only on growing
bacteria [3], which is not the case for cecropins or sarcotoxins I.
Studies with cecropin analogues indicate the importance of specific residues in the molecule [20]. The bactericidal effect of the cecropin-like sarcotoxin
Mode of Action of Diptericin A
Time (h)
Fig. 6. Effect of Triton X-I00 o n the absorbance of E. coli in the presence of diptericin A: 1.5
pg of SP-diptericin A was added to 500 pl of an exponentially growing culture of E. coli BZB
1011 containing 5 x lo7 bacteria. Triton X-100 at a final concentration of 0.1%was added to the
culture after either 15,35,45, or 60 min, and the absorbance at 600 nm was recorded. C, C’:
control in the presence or in the absence of Triton; A: diptericin A alone.
I is probably due to its ionophore activity and to a block of ATP generation
caused by the dissipation of the proton gradient [18,21]. The bacteriostatic
properties of the attacins [22] isolated from immune hemolymph of H.cecropia
rule out a possible analogy with the mode of action of diptericin A,
initially bacteriostatic but becomes bactericidal after 30 min. Likewise, a similarity with lysozyme, which has been isolated from many insect species, was
ruled out by the specific action of this enzyme, which hydrolyses p-(1-4)glycosidicbonds between N-acetylglucosamine and N-acetyl-muramicacid units
of the bacterial wall. Diptericin A does not exhibit this activity (not shown).
Thus, we conclude that diptericin A probably acts at the level of the cytoplasmic membrane of susceptible bacteria, leading eventually to death and
cell lysis. Interaction with a component involved in active amino acid uptake
would be consistent with the absence of action of diptericin A on resting cells.
The effects of the diptericin appear to be reversible during the first 30 min,
implying that diptericin is diluted out when the cells are plated out. We note
that high concentrations of diptericin A (relative to other characterized antibacterial agents of insect origin) are required to kill even the highly sensitive
Keppi et at.
Time (min)
Fig. 7.
Kinetics of the effects of diptericin A o n f. coli cultures.
E. coIi K12 strains such as D31. Other antibacterial agents with similar models
of action (e.g., colicin A, E l and I [23], magainins [24], cecropins [25], and the
cecropin-like sarcotoxins [191have either hydrophobic a-helices or amphiphilic
helices in their interactive domains. These helices are proposed to span the
cytoplasmic membrane and in some cases are known to form channels through
which ions diffuse across the membrane. Structure predictions for diptericin
A, the primary sequence of which has been determined [3], show the complete absence of segments of significant hydrophobicity. Helix-breaking proline residues are present along the entire length of the molecule, making it
unlikely that any part of it could span the cytoplasmic membrane as an a-helix
(data not shown). Thus, the mode of action of diptericin A may be different
from that of any previously characterized, membrane-active antibacterial agent.
1. Keppi E, Zachary D, Robertson M, Hoffmann D and Hoffmann J: Induced antibacterial proteins in the hemolymph of Pkormia terranovae (Diptera). Insect Biochem 26,395 (1986).
2. Keppi E, Dimarcq JL, Lambert J, Zachary D, Reichhart JM, Hoffmann D, Keller R, Hoffmann
J: Recherches sur les mecanismes de defense antibacterienne chez les Insectes: Isolement
de peptides antibacteriens dans I'hemolymphe du diptPre Pkormia terranovae. CR Acad Sci
(Paris) ser. 111303. 155 (1986).
3. Dimarcq JL, Keppi E, Dunbar B, Lambert J, Reichhart JM, Hoffmann D, Rankine SM, Fothergd
JE, Hoffmann JA: Insect immunity. Purification and characterization of a family of novel
inducible antibacterial proteins from immunized larvae of the dipteran Pkormia terranovae
Mode of Action of Diptericin A
and complete amino-acid sequence of the predominant member, diptericin A. Eur J Biochem
171,17 (1988).
4. Engstrom A, Xanthopoulos KG, Boman HG, Bennich H: Amino acid cDNA sequences of
lysozyme from Hyalophora cecropia. EMBO J 4,2119 (1985).
5. Hultmark D, Engstrom A, Andersson K, Steiner H, Bennich H, Boman HG: Insect immunity. Attacins, a family of antibacterial proteins fromHyaZophoracecropia.EMBO J 2,571 (1983).
6. Hultmark D. Steiner H, Rasmuson T, Boman HG: Insect immunity. Purification and properties of three inducible bactericidal proteins from hemolymph of immunized pupae of
Hyalophora cecropia. Eur J Biochem 106,7 (1980).
7. Okada M, Natori S Purification and characterizationof a bactericidal protein from hemolymph
of Sarcophagu peregrinu (flesh-fly)larvae. Biochem J 211, 727 (1983).
8. Dunn PE: Biochemical aspects of insect immunology. Annu Rev Entomol31,321(1986).
9. Boman HG, Hultmark D: Cell-free immunity in insects. Annu Rev Microbiol41, 103 (1987).
10. Miller JH: Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Cold Spring
Harbor, NY, (1972).
11. Pugsley AP, Oudega B: Methods for studying colicins and their plasmids. In: Plasmids, Practical approach. Hardy K, ed. IRL Press, Oxford, UK, p 105 (1987).
12. Boman HG, Steiner H: Humoral immunity in Cecropia pupae. Curr Top Microbiol Immunol
94-95,75 (1981).
13. Boman HG, Jonsson S, Monner D, Nomark S, Bloom GD: Cell-surface alterations in Escherichia coli K-12 with chromosomal mutations changing ampicillin resistance. Ann NY Acad
Sci 182, 342 (1971).
14. van der Ley P, de Graaf P, Tommassen J: Shielding of Escherichiu coli outer membrane proteins as receptors for bacteriophages and colicins by 0-antigenic chains of lipopolysaccharide. J Bacterioll668,449 (1986).
15. Pugsley AP, Schwartz M: Colicin E2 release: Lysis, leakage or secretion? Possible role of a
phospholipase. EMBO J 3,2393 (1984).
16. Lambert J, Keppi E, Dimarcq JL, Wicker C, Reichhart JM, Dunbar B, Lepage P, Van Dorsselaer
A, Hoffmann J, Fothergill J, Hoffmann D: Insect immunity: Isolation from immune blood of
the Dipteran Phormia terranovae of two insect antibacterial peptides with sequence homology to rabbit lung macrophage bactericidal peptides. Proc Natl Acad Sci USA 86,262 (1989).
17. de Lorenzo V, Pugsley AP: Microcin E492, a low molecular-weight peptide antibiotic which
causes depolarization of the Escherichiu coli cytoplasmic membrane. Antimicrob Agents
Chemother 27,666 (1985).
18. Okada M, Natori S: Mode of action of a bactericidal protein induced in the hemolymph of
Sarcophaga peregrinu flesh-fly) larvae. Biochem J 222,119 (1984).
19. Okada M, Natori S: Primary sturcture of sarcotoxin I, an antibacterial protein induced in
hemolymph of Sarcophugu peregrinu. Biochem J 260,7174 (1985).
20. Andreu D, Merrifield RB, Steiner H, Boman HG: N-terminal analogues of cecropin A: Synthesis, antibacterial activity, and conformational properties. Biochemistry 24,1683 (1985).
21. Okada M, Natori S: Ionophore activity of a sarcotoxin I, a bactericidal protein of Sarcophaga
peregrinu. Biochem J 229,453 (1985).
22. Engstrom P, Carlsson A, Engstrom A, Tao ZJ, Bennich H: The antibacterial effect of attacins
from the silk moth Hyalophoru cecropia is directed against the outer membrane of Escherichiu
coli. EMBO J 3,3347 (1984).
23. Pattus F, Hertz F, Martinez C, Provencher SW, Lazdunski C: Secondary structure of the poreforming colicin A and the C-terminal fragment. Experimental fact and structure prediction.
Eur J Biochem 152,681 (1985).
24. Zazloff M: Magainins, a class of antimicrobial peptides from Xenopus skin: Isolation, characterization of two active forms and partial cDNA sequence of a precursor. Proc Natl Acad
Sci USA 84, 5449 (1987).
25. Steiner H: Secondary structure of cecropins: antibacterial peptides from the moth Hyalophoru
cecropia. FEBS Lett 137,283 (1982).
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