(Received for publication, May 5, 1995; and in revised form, August 11, 1995)
From the
A novel antibacterial peptide that shows antibacterial activity
against Staphylococcus aureus was isolated from the hemolymph
of the silkworm, Bombyx mori. The novel peptide consisted of
42 amino acids and was highly basic. This peptide indicated no
significant similarity with other antibacterial peptides. The peptide
showed antibacterial activity against several Gram-negative and
-positive bacteria and had a higher activity against Gram-positive
bacteria than cecropin B, a major antibacterial peptide of B. mori. The novel peptide was inducible by bacterial
injection. These results suggest that the peptide is responsible for
the antibacterial activity in B. mori against Gram-positive
bacteria. The effects of the peptide on bacterial and liposomal
membranes showed that a target of the peptide is the bacterial
cytoplasmic membrane. The results also suggest that the N-terminal
portion of the peptide, containing a predicted
-helix, is
responsible for an increase in the membrane permeability. We propose
the name ``moricin'' for this novel antibacterial peptide
isolated from B. mori.
Although insects do not have immune systems that involve antigen-antibody reactions, they do have efficient self-defense mechanisms against bacterial infection. For example, they can induce antibacterial proteins upon bacterial infection (Boman and Hultmark, 1987). To date, many antibacterial proteins have been isolated from different species of insects (Cociancich et al., 1994a) and can be classified into five major groups (Hultmark, 1993): cecropins, insect defensins, attacin-like (glycine-rich) proteins, proline-rich peptides, and lysozymes. Cecropins and insect defensins belong to a small group of antibacterial proteins showing high antibacterial activity and rapid bactericidal effects. The antibacterial mechanisms of cecropins and insect defensins have also been studied (Okada and Natori, 1984, 1985; Christensen et al., 1988; Matsuyama and Natori, 1990; Cociancich et al., 1993; Yamada and Natori, 1994).
Cecropins are thought to be primarily responsible for the antibacterial activity in some insects since they show antibacterial activity against many kinds of Gram-negative and -positive bacteria. Although cecropins have been isolated from several species of lepidopteran and dipteran insects (Cociancich et al., 1994a), they have not been found in other orders of insects. This suggests that cecropins are not general antibacterial proteins in insects.
Insect defensins are highly effective against Gram-positive bacteria, including human pathogenic bacteria such as Staphylococcus aureus, whereas they do not exhibit strong activity against Gram-negative bacteria. Contrary to cecropins, insect defensins are more common in insects and have been isolated from several orders of insects such as dipteran, hymenopteran, coleopteran, trichopteran, hemipteran, and odonata (Hoffmann and Hetru, 1992; Cociancich et al., 1994b). However, insect defensins have yet to be observed in lepidopteran insects. Except for the insect defensins, all types of antibacterial proteins have been reported in lepidopteran insects (Boman et al., 1991; Hara and Yamakawa, 1995). All five antibacterial protein groups have been isolated from dipteran insects (Hultmark, 1993). Considering that all cecropins are not effective against some genera of Gram-positive bacteria, such as Staphylococcus and Bacillus (Hultmark et al., 1982; Qu et al., 1982; Teshima et al., 1987; Tu et al., 1989; Boman et al., 1989), we hypothesize that there is an as yet unidentified antibacterial protein(s) in lepidopteran that shows a strong activity against Gram-positive bacteria.
Previously, we investigated the
antibacterial activity of the hemolymph from Bombyx mori against Gram-negative bacteria (Yamakawa et al., 1990;
Hara et al., 1994; Hara and Yamakawa, 1995). In the present
work, we surveyed the antibacterial activity of the hemolymph against
one of Gram-positive bacteria, S. aureus, and isolated a novel
antibacterial peptide. The peptide showed antibacterial activity
against both Gram-negative and -positive bacteria. This peptide
indicated a higher activity against Gram-positive bacteria than
cecropin B, a major antibacterial peptide of B.
mori. Our results also suggested that a target of the peptide was
the bacterial cytoplasmic membrane. Moreover, the N-terminal half of
the peptide, containing a predicted
-helix, was shown to be
responsible for the increase in membrane permeability.
Next, a question if the activity against S. aureus derived from cecropins, the major antibacterial peptide in B. mori, was examined. Antibacterial substances from immunized hemolymph with E. coli and from non-immunized control hemolymph were fractionated by cation exchange column chromatography and subsequently by reverse-phase HPLC. The activity against S. aureus of each HPLC fraction was assayed. Fractions from the immunized hemolymph showed two peaks indicating antibacterial activity (Fig. 1a). The two peaks were not found in the fractions from non-immunized hemolymph, suggesting they are induced by bacterial infection (Fig. 1b). The first antibacterial peak (41 min) contained cecropin B as described below. The second peak (44 min) contained a previously unreported antibacterial substance(s).
Figure 1:
Antibacterial activity against S.
aureus in the fractions from the hemolymph of B. mori. 1
ml of the hemolymph from the immunized larvae with E. coli (a) or the hemolymph from normal larvae (b) was
diluted with 10 ml of buffer A/buffer B (9:1) and loaded onto a column
of CM-Sepharose FF (1 0.3 cm) previously equilibrated with
buffer A/buffer B (9:1). After washing the column with 2 ml of the
buffer mixture, substances were eluted with 1 ml of buffer B. A half of
the eluate was applied to a Capcell Pak C8 SG300 column (4.6
250 mm), and substances were eluted for about 60 min with a linear
gradient of 12-60% acetonitrile (dotted line) containing
0.1% trifluoroacetic acid. Absorbance at 220 nm was monitored (solid line). Fractions of 0.5 ml were collected, lyophilized,
and dissolved in 20 µl of distilled water. A half of the solution
was poured into wells (4 mm in diameter) on a nutrient medium plate
containing an additional 150 mM NaCl. Diameters of inhibition
zones in the assay plate for antibacterial activity were measured after
incubating the plate at 37 °C overnight (indicated with bars). No activity was observed in the fractions from the
hemolymph of non-immunized larvae. An arrow shows the as yet
unidentified peak.
The peak fractions corresponding to the induced antibacterial activity (44 min) in the analytical HPLC were pooled (Fig. 1a). The pooled sample was again subjected to reverse-phase HPLC, and the peak fraction showing antibacterial activity was collected. A typical HPLC elution pattern is shown in Fig. 2. The unidentified substance showed a single stained band in SDS-polyacrylamide gel electrophoresis for low molecular weight proteins (Schägger and von Jagow, 1987), suggesting the presence of a peptide (data not shown). After the final purification step, about 150 µg of the antibacterial peptide was obtained.
Figure 2: Final purification of a novel antibacterial peptide by reverse-phase HPLC. Experimental details were as described under ``Materials and Methods.'' The peak containing induced antibacterial activity is indicated with an arrow.
In the first HPLC, the fractions containing
antibacterial samples (41 min) were also collected and pooled (Fig. 1a). Three types of cecropin B (Teshima et
al., 1986; Morishima et al., 1990) were purified by
further HPLC. Namely, the amino acid sequences determined using a
protein sequencer revealed that they were cecropin B (Lys-21 was hydroxylated), B
(no Lys was
hydroxylated), and a molecule having two hydroxylated Lys residues
(Lys-21 and Lys-33), respectively (data not shown). We designated the
third molecule as ``cecropin B
.''
A sequence of 33 amino acid residues was initially obtained by the Edman degradation method (Fig. 3). To obtain additional sequence information, the peptide was digested with an endoproteinase Asp-N, which cleaves the peptide bond on the N-terminal side of Asp residue (Gabriel, 1980). The resultant fragments were purified by reverse-phase HPLC (the fragments were designated as D1 and D2 based on their elution), and their sequences were determined (Fig. 3). The combined sequences showed that the peptide consists of 42 amino acid residues as shown in Fig. 3. The amino acid composition calculated from the sequence was agreed well with the analyzed composition (Table 1).
Figure 3: Amino acid sequence of the peptide. Amino acid sequences were determined on an Applied Biosystems model 473A protein sequencer. Fragments D1 and D2 were obtained by digesting the peptide with endoproteinase Asp-N. Positively and negatively charged amino acids are indicated with + and -, respectively.
For further confirmation, the molecular mass of the peptide was measured by ion spray mass spectrometry on a Sciex model API-III triple quadrupole spectrometer. The obtained value, 4543.1 ± 0.6 Da, was coincident with the calculated value, 4543.5 Da, assuming that the C terminus of the peptide is unmodified.
The C termini of cecropins are generally amidated (Boman and Hultmark, 1987; Hara et al., 1994). Contrary to cecropins, the analysis of the peptide by mass spectrometry suggested that the C terminus was unmodified. To confirm the C terminus is unmodified, the peptide was digested with carboxypeptidase A, which can release amino acids from unmodified C termini (Neurath, 1960). The carboxypeptidase A did cleave the peptide, suggesting that the C terminus of the peptide is unmodified (data not shown).
Since S. aureus did not grow when more than 2 µg/ml (0.44 µM) of the peptide was present in a liquid medium of brain heart infusion, the time course of the viability of S. aureus cells incubated with 3 µg/ml of the peptide was examined. The results demonstrated that the reduction of the viability of S. aureus cells occurred within a few min after addition of the peptide (Table 3), suggesting that the peptide has strong bactericidal activity against S. aureus.
Figure 4: Effect of the novel peptide and its proteolytic fragments on the permeability of liposomal membrane. S. aureus type of liposome with entrapped glucose was prepared (see ``Materials and Methods'') and incubated with the indicated concentration of the peptides and proteolytic fragments. After the incubation, the amount of glucose released from the liposome was measured. The amount of glucose released by 1% Triton X-100 was defined as 100%. The peptide samples used were the novel peptide (closed circles), fragment D1 (open circles), and fragment D2 (closed squares).
We isolated a novel type of antibacterial peptide from the hemolymph of B. mori by monitoring its antibacterial activity against S. aureus. We propose the name ``moricin'' for this novel peptide.
A search of the Protein Identification
Resource data base yielded no peptides or proteins with significant
similarity to moricin. Like other antibacterial peptides, moricin is
highly basic, and the higher basicity tends to correlate with the
higher antibacterial activities (Fink et al., 1989; Bevins and
Zasloff, 1990; Hoffmann and Hetru, 1992; Cociancich et al.,
1994a; Kagan et al., 1994; Gabay, 1994; Iwanaga et
al., 1994). The basicity is thought to be responsible for the
attachment of positively charged peptides to the negatively charged
bacterial surface through the electrostatic interaction (Christensen et al., 1988; Gabay, 1994). The value of isoelectric point of
moricin was calculated to be 12.0, higher than those of cecropins (pI
= 8.2-9.6). Second, moricin has a predicted amphipathic
-helix. Generally,
-helical structures are responsible for
the expression of antibacterial activity. For example, NMR analysis
indicated that cecropins consist of two amphipathic
-helixes
(Holak et al., 1988; Iwai et al., 1993). Recently,
the
-helical region of an insect defensin was found to be
responsible for both antibacterial activity and the increase of
permeability of liposomal membrane (Yamada and Natori, 1994). In the
N-terminal half of moricin, charged amino acids appear at intervals of
three or four amino acid residues, indicating a characteristic
structure in antibacterial proteins containing the amphipathic
-helix (Cociancich et al., 1994a; Kreil, 1994). The
-helical wheel projection (Schiffer and Edmundson, 1967) of amino
acid
residues(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) of
moricin resulted in a clear separation of hydrophobic and hydrophilic
faces (Fig. 5), suggesting the existence of an amphipathic
-helix in this region.
Figure 5: Helical wheel projection of moricin amino acid residues. Helical wheel projection of moricin residues was analyzed by the method of Schiffer and Edmundson(1967). Positively charged amino acids are indicated by +.
The results indicated that a target of
moricin is the bacterial membrane. The N-terminal fragment (fragment
D2, 29 amino acid residues) seems to be partially responsible for the
increase of the membrane permeability because the fragment itself
increased the permeability of liposomal membrane (Fig. 4).
Hence, the active center of moricin may be in the predicted
-helical region (residues 5-22). On the other hand, it is
characteristic that basic amino acid residues cluster in the C-terminal
region. Although the effects of the two fragments on the viability of S. aureus and on the permeability of cytoplasmic membrane
require further investigation, the results suggest that the basic
C-terminal region interact with the surface of bacterial membrane and
then changes the permeability of the membrane by the N-terminal
amphipathic
-helix.
Since moricin is induced by bacterial injection and shows a strong antibacterial activity against bacteria, we assume that moricin plays an important role in the self-defense against bacterial infection in B. mori. Our results suggest that the main target of moricin is bacteria, because moricin showed only slight antifungal activity against some strains of yeast and no hemolytic activity against murine erythrocyte (data not shown). Although both moricin and cecropins show antibacterial activity against several species of bacteria, moricin tends to have higher activity against Gram-positive bacteria. On the contrary, cecropins have higher activity against Gram-negative bacteria. This suggests that moricin and cecropins are simultaneously induced upon bacterial infection, and they can efficiently eliminate the wide variety of invading bacterial species. Furthermore, the existence of moricin in B. mori may be a reason why the defensin-type antibacterial peptides are missing in lepidopteran insects. To confirm this hypothesis, the presence of moricin-type antibacterial peptides in other lepidopteran insects should be investigated.
Interestingly, moricin has no modified amino
acid residues such as the -amidation of C termini in cecropins,
the hydroxylation of Lys residues in B. mori cecropins, the O-glycosylation of Thr residues in proline-rich antibacterial
peptides (Bulet et al., 1993; Cociancich et al.,
1994b; Hara and Yamakawa, 1995), or formation of intramolecular
disulfide bonds in defensins (Kuzuhara et al., 1990; Lepage et al., 1990). These unique properties of moricin provide a
favorable condition for the production of the peptide by chemical
synthesis or by biotechnology using suitable protein expression
vectors. For this reason, we are presently expressing an artificial
moricin gene in mass scale to obtain a sufficient quantity for further
analysis of its precise role in self-defense system.