From the School of Biological Sciences and § Macquarie University Centre for Analytical Biotechnology, Macquarie University, Sydney, New South Wales 2109, Australia
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ABSTRACT |
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Reported here is the isolation and characterization of two antibacterial peptides synthesized in an ant Myrmecia gulosa in response to bacterial challenge. The peptides were purified by reversed-phase high performance liquid chromatography and characterized by peptide sequencing and mass spectrometry. Both peptides were formed from 16 amino acids, were rich in proline (~30%), and had N-acetylgalactosamine O-linked to a conserved threonine. The activity of a synthetic non-glycosylated isoform was markedly reduced demonstrating that glycosylation was necessary for maximum activity. The peptides were active only against growing Escherichia coli. They were inactive against stationary cells, Gram-positive bacteria, the yeast Candida albicans, two species of mammalian cells, and bovine pestivirus.
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INTRODUCTION |
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Based on primary and secondary structure, four main classes of
cationic peptide antibiotics are derived from metazoa (1, 2) as
follows: (i) linear, mostly helical, peptides without cysteine; this
group includes the cecropins from insects and pigs, the magainins from
the frog Xenopus laevis, and pardaxin from Moses sole fish
(1, 3). (ii) Peptides with an antiparallel -sheet structure
stabilized by two or three intramolecular disulfide bonds. Principal
among these are the defensins, insect defensins and protegrins.
Defensins have broad spectrum activity and are compartmentalized within
vesicles in mammalian neutrophils and macrophages. Insect defensins, on
the other hand, are exported from the cell into the insect blood and
are active against Gram-positive bacteria (4). The protegrins occur in
porcine leukocytes and have broad antimicrobial and antiviral activity
(5). (iii) Peptides with one intramolecular disulfide bond are the
third class. The bond typically joins together the two ends of a
secondary loop structure; bactenecin isolated from bovine neutrophils,
for example, has a loop formed of 9 residues with 1 and 2 residues on
each end (6). (iv) Linear peptides containing high proportions of 1 or
more amino acids comprise the fourth class. These linear peptides
include indolicidin, a tryptophan-rich peptide, PR-39 which is rich in
both arginine and proline, and the proline-rich peptide antibiotics
including drosocin from Drosophila melanogaster and
apidaecin from honeybees (2, 7-9).
Most cationic peptide antibiotics disrupt the cell membrane via a peptide-lipid interaction. The mechanism of action of drosocin and apidaecin, however, appears to involve a peptide-receptor recognition process. Unlike enantiomers of cecropin and mellitin synthesized using D amino acids, enantiomers of apidaecin and drosocin lacked antibacterial activity. This indicated stereospecific interaction between the peptide and an unknown target (10, 11). Other proline-rich peptide antibiotics isolated from insects include abaecin, pyrrhocoricin, the metalnikowins, and the lebocins (12-15).
Disease suppression is of paramount importance in living organisms. The Red Queen hypothesis states that sex is an adaptation to escape parasites. The genetic diversity of offspring as a result of recombination facilitates selection for minimization of parasite-induced loss of fitness (16). From this perspective it can be seen that disease suppression must be of particular importance in social insects (17) where there is high genetic similarity between workers. We hypothesized that as a result of both their genetic structure and their ecology ants must have evolved effective mechanisms for combating microbial infection.
Here we report the structure and antibacterial activity of two O-glycosylated proline-rich peptides synthesized in the bulldog ant Myrmecia gulosa in response to bacterial infection. Because they are the first inducible peptide antibiotics recovered from Formicidae, the name formaecins is proposed for these peptides. The formaecins were tested against a phylogenetically diverse range of microbial species. They were active only against growing Escherichia coli but were inactive against other Gram-negative bacteria, Gram-positive bacteria, a yeast, two species of mammalian cells, and pestivirus.
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EXPERIMENTAL PROCEDURES |
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Immunization-- Adult workers of M. gulosa were collected from five field colonies in New South Wales. The ants were maintained in the laboratory at 25 °C on an artificial diet for at least 2 weeks prior to experimentation. E. coli NCTC 8196 were grown overnight at 37 °C with agitation in 1/2 strength nutrient broth (Oxoid), centrifuged, washed in 150 mM NaCl, and pelleted (15,000 × g, 10 min, 4 °C). The femur of either anterior leg was pricked with a glass micro-capillary drawn to an ultrafine point to produce a shallow wound. By using the point of a glass capillary the exposed hemolymph was infected with approximately 4 × 106 bacteria. Sixty M. gulosa larvae, derived from two different colonies, were challenged using essentially the same method.
Extraction of Hemolymph--
After 7 days bacterially challenged
ants were snap-frozen in liquid nitrogen. The gaster was removed from
each ant immediately prior to snap freezing. Eight bacterially
challenged ants from each colony (42 ants in total) were ground to a
powder under liquid nitrogen using a mortar and pestle. The powder
(2.17 g) was mixed with an equal volume (w/v) of cold aqueous aprotinin
(10 µg/ml), incubated in an ice-bath for 40 min, and centrifuged 3 times (15,000 × g, 4 °C, 15 min). The supernatant
was extracted with an equal volume of cold n-hexane (v/v)
(Ajax) to remove metapleural gland secretions (18) and recentrifuged
(15,000 × g, 4 °C, 15 min). The aqueous phase was
pre-filtered (AP20 glass fiber; Millipore), post-filtered (0.45 µm),
and stored at 80 °C. Unchallenged control ants from the same
colonies were processed by the same method. Hemolymph was recovered
from challenged and control larvae as described (19). For all assays
the whole ant extract (hemolymph) was heat-treated prior to use. The
supernatant was recovered after boiling (5 min) and centrifugation
(15,000 × g, 10 min).
Reversed-phase Chromatography-- A Pharmacia Biotech SMART high performance liquid chromatography system was used for separation. Twenty-µl aliquots of hemolymph were separated by a Sephasil C8 column (2.1 mm × 10 cm) equilibrated with acidified water (0.05% trifluoroacetic acid). The column was washed with acidified water for 5 min. Elution proceeded with a linear gradient of 0-59.5% acetonitrile in acidified water (0.045% trifluoroacetic acid) over 60 min at a flow rate of 100 µl/min. Fractions were collected into 150-µl volumes. Two fractions with activity against E. coli were independently concentrated (~25 µl) and loaded onto a Sephasil mixed C2/C18 column (2.1 mm × 10 cm) equilibrated with acidified water (0.05% trifluoroacetic acid). Elution was done at a flow rate of 100 µl/min and collected into 100-µl fractions as follows: from 0 to 10 min the column was washed with acidified water (0.05% trifluoroacetic acid), and between 10 and 60 min the acetonitrile concentration (containing 0.045% trifluoroacetic acid) was increased linearly from 0 to 34%.
Peptide Sequencing and Amino Acid Analysis-- Peptides were sequenced using a Beckman Glycosite sequenator as described (20). This method identifies sites of glycosylation. Protein was quantified by amino acid analysis as described (21, 22).
Mass Spectrometry-- Electrospray ionization spectra were recorded using a Quattro II triple quadrupole mass spectrometer (Micromass) fitted with an electrospray source. On-line LC-MS1 was performed using the 2nd step separation procedure (C2/C18 column). A post-column split of approximately 10:1 (fraction collector, electrospray ionization-mass spectrometer) was employed to enable parallel fractionation and mass determination. Two alternating scan functions were used as follows: one at a sampling cone potential of 35 V to give the molecular weight of the peptides, and a second scan at a sampling cone potential of 100 V to produce low mass fragment ions to enable the determination of any post-translational modification that might be present, such as glycosylation. The primary UV wavelength of the SMART system was also recorded by the data system of the mass spectrometer so that the UV and total ion chromatogram outputs could be aligned.
Monosaccharide Analysis-- To identify the nature of the monosaccharides on the glycopeptides they were hydrolyzed with 100 ml of 2 M trifluoroacetic acid by heating for 4 h at 100 °C. The samples were lyophilized and redissolved in water, and the monosaccharides were separated by high performance anion exchange chromatography on a Dionex DX 500 Carbohydrate System. A CarboPac PA10 column (4 × 250 mm) was eluted isocratically in 12 mM NaOH at 1 ml/min, and the monosaccharides were monitored by pulsed amperometric detection. The retention of the monosaccharides was compared with standards, and quantitation was by the addition of an internal standard (2-deoxyglucose).
Data Base Search-- Peptide sequence data were subjected to a FASTA search (23) using the current version on the Australian National Genomic Information Service2 server and the Non-redundant Protein Data base.
Synthetic F1-- A non-glycosylated isoform of formaecin 1 (F1-SYN) was synthesized by a commercial firm (Auspep). Purity and structure were validated by reversed-phase high performance liquid chromatography, amino acid analysis, and mass spectrometry.
Microorganisms-- E. coli NCTC 8196, Staphylococcus aureus NCTC 4163, Arthrobacter globiformis ACM 2455, Acinetobacter calcoaceticus ACM 673, and Bacillus thuringiensis ACM 453 were obtained from the Australian Collection of Microorganisms at the University of Queensland. E. coli D22 was obtained from Dr. M. Hellers at the University of Adelaide. D22 has a defective lipopolysaccharide layer and displays high susceptibility to many peptide antibiotics (9-11). E. coli BLR was obtained from C. Moss at Macquarie University Center for Analytical Biotechnology. Curtobacterium flaccumfaciens pathovar flaccumfaciens DAR 58705 was obtained from the Biological and Chemical Research Institute, New South Wales Agriculture. Candida albicans AMMRL 36.42 was obtained from the Australian Mycological Medical Reference Library at the Royal North Shore Hospital, New South Wales. Bacteria were grown in 1/2 strength nutrient broth at 37 °C (E. coli strains and S. aureus), 30 °C (B. thuringiensis), and 25 °C (A. calcoaceticus, A. globiformis, and C. flaccumfaciens pv flaccumfaciens). C. albicans was grown at 37 °C in 1/10 strength Sabouraud liquid medium (Oxoid). All microorganisms were grown, with agitation, in 100 ml of culture in 250-ml Erlenmeyer flasks. Growth parameters were determined for all microbial species enabling the doubling time and major phases of growth to be identified.
Antimicrobial Assays-- The liquid inhibition growth assay used to detect antimicrobial activity was as follows: mid-exponential phase bacteria were suspended in 1/10 strength nutrient broth in 25 mM sodium phosphate buffer (pH 7) containing 15 mM NaCl at a concentration of approximately 200 cfus/100 µl. Mid-exponential phase C. albicans were suspended in 1/20 strength Sabouraud broth in the same buffer at a concentration of approximately 40 cfus/100 µl. Acetonitrile and trifluoroacetic acid were removed by vacuum centrifuge (Savant) prior to assay. Aliquots (100 µl) of cell suspension were combined with fractionated protein and incubated for 2 or 4 h (the latter was used for A. globiformis and C. albicans) with agitation at growth temperature. The experimental periods were designed to allow two or more cycles of cellular division. When testing hemolymph (20 µl) the bacterial concentration was doubled to approximately 400 cfus/100 µl. Following incubation cells were diluted 10-fold in 150 mM NaCl; 20-µl aliquots were plated in quadruplicate on nutrient agar plates and incubated at growth temperature for 1 or 2 days (C. albicans and A. globiformis). Numbers of cfus were then determined.
The inhibitory kinetics of F1 were assayed as follows: 100-µl aliquots of mid-exponential phase E. coli NCTC 8196 (~400 cfus) suspended in 50 mM sodium phosphate buffer (pH 7) were pipetted into nine microcentrifuge tubes; six of the tubes also contained 1.5 µg of F1. Cells were incubated at 37 °C with agitation, sampled (10 µl) at various time points (Fig. 5), diluted, and grown on nutrient agar as described above. After 250 min an equal volume (50 µl) of 1/5 strength nutrient broth was added to three tubes containing cells and F1 and to three tubes containing cells only. To the remaining three tubes, containing cells and F1, was added 50 µl of 17 mM NaCl (an NaCl concentration equal to that in 1/5 strength nutrient broth). Cells were reincubated and sampled as described. The activity of F1 and F1-SYN were compared. Peptides were suspended in 20 µl of 1/10 strength nutrient broth in 25 mM phosphate buffer (pH 7). Aliquots (100 µl) of exponential phase E. coli NCTC 8196 (1.75 × 106 cfus) in 1/2 strength nutrient broth were combined with peptide and incubated at 37 °C with agitation. Cells were sampled (20 µl) at 4 and 24 h as described.Protease Assay-- Twenty-µl aliquots of challenged and control hemolymph were preincubated (37 °C, 18 h) with 20 µg of protease V (Sigma) in 4 mM CaCl2. As controls hemolymph were also incubated in 4 mM CaCl2 with and without bovine serum albumin (20 µg). Samples were tested for antimicrobial activity against E. coli NCTC 8196 using the standard inhibition assay. In a further experiment hemolymph (20 µl) was tested against E. coli NCTC 8196 in the presence of 2 mM dithiothreitol prepared according to Ref. 24.
Antiviral and Cell Culture Assays-- A modified virus neutralization peroxidase-linked assay was carried out in 96-well cell culture plates (25). The virus was a non-cytopathogenic Australian bovine viral diarrhea virus (pestivirus) isolate (Trangie) (26). Pestiviruses are small enveloped positive-sense RNA viruses (Flaviviridae). Peptide was suspended in 20 µl of 50 mM sodium phosphate buffer (pH 7). This, challenged, and control hemolymph (20 µl) were serially diluted 2-fold in cell culture medium starting at a dilution of 1/5 in a final volume of 50 µl. When testing a bovine turbinate cell line Basal Medium Eagle with Hank's salts (Life Technologies, Inc.) and 10% (v/v) bovine serum were used. For lamb testis cells (passage 5) the medium was minimum essential medium (Life Technologies, Inc.) and 10% (v/v) bovine serum. An equal volume (50 µl) of media containing either 100, 10, or 0 TCID50 of virus was added to wells. 100 TCID50 is equivalent to 50 infectious particles. Virus and test material were incubated for 1 h at 37 °C. Cells in 100 µl of media were added at approximately 2 × 104 cells/well. Cells, control positive bovine sera, and virus were independently titrated to ensure viral calibration was correct. Plates were incubated at 37 °C in 5% CO2 for 5 days. Cells were stained by immunoperoxidase using pestivirus-specific monoclonal antibodies (27) and horseradish peroxidase-conjugated goat anti-mouse IgG and visualized with 3-amino-9-ethyl carbazole chromogen.
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RESULTS |
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Antimicrobial Activity of Hemolymph-- Control hemolymph was free of activity against all species tested. Immune hemolymph was active against E. coli but was inactive against two species of mammalian cell, pestivirus, the yeast C. albicans, and Gram-positive bacteria (Table I). The susceptible strain E. coli NCTC 8196 was used to challenge the ants; the other susceptible strain, E. coli BLR, was randomly obtained for use as a control. The growth of E. coli D22 was partially inhibited by immune hemolymph (Table I). The activity of immune hemolymph was destroyed by preincubation with protease but was unaffected by dithiothreitol (2 mM).
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Purification of the Active Peptides-- The chromatographic profiles of immune and control hemolymph were different. A number of peaks observed in either hemolymph were absent in the other (Fig. 1). All fractions were tested against E. coli NCTC 8196. Two active peptides present in immune hemolymph were purified (Fig. 2). Both F1 and F2 were consistently observed when challenged hemolymph obtained from independent field colonies was fractionated and tested (results not shown). It is not known if both peptides occur within individual ants. F1 and F2 were also recovered from hemolymph collected from bacterially challenged larvae. F1-SYN eluted 160 s later than F1 when fractionated using the C2/C18 separation protocol.
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Primary Structure-- Formaecins 1 and 2 were formed of 16 amino acid residues, were free of cysteine, and contained a high proportion of proline (~30%). The primary structure of F1 and F2 differed at two sites (Fig. 3). Formaecin 1 and 2 have 50 and 44% sequence identity with drosocin (7). Both peptides were cationic; the theoretical pI of F1 is 12.0 and of F2 is 11.0. At physiological pH F1 and F2 have a net charge of (+4) and (+3), respectively. Sequencing indicated N-acetylhexosamine linked to Thr-11 on both F1 and F2. To determine the mass of the peptides, LC-MS was conducted on purified F1 and F2 using the C2/C18 column separation protocol. F1 had a mass of 1997 daltons and F2 had a mass 2010 daltons (Fig. 4). These masses were precisely 203 Da higher than the mass of F1 and F2 deduced using the sequence data. Small peaks were observed at these deduced masses (Fig. 4). This was attributed to collision-induced loss of the sugar in the electrospray source of the mass spectrometer. This indicated HexNAc on formaecins 1 and 2 validating the findings obtained by sequencing. N-Acetylgalactosamine was identified as the O-linked sugar following hydrolysis and anion exchange chromatography.
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Antimicrobial Activity of F1 and F2-- Purified F1 and F2 killed or inhibited the growth of two strains of E. coli but were inactive against Gram-positive bacteria, C. albicans, eukaryotic cells, and pestivirus (Table I). The limited antibacterial range is expected to reflect the wide genetic and phenotypic diversity of the test bacteria. The standard inhibition assays were done at low ionic strength (15 mM NaCl), but F1 and F2 were equally active when tested in 150 mM NaCl.
A time course analysis of the inhibitory activity found that it took an experimental period up to 2 h at a concentration of 7.5 µM for either peptide to completely inhibit the subsequent growth of E. coli on agar (results not shown). This is considerably lower than the in vivo concentration of either peptide. From 20-µl aliquots of heat-treated hemolymph extract, 1.5 and 1 µg of F1 and F2 were purified. This indicates a minimum concentration of 38 and 25 µM, respectively. Because an equal volume of water was combined (v/w) with the crushed ants the concentration in vivo was more than twice these values. The data suggested only growing cells were susceptible to the effects of formaecin. To test this hypothesis E. coli was suspended in nutrient-free buffer with and without F1 (7.5 µM) and sampled over 4 h. There was essentially no change in cell numbers over this period. The subsequent addition of nutrient media to cells demonstrated that only growing cells were affected by F1 (Fig. 5).
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DISCUSSION |
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The biological and structural properties of the formaecins, a new
family of O-glycosylated proline-rich peptide antibiotic, are described here. Both formaecins were small (2 kDa), heat stable, and cationic. The O-glycosylation substitution was clearly
important for activity. The concentration of F1 required for complete
killing or inhibition of cell growth was 75 times less than the
non-glycosylated isoform. Relative to control cells grown in the
absence of peptide, the IC99 of F1 was 16 times less than
F1-SYN. Glycosylation is also necessary for maximal activity of the
other proline-rich peptides of comparable mass, drosocin and
pyrrhocoricin (7, 13). In both the cases the O-glycosylation
substitution is at Thr-11 although drosocin is glycosylated with the
disaccharide GalGalNAc. Interestingly, the minimum inhibitory
concentration of synthetic drosocin glycosylated with GalNAc was
comparable with that of F1 (11). An analysis of the structure and mass of apidaecin-type peptides isolated from other Hymenopteran insects (bumblebees, bees, wasps and hornets) demonstrated they have a conserved structure and are not glycosylated (28, 29).
E. coli D22 was resistant to F1 and F2 when tested under these conditions. It was, however, partially sensitive to immune hemolymph. The comparatively low sensitivity of D22 to formaecins suggests their activity was not limited by their ability to access the cell membrane but may instead be limited by the cellular concentration of a target receptor. Alternatively, other unidentified compounds in the hemolymph may have accounted for the partial activity against E. coli D22.
It was found here that non-growing cells were fully resistant to
formaecin at a concentration well in excess of the minimum inhibitory
concentration. To our knowledge this is the first report of such an
antibacterial effect by an insect-derived proline-rich peptide. The
activity of apidaecin was partly, although not completely, reduced when
tested in nutrient-free buffer (8). The finding that only growing cells
were susceptible to F1 suggests the activity may be
voltage-dependent in a manner similar to that described for
indolicidin, a 13-residue peptide rich in tryptophan (39%) and proline
(23%) (2). Indolicidin was inactive on cell membranes with a
transmembrane potential less than 70 to
80 mV. The energy transducing membranes of growing bacteria have potentials in excess of
140 mV. Using circular dichroic spectroscopy indolicidin was found to
adopt a type II polyproline extended helix upon interaction with
liposomes. Indolicidin acts by forming channels across the cell
membrane, and unlike the apidaecins and drosocin, its action did not
involve a receptor-mediated recognition process.
As more peptide antibiotics are discovered, it becomes more apparent that in many cases similarities between compounds are the result of convergent evolution rather than common ancestry (4). Thus the structure and function of peptide antibiotics is shaped by both the historicity and ecology of the host species. Several factors led us to think that ants should produce effective inducible peptide antibiotics. Conditions of relatively high population density increase opportunity for the spread of infectious disease. One can easily see that this will be exacerbated when the individuals are closely related. In an idealized Hymenopteran colony, the mean genetic identity by descent between workers is 75% although in practice the level may be lower. Another factor is the longevity of M. gulosa that can survive a decade or more. Other things being equal, the longer the life span the more likely it is an animal will be injured or contract infectious disease. The habitat is also important as soils contain a large and diverse range of bacterial and fungal species (30). We suggest consideration of the ecology of a species and its ability to shape immune defenses could assist in the discovery of new compounds for treating infectious or other disease.
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ACKNOWLEDGEMENTS |
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We thank the people mentioned under "Experimental Procedures" for the supply of microorganisms. We also thank Susan Mackintosh at Elizabeth Macarthur Agricultural Institute for facilitating the cell culture and virological assays. We are indebted to Dr. Daniel Jardine for mass spectrometry; Dr. Nicolle Packer for monosaccharide analysis; Craig Angus for help with the field work; Natasha Zachara for technical advice; and Dr. Andrew Holmes for comments on the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by an Australian Postgraduate Research Award (Industry).
To whom correspondence should be addressed: School of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia. Tel.: + 612 9850 6950; Fax: + 612 9850 8245; E-mail: jmackint{at}rna.bio.mq.edu.au.
1 The abbreviations used are: LC-MS, liquid chromatography mass spectrometry; cfus, colony-forming units.
2 ANGIS, available at the URL, http://morgan.angis.su.oz.au.
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REFERENCES |
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