of Antimicrobial Peptides of Invertebrates. Charles Hetru and Philippe Bulet. 1. Introduction. Resistance of bacteria to antibiotics has become one of the main ...
strategies for the Isolation and Characterization of Antimicrobial Peptides of Invertebrates Charles Hetru and Philippe Bulet 1. Introduction Resistance of bacteria to antibiotics has become one of the main problems in human health. In addition, m agronomy, microbial diseases are largely responsible for the decrease m agricultural production. The discovery of new antibiotic families is a way to circumvent such problems and antimicrobial peptides may represent a new type of such antibiotics. In animals, antimicrobial peptides are important effectors of the innate immune response (nonadaptive immunity). In mammals, they are produced by neutrophils or macrophages and kill microbial invaders in a first barrier of host defense. The acquired and specific immune responses with production of antibodies occur later. In insects, there is no specific and adaptive immunity but only an innate response that includes cellular and humoral factors. The cellular response consists mainly of phagocytosis and encapsulation. The humoral immune response includes the rapid synthesis of a battery of antimicrobial peptides. A recent review lists the antimicrobial peptides/polypeptides isolated from insects and shows the rapid increase in the number of molecules that have been characterized (/). To date, from only 22 species, more than 100 different peptides/polypeptides have been fully characterized. Invertebrates, which include the insects, present an extreme diversity and a potential source of a large variety of antimicrobial substances. Since the discovery of the first antibacterial peptide in insects in 1981 (2), a large variety of techniques have been used to isolate, purify, and characterize such molecules.
From Methods in Molecular Biology, Vol 78 Antibacterial Peptide Protocols Edited by W M Shafer, Humana Press Inc , Totowa, NJ
35
36
Hetru and Bulet
In this chapter, we describe a number of methods for the identification, purification, and characterization of antimicrobial peptides/polypeptides. Several of the topics of the present chapter have certainly been covered in earlier reviews; however, to be complete and for the reader's autonomy, we have chosen to describe all the techniques needed. We have focused our descriptions and advice to specific aspects of invertebrate antimicrobial peptides/polypeptides: induction to the antimicrobial peptides, preparation of the samples, and, as the material will be obtained in very small amounts, on modified methods for chemical characterizations and antimicrobial tests The principal group of invertebrates that has been used until now, for the isolation of antimicrobial molecules, is the insect class (3). In this class, most of the antimicrobial peptides are not present in the hemolymph of normal animals, but are induced by an injury or an injection of microbes (4). Thus the first operation, prior to extraction and purification, is the induction of the production of antimicrobial peptides by the animals. Various kind of inducers have been injected; living bacteria from lO'^-lO^ cells per animal (5-11), heat-killed microbes (12), and components of the cellwall of bacteria (13,14). However, to obtain a more complete induction of antimicrobial peptides, the injection of a mixture of living Gram-negative and Gram-positive bacteria at sublethal doses is recommended. The main source of antimicrobial peptides in invertebrates is hemolymph, and the first step after collection is centrifugation to separate plasma from hemocytes. In order to remove from plasma, compounds other than the peptides of interest, several treatments have been proposed: heat treatment (6) or plasma acidification (15). For very small animals, to simplify the collection of material the protein extraction is directly performed from the total body of the animals. Recently, solid-phase extraction (SPE) on reversed-phase (CI8) has been used to prepare peptide samples (11,16-21). This procedure consists of a kind of crude chromatography. Before loading on this support, the sample should be acidified, typically with trifluoroacetic acid (TFA) or acetic acid. A sequential elution with low, medium, and high percentage of acetonitrile (or methanol) in acidified water leads to a prepurification of the sample. Salts, sugars, and most hydrophilic proteins are eliminated during the washing cycle, whereas lipids and most hydrophobic proteins are retained on the solid-phase. Antimicrobial peptides from arthropods have also been isolated from hemocytes (22,23) that are homogenized, centrifuged, and the supernatant is extracted as for the plasmatic fraction. In the case of cultured cells, the medium can be submitted directly to purification without any concentration (24,25).
Invertebrate Peptide Strategies
37
After sample preparation, the extracts are concentrated by lyophilization or in a vaccum centrifuge and then resuspended in the appropriate buffers for purification The principle mode of purification used for antimicrobial peptides is chromatography mainly on reverved-phase and size-exclusion columns. The characterization is mainly performed by combination of sequencing by automated Edman degradation, mass spectrometry analysis, and enzymatic cleavage. Monitoring of antibacterial activity by the paper disk method is the oldest protocol available and is still currently used for antibiograms (medical and pharmaceutical diagnostics). A variation of this method, often used, is the inhibition zone assay (5,8,9,12,26-31). Recently, a sensitive liquid growth inhibition assay has been described (17,20). Antifungal assay agamst filamentous fungi can novi' be easily conducted with a very good sensitivity also m a liquid growth inhibition assay
2. Materials 2.1. Insect Immunization 1 Gram-positive bacteria Micrococcus luteus. 2. Gram-negative bacteria: Escherichia call 1106 or any nonpathogen wild type. 3 Luria Broth medium (LB): 15 5g of Millers's modification of Luria Broth, GibcoBRL, IL of H2O, pH 7 4 or 1% bactotrypton, 0.5% yeast extract, 0.9% NaCI w/v. 4 Eppendorf tubes, 1 5- or 2 5-mL for bacterial dilutions or to put the bacterial pellet into the cap 5 Hamilton syringe (5 or 10 |jL) or stainless steel needle (ultrafme)
2.2. Extraction 1. 2. 3 4.
Phenylthiourea (Sigma, St Louis, MO; PTU. stock solution 20 xx\M in ethanol) Aprotinin (protease inhibitor; Sigma). Trifluoroacetic acid, sequenal grade (Pierce, Rockford, IL) Filter units, 0.8-|am membrane (Millex unit, Millipore, Bedford, MA).
2.3. Purification of Antimicrobial Peptides 1. Solid-phase extraction cartridges (Sep-Pak CI8 cartridges. Waters, Milford, MA). Several sizes are available according to the quantity of extract 2. Methanol for HPLC (Carlo Erba, Rodano, Italy) 3 Acetonitrile for HPLC (Merck, Rah way, NJ). 4. HPLC water or any ultrapure water (MilliQ water, Millipore) 5. Polypropylene tubes Minisorp (NUNC Immuno tubes, 75 x 12 mm, Roskilde, Denmark). 6 HPLC columns' Porosity 300 A, granulometry 7 pm, re versed-phase C8 or C18, analytical columns (2 1-4 6 mm id).
38
Hetru and Bulet
1. Size-exclusion column (SEC 2000 and SEC 3000, Beckman, Fullerton, CA) and a precolumn (Beckman). 8 HPLC system pump (one or two), UV detector (detection at 225 nm) with two output (analogical for paper recorder and digital for computer) and a paper recorder. 9. Centrifuge vacuum drier (Speed Vac, Savant, Hicksville, NY)
2.4. Microsequencing Analysis 1. Automated Edman degradation of the pure peptide and detection of phenylthiohydantoin derivatives are performed on a pulse liquid automatic sequenator (e.g., Perkin Elmer Applied Biosystems, model 473A) Reagent and solvants are purchased to manufacturer (Perkin Elmer, Applied Biosystem Division, Norwalk, CT)
2.5. Mass Spectrometry 1 Electrospray ionization mass spectrometer with an electrostatic ion spray source operating at atmospheric pressure followed by a quadrupole mass analyzer (mass range 1-4000, scanning from m/z 500 to m/z 1500 in 10 s) (VG Biotech BioQ mass spectrometer, Manchester, UK) 2 Multichannel analyzer as data system 3 Calibration with heart myoglobin 4. Acetic acid and methonol for analysis quality 5. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometer (Bruker, Bremen, Germany) 6. a-cyano-4-hydroxycinnamic acid (Sigma) 7 Moderate vacuum pump (membrane pump) 8 Standard peptides for calibration angiotensin II, ACTH 18-39 and bovine insulin
2.6. Enzymatic Cleavage 1 Water bath (37°C) 2. Polypropylene tubes 3 Protease of sequencing grade (Proteases from Boehnnger, Mannheim or residuespecific protease kit from Takara, Japan). 4 Reaction buffersa. Lysyl endoproteinase {Achromobacter protease I), 10-25 mM Tns-HCl, pH 8 0, 0.01% Tween-20 with or without 1 mM EDTA. b Arginyl endoproteinase, 10 mM Tns-HCl, pH 8 0, 0 01 % Tween-20 c Trypsin, 10 mM Tns-HCl, pH 8 0, 0.01% Tween-20, 10 mM CaCl2 d. Staphylococcus aureus V8 proteinase, 50 mM ammonium carbonate, pH 7 8, 0 01% Tween-20 e Asparaginyl endopeptidase, 20 mM sodium acetate buffer, pH 5 0, 0.01%, Tween-20, 1 mM DTT, 1 mM EDTA.
Invertebrate Peptide Strategies
39
f. Pyroglutamate ammopeptidase, 100 mM sodium phosphate buffer, pH 8.0, 10 mM EDTA, 5% glycerol, 5 mM dithiothreitol g. Carboxypeptidase Y and P, 50 mM sodmm citrate buffer, pH 6 0 (for Y) and pH 4.0 (for P).
2.7. Bioassays 2.7.1. Preparation of Spore Suspension of Neurospora crassa 1 Six cereal agar: 20 g of six cereal instant flakes (Nestle) and 15 g agar in 1 L of sterilized water 2. Autoclaved water. 3 Autoclaved 50% glycerol. 4. Sterile microtubes (Eppendorf tubes). 5. Broad spatula 6 Glass funnel plugged with glass wool wrapped in aluminium foil and autoclaved.
2.7.2. Antifungal and Antibacterial Tests 1. 2. 3 4. 5 6. 7
Potato dextrose broth (DIFCO) 12 g PDB for 1 L water Tetracychne (Sigma) stock solution. 10 mg/mL m DMSO. Cefotaxim (Sigma) stock solution 100 |Jg/mL in water Luna Bertani's nch medium. 1 % bactotrypton, 0.5% yeast extract, 0.9% NaCl w/v Poor broth medium: 1% bactotrypton, 0.9% NaCl w/v. 96-well microliter plates for cell culture (Nunc, Wiesbaden-Biebrich, Germany). Microplate reader
2.8. Reduction and Alkylation 1. Reaction buffer- 0 5M Tns-HCl, 2 mM EDTA, pH 7 5 containing 6M guanidmium chloride 2. Dithiothreitol stock solution 2 2M. 3. Water bath, 45°C 4 4-Vinylpyridine (4-VP) is distilled under reduced pressure (vacuum water pump) At the end of the distillation the apparatus is filled with inert gas (N2 or Ar) to avoid oxidation of the colorless pure 4-VP The reactive can be stored as pure liquid in sealed vials under inert gas and conserved for months at -20°C 5. Nitrogen or Ar gas tank
3. Methods This chapter has been organized in a way that the reader can start from animals and end with structural information about the antimicrobial peptides present in the hemolymph or in the total body of the organism they are studying. We have chosen to describe the procedures in the order of normal execution, from induction of the antimicrobial peptides in the animal to sequencing and mass spectrometry.
40
Hetru and Bulet
3.1. Insect Immunization 1. Choose insects at the same developmental stage or animals of similar size 2 Prepare overnight cultures of Micrococcus luteus (Gram-positive strain) and Escherichia coli wild-type (Gram-negative strain) 3 Anesthesize the animals by chilling or with CO2 4 Inject a mixture of living M luteus and E. coli (1000 cells of each/|jL injected) or, for small-size insects, replace the injection by simple pricking of individuals with a fine stainless steel needle previously dipped into a moist combined bacterial pellet. 5. Keep the bacteria-challenged insects in appropriate conditions for 24-48 h {see Note 1)
3.2. Extraction 3.2 1. Extraction from Hemolymph 1. Collect the hemolymph (through an incision, a leg, or an antenna) in precooled polypropylene tubes containing a protease inhibitor (for example aprotinin) at a final concentration of 10 ng/mL of hemolymph and an inhibitor of melanization (phenylthiourea, PTU, 1 |ig/mL, see Note 2) 2 Recover cell-free hemolymph after centnfugation at 15,000g at 4°C for a short period to avoid coagulation. 3 Acidify the cell-free hemolymph with a solution of 0.1 % tnfluoroacetic acid (v v) and, after incubation for 30 min in an ice-cold water bath under gentle shaking, centrifuge at 15,000g for 30 min, and collect the supernatant
3.2.2. Extraction from Small-Sized Insects 1 Freeze insects in liquid nitrogen and reduce to a fine powder in a mortar in the continuous presence of liquid nitrogen (see Note 3). 2 Transfer the powder to 10 vol (w/v) of acidified water (0 1% TFA) containing a protease inhibitor (aprotinin, final concentration 10 ^ig/mL of medium) and an inhibitor of melanization (PTU, 1 |ug/mL). 3 Extract as for the hemolymph, 30 min in a ice-cold water bath. 4 Centrifuge the extract, clarify through 0.8-|jm filters and immediately (without freezing) submit to purification.
3.3. Purification of Antimicrobial Peptides 3.3.1. Solid-Phase Extraction (SPE) on Sep-Pak C18 Cartridges 1 Prepare the cartridge by washing with methanol and equilibrate with acidified water (0.05% TFA) Usually one cartridge is sufficient for 2-3 g of total animals or 1-2 mL of hemolymph. 2 Load the sample after verification of the pH of the extract, which should be acidic, pH 25 mL) 5. Reconstitute the fractions in HPLC water and keep at -20°C until use.
3.3.2. Reversed-Phase HPLC 1. Load the sample on an analytical reversed-phase column (300 A, 3 8-4 6 mm id, C18 or C8) equilibrated in 2% acetonitrile m acidified water (0 05% TFA) 2 Elute with an appropriate linear gradient of acetonitrile in acidified water (0 05% TFA; see Note 5) at a flow rate of 1 mL/min (see Note 6) a. For the 10% SPE fraction 2-20% acetonitrile in 60 min. b. For the 40% SPE fraction- 2-60% acetonitrile in 120 min c. For the 80% SPE fraction- 10-80% acetonitrile in 120 min. 3. Collect fractions manually according to the absorbance measured at 225 nm (best ratio signal/solvent) Using this collecting procedure, each fraction corresponds to an individual peak (see Note 7). 4. Dry the eluted fractions in vacuum centrifuge, dissolve in sterile distilled water, and monitor the antimicrobial activity on aliquots
3.3.3. Size-Exclusion HPLC 1 Load the active fraction on two serially linked Beckman SEC 3000 and SEC 2000 columns, 300 x 7 5 mm (or equivalent columns) protected by a precolumn. The injected volume should be
