Br. J. exp. Pith. (1978) 59, 416

THE PREPARATION, PROPERTIES AND ACTION ON STAPHYLOCOCCUS AUREUS OF PURIFIED FRACTIONS FROM THE CATIONIC PROTEINS OF RABBIT POLYMORPHONUCLEAR LEUCOCYTES E. WALTON From The Sir W97illiam Dunn School of Pathology, University of Oxford Received for publication March 17, 1978

Summary.-A micropreparative electrophoresis system for purifying the major staphylocidal fractions of cationic proteins from rabbit polymorphonuclear leucocytes is described. The most staphylocidal fraction prepared is also the most cationic and contains two bands migrating immediately behind protamine sulphate on analytical acid gel electrophoresis. SDS gel electrophoresis indicates that these proteins have low molecular weights between 3,500 and 14,400. The staphylocidal activity of the fraction is affected in the same manner as a crude extract of rabbit PMN granules by iron compounds, respiratory inhibitors, and compounds affecting energy transfer and oxidative phosphorylation. It is stable to heating up to 800 and amino acid analysis shows that it contains 24% arginine. Electron microscopy of staphylococcal spheroplasts treated with the purified fraction or with the crude extract shows that they both have a very marked "blebbing" and distorting action on the double membrane. Comparisons are made between the action of the purified fraction and protamine, and it is concluded that they have very similar, although not identical, properties and actions on staphylococci.

A NUMBER of studies have described the fractionation of cationic bactericidal components from the lysosomes of rabbit and human polymorphonuclear (PMN) leucocytes. In 1968, Zeya and Spitznagel resolved the cationic antibacterial proteins, extracted under acid conditions from rabbit PMN lysosomes, into at least 5 fractions, using sucrose density gradient electrophoresis. These fractions migrated ahead of lysozyme, and were denoted I-V in order of decreasing cationic character. Each fraction showed substantial selectivity in its antibacterial action against several pathogenic bacteria (strains of Escherichia coli, Staph. aurewu, Staph. albus, Proteus and Streptococcus). Fraction III was most active in preventing growth of Staph. aureus. Hibbitt, Cole and Reiter (1969) extracted antimicrobial proteins from the teat canal keratin of cows by treatment under acid conditions followed by chroma-

tography on carboxymethylcellulose. The single peak of protein which emerged inhibited growth of Staph. aureus and Strep. agalactiae, and was resolved into 6 bands by analytical gel electrophoresis at pH 3 0. Later studies (Hibbitt, Brownlie and Cole, 1971), using similarly extracted cationic proteins from cells in bulk milk samples, demonstrated that these had greater antibacterial activity against the same test organisms. At pH 3 0, analytical gel electrophoresis demonstrated the presence of at least 9 bands migrating towards the cathode, although electrophoresis at pH 7 0 showed that the majority of these had isoelectric points of less than 9 0. The components were not tested separately for antibacterial activity. Lysozyme did not appear to be present in preparations of antimicrobial proteins from either source. Weiss et al. (1975) described the partial purification of a factor obtained by acid

PROPERTIES OF POLYMORPHONUCLEAR PROTEIN FRACTIONS

extraction of a whole rabbit granulocyte homogenate, followed by chromatography on CM-Sephadex. This study and subsequent ones (Weiss et al., 1976; van Houte et al., 1977; Weiss and Elsbach, 1977) have investigated various aspects of the effects of the granulocyte fraction on Escherichia coli. Olsson and Venge (1974) described the acid extraction of highly cationic proteins from the cytoplasmic granules of leucocytes ftrom patients with chronic myeloid leukaemia. The proteins were purified by chromatography on Sephadex G-75 and E-aminocaproic acid-Sepharose ion adsorbant, followed by preparative electrophoresis on agarose. Seven components were identified. In the 4 most cationic components bactericidal activities were found against Staph. aureus, Streptococcust faecalis, E8cherichia coli and Pseudomonac8 aeruginosa (Odeberg and Olsson, 1975). Further studies (Odeberg and Olsson, 1976) demonstrated mechanisms for this microbicidal activity. Their work will be discussed further in relation to the results described in this paper. In this paper, a micropreparative electrophoresis system is described for the cationic components of rabbit PMN leucocyte lysosomes which kill Staph. aureus. The killing action of the purified components is compared with that of protamine (to which they appear to bear some similarity), and other characteristics of the purified components are also investigated. MATERIALS AND METHODS

Staphylococci.---All of the work described here has been carried out with Staphylococcus aureus, Strain P66. Organisms were grown overnight in a T-tube in brain heart infusion broth (BHI) and prepared for use as previously described (Gladstone, Walton and Kay, 1974). Materials.-Sources and stock solutions of most inaterials used are as previously described (Walton and Gladstone, 1976). Haematin was obtained from Dr W. E. van Heyningen, protainiie sulphate froin Sigina, egg white lysozyme from Armour and lysostaphin from Schwartz/MIanin. Components of polyacrylamide

417

gels were specially purified for electrophoresis and obtained from B.D.H. Preparation of cationic proteins from lysosomes of rabbit polymorphs (crude granular extract: GE). Rabbit polymorphs were obtained from the peritoneal exudates of about 8 rabbits and granules were prepared from these as previously described (Gladstone, Walton and Kay, 1974). Generally, the broken cell residue was resuspended after removing the supernatant containing the first lot of granules, so that a second lot of granules could be prepared. This procedure increased the total yield of granules considerably. Packed granules were stored at 4° for up to 2-3 weeks before acid extraction. Acid extraction and freeze-drying: Two or more preparations of granules were pooled and resuspended to about 20 ml in 0-32 mol/I sucrose0 05 mol/l acetate buffer at pH 4 0 at 4°. Clumps were broken up in a glass homogenizer. The pH of the suspension was adjusted to 2-0 by addition of 1 mol/l HCI, it was left to stand for several minutes on ice and then centrifuged at 25,000 g for 20 min. The supernatant, which contains the cationic proteins, was removed and concentrated in an Amicon 52 ultrafiltration cell fitted with a PM 10 membrane (exclusion MW 10,000). It was dialysed by washing 4 times with distilled water. The concentrated and washed acid extract was freeze-dried overnight and stored at 4°. The solid material was soluble in water or sucrose to give a solution containing about half its dry weight in protein. The yield from 16 rabbits was of the order of 150 mg. 7fMicro-preparative polyacrylamtde gel electrophoresis of GE.-Separation of the components of GE into small groups was carried out in a 10 cm 7 0% sinall pore polyacrylamide gel at pH 4 3, overlaid by 0 5 cm of large pore spacer gel at pH 658, as described by Reisfeld, Lewis and Williams (1962). The gel was poured in a 12 cm gel tube (inner diameter 0-6 cm, outer diameter 0 9 cm), the bottom of which was attached to an elution device made according to the pattern described by Foissy and Summer (1976). At first the semipermeable membrane used in the elution cell was a disc of dialysis tubing, but this was later replaced by a disc of Amicon UM 2 ultrafiltration membrane (exclusion MW 1,000), in order to prevent loss of low-molecular-weight proteins. To avoid leaks, the apparatus was also modified by using PTFE washers (0.05 mm thick), which were placed on either side of the membrane and beneath the upper vulcanite ring on the nylon support net for the gel. All joints were well greased with silicone high-vacuum grease. The elution device, fixed to the gel tube, was supported in position in the lower buffer vessel (12 cm high), on a perspex tripod (3 cm high). It was also found that it is essential to place the whole elution device, and with it the lower part of the gel tube, in a crystallizing dish

418

E. WALTON

on the tripod. This diverts hydrogen formed (about 10 mamps) for 6 h, until the marker had during electrophoresis up the side of the lower migrated to within 1 cm of the bottom of the buffer vessel away from the base of the elution gel. Equally good separations were obtained with cell. The upper (12 cm high) and lower buffer or without urea so this additive was not routinely vessels were filled with f-alanine-acetic acid used. "tray buffer", pH 4-5, and this buffer, after deS.D.S. polyacrylamide gel electrophoresis.-The gassing, was also used for the elution buffer. The system used was based on that of Laemmli ( 1970). elution buffer flow was actuated by an LKB The separating gel (14 x 10 cm, 1 mm thick) 12 000 variospeed peristaltic pump which was contained 20% acrylamide, and the sample gel set up to give a flow rate of 2 ml/15 min. (14 x 2 cm, 1 mm thick), which incorporated 10 The gel was pre-electrophoresed to constant sample slots, contained 10% acrylamide. Both voltage (150 V) overnight at 8 mamps, before were poured from a stock solution of 60% attachment to the elution device. The semi- acrylamide and 0-4% N,N'-bis-methylene acrylapermeable membrane in the device was equili- mide. Final concentrations of other gel combrated with the elution buffer by sealing the ponents were as follows: 0375 mol/1 Tris-HCl elution cell off from the lower buffer vessel with a (pH 8-8), 0-1% SDS, 0-05% (v/v) tetramethylrubber bung and letting buffer flow over the ethylenediamine, 0-1 % ammonium persulphate. membrane overnight. After connection of the The electrode buffer (pH 8-3) contained 0-05 mol/ elution device to the gel a further "pre-run" of 1 Tris, 0-386 mol/I glycine and 0-1% SDS. The w h was needed to stabilize to constant voltage samples (5-50 ,il) contained 5-10 ,ig protein (about 220 V, using a UM 2 membrane), before (15 ,ug for GE) in 10 mmol/l phosphate (pH 8 3) application of the sample. The sample was containing (10% v/v) glycerol, 2% SDS, 5% 2applied as 150 ,u solution in 20% sucrose. For mercaptoethanol, and 0-1% bromophenol blue micropreparative runs 20 mg GE (approx. as marker. They were heated in a boiling water 10 mg protein) was added, for investigative bath for 3 min before loading on to the gel, which runs, 1-5 mg each of protamine sulphate and was run at the constant voltage of 100 V for 6 h, lysozyme was added. The electrophoretic separa- until the marker had migrated to within 2 cm tion was carried out at a constant current of of the end of the gel. 8 mamps. The eluate was monitored by an LKB Fixation, staining and destaining of gels.Uvicord ultraviolet absorptiometer, and 1 ml After electrophoresis slab gels were placed in fractions were collected by an LKB Ultrorac 5: 5: 1 methanol: water: acetic acid containing fraction collector in siliconized tubes. Pooled 0 25% Coumassie Brilliant Blue and left overfractions were desalted and concentrated by night to fix and stain the bands of protein. ultrafiltration in an Amicon ultrafiltration cell Destaining was carried out by repeated washcontaining a UM 2 membrane. ing in the same solvent. Test of the staphylocidal activity of GE and its Analytical polyacrylamide gel electrophoresis.In early attempts to resolve the components of fractions, and protamine.-The technique is GE the discontinuous disc system of Reisfeld, described in a previous paper (Walton and Lewis and Williams (1962) described in the Gladstone, 1976). previous section was used. Using 8 cm gels, and a Heat stability of a purified fraction of GE. current of 8 mamps per tube only 3 major 5 ,ug samples of Fraction 1 from GE, which concationic bands running before lysozyme were tains the 2 most cationic components (Bands I identified. Separation was improved by pre- and II), were heated for 1 h in a water-bath at running the gels to constant voltage overnight, temperatures ranging from 500 to 900, using by reducing the current to 2 mamps per tube and 100 ,ul of aqueous solution. The staphylocidal by increasing the acrylamide concentration in the activity of the heated samples was tested in the small pore gel to 15%. These changes did not usual system. cause resolution of any more bands, and the Amino acid analysis.-Freeze-dried samples of system was unsatisfactory as a slab gel, so for Fraction 1, which contained about 65 ,ig of this purpose the system of Panyim and Chalkley protein, were hydrolysed in va0uo in 6 mol/l (1969) was tried. This uses a 15% gel at pHs of aristar HC1 for 17 or 24 h at 110°. Analyses were 2-4 (without urea) or 2-7 (with 2-5 mol/I urea), carried out in an LKB 4101 amino acid analyser. after pre-electrophoresis to constant voltage. Preparation of staphylococcal spheroplasts and Separations were carried out in slab gels 14 cm treatment with cationic proteins.-4 h cultures of wide x 12 cm high and 1 mm thick, poured with Staph. aureus (P66) were grown in T-tubes in 9-11 sample slots. Samples (10-50 [lI) were BHI. The organisms were washed twice in applied in 15% sucrose and contained 30 ,ug GE phosphate-buffered saline and resuspended in the or under 10 ,ug fractionated GE, measured as minimum volume of 0-05 mol/l Tris-HCl buffer protein. 2-5 Hg each of lysozyme and protamine containing 0-045 mol/l NaCl at pH 7-4 (Tris sulphate with 20 ug of methyl green were run as buffer). Spheroplasts were prepared from these standards and marker. Electrophoresis was organisms by adding 3 x 101 colony forming carried out at the constant voltage of 100 V units (cfui) to 15 ml Tris buffer containing 30%

PROPERTIES OF POLYMORPHONUCLEAR PROTEIN FRACTIONS

sucrose and 1 unit lysostaphin/ml (Schuhardt and Klesius, 1968), and incubating for 10 min at 37°. The resulting spheroplasts were loosely pelleted by centrifuging for 5 min at 8,600 g at 4°. The pellet was resuspended to 9 ml and 2 x 1 ml portions were further incubated for 15 min at 37° with 150 ,tg each of GE, purified fraction of GE, or protamine sulphate in Tris buffer+ 30%o sucrose, or with buffer containing no added protein. Early studies used up to 10 times as mnuch GE. The treated spheroplasts were loosely pelleted as above before fixation and preparation for electron microscopy. Preparation of staphylococcal spheroplasts for electron microscopy. Treated and control spheroplasts were fixed in two different osmium tetroxide fixatives. The spheroplast pellets were resuspended in either 1 ml Kellenberger's standard bacterial fixative (Glauert, 1965a), or 3 ml Millonig's fixative (Glauert, 1965b). The latter procedure is good for preservation of the fine structure of cells. Similarly fixed Escherichia coli provided a marker. After fixation specimens were embedded in Araldite and sectioned on an LKB microtome. Before examination in the electron microscope sections were stained in lead citrate (Glauert, 1965c) for 5 min. Protein determination.-Rapid monitoring of the micropreparative polyacrylamide gel electrophoresis eluate was carried out by using the Coumassie Brilliant Blue G-250 binding technique of Bradford (1976). The most cationic fractions from GE, like protamine sulphate, give about twice the colour intensity at 595 nm as that given by bovine serum albumin, and more than twice that given by lysozyme (Walton, unpublished observations), whereas their extinction at 280 nm is almost zero. Routine protein estimations were carried out by the method of Lowry inodified by Bailey (1962). RESULTS

Fractionation of components of GE by micropreparative polyacrylamide gel electrophoresis Preliminary micropreparative electrophoretic runs with crude granular extracts yielded only very small amounts of fractions capable of killing staphylococci. These fractions seemed to contain the most cationic components of GE, and studies of the properties of the killing activity (e.g. dose required to kill, agents inhibiting killing) in comparison with protamine sulphate, a potent cationic bactericidal protein, showed that these seemed very similar. Huge losses of the material were

419

found on dialysis using ordinary dialysis tubing, indicating that like protamine the material had low-molecular-weight components. This finding was in contrast to the apparent non-dialysability of the crude GE before electrophoresis. Consequently, investigative micropreparative runs were carried out with protamine sulphate and lysozyme as test proteins. When ordinary dialysis tubing was used in the elution cell only about 50% of the protamine (MW 10,000 or below) was retrieved after electrophoresis, although lysozyme (MW 14,400) was completely recovered. When the dialysis tubing was replaced by a PM 10 membrane (exclusion MW 10,000) the recovery was increased to 80%, but when it was replaced by a UM 2 membrane (exclusion MW 1,000) complete recovery was obtained. This membrane was therefore used in all subsequent micropreparative runs, and dialysis of fractions after micropreparative electrophoresis was carried out in an Amicon ultrafilter containing a UM 2 membrane. A typical elution profile obtained from 20 mg freeze dried GE is shown in Fig. 1. Superimposed on the Uvicord trace are estimations of protein in the eluate obtained by the Coumassie Blue method. This method demonstrates the presence of the first eluted highly cationic components of GE, which show virtually no absorption at 280 nm. The following large and welldefined peak given by the Uvicord trace is probably lysozyme since it elutes from the gel in exactly the same place as test egg-white lysozyme, and like this protein gives a high absorption at 280 nm and a low colour intensity by the Coumassie Blue method. 1 ml fractions of eluate were pooled as indicated to give 5 major fractions, numbered 1-5, and analysed by electrophoresis on a 15% polyacrylamide gel at pH 2-4 (top, Fig. 1). A sample of crude GE run at the same time shows 5 fast-migrating bands (numbered I-V in order of decreasing cationic character) running before a compact band, VI, which migrates in almost the same position as the egg-white lysozyme marker.

420(

E. WALTON

FIG. 1. Bottom: typical elution profile obtained from micropreparative polyacrylamide gel electro, Uvicord trace (E280); - -- - -, E595 phoresis of 20 mg freeze-dried crude GE, at pH 4-3. obtained from Coomassie blue microassay using 10 pi sample (Scale 1); -- - -, E595 obtained from Coomassie blue microassay using 100 ILI sample (Scale 2). Top: analytical polyacrylamide gel electrophoresis at pH 2-4 of fractions pooled as indicated.

Staphylocidal activity.-The staphy- TABLE I. Killing of Staph. aureus (P66) locidal activity of 1,5,10,50 jug of Fractions by Fractions of Cationic Proteins from 1-5, unfractionated and recombined GE, Rabbit Leucocytes and protamine sulphate is shown in Table Minimum 00 organisms killing I. Only the fractions containing combinakilled in 4 h (lose (ug) Protein* tions of the 5 most cationic components Unfractionated GE 99-8 5 exhibit killing activity, and the most Fraction 1 63 1 5 99 7 lethal of these is Fraction 1, which con- Fraction 2 62 50 3 tains principally Bands I and II. These Fraction 0 Fraction 4 >50 bands migrate immediately behind the Fraction 5 0 >50 5 99-8 highly cationic protamine marker and, Recombined GE 1 74 combined as Fraction 1, show a similar Protamine * The components of these protein samples are level of staphylocidal activity. Fraction 3, in the top of Fig. 1. The maximum dose of which contains the lysozyme-like Band shown Fractions 4 and 5 tested was 50 jig. Recombined GE VI, and the two preceding cationic bands, was made by combining equal concentrations of IV and V, has very little killing activity. fractions 1-5. Recombined GE has a killing activity locidal activity and the acid gel analysis of similar to that of crude GE. A further definition of the staphylocidal 5 fractions obtained in this manner from a components of GE was achieved by pool- typical micropreparative run. These results ing smaller fractions before the lysozyme- clearly show that the three most cationic like Band VI. Fig. 2 shows the staphy- components of GE (Bands I-III, present in

PROPERTIES OF POLYMORPHONUCLEAR PROTEIN FRACTIONS

421

Hours

FIG. 2.-Bottom: Staphylocidal activity of the 5 most cationic fractions of GE obtained by micropreparative polyacrylamide gel electrophoresis at pH 4-3. Doses of protein are given as pg/025 ml test medium: (a) crude GE; (b) Fraction 1; (c) Fraction 2; (d) Fraction 3; (e) Fraction 4; (f) Fraction 5; (g) protamine sulphate (see text). Diagram above each set of graphs shows analytical polyacrylamide gel electrophoresis at pH 2-4 of each protein sample. The degree of band intensity is indicated by the shading.

Fractions 1 and 2) are the most effective staphylocidal agents, killing at doses between 1 and 5 ,ug. However, the less cationic components (Bands IV and V, present in Fractions 4 and 5) can kill if enough protein (50 fg) is used. Fraction 3, which contains as its principal component Band IV, together with smaller amounts of Bands II and III, kills at an intermediate dose of 10 ,ug. The purification of killing activity which has occurred in the preparation of Fraction 1 is not impressive and is only expressed in terms of minimum killing dose- 1,ug of Fraction 1 compared with 5 ,ug of GE. Some experiments with different combinations of fractions of GE have indicated that killing by Fraction 1 can be enhanced by the presence of ,ug amounts of practically any other protein, for example any fraction of GE, bovine serum albumin or lysozyme. However, the

results are not at all consistent, on some occasions added protein gives no enhancement of killing.

S.D.S. gel electrophoresis Fractions 1-5 described above and shown in Fig. 2 were analysed by S.D.S. gel electrophoresis, and the results are shown in Fig. 3. Fractions 1, 2 and 4, which contain respectively Bands I and II, Bands I, II and III, and Bands IV and V on acid gel electrophoresis, do not give discrete bands on S.D.S. gel electrophoresis. These fractions are, therefore, probably made up of components with a range of molecular weights from 3,500 (given by the glucagon marker), to below 14,000 (given by the RNAase and lysozyme markers). Since Fraction 1 migrates slightly more slowly than Fractions 2 and 4, and Fraction 2 slightly more slowly than Fraction 4, it is

422

E. WALTON

FiG. 3.-SDS gel electrophoresis of Fractions 1, 2, 4 and 5 shown in Fig. 2. Bands from left to right are: myoglobin (M, MW 17,000); lysozyme (L, MW 14,400); RNAase (R, MW 13,700); trypsin (MW 23,800) plus glucagon (MW 3,550), T + G; Fraction 1; Fraction 2; Fraction 4; Fraction 5; crude GE.

likely that Bands I and II, Bands I, II and haematin, at concentrations of 4*6 and III, and Bands IV and V contain pro- 0-24 mmol/I respectively, inhibit completely the staphylocidal activity of crude gressively lighter components. GE, Fractions 1 and 2, and protamine. Use Comparison of the properties of the major of a lower concentration of haematin staphylocidal fractions of GE with those of (0.03 mmol/1) differentiates between the crude GE and protamine sulphate less lethal crude GE and Fraction 2, which Previous studies have investigated the both contain all 5 staphylocidal comeffect of iron-containing compounds, re- ponents, and do not kill at doses below ducing agents, respiratory inhibitors and 5 /g, and the more lethal Fraction 1 and agents affecting energy transfer and oxida- protamine, which contain only highly tive phosphorylation, on the staphylocidal cationic components and kill at doses action of crude GE (Walton and Gladstone, around 1 jug. The former killing agents are 1976). The effects of these reagents on completely inhibited by 0 03 mmol/I samples of fractionated GE containing haematin and the latter are unaffected. Bands I and II, with sometimes a small However, this similarity between Fracamount of Band III (Fraction 1), or tion 1 and protamine does not extend to Bands I-V (Fraction 2), (cf. Fractions 1 their reaction to the presence of dithioand 2, Fig. 1) is now described and com- threitol. This reagent completely inhibits pared with their effects on crude GE and killing by crude GE and Fractions 1 and 2, protamine. but has no effect on the killing action of Effects of iron, haematin and dithio- protamine sulphate (Table II). threitol.-Table II shows that iron and Effects of respiratory inhibitors.-Table

423

PROPERTIES OF POLYMORPHONUCLEAR PROTEIN FRACTIONS

TABLE II.-Effect of Iron, Haematin and Dithiothreitol on the Killing of Staph. aureus (P66) by Cationic Proteins from Rabbit Leucocytes and by Protamine Sulphate Effect

on

killing in 4 h A-

Concentration

Compound

Crude GE

A

Fraction 2* Fraction 1 * (principally Bands I & II) (Bands I-V) Protamine

(mmol/l) 4-6 0 03 0-24 10 0

FeSO4 Haematin

-I0

+

±

0 +

0 Dithiothreitol * For components of Fractions 1 and 2 see text. Tests utilized 5 jig protein in the presence of FeSO4 and 10 ,ug protein in the presence of haematin and dithiothreitol. + =complete inhibition of killing; 0=no effect on killing.

III shows that all the respiratory inhibitors tested have similar actions on the staphylocidal activity of crude GE, Fractions 1 and 2, and protamine. Malonate and cyanide both completely inhibit killing, amytal and rotenone both enhance killing. HOQNO is again shown to have a dual effect on killing by crude GE, enhancing it

at a low concentration (0.1 ,umol/l) and inhibiting it at a higher concentration (100 ,umol/l). Killing by Fractions 1 and 2 responds similarly, but although killing by protamine can be enhanced by lower concentrations of HOQNO (> 1 ,mol/l), no inhibition of killing was demonstrated at the higher concentration of 100 tumol/l.

TABLE III.-Effect of Respiratory Inhibitors on the Killing of Staph. aureus (P66) by Cationic Proteins from Rabbit Leucocytes and by Protamine Sulphate Effect on killing by 10* ,ug protein in 4 h \-

-

Inhibitor Malonate

Concentration 0 * 1 mol/l

Amytal

10 mmol/l

Rotenone

Saturated solution 0 *05 mmol/l 01 ,umol/l

HOQNO

1

,umol/l

100 ,umol/l

Crude GE Inhibits completely (40-fold) Enhances (10-fold) Enhances (10-fold)

Fraction 1 Fraction 2 (principally Bands I & II) (Bands I-V) Inhibits Inhibits completely completely (25-fold) (20-fold) Enhances Enhances (2-fold) (45-fold) Enhances Enhances (10-fold) (20-fold)

Enhances (2-fold)

No effect

No effect

Enhances (2-fold) Inhibits (4-fold)

Enhances (3-fold) or inhibits (6-fold) Inhibits (5-fold) Inhibits completely

Protamine Inhibits completely (30-fold) Enhances (200-fold) Enhances (10-fold)

Enhances (4-fold) Enhances (10-fold) No effect

Inhibits completely (10-fold) (50-fold) 20 mmol/l Inhibits Inhibits Inhibits Inhibits Cyanide completely completely completely completely (20-fold) (30-fold) (40-fold) (20-fold) * 5 ,ug protein in the presence of rotenone as this reagent is added in ethanolic solution and ethanol alone enhances killing.

424

E. WALTON

TABLE IV.-Effects of Inhibition of Energy Transfer and of Uncoupling or Inhibition of Oxidative Phosphorylation on the Killing of Staph. aureus (P66) by Cationic Proteinns from Rabbit Leucocytes and by Protamine Sulphate

Inhibitor DCCD DNP

FCCP

Concentration 0 5 mmol/l 10 mmol/l

10

timol/l

Valinomycin

0 5 pg/ml

Gramicidin

0

Crude GE Enhances (3-fold) Inhibits completely (25-fold) Inhibits completely (250-fold) Inhibits

Effect on killing by 5* jig protein in 4 h Fraction 1 Fraction 2 (principally Bands I & II) (Bands I-V) Enhances Enhances (5-fold) (6-fold) Inhibits Inhibits

(10-fol(l)

* 5 ,ug/ml

No effect or inhibits up

(20-fold) Inhibits completely (700-fold) [nhibits (40-fold) Inhibits (10-fold)

Protamine Enhances

(8-fold) Inhibits

completely (40-fold)

(25-fold)

Inhibits

Inhibits

completely (150-fold)

completely

Inhibits

No effect

(6000-fold)

(15-fold) Inhibits

(15-fold)

Inhibits (10-fold)

to

90-fold * 5 [kg protein where reagents added in ethanol (DCCD, FCCP, valinomycin, gramicidin; 10 [kg protein where reagent added in aqueous solution (DNP).

Effects of inhibition of energy transfer and of uncoupling or inhibition of oxidative phosphorylation.- Studies with these reagents are summarized in Table IV. The enhancing action of DCCD, and the inhibitory actions of DNP, FCCP and valinomycin, on killing by crude GE, were all confirmed in this study. However, it has not been possible to demonstrate again the enhancing action of gramicidin on killing by GE. In this series of experiments gramicidin either inhibited killing or did not affect it at all. Killing by Fractions 1 and 2, and protamine, responds like GE to the presence of DCCD, DNP, FCCP and gramicidin. However, although valinomycin inhibits killing by GE and its fractions, it has no effect on killing by protamine.

Further properties of a fraction of GE containing only Bands I and II Since the fraction of GE which contains only Bands I and II (Fraction 1) is the most lethal to staphylococci, this fraction only was subjected to further investigations. Heat stability. Previous studies in this laboratory have shown that the staphylocidal activity of crude GE is stable to

see

Table II),

heating at 60° for 1 h. Samples of Fraction 1 were heated for 1 h at temperatures ranging from 500 to 90° and then tested for staphylocidal activity. The results are shown in Fig. 4 and clearly indicate that this fraction of GE is stable to heating up to 80°, but that between 800 and 90° its killing action is completely destroyed. Action of Fraction 1 on staphylococcal spheroplasts, and comparison with the actions of crude GE, and protamine sulphate.-Staphylococcal spheroplasts were originally prepared in order to see if crude GE could be shown to have any action on the cell membrane as observed under the electron microscope. The results were of sufficient interest to undertake studies with Fraction 1, in order to see whether the effects on the cell membrane were associated with the killing activity of the purified fraction, and for comparative purposes, with that of protamine. In the first set of experiments very high concentrations of GE (up to 1-5 mg protein/ml spheroplast suspension) were used, and after treatment spheroplasts were fixed in Kellenberger's fixative. Fig. 5, f and g show the characteristic blebs which are seen on both inner and outer membrane surfaces after treatment with 1-5 mg/ml GE. The

PROPERTIES OF POLYMORPHONUCLEAR PROTEIN FRACTIONS

425

TABLE V. Blebbing

of Staphylococcal Spheroplast Membranes after Treatment with Cationic Proteins from Rabbit Leucocytes Total number of Treatment

None

plus 0 15 mg/ml GE plus 0 75 mg/ml GE plus 15. mg/ml GE

spheroplasts counted 205 214 200 203

With Without blebs blebs 41 164 209 5 197 3 1 202

c 0

S0

Hours

Fic. 4. Heat stability of Fraction 1 from crtude GE. Graphs show the staphylocidal activity of 5 jig Fraction I in 0-25 ml test me(lium after heating for 1 h at the temperatures in(licated. Control incubations containe(i no protein, and untreated Fraction I storetd at 4°.

spheroplasts also seem very much more empty than those from control incubations with no added protein (Fig. 5, a and b). Table V shows that in the presence of concentrations of GE ranging from 0-15 to 1-5 mg/ml, nearly all the spheroplasts in a representative count of 200 show blebs, whereas in the absence of GE only 20% show blebs. This table is, however, somewhat misleading, since although the number of cells with blebs is nearly independent of the concentration of GE, the number of blebs per cell is dependent on the concentration: the degree of "blebbing" on the spheroplasts shown in Fig. 5, c, d and e, which have been treated with 0- 15 mg/ml GE, is very much less than that shown in Fig. 5, f and g, where the spheroplasts have been treated with 10 times this concentration of GE.

Treatment of spheroplasts with 0d15 mg/ ml of Fraction 1 protein (Fig. 5, h, i, j) followed by fixation in Kellenberger's fixative shows that changes in the membrane and spheroplast are similar to those occurring with crude GE, although the spheroplasts appear also to be somewhat shrunken and blebs are seen on both empty and full spheroplasts. There are also larger distortions and separations of the double structure of the membranes. Treatment with 0415 mg/ml protamine (Fig. 5, k, 1, m) gives a preparation containing much cellular debris in which blebs are seen on the spheroplasts but there also appear to be large areas where the double membrane is parted. Fig. 6 shows a similar sequence of spheroplasts after fixation in Millonig's fixative. These preparations, especially in control samples (Fig. 6, a and b), and in those treated with 0-15 mg/ml Fraction 1 or protamine (Fig. 6, f and g, and h, respectively) show many more full or partially full spheroplasts suggesting that this fixative is superior to Kellenberger's in preserving the structure of the spheroplasts. In empty spheroplasts it can also be seen that the double membrane shows up more clearly. The membrane changes brought about by addition of 0-15 mg/ml crude GE (Fig. 6, c, d and e), Fraction 1 (Fig. 6, f and g), or protamine (Fig. 6, h) are similar to those depicted in Fig. 5, but often more clear. The emptiness of many of the spheroplasts treated with crude GE suggests that the crude preparation may promote a more drastic disintegration of

426

E. WALTON

:.

"IW

9-0,10.6 PM

I

FIG. 5.-Electron micrographs of sections of staphylococcal spheroplasts treated with crude GE and with purified Fraction 1 and fixed in Kellenberger's fixative. a and b, untreated spheroplasts; c, d and e, plus 0-15 mg/ml GE; f and g, plus 1-5 mg/ml GE; h, i and j, plus 0-15 mg/ml Fraction 1 (containing cationic bands I and II); k, 1 and m, plus 0-15 mg/ml protamine sulphate.

PROPERTIES OF POLYMORPHONUCLEAR PROTEIN FRACTIONS

FIG. 6.-Electron micrographs of sections of staphylococcal spheroplasts treated with crude GE and with purified Fraction 1 and fixed in Millonig's fixative. a and b, untreated spheroplasts; c, d and e, plus 0-15 mg/ml GE; f and g, plus 0-15 mg/ml Fraction 1; h, plus 0-15 mg/ml protamine sulphate.

427

428

E. WALTON

TABLE V71. Amino acid Composition of Fraction 1 from Cationic Proteins of Rabbit Leucocytes Expressed as Percentage of Total Moles Recovered Basic amino aci(Is

Acidic amino acids

Arg

Asp Glti

Lys His

24 * 2 1 3 2 6

2 8 5 8

Others

Tyr Pro

Cys

2 7 8 2

7 9 7 3 7

Ser Thir G1l Ala Val1

10-2 4-1

LeILi

10-0

Tlti u

7-7

the cell membrane than does the purified Fraction l.

1

8-4

more

Amino acid analysis The amino acid analysis of Fraction 1 is shown in Table VI. The figures given are the mean of two determinations. Arginine alone constitutes 24% of the total amino acids and hence is the dominant cause of the cationic character of the protein. Histidine and lysine together (3*9 00) further contribute to this character. The acidic amino acids, glutamic and aspartic acids, comprise only 866% of the total. Fraction I also contains 807% cysteine (or cystine, seen in the analyser trace as cysteic acid) but only 2 7 % tyrosine and no phenylalanine. DISCUSSION

This study has been limited to the staphylocidal action of the different components of a crude extract of lysosomes from rabbit polymorphonuclear leucocytes, and hence conclusions cannot be drawn regarding their spectrum of antibacterial activity. However, it is of interest to compare these results with those of Zeya and Spitznagel (1968) and Odeberg and Olsson (1975) on rabbit and human granulocyte extracts respectively. Zeya and Spitznagel (1968) using sucrose density gradient electrophoresis, prepared 5 cationic fractions each containing

one cationic band in a high state of purity, and running before a fraction containing both RNAase (Band VI) and lysozyme (Band VII). They estimated antibacterial activity by measuring the amount of cationic fraction required to reduce bacterial growth by 5000 in 1 h at pH 5*6. Growth was measured by an optical density method. Antibacterial activity was found in Bands I-V, and slightly in bands VI and VII. However, in contrast to the results described in this paper, of the 4 strains of staphylococci tested, only one, a penicillinase-producer, was sensitive to Bands I and II. This strain was also sensitive to Bands III-V, and to a lesser extent to Bands VI+ VII. The other strains were sensitive only to Band III. The P66 strain used in this study was maximally sensitive to Bands 1+11. This difference could be ascribed to the different methods of determination of antibacterial activity (killing vs growth inhibition), to different test conditions (different pH), or to the fact that only one of Zeya and Spitznagel's strains (the penicillinase-producer) was similar to the Staph. aureus strain used here. Differences in the method of producing the antibacterial fraction may also be responsible. Olsson and Venge (1974), using chromatographic methods followed by preparative electrophoresis on agarose, resolved 7 cationic proteins. Odeberg and Olsson (1975) estimated antibacterial activity in the first 4 components (1-4) by measuring killing. Bacteria surviving incubation with cationic protein at pH 7*4 were determined by the pour-plate method. Staphylocidal activity was found in Fraction A (Bands 1+2, with 2 predominating) and Fraction B (Bands 3+4, with 4 predominating) but it was slightly greater in Fraction A. Bands I and 2 run ahead of lysozyme, and Bands 3 and 4 are nearly coincident with it. Killing could be detected after 1 h if 510 jug cationic protein was added. The level and pattern of the staphylocidal activity described by Odeberg and Olsson is, therefore, similar to that described in this paper.

PROPERTIES OF POLYMORPHONUCLEAR PROTEIN FRACTIONS

The dependence of the staphylocidal action of a crude granular extract on bacterial oxidative energy metabolism has been previously shown (Walton and Gladstone, 1976). This paper demonstrates that killing of staphylococci by Fractions 1 (Bands I and II) and 2 (Bands I-V) is similarly dependent. A parallel has already been drawn between the effects of agents altering oxidative energy metabolism on the action of histones on mitochondria (Johnson, Goldstein and Schwartz, 1973), and on the action of cationic proteins (GE) on staphylococci (Walton and Gladstone, 1976). Here, the similarity of the action of GE on staphylococci to that of another highly cationic protein, protamine, is shown. Killing by protamine shows a pattern of sensitivity to agents altering energy metabolism which is practically identical to that given by crude GE and its two most cationic fractions. The only agent tested to which it is not sensitive is valinomycin. The role of metabolic energy in the lethal action of basic proteins on Candida albicans has also recently been demonstrated by Olson, Hansing and McClary (1977). The general similarity of protamine, histones and other basic proteins to GE and its fractions suggests that the role of cationic proteins extracted from leucocyte granules in bacterial killing and in membrane respiratory or energy-producing activities may be quite nonspecific and entirely dependent on their basic character. Killing by protamine is also similar to killing by GE and its cationic fractions in its sensitivity to Fe and haematin, but it is quite different in its lack of sensitivity to dithiothreitol. This difference can be explained by the differing amino acid compositions of protamine and Fraction 1 of GE. Protamines do not generally contain sulphur-containing amino acids (Felix, 1960). Fraction 1 contains cystine/cysteine and the inactivation of its killing activity by dithiotreitol suggests that its action may depend on the presence of disulphide bonds. Protamines also contain much larger percentages of arginine (66 % or

429

more) than Fraction 1 (24%) and very little or no tryptophan, tyrosine and phenylalanine. The amino acid analysis of Fraction 1 is similar to those given by Zeya and Spitznagel (1968) for the analysis of their most basic fractions (Bands I, II, III) although their samples contain more arginine (32-37%). The two most cationic components isolated by Olsson and Venge (1974) from human cells, although containing a high percentage of arginine (14%) also contain a high percentage (20%) of acidic amino acids. Other properties of the fractions of GE described here have also been demonstrated by other workers. The molecularweight values given by SDS gel electrophoresis for Fractions 1-5 are in agreement with similarly low values found using gel filtration chromatography by Ranadive and Cochrane (1968). These workers isolated 4 cationic proteins (Bands 1-4) from rabbit neutrophils which increased vascular permeability. Bands 1 and 2, which had the highest arginine contents (10 and 18% respectively) had molecular weights around 5,000, Band 3 was similar, and Band 4 had a molecular weight of 12,000. The molecular weight of Band 2 protein (as mast-cell rupturing factor) was also estimated by Seegers and Janoff (1966) as between 1,200 and 2,400. However, Olsson and Venge (1974), using SDS gel electrophoresis, demonstrated much higher molecular weights for cationic Components 1-4 (25,500-29,500) and 5-7 (21,000-26,000). Several workers have demonstrated the heat stability of the killing action of leucocyte extracts (Hirsch, 1956; Skarnes and Watson, 1956; Hibbitt, Brownlie and Cole, 1971; Odeberg and Olsson, 1975; Weiss et al., 1975). These studies confirm this finding: Fraction 1 from crude GE is stable to heating up to 80° for 1 h. The action of leucocyte extracts on bacterial spheroplasts has not previously been studied, although there have been studies with whole bacteria. Macmillan and Hibbitt (1969) demonstrated changes in both cell wall and membrane of staphy-

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lococci treated with antimicrobial cationic proteins from the teat canal of the cow, although the most marked change was the lack of a discrete cell wall. Changes induced by calf thymus histone were similar, but those induced by protamine sulphate or polylysine were quite different: in these cases the cell wall remained intact but membranous bodies were found associated with it and with the septum between dividing organisms. These bodies bear a distinct similarity to some of those demonstrated in the spheroplast preparations described in this paper. Later work by Hibbitt and Benians (1972) on the site of action of cationic proteins on staphylococci demonstrated that fluorescent calf thymus histones bound to membrane fractions isolated from staphylococci, and to whole staphylococcal spheroplasts, although actual changes in the membrane were not reported. Studies by Johnson et al. (1973) demonstrated that addition of histone to mitochondria caused a marked unfolding of the usually highly folded inner membrane. The electron micrographs in this paper seem to indicate a similar unfolding or spreading, rather like the action of a detergent on a lipid film. Weiss, Elsbach and their co-workers (Beckerdite et al., 1974; Weiss et al., 1975; Weiss et at., 1976) have studied the killing of Escherichia coli by a highly purified fraction from rabbit polymorphonuclear leucocytes, high in phospholipase A2 activity, and devoid of lysozyme, myeloperoxidase and protease activities. They conclude that killing occurs with minimal structural and functional disorganisation: changes in the cell envelope appear to be discrete and reversible (e.g. permeability and phospholipid hydrolysis), and electron microscope studies of thin sections of E. coli exposed to the purified fraction show slight changes only in the outer membrane. One of the most recent papers by these workers (van Houte et al., 1977) uses freeze-fracture electron microscopy, a method designed to detect alterations in both cytoplasmic and outer membranes of Gram-negative organisms. The study

demonstrates no recognisable morphological changes in E. coli treated with purified fraction. However, the constitution of this purified fraction is to some degree unknown as no electrophoretic studies or other chemical analyses have been reported, although it has many of the properties of a pure phospholipase A2 preparation. It is therefore difficult to compare results, especially as the organism being studied is the Gram-negative E. coli, and Odeberg and Olsson (1975) have demonstrated that this organism, along with the Gram-negative Pseudomonas aeruginosa, is considerably less sensitive to the killing action of the 4 most cationic proteins isolated from human granulocytes. I wish to record thanks to Dr G. P. Gladstone for help and advice, to Mr H. Stroud and his staff for construction of the preparative electrophoresis apparatus, and to Mr A. J. Hayle for able technical assistance. REFERENCES BAILEY, J. L. (1962) In Techniques in Protein Chemistry. New York: Elsevier Publishing Co. p. 293. BECKERDITE, S., MOONEY, C., WEISS, J., FRANSON, R. & ELSBACH, P. (1974) Early and Discrete Changes in Permeability of Escherichia coli and Certain Other Gram-negative Bacteria During Killing by Granulocytes. J. exp. Med., 140, 396. BRADFORD, M. M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochem., 72, 248. FELIX, K. (1960) Protamines. Adv.Prot. Chem., 15, 1. FoIssY, H. & SUMMER, K. (1976) Electrophoretic Fractionation of Minute Protein Samples Using a Micropreparative Device. Science, N. Y., 25, 582. GLADSTONE, G. P., WALTON, E. & KAY, U. (1974) The Effect of Cultural Conditions on the Susceptibility of Staphylococci to Killing by the Cationic Proteins from Rabbit Polymorphonuclear Leucocytes. Brit. J. exp. Path., 55, 427. GLAUERT, A. M. (1965a) In Techniques in Electron Microscopy. D. H. Kay, Ed. Oxford: Blackwell Scientific Publications Ltd., Ch. 7, p. 176. GLAUERT, A. M. (1965b) Ibid. Ch. 7, p. 174. GLAUERT, A. M. (1965c) Ibid. Ch. 9, p. 269. HIBBITT, K. G., COLE, C. B. & REITER, B. (1969) Antimicrobial Proteins Isolated from the Teat Canal of the Cow. Biochemn. J., 56, 365. HIBBITT, K. G., BROWNLIE, J. & COLE, C. B. (1971) The Antimicrobial Activity of Cationic Proteins Isolated from the Cells in Bulk Milk Samples. J. Hyg., Camb., 69, 61.

PROPERTIES OF POLYMORPHONUCLEAR PROTEIN FRACTIONS HIBBITT, K. G. & BENIANS, M. (1972) The Site of Action of Antimicrobial Cationic Proteins on Staphylococci. Biochem. J., 126, 26P. HIRSCH, J. G. (1956) Phagocytin: a Bactericidal Substance from Polymorphonuclear Leucocytes. J. exp. Med., 103, 589. JOHNSON, C. L., GOLDSTEIN, M. A. & SCHWARTZ, A. (1973) Biochemical and Ultrastructural Studies on the Interaction of Basic Proteins with Mitochondria: a Primary Effect on Membrane Configuration. Arch. biochem. Biophys., 157, 597. LAEMMLI, U. K. (1970) Cleavage of Structural Proteins During the Assembly of the Head of Bacteriophage T4. Nature (Lond.), 227, 680. MACMILLAN, W. G. & HIBBITT, K. G. (1969) The Effect of Antimicrobial Proteins on the Fine Structure of Staphylococcus aureus. J. gen. Microbiol., 56, 373. ODEBERG, H. & OLSSON, I. (1975) Antibacterial Activity of Cationic Proteins from Human Granulocytes. J. clin. Invest., 56, 1118. ODEBERG, H. & OLSSON, I. (1976) Mechanisms for the Microbicidal Activity of Cationic Proteins of Human Granulocytes. Infectn. & Immun., 14,

269. OLSON, V. L., HANSING, R. L. & MCCLARY, D. 0. (1977) The Role of Metabolic Energy in the Lethal Action of Basic Proteins on Candida albicans. Can. J. Microbiol., 23, 166. OLSSON, I. &. VENGE, P. (1974) Cationic Proteins of Human Granulocytes. II. Separation of the Cationic Proteins of the Granules of Leukemic Myeloid Cells. Blood, 44, 235. PANYIM, S. & CHALKLEY, R. (1969) High Resolution Acrylamide Gel Electrophoresis of Histones. Arch. biochem. Biophys., 130, 337. RANADIVE, N. S. & COCHRANE, C. G. (1968) Isolation and Characterization of Permeability Factors from Rabbit Neutrophils. J. exp. Med., 128, 605. REISFELD, R. A., LEWIS, U. J. & WILLIAMS, D. E. (1962) Disk Electrophoresis of Basic Proteins and Peptides on Polyacrylamide Gels. Nature (Lond.), 195, 281.

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SCHUHARDT, V. T. & KLESIUS, P. H. (1968) Osmotic Fragility and Viability of Lysostaphin-Induced Staphylococcal Spheroplasts. J. Bact., 96, 734. SEEGERS, W. & JANOFF, A. (1966) Mediators of Inflammation in Leukocyte Lysosomes. VI. Partial Purification and Characterization of a Mast CellRupturing Component. J. exp. Mled., 124, 833. SKARNES, R. C. & WATSON, D. W. (1956) Characterization of Leukin: an Antibacterial Factor from Leucocytes Active against Grampositive Pathogens. J. exp. Med., 104, 829. vAN HOUTE, A. J., ELSBACH, P., VERKLEIJ, A. & WEISS, J. (1977) Killing of E8cherichia coli by a Granulocyte Fraction Occurs Without Recognizable Ultrastructural Alterations in the Bacterial Envelope as Studied by Freeze-Fracture Electron Microscopy. Infectn. & Immun., 15, 556. WALTON, E. & GLADSTONE, G. P. (1976) Factors Affecting the Susceptibility of Staphylococci to Killing by the Cationic Proteins from Rabbit Polymorphonuclear Leucocytes: the Effects of Alteration of Cellular Energetics and of Various Iron Compounds. Brit. J. exp. Path., 57, 560. WEISS, J., FRANSON, R. C., BECKERDITE, S., SCHMEIDLER, K. & ELSBACH, P. (1975) Partial Characterization and Purification of a Rabbit Granulocyte Factor that Increases the Permeability of Escherichia coli. J. clin. Inve8t., 55, 33. WEISS, J., FRANSON, R. C., SCHMEIDLER, K. & ELSBACH, P. (1976) Reversible Envelope Effects During and After Killing of E8cherichia coli by a Highly-Purified Rabbit Polymorphonuclear Leukocyte Fraction. Biochem. biophys. Acta, 436, 154. WEISS, J. & ELSBACH, P. (1977) The Use of a Phospholipase A-Less Escherichia coli Mutant to Establish the Action of Granulocyte Phospholipase A on Bacterial Phospholipids During Killing by a Highly Purified Granulocyte Fraction. Biochem. biophy8. Acta, 466, 23. ZEYA, H. I. & SPITzNAGEL, J. K. (1968) ArginineRich Proteins of Polymorphonuclear Leucocyte Lysosomes. Antimicrobial Specificity and Biochemical Heterogeneity. J. exp. Med., 127, 927.