Br. J. exp. Path. (1975) 56, 459

A STUDY OF THE ACTION OF THE CATIONIC PROTEINS FROM RABBIT POLYMORPHONUCLEAR LEUCOCYTES ON THE STAPHYLOCOCCAL CELL MEMBRANE E. WALTON AND G. P. GLADSTONE From the Sir William Dunn School of Pathology, Univer8ity of Oxford Received for publication May 7, 1975

Summary.-The inhibitory effect of cationic proteins from rabbit polymorphonuclear leucocytes on the oxidation of NADH by staphylococcal membrane preparations is described. Both cyanide and haematin are shown to interfere with the inhibitory process, by different mechanisms. Other authors have shown that glucose repressed staphylococci are diverted to a fermentative mode of metabolism. These findings were confirmed by demonstrating that membrane preparations from staphylococci grown in the presence of glucose have diminished cytochrome and succinic dehydrogenase levels. From a comparison of the effect of the cationic proteins on NADH oxidation in membrane preparations from organisms grown normally and under conditions of glucose repression, and from knowledge of the different susceptibility to the cationic proteins of the two types of organisms, it is suggested that the cationic proteins exert their bactericidal action on staphylococci following an energy dependent binding to the membrane.

VARIOUS studies have implicated the cell membrane as the site of action of polycationic substances both in animal cells, to which they are cytotoxic, and in bacteria, to which they are bactericidal. Alterations in cellular and mitochondrial permeability, oxygen consumption and adenosine triphosphatase activity (Schwartz, 1965a, b; Zeya and Spitznagel, 1966; Harold, 1970; Mayhew, Harlos and Juliano, 1973; Rosenthal and Buchanan, 1974) have been demonstrated. The two processes of transport and energy generation are intimately linked at the level of the membrane (Harold, 1972). Penniall and Zeya (1971) have shown that the bactericidal proteins from rabbit polymorphonuclear leucocyte lysosomes exert a biphasic effect on the respiratory activity of isolated rat liver mitochondria, stimulating it at low concentrations and inhibiting at high concentrations. The site of inhibition has been shown to be the membrane bound cytochrome oxidase which was extracted using Tritons X-114

and X-100 and measured by the oxidation of cytochrome c. There was some evidence for a precipitation of the oxidase by one of the protein fractions (Penniall, Holbrook and Zeya, 1972). In bacteria, very few studies have been carried out at this level. Harold (1964) has shown that spermine and other polycations stabilize protoplasts of Streptococcusfaecalis against gross lysis, probably by binding to acidic surface groups. Hibbitt and Benians (1972) demonstrated that fluorescent labelled calf thymus histone binds to the cell membrane fraction of staphylococci and can also be seen attached to staphylococcal protoplasts. Gooch and Donaldson (1974) showed that f8-lysin from rabbit serum reacts with the cytoplasmic membrane of Bacillus megaterium, and induces a change in its structure. Previous work from this laboratory (Gladstone, Walton and Kay, 1974) suggested that the respiratory enzymes of the staphylococcal cell membrane are the sites of action of the bactericidal cationic

460

E. WALTON AND G. P. GLADSTONE

twice in cold phosphate buffered saline before resuspension in 0-01 mol/l Tris-HCl buffer, pH 8-0, containing 0-1 mol/l KCI, to give 30-35 ml of a thick suspension. Preparation of membranes. The majority of the staphylococci were broken up by shaking 11-ml portions of the cold thick suspension with 14 ml of Ballotini beads in a Braun disintegrator for a total of 10 min. The disintegration time was divided into 3, with cooling after each shaking. The beads were removed by filtration at a sintered glass Buchner funnel. Unbroken staphylococci and cell wall fragments were removed from the filtrate by centrifugation at 10,000 g for 15 min at 4°. The membrane fragments were removed from the supernatant by centrifugation at 100,000 g for 1 h in the cold. After washing twice in Tris-KCl buffer they were stored frozen at -20° as pellets. Spectral analysis for cytochrome content was carried out the next day but enzymic investigations could be undertaken up to several weeks after preparation. Analysis of cytochrome content of membranes. The membrane pellets were suspended in a small volume of Tris-KCl buffer. After removal of any lumps which would not resuspend by brief centrifugation, the suspension was diluted with twice its volume of glycerol. The cytochrome content was investigated with a Cary model 14 recording spectrophotometer using the sensitive slide wire. Reduced minus oxidized and carbon monoxide reduced minus reduced difference spectra were obtained, using dithionite as reducing agent. Enzyme assays.-The activities of 3 enzymes in the membranes were measured in a Unicam SP 800 recording spectrophotometer in the MATERIALS AND METHODS following systems after resuspension of the memStaphylococci.-All the work described here brane pellets in a small volume of Tris-KCl has been carried out with Staphylococcus aureus, buffer and removal of any non-resuspended strain P66 (Gladstone and Walton, 1971). material: Mlembranes were prepared from 6 h cultures of Succinic dehydrogenase: Na succinate, 13-3 staphylococci grown at 370 in 2-3 1 of 10% mmol/l; KCN, 1 mmol/l; 2,6-dichlorophenolproteose peptone broth, made up using 0-067 indophenol, 2-5 mg/100 ml; phenazine methoinol/l phosphate buffer, pH 7 4, with added sulphate, 13-3 mg/100 ml; potassium phosphate lactate or glucose (0-067 mol/l). The culture buffer, pH 8-0, 0-167 mol/l; water to a total xessel was a Biotec Fermenter (obtained from volume of 3 ml; 10-20 ,ul appropriately diluted LKB Instruments) in which rates of stirring membrane preparation. The change in extincand aeration were kept constant. No control tion at 600 nm after addition of enzyme was of pH was necessary in the lactate cultures but measured. the pH of the glucose cultures was monitored NADH dehydrogenase: NADH, 0-1 mg/ml; throughout the period of growth and auto- 2,6-dichlorophenol-indophenol, 2-5 mg/100 ml: inatically adjusted to 7-4 by addition of 1 mol/l KCN, 1 mmol/l; potassium phosphate buffer, NaOH. The inoculum was 250 ml of stagnant pH 8-0, 0-167 mol/l; water to a total volume of 6-h culture in the same medium. When har- 3 mi; 10-20 ul appropriately diluted membrane -ested, the organisms were approaching the end preparation. The change in extinction at 600 of the log phase and the numbers developing in nm after addition of enzyme was measured. each type of culture were similar and reproNADH oxidase: Potassium phosphate buffer, duicible (mean viable count 2-4 x 109/ml). The pH 8-0, 0-167 mol/l; water to a total volume of organisms were collected by centrifugation at 3 ml; 10-20 ,ul appropriately diluted membrane 6000 g for 5 min in the cold. They were washed preparation; NADH, 0-1 mg/ml. The change

proteins from rabbit polymorphonuclear leucocytes. Bacteria without cytochromes, such as Streptococcus faecalis, which obtain their energy anaerobically were found to be resistant to the cationic proteins. Moreover, staphylococci grown under conditions of repression by glucose leading to enhanced glycolysis, suppressed Krebs cycle, decreased pentose cycle and suppression or decrease of various enzymes including the cytochromes (Collins and Lascelles, 1962; Strasters and Winkler, 1963) were also shown to be resistant. Similarly, staphylococci were resistant when grown under anaerobic conditions in which they become deficient in cytochromes (Jacobs and Conti, 1965). It was also found that aerobic respiration is essential during the killing process, for if the conditions of the test prevent or reduce aerobic respiration, as in anaerobiosis or in the presence of cyanide, the bactericidal action of the cationic proteins is inhibited (Walton and Gladstone, unpublished observations). In this paper an investigation of the cytochrornes and respiratory enzymes of staphylococcal niembranes, and their interaction with the cationic proteins from rabbit polymorphonuclear leucocyte lysosomes, are described.

461

A STUDY OF THE ACTION OF CATIONIC PROTEINS

in extinction at 340 nm after addition of NADH was measured. JMaterials.-NADH, 2,6-dichlorophenol-indophenol and phenazine methosulphate were obtained from Sigma Chemical Corp. Other chemicals were reagent grade obtained from commercial souirces. Proteose peptone was obtained from Oxoid. Solutions of glucose, sodiuim lactate and haematin were prepared as previously described (Gladstone et al., 1974). Cationic proteins (granular extract: GE) from the lysosomes of rabbit polymorphonuiclear leucocytes (PMN) were prepared as previously described (Gladstone et al., 1974). l'rotein estimcation. -This was ctarried otut by the miethod of Lowry modified by Bailey (1962). RESULTS

The cytochrome content of the membranes The reduced minus oxidized difference spectra in Fig. 1 show the cytochrome contents of membrane preparations from Staph. aureus P66 grown in lactate-or glucose-containing media. The pattern of peaks is the same in both preparations and similar to that given by Jacobs and Conti (1965) and Taber and Morrison (1964). It indicates that presence of an a type cytochrome with peaks at 602 and 442 nm, and a b1 type with peaks at 558, 0.15

_

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-

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Wavelength (nm)

FIG. 1. Reduced minus oxidized difference spectra of membrane preparations from Staph. aureus (P66) grown aerobically in proteose peptone broth with added lactate or glucose (0-067 mol/l). Membrane preparation from lactate-grown organisms (4.9 mg protein/ml); -----membrane preparation from glucosegrown organisms (4.7 mg protein/ml).

0.15

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400

450

500

550

600

Wavelength (nm)

Carbon monoxi(le reduceed minus reduced(ldifference spectra of membrane preparations from Staph. aureus (P66) grown aerobically in proteose peptone broth with added lactate or glucose Membrane pre(0-067 mol/l). paration from lactate-grown organisms as above, (5-2 mg protein/ml); but with added GE (0.7 mg/ml); -----membrane preparation from glucose-grown organisms (4-7 mg protein/ml).

FYi. 2.

526 and 428 nm. The levels of the 2 cytochromes are very much lower in the glucose grown membranes. The carbon monoxide reduced minus reduced difference spectra from the 2 preparations are shown in Fig. 2. There is only one clear peak shown, at 416 nm, although other workers have demonstrated small absorption bands in the visible region. This peak indicates the presence of a carbon monoxide binding cytochrome of the o type (Jacobs and Conti, 1965). The level of this cytochrome is also very low in the glucose grown membranes. Figure 2 also shows the effect of adding cationic proteins from rabbit PMN (GE) on this difference spectrum. The absorption peak due to cytochrome o is altered and there is a general increase in absorption in the visible region. This is probably due to increased light scattering, since precipitation visible to the naked eye occurred when GE was added.

E. WALTON AND G. P. GLADSTONE

462

TABLE.-Activities of Enzymes Connected with the Cytochrome Chain in Membrane Preparations from Staph. aureus (P66) Grown Aerobically in 1% Proteose Peptone Broth with Added Lactate or Glucose Specific activity of enzyme* A

I

Conditions of growth With lactate (0 067 mol/l) With glucose (0 * 067 mol/l) -

NADH oxidase 0-152 ±0 038 0- 162 ±0-031

NADH dehydrogenase 0 34 0 056 0 358 ±O* 084

Succinic dehydrogenase 0 226 ±0 016 0 038 ±0 007

Ratio of enzyme activities 1: 2-23: 1*49 1: 2-22: 0 23

* umol substrate oxidized/mg protein/min; mean of assays from 3 membrane preparations. Differences in sample variances are not significant (values of F give P > 0 -1). Differences between NADH oxidase and dehydrogenase activities are not significant (P > 0 8). Difference between succinic dehydrogenase activities is significant (P < 0-001).

The enzymic content of the membranes The Table shows the levels of the 3 enzymes investigated in glucose- and lactate grown membranes. The specific activity of succinic dehydrogenase is reduced by 83% in the glucose grown membranes but, despite their differences in cytochrome content, the 2 preparations show no significant difference in the specific activities of either NADH dehydrogenase or NADH oxidase. The total activity of these 2 enzymes is, how-

ever, reduced in the glucose grown membranes since they contain only 70% of the protein found in lactate grown membranes.

The effect of the cationic proteins on the membrane enzymes An investigation of the effect of GE on the 3 enzymes showed that it has a marked

C

c

'C CZ

Time (sec)

Fio. 3.- The inhibition of NADH oxidase in a membrane preparation from Staph. aureu8 (P66) by cationic proteins from rabbit leucocytes. Oxidation of NADH was measured in the systsm described using 0-041 mg/ml membrane protein from a culture grown in lactate, alone, and in the presence of 0-27 mg/ml GE.

GE(mg/ml)

FIG. 4.-The inhibition of NADH oxidase in a membrane preparation from Staph. aureus (P66) grown in the presence of lactate, by cationic proteins from rabbit leucocytes.

A STUDY OF THE ACTION OF CATIONIC PROTEINS

463

effect only on the oxidase. It slightly stimulates NADH dehydrogenase and slightly inhibits succinic dehydrogenase but markedly inhibits NADH oxidase (Fig. 3). A turbidity develops on addition of GE to the membrane preparation. This is reflected by an increase in absorption at 340 nm, which obscures the initial rate given in the presence of GE. When the development of turbidity has ceased (within about 75 s), a steady inhibited rate of NADH oxidation is seen. This can reach 70% at high concentrations of GE (0-2-0-3 mg/ml). Figure 4 shows that the relationship between concentration of GE and rate of NADH oxidation is not linear. A plot of the reciprocal of the rate against concentration of GE also shows a non-linear relationship, indicating that the inhibition caused by GE is more complex than the simple competitive or ion-competitive types. The effect of GE 63.2 Time (1 division = 50sec)

50.6 GE

(65Vtg/ml)

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has not yet been further investigated but it appears to be similar whether the oxidation of NADH is studied using glucose- or lactate grown membranes.

The effect of potassium cyanide At a concentration of 6 mmol/l, KCN 179 ~~~~~~9.4 inhibits \p 17.9f> membrane NADH oxidase activity KCN by 90%; at 0-6 mmol/l it still inhibits it by - (1.5 mmol/i) 40%. Figure 5 shows that GE and KCN GE together inhibit NADH oxidase less than (130 pg/ml) the sum of their separate inhibitions; it Time (1 division = 50 sec) appears that they interfere with one The effect of cyanide on the inhibianother's inhibitory activities.

CL

GE

63.2

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FIG. 6.-The effect of haematin on the inhibition of NADH oxidase in a membrane preparation from Staph. aureus (P66) grown in the presence of lactate, by cationic proteins from rabbit leucocytes. Figures give rates as ,umol NADH oxidized/mg

(130g/mI)

FiG. 5. tion of NADH oxidase in

a membrane preparation from Staph. aureus (P66) grown in the presence of lactate, by cationic proteins from rabbit leucocytes. Figures give rates as nmol NADH oxidized/mg protein/min.

The effect of haematin Figure 6 shows that haematin, at a concentration of 30 ,umol/l, stimulates

E. WALTON AND G. P. GLADSTONE

464

NADH oxidase by 30%O. If GE is added to the stimulated oxidase, the rate returns to the original value but no inhibition develops. If haematin is added to the enzyme system when it is already inhibited by GE, the inhibition is partially reversed. Haematin is inhibitory to the 2 dehydrogenases studied. At 0-1 mmol/l their activity is inhibited up to 90%o and some inhibition is still evident at 30 /imol/l, the concentration used in studies with the oxidase. DISCUSSION

These studies with staphylococcal membranes are in conformity with the results of other investigators, who have shown that growth in a glucose containing medium enhances a fermentative mode of metabolism. The changes demonstrated here are decreased levels of cytochromes and succinic dehydrogenase but no decrease in the specific activities of NADH oxidase and dehydrogenase, although their total activities were slightly reduced. However, in the glucose repressed cell the source of electrons to the respiratory chain would be substantially diminished in the absence of operation of the Krebs cycle. Taber and Morrison (1964) have suggested the following scheme for the electron transfer sequence of staphylococci:

The spectral analysis showed that GE altered the Soret absorption peak of the cytochrome o-CO complex. The inhibitory effect of GE on NADH oxidase, but not on succinic or NADH dehydrogenases, could therefore be explained by its combination with the membrane at the site of the terminal respiratory enzyme of the chain, cytochrome o. The experiments with cyanide and haematin confirm this idea. Cyanide is an efficient inhibitor of staphylococcal aerobic respiration and it also inhibits the staphylocidal action of GE. At low concentrations it is a specific inhibitor of cytochrome oxidase, although there is no definite evidence for the formation of a cytochrome-cyanide complex (Taber and Morrison, 1964). It has been shown here that cyanide and GE interfere with one another's inhibitory actions on membrane NADH oxidase, suggesting again that GE acts at the end of the respiratory chain. Haematin and iron precipitate GE and also inhibit its bactericidal action (Gladstone and Walton, 1970). In the staphylococcal membrane system, haematin can reverse the inhibition of NADH oxidation caused by GE and, if added to the system first, can prevent it from developing. These results suggest that GE may bind specifically to the protoheme prosthetic group of cytochrome o, but that it binds

light sensitive substrate

--

flavoprotein

--

naphthoquinone --4 cyt b (vitamin K2)

cyt

a --+

NO3

cyt

o -°-+

02

--

CO

The light sensitive naphthoquinone of their preferentially to free haematin. If the scheme has been identified as vitamin K2 environment of the prosthetic groups of by Bishop, Pandya and King (1962). the other 2 cytochromes are accessible, Electrons feed into the chain from mem- GE could also bind here. Any membrane brane bound primary flavoprotein de- distortion resulting from the binding hydrogenases which obtain electrons from could have far-reaching effects on such various substrates, either directly (e.g. processes as oxidative phosphorylation lactate) or via NAD. Vitamin K2 links and membrane transport. these dehydrogenases with the cytoIn organisms possessing no cytochromes but does not appear to be in- chromes, or in those with very low levels volved in electron transfer from succinic of cytochromes, such as streptococci or dehydrogenase (Goldenbaum, Keyser and staphylococci grown anaerobically or with glucose, little or no killing would occur White, 1975).

A STUDY OF THE ACTION OF CATIONIC PROTEINS

because very little binding and distortion by GE would be possible. However, further consideration of the resistance of glucose grown staphylococci to GE shows that this hypothesis requires modification. Membrane preparations from these organisms can carry out a nearly normal level of NADH oxidation despite the fermentative mode of metabolism of the whole organism. It is therefore necessary to suggest that a functional cytochrome chain, which is not possessed by glucose grown staphylococci or by those treated with cyanide, is essential for the binding and subsequent action of GE. Johnson, Goldstein and Schwartz (1973) have put forward a similar hypothesis, suggesting that the primary action of cationic proteins on the mitochondrial membrane involves an energy dependent structural alteration.

We wish to record our thanks to Dr D. A. Broadbent of the Department of Microbiology, University of Oxford, for help and advice with the staphylococcal membrane preparation, and with cytochrome and enzyme estimations; and to Mr A. J. Hayle for able technical assistance. REFERENCES BAILEY, J. L. (1962) In Techniques in Protein Chernistry. New York: Elsevier Publishing Co. p. 293. BISHOP, D. H. L., PANDYA, K. P. & KING, H. K. (1962) Ubiquinone and Vitamin K2 in Bacteria. Biochem. J., 83, 606. COLLINS, F. M. & LASCELLES, J. (1972) The Effect of Growth Conditions on Oxidative and Dehydrogenase Activity in Staphylococcus aureus. J. gen. Microbiol., 29, 531. GLADSTONE, G. P. & WALTON, E. (1970) Effect of Iron on the Bactericidal Proteins from Rabbit Polymorphonuclear Leucocytes. Nature, Lond., 227, 849. GLADSTONE, G. P. & WALTON, E. (1971) The Effect of Iron and Haematin on the Killing of Staphylococci by Rabbit Polymorphs. Br. J. exp. Path., 52, 452. 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. Br. J. exp. Path., 55, 427. 33

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GOLDENBAIJM, P. E., KEYSER, P. D. & WHITE, D. C. (1975) Rolo of Vitamin K2 in the Organization and Function of Staphylococcus aureus Membranes. J. Bact., 121, 442. GOOCH, G. T. & DONALDSON, C. M. (1974) Reactions of fl-Lysin with Purified Cytoplasmic Membranes of Bacillus megaterium. Infect. Immun., 10, 1180. HAROLD, F. M. (1964) Stabilization of Streptococcus faecalis Protoplasts by Spermine. J. Bact., 88, 1416. HAROLD, F. M. (1970) Antimicrobial Agents and Membrane Function. Adv. Microbial Physiol.,

4, 45. HAROLD, F. M. (1972) Conservation and Transformation of Energy by Bacterial Membranes. Bact. Rev., 36, 172. HIBBITT, K. C. & BENIANS, M. (1972) The Site of Action of Antimicrobial Cationic Proteins on Staphylococci. Biochem. J., 126, 26P. JACOBS, N. J. & CONTI, S. F. (1965) Effect of Haemin on the Formation of the Cytochrome System of Anaerobically Grown Staphylococcus epidermidis. J. Bact., 89, 675. 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. Archs biochem. Biophys., 157, 597. MAYHEW, E., HARLOS, J. P. & JULIANO, R. L. (1973) The Effect of Polycations on Cell Membrane Stability and Transport Processes. J. memnb. Biol., 14, 213. PENNIALL, R. & ZEYA, H. I. (1971) The Effects of Cationic Proteins of Rabbit Polymorphonuclear Leukocyte Lysosomes on the Respiratory Activity of Liver Mitochondria. Biochem. biophys. Res. Commun., 45, 6. PENNIALL, R., HOLBROOK, J. P. &ZEYA, H. I. (1972) The Inhibition of Cytochrome Oxidase by Lysosomal Cationic Proteins of Rabbit Polymorphonuclear Leukocytes. Biochem. biophys. Res. Commun., 47, 1270. ROSENTHAL, S. L. & BUCHANAN, A. M. (1974) Influences of Cationic Bactericidal Agents on Membrane ATPase of Bacillus subtilis. Biochim. biophys. Acta, 363, 141. SCHWARTZ, A. (1965a) The Effects of Histones and Other Polycations on Cellular Energetics. I. Mitochondrial Oxidative Phosphorylation. J. biol. Chem., 240, 939. SCHWARTZ, A. (1965b) The Effects of Histones and Other Polycations on Cellular Energetics. II. Adenosine Triphosphatase and Adenosine DiphosExchange phate-Adenosine Triphosphate Reactions of Mitochondria. J. biol. Chem., 240, 944. STRASTERS, K. C. & WINKLER, K. C. (1963) Carbohydrate Metabolism of Staphylococcus aureus. J. gen Microbiol., 33, 213. TABER, H. W. & MoRRISoN, M. (1964) Electron Transport in Staphylococci. Properties of a Particle Preparation from Exponential Phase Staphylococcus aureus. Archs biochem. Biophys., 105, 367. ZEYA, H. I. & SPITZNAGEL, J. K. (1966) Cationic Proteins of Polymorphonuclear Leukocyte Lysosomes. II. Composition, Properties and Mechanism of Antibacterial Action. J. Bact., 91, 755.