INFECTION AND IMMUNITY, Aug. 1976, P. 555-563Copyright ©D 1976 American Society for Microbiology

Vol. 14, No. 2Printed in U.S.A.

Lysis and Killing of Bacteria by Lysosomal ProteinasesKAREEN J. I. THORNE,* RHONDA C. OLIVER, AND ALAN J. BARRETT

Strangeways Research Laboratory, Cambridge CB1 4RN, United Kingdom

Received for publication 17 February 1976

The bacteriolytic and bactericidal effects of the human proteinases cathepsinB, cathepsin D, cathepsin G, and elastase were investigated. Cathepsin G andelastase were 5 to 10% as active as egg white lysozyme in the lysis ofMicrococcuslysodeikticus. All four enzymes slowly lysed the lysozyme-resistant Staphylococ-cus aureus. The gram-negative Acinetobacter 199A was rendered sensitive tolysozyme by all of the proteinases. Only elastase caused marked proteolysis ofthe outer membrane, which would permit access of lysozyme to the underlyingpeptidoglycan. When the surface layer of regularly arranged a protein was

removed, however, the outer membrane proteins became susceptible to the otherproteinases. Cathepsin G, elastase, and cathepsin D were bactericidal to Acine-tobacter 199A. The bactericidal activity of cathepsin D was shown to be depend-ent on enzymatic activity, unlike that of cathepsin G, which was related to itscationic nature.

One of the defense mechanisms of the hostagainst infection is the phagocytosis and killingof invading bacteria by polymorphonuclear leu-kocytes and macrophages (20). After phagocyto-sis, enzymes from polymorphonuclear leuko-cyte storage granules are discharged into thephagocytic vacuole (2) where killing and diges-tion subsequently occur (11). The lysosomalazurophil granules contain an elastase and achymotrypsin-like enzyme (14), and both havebeen purified from human spleen (31a, 31b,31c). The elastase was shown by Janoff andBlondin to lyse heat-killed bacteria (18). Achymotrypsin-like esterase from human poly-morphonuclear leukocytes was found to bebactericidal (26). The relative importance of theseveral individual proteinases of leukocytelysosomes in the digestion of bacteria has notbeen assessed previously, however, and thepurpose of the present paper is to report theresults of tests of the activity of the separatepurified human lysosomal enzymes and to com-pare them with some well-characterized pro-teinases from other sources.

MATERIALS AND METHODS

Materials. Acinetobacter sp. strain MJT/F5/199A(NCIB 10885) and Staphylococcus aureus (NCIB6571) were grown to early logarithmic phase in heartinfusion broth (Difco Laboratories, Detroit, Mich.),to which 0.01% CaCl2 was added, with maximalaeration. Micrococcus lysodeikticus was obtained asa freeze-dried powder from Boehringer Corp.(Lewes, Sussex BN7 1LG, U.K.).Human cathepsin B (EC 3.4.22.1), cathepsin D

(EC 3.4.23.5), lysosomal elastase (EC 3.4.21.2), andcathepsin G (EC 2.3.21._) were purified as describedpreviously (6, 29). Bovine pancreatic trypsin type I(EC 3.4.21.4), hog pancreatic elastase type I (EC3.4.21.11), papain (EC 3.4.22.2), lactoperoxidase (EC1.11. 1.7), cytochrome c, polymyxin B sulfate, diiso-propyl phosphofluoridate (Dip-F), and phenylmeth-ylsulfonyl fluoride (Pms-F) were obtained fromSigma Chemical Co. (Kingston upon Thames, Sur-rey KT2 7BH, U.K.). Egg white lysozyme (EC3.2.1.17) was obtained from Armour PharmaceuticalCo. (Eastbourne, Sussex, U.K.). Pepstatin waskindly given by H. Umezawa, Institute of MicrobialChemistry, Tokyo.

Inhibited cathepsin G was prepared by incubationofthe enzyme (35 U in 0.5 ml) with 10 mM Dip-F or 1mM Pms-F in 20 mM sodium acetate (pH 6.0) at 22°Cfor 4 h. Enzyme and inhibitor were incubated sepa-rately in parallel, and all three samples were thendialyzed against 1,000 volumes of 0.1 M sodiumphosphate (pH 7.4) containing 0.2 M NaCl. The sam-ples were then adjusted to identical volumes withthe buffered saline and assayed for activity (seeResults).Enzyme assays. Assays of proteolytic activity

were made with azocasein as the substrate. Thepreparation of azocasein was as described by Char-ney and Tomarelli (10) except that sulfanilamidewas replaced by sodium sulfanilate. Incubation mix-tures (1 ml) contained 15 mg of azocasein, bufferedwith 0.1 M tris(hydroxymethyl)aminomethane-hy-drochloride (pH 7.5) (for elastase, cathepsin G, andtrypsin) or 0.1 M sodium phosphate (pH 6.0) contain-ing 1 mM disodium ethylenediaminetetraacetate(EDTA) and 2 mM cysteine (for cathepsin B andpapain). The reaction was for 30 min at 40°C and wasstopped by the addition of 5 ml of3% (wt/vol) trichlo-roacetic acid solution. The extinction at 366 nm(E366) of the filtrates was measured, and AE366, the

555

on April 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

556 THORNE, OLIVER, AND BARRETT

change due to enzymatic activity, was calculated.NE values up to 0.3 were found to be linear withenzyme concentration, and such values are referredto as units of enzymatic activity per tube. Unlessotherwise stated, values given for enzymatic activ-ity in the present paper refer to the azocasein assay.Cathepsin D was assayed with hemoglobin as thesubstrate as described previously (6).Bacterial lysis. Suspensions of bacteria were in-

cubated with enzyme in 0.05 M sodium phosphate atpH 7.4 or 6.4, or in 0.05 M sodium acetate at pH 4, at22°C and at an initial E600 of 0.5 to 0.7. The initialrate of fall ofextinction (AE60o) was measured by useof a Unicam SP 700 spectrophotometer (Cambridge,U.K.).

Proteolysis of Acinetobacter sp. outer mem-brane. Cell walls consisting of outer membrane andan underlying peptidoglycan layer but lackingplasma membrane were prepared as described pre-viously (34). The regularly arranged surface pro-tein, the a layer, was removed by treatment with 2M urea at 37°C for 1 h (35). Walls (0.5 mg/ml), withor without the a layer, were incubated in 0.025 Msodium phosphate overnight at 37°C with papain(0.20 U/ml), cathepsin B (0.23 U/ml), and cathepsinD (0.22 U/ml, against hemoglobin) at pH 6.4, andwith trypsin (0.25 U/ml), spleen elastase (0.25 U/ml), and cathepsin G (0.28 U/ml) at pH 7.4. Sampleswere then taken for discontinuous electrophoresis inthe presence of sodium dodecyl sulfate (SDS) by themethod of Neville (24) in polyacrylamide gel slabs(11% T x 0.9% C) (24).

Effect of proteinases on viability. Logarithmic-ally growing cells of Acinetobacter 199A were sus-

pended in 50 mM sodium phosphate (pH 7.4 or 6.4)and incubated for 30 min at 30°C with the followingenzymes: cathepsin G (1.1 U/ml), spleen elastase(1.3 U/ml), trypsin (1.4 U/ml), and cytochrome c (20,ug/ml) at pH 7.4; and cathepsin B (0.9 U/ml), papain(0.9 U/ml), and cathepsin D (1.2 or 9.6 U/ml) at pH6.4. All samples were then serially diluted withsterile phosphate-buffered saline (8 g of NaCl, 0.2 gof KCl, 0.2 g of KH2PO4, and 1.15 g of Na2HPO4 per

INFECT. IMMUN.

liter), and 0.02 ml of each dilution was spotted ontoDifco heart infusion agar plates by the method ofMiles and Misra (23). Plates were incubated at 25°Cfor 24 h and the colonies were then counted.

Electron microscopy. Rabbit peritoneal polymor-phonuclear leukocytes (4 x 106/ml) were incubatedwith Acinetobacter sp. (1.5 x 107/ml) for 1 h at 37°C(22). The polymorphonuclear leukocytes were thenharvested by centrifuging and washed once withHanks salts buffered with 10 mM HEPES (N-2-hy-droxyethylpiperazine-N'-2-ethanesulfonic acid), pH7.6 (22). The pellets were fixed in 2.5% glutaralde-hyde in 90 mM cacodylate buffer (pH 7.2) containing3 mM CaCl2 for 1 h at room temperature and thenwere washed overnight or longer in cacodylatebuffer at 4°C. The pellets were postfixed in veronal-acetate-buffered OS04 (pH 7.2) for 1 h at room tem-perature, stained with 0.5% uranyl acetate in ve-ronal-acetate buffer for 1 h at room temperature,dehydrated in ethanol, and embedded in Araldite.Thin sections were cut with glass knives on an LKBUltrotome III or a Cambridge Huxley ultramicro-tome and stained with lead citrate. Specimens were

examined in an AEI EM6B electron microscope op-erating at 60 kV with a 50-tm objective aperture.

RESULTS

Lysis of bacteria by proteinases. Lysis ofthegram-positive bacteria M. lysodeikticus and S.aureus and the gram-negative Acinetobacter199A was investigated. M. lysodeikticus is sen-

sitive to lysis by egg white lysozyme and wasused for comparison of the lytic activity of pro-teinases with that of lysozyme. S. aureus isresistant to egg white lysozyme; proteolyticlysis could provide, therefore, an alternativelytic mechanism. Acinetobacter 199A, likeother gram-negative bacteria, is protected fromlysozyme by an outer membrane, which mightbe rendered permeable by proteolytic attack. A

TABLE 1. Lysis of bacteria by proteinases and lysozymeConcn Lysisa (AEw/min)

Enzyme pH,ug/ml U/ml {M. lysodeikticus S. aureus Acinetobacter

Lysozyme 14 7.4 0.31 ± 0.07 0 0Cathepsin G 7 0.32 7.4 0.014 ± 0.002 0.001 ± 0.0002 0Lysosomal elastase 14 0.95 7.4 0.009 ± 0.002 0.002 ± 0.0004 0.006 ± 0.002Trypsin 0.6 0.8 7.4 0.001 ± 0.0003 0 0.001 ± 0.0003Pancreatic elastase 28 0.5 7.4 0.002 ± 0.0002 0.002 + 0.0004 0Lysozyme 14 6.4 0.56 ± 0.05 0 0.08 ± 0.03Cathepsin B 100 0.3 6.4 0.002 ± 0.0004 0.001 ± 0.0002 0.01 ± 0.003Cathepsin D 0.3 0.2b 6.4 0 0.004 ± 0.0007 0Papain 28 1.0 6.4 0.034 ± 0.004 0.002 + 0.0004 0.022 + 0.005Lysozyme 14 4.0 0.11 ± 0.02 0 0Cathepsin B 100 0.3 4.0 0 0 0Cathepsin D 0.3 0.2b 4.0 0 0 0Papain 28 1.0 4.0 0.028 + 0.004 0 0a Values represent the mean of two to six determinations. Errors are standard errors of the mean.b Against hemoglobin.

on April 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

LYSOSOMAL PROTEINASES 557

comparison of the lysis of these bacteria bylysozyme and proteinases is shown in Table 1.Lysis was measured by the fall in E,,4 per min-ute. The lysosomal proteinases cathepsin G,cathepsin D, cathepsin B, and elastase werecompared with the pancreatic proteinases elas-tase and trypsin, the plant proteinase papain,and egg white lysozyme.Against M. lysodeikticus none of the protein-

ases tested was as active as lysozyme, butcathepsin G and, to a lesser extent, lysosomalelastase had considerable lytic activity andwere much more active than the nonlysosomalenzymes trypsin and pancreatic elastase. Cath-epsin B had little activity at pH 6.4 or 4.0, butthe plant proteinase papain was as active ascathepsin G.S. aureus, although completely resistant to

lysozyme, was lysed very slowly by cathepsin Gby lysosomal and pancreatic elastase at pH 7.4,and by cathepsin D, cathepsin B, and papain atpH 6.4. Although cathepsin D has a pH opti-mum of 3.5 against hemoglobin, it had no activ-ity against S. aureus at pH 4.Acinetobacter 199A was lysed most effec-

tively by the lysosomal enzymes cathepsin Band elastase but not by cathepsin G or cathep-sin D. It was also lysed by papain and veryslowly by trypsin. The addition of cysteine andEDTA did not increase the lytic activity of cath-epsin B or papain.

Sensitization ofAcinetobacter 199A to lyso-zyme. The outer membrane of gram-negativebacteria is a barrier that prevents the access oflysozyme to the underlying peptidoglycan.Lysis of gram-negative bacteria can beachieved, however, if the outer membrane isrendered permeable by removal of the lipopoly-saccharide with EDTA (28) or by partial hydrol-ysis of the membrane protein with trypsin (8,25). Lysosomal proteinases could be importantin the hydrolysis of the outer membrane pro-tein of gram-negative bacteria, which wouldallow subsequent lysis with lysozyme. The ef-fect of pretreatment with various proteinaseson the sensitivity ofAcinetobacter 199A to lyso-zyme was compared with cell treatments thatare known to permit access of lysozyme to thepeptidoglycan layer: extraction with EDTA andreaction with the surface-active polybasic pep-tide polymyxin (36). The combination of peroxi-dase and hydrogen peroxide was tested since, inthe presence of iodide or chloride ions, this hasa lethal effect on bacteria (21) which is, at leastpartially, explained by peroxidation of the bac-terial membrane lipid (31).A suspension ofAcinetobacter 199A was pre-

treated for 4 min at 22°C with the appropriateenzyme before the addition of lysozyme. All of

the proteinases tested were effective in poten-tiating lysis ofAcinetobacter 199A by lysozyme(Table 2). The lysosomal proteinases cathepsinG, elastase, cathepsin B, and cathepsin D in-creased the sensitivity ofAcinetobacter 199A tolysozyme up to 20 to 30% of the value obtainedfor lysis ofM. lysodeikticus by lysozyme (Table1). The effect, however, was not restricted tolysosomal enzymes, since trypsin (at a highconcentration), papain, and pancreatic elastasewere also active. Modification of the outermembrane with the peroxidase system, withEDTA above pH 7, or with polymyxin had asimilar effect.

Certain gram-positive bacteria have an outerlayer of protein that protects them from lyso-zyme. This protein layer can be removed withEDTA (32) or with trypsin (3, 9). Proteinases,however, did not increase the sensitivity of S.aureus to egg white lysozyme. The resistance ofS. aureus to lysozyme is believed to be due tothe presence of 6-O-acetyl groups on the mur-

TABLE 2. Sensitization ofAcinetobacter 199A to

Pretreatment

NoneCathepsin G (0.32 U/

ml)Lysosome elastase

(0.9 U/ml)Pancreatic elastase

(0.5 U/ml)Trypsin (40 U/ml)EDTA (1 mM)Peroxidase (28 ,lg/

ml), H202 (2 mM),NaCl (4 mM)

Polymyxin (20 ug/ml)

NoneCathepsin B (0.3 U/

ml)Cathepsin D (0.2 U/

mI)bPapain (1 U/ml)Trypsin (40 U/ml)EDTA (1 mM)Peroxidase (28 jAg/ml)H202 (2 mM), NaCl(4 mM)

NoneCathepsin B (0.3 U/

ml)Cathepsin D (0.2 U/

mI)bPapain (1 U/ml)EDTA (1 mM)

lysozyme'

pH

7.47.4

7.4

7.4

7.47.47.4

Lysis by lysozyme(AEN}min)

00.065 + 0.01

0.065 + 0.005

0.035 + 0.005

0.16 + 0.080.13 + 0.020.11 + 0.02

7.4 0.11 t 0.02

6.4 0.08 t 0.036.4 0.16 t 0.02

6.4 0.12 t 0.02

6.4 0.17 t 0.036.4 0.17 t 0.086.4 0.065 t 0.016.4 0.06 t 0.01

44

4

44

00

0

00

a Acinetobacter 199A was pretreated for 4 min and thentested for sensitivity to lysozyme (14 iLg/ml). Values quotedrepresent the mean of two determinations. Errors arestandard error of the mean.

b Against hemoglobin.

VOL. 14, 1976

on April 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

558 THORNE, OLIVER, ANDBARRETTI

amic acid residues ofthe peptidoglycan (29) andnot to an impeding protein layer.Proteolysis of Acinetobacter outer mem-

brane. To determine ifthe potentiating effect ofthe proteinases on lysozyme was due to break-down of the outer membrane, isolated cell wallswere incubated ovemight with the proteinasesand their protein composition was determinedby electrophoresis on SDS-acrylamide gels(Fig. 1). The main proteins of the outer mem-brane are the regularly arranged surface a pro-tein (a) and three others, designated 1, 2, and3 (33). Although all of the proteinases testedpotentiated the action oflysozyme, only papain,lysosomal elastase, and cathepsin G produceddetectable modifications in the proteins of theouter membrane. Papain hydrolyzed a protein(molecular weight, 65,000) and proteins 1 and3 (molecular weight, 49,000 and 31,500, re-spectively), with the production of five new pep-tides ranging in size from 20,000 to 30,000 andtwo of less than 5,000 daltons. Spleen elastasealso hydrolyzed a protein and proteins 1 and 3but produced a different pattern of fragments:molecular weight, 20,000 and 5,000. Cathepsin

G only attacked the a protein. The main frag-ments had molecular weights of 25,000 and5,000.

Protein 2 in the cell walls was resistant to allof the proteinases tested. This protein may beinherently resistant to proteolytic attack, or itmay be inaccessible to the added proteinases. Ithas been shown that all of the outer membraneproteins, except for protein 2 and surface aprotein, become extractable with dilute bufferafter treatment of the membrane with phospho-lipase C (K. J. I. Thorne, R. C. Oliver, and A.M. Glauert, unpublished data). This suggeststhat protein 2 is less accessible to externalagents than the other cell wall proteins.One possible function of the regularly ar-

ranged surface a protein is as a protectivelayer. Susceptibility of isolated cell walls toproteolytic attack in the presence and absenceofthe a protein was investigated therefore. Fig-ure 1 illustrates the effect of proteinases on cellwalls with an intact a layer, whereas Fig. 2shows proteolysis of cell walls from which the alayer had been removed with 2 M urea. Theeffects of papain and spleen elastase were the

a

I_4.

2 _M, .3

a bc de f g h i j k IFIG. 1. Proteolysis ofAcinetobacter 199A outer membrane proteins. Isolated cell walls (0.5 mg ofprotein

per ml) were incubated overnight at 37°C with proteases and then analyzed by discontinuous SDS-gelelectrophoresis in 11% acrylamide. (a) Cell wall and cathepsin B (0.23 U/ml), (b) cathepsin B alone, (c) cellwall and trypsin (0.25 U/ml), (d) trypsin alone, (e) cell wall and papain (0.20 U/ml), (t) papain alone, (g) cellwall and spleen elastase (0.25 UIml), (h) spleen elastase alone, (i) cell wall and cathepsin G (0.28 U/ml), (j)cathepsin G alone, (k) cell wall alone at pH 7.4, and (1) cell wall stored at 4°C.

INFECT. IMMUN.

_mtMk _ii on April 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

LYSOSOMAL PROTEINASES 559

I

2..- .....

3

a b c d e f h i i

FIG. 2. Proteolysis ofAcinetobacter 199A outer membrane proteins in the absence ofthe regularly arrangedsurface a layer. Experimental procedure as in Fig. 1. (a) Cell wall stored at 4°C, (b) cell wall alone atpH 7.4,(c) cell wall and papain (1.8 U/ml), (d) papain alone, (e) cell wall and cathepsin B (1.1 UIml), (f) cathepsinB alone, (g) cell wall and spleen elastase (0.4 U/ml), (h) spleen elastase alone, (i) cell wall and cathepsin G(0.28 UIml), and (j) cell wall and trypsin (34.6 U/ml).

same in the presence or absence of a protein.Removal of a protein allowed cathepsin G toattack protein 1 (molecular weight, 49,000),with the production of new peptides of molecu-lar weights 48,000, 46,000, 24,000, and 21,000.The outer membrane proteins also became sus-ceptible to cathepsin B. All of the proteins,including the resistant protein 2, were at-tacked. In this experiment, higher concentra-tions of trypsin were used. These were suffi-cient to hydrolyze protein 1, which could ex-plain the potentiating effect of high concentra-tions of trypsin for lysozyme. Results for ca-thepsin D are not shown, but it had no detecta-ble effect in the presence or absence of a pro-tein.

Killing of bacteria by proteinases. One ofthe agents responsible for the killing of phago-cytosed bacteria is a group of lysosomal cationicproteins (15, 37). Evidence is accumulating thatsome of the cationic proteins are identical tocathepsin G. A chymotrypsin-like proteinasefrom human granulocytes kills S. aureus evenafter heat inactivation of the enzyme (26). Theeffect of proteinases on the viability ofAcineto-bacter was investigated (Table 3). The cationiclysosomal proteinase cathepsin G reduced theviability of Acinetobacter 199A by two-thirdsafter 30 min. The bactericidal effect of cathep-

sin G was not due to its enzymatic activity sincethe bacteria were also killed when the enzymewas inactivated with Dip-F or with PmsF. Cy-tochrome c, a nonlysosomal cationic protein,was as effective as cathepsin G in killing Acine-tobacter.Cathepsin D was also bactericidal. Its ability

to kill Acinetobacter was dependent, however,on its activity as an enzyme. Inhibition of theenzyme with pepstatin reversed its ability tokill the bacteria (Table 4). The amount of pep-statin required to inhibit completely cathepsinD at low pH values is 25 ng/U (7), althoughhigher concentrations are needed at pH 6.4. Atconcentrations of pepstatin below 25 ng/U (5.2and 10.4 ng/U), the killing was reversed onlypartially. At the high pepstatin concentrationof 52 ng/U, cathepsin D was no longer bacteri-cidal. Lysosomal elastase was bactericidal, butcathepsin B was not.Effect of lysosomal enzymes on the bacte-

rial ultrastructure. The destruction of the bac-terial cell envelope by the enzymes of leukocytegranules was illustrated by using rabbit poly-morphonuclear leukocytes (Fig. 3). A thin sec-tion through Acinetobacter 199A (Fig. 3A)shows the typical gram-negative envelope ofplasma membrane, peptidoglycan layer, andouter membrane. After phagocytosis and diges-

VOL. 14, 1976

on April 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

560 THORNE, OLIVER, AND BARRETT

TABLE 3. Killing ofAcinetobacter 199A by proteinasesaProtein concn Viable count (col- Inhibi-

Enzyme pH ony-forming units/ tion (Atg/ml U/mi ml)

Expt 1None 0 0 7.4 7.4 x 105Cathepsin G 24 1.1 7.4 1.3 x 105 82Dip-cathepsin G 24 0.4 7.4 0.15 x 105 98Dip-F controlb 0 0 7.4 6.8 x 105 8Elastase 19 1.3 7.4 5.2 x 105 30Cathepsin D 1.8 1.2 6.4 3.8 x 105 48Cathepsin D and pepstatin (1 ug/ml) 1.8 0 6.4 7.6 x 105 0Pepstatin (1 Ag/ml) 0 0 6.4 6.9 x 105 7Cathepsin B 300 0.9 6.4 10.5 x 105Trypsin 1 1.4 7.4 1.3 x 105 82Papain 31 0.9 6.4 4.9 x 105 34

Expt 2None 0 0 7.4 1.9x 107Cathepsin G 22 1.0 7.4 1.47 x 107 23Pms-cathepsin G 16 0.1 7.4 1.61 x 107 15Cathepsin D 1.8 1.2 6.4 0.99 x 107 48Cytochrome c 20 0 7.4 1.49 x 107 22

a Bacteria were incubated for 30 min with the appropriate enzyme in 50mM sodium phosphate at 30°C.b

c Dip-F incubation without cathepsin G and then dialyzed to remove reagent (see Materials and Methods).

TABLE 4. Inhibition of bactericidal effect of cathepsin D by pepstatina

PepstatinPepstatin

Viable count (104 colony- Inhibition Significant byEnzyme ng/U of cath- forming units/ml + SE)' (%) Student's t testng/mlepsin D

Control 0 0 8.65 + 0.16 (6) 0Cathepsin D 0 0 6.84 ± 0.30 (6) 21 0.01Cathepsin D 50 5.2 7.62 ± 0.35 (6) 12 0.05Cathepsin D 100 10.4 8.08 ± 0.18 (6) 6.5 0.05Cathepsin D 500 52 9.10 ± 0.37 (6) -5 NSC

a Bacteria were incubated for 30 min in 50mM sodium phosphate at 30°C with cathepsin D (9.6 U/ml). Thesignificance of the differences between means for the control (cathepsin D that had been heated for 3 min at100°C) and the tests is given.

b SE, Standard error.c NS, Not significant.

tion by the polymorphonuclear leukocyte (Fig.3B), the outer membrane is fragmented anddigestion of the peptidoglycan layer has begun.

DISCUSSIONThe major proteinases that have been identi-

fied in the lysosomes ofpolymorphonuclear leu-kocytes seem to be cathepsins D and B (activeprimarily at acid pH values) and elastase andcathepsin G (which are most active at neutraland alkaline pH). The relative amounts of theenzymes vary between species. In rabbits, theactivity of the acid proteinases is far greaterthan that of the neutral ones (16), although a

neutral proteinase is present and has been puri-fied partially (12). In human polymorphs, it isthe neutral proteinase activity of elastase andcathepsin G that is most prominent (16), al-though cathepsin B has also been identified (P.Davies, personal communications). Since theproteinases used in this study were isolatedfrom human rather than rabbit tissues, theresults are more directly relevant to the han-dling of bacteria by human leukocytes. Never-theless, rabbit cathepsin D is closely similar tothat ofhumans (4), and rabbit cathepsin B alsohas many properties in common with the hu-man enzyme (27). The work of Jensen andBainton (19) indicates that the pH value within

INFECT. IMMUN.

on April 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

LYSOSOMAL PROTEINASES 561

a phagosome containing bacteria is likely to beneutral initially but acidic later. It is likely,therefore, that proteinases of both groups havean opportunity to participate in lysis of thebacteria. It was of interest, therefore, to use the

A

isolated proteinases in tests of their capacity tocleave peptide bonds ofeither the peptidoglycanor, where appropriate, the outer membraneprotein of the cell envelope of various classes ofbacteria, and to kill the bacterial cell.

I,r

.N..

-md< pm

If

I:,t , _

I.

St,

V:-

7/ '~~~~~~

14Bk .6 tt-

<i;i;,211

1.8"'0; X.4''

FIG. 3. EIC/ect ot lysosomal enzymes on the bacterial ultrastructure. (A) Thin section through a normalAcinetobacter 199A cell. (B) Thin section through Acinetobacter inside a rabbit polymorphonuclear leukocyte.pm, Plasma membrane; d, peptidoglycan layer; om, outer membrane. The bar represents 0.1 ,um.

-B

t.*

VOL. 14, 1976

to,.,

'PI'I..l- .I

.4I

16.

on April 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

562 THORNE, OLIVER, AND BARRETT

M. lysodeikticus was attacked by cathepsin Gand spleen elastase at 5 to 10% of the rate of anequivalent amount of lysozyme; the nonlysoso-mal enzymes trypsin and pancreatic elastasewere inactive. In the presence of lysozyme, thelysosomal proteinases are probably of only sec-ondary importance, allowing further degrada-tion of cell wall fragments after the preliminarymuramidase action of the lysozyme. S. aureusis resistant to lysozyme, however, and is slowlylysed by cathepsin D, cathepsin B, cathepsin G,and elastase. Although the rate is slow, in theabsence of an effective lysozyme it could besignificant. The concentrations of lysosomalproteinases in digestive vacuoles exceed thatattained in in vitro experiments by orders ofmagnitude (12), and Cohn found that the diges-tion of staphylococci in leukocytes took severalhours (11). Janoff and Blondin (18) reportedthat both elastase and leukocyte granules lyseautoclaved S. aureus. However, Salton (30)showed that the increased lysis ofheated bacte-ria by proteinases represents digestion of re-leased cytoplasm and does not reflect hydrolysisof the cell wall.

Direct lysis ofthe gram-negative Acinetobac-ter 199A was found to be less important thanpotentiation of lysis by lysozyme. Pretreatmentwith cathepsin G, elastase, cathepsin D, orcathepsin B increased the susceptibility ofAci-netobacter to lysozyme. The proteinases wereas effective at sensitizing Acinetobacter to lyso-zyme as was treatment with EDTA to removelipopolysaccharide (33) or with peroxidase andhydrogen peroxide or polymyxin to attack thelipid of the outer membrane (31). Nonlysosomalproteinases were also effective.Although all four lysosomal enzymes poten-

tiated the action of lysozyme, they had differenteffects on the outer membrane proteins. Onlythe elastase was markedly proteolytic. Cathep-sin G attacked the regularly arranged surface aprotein but not the other proteins. The a pro-tein seemed to protect the outer membranefrom cathepsin G and cathepsin B, since afterits removal the proteins became susceptible toproteolysis. The selective susceptibility of outermembrane proteins to protease attack was firstobserved by Inouye and Yee (17). They foundthat trypsin only hydrolyzed some of the pro-teins of the outer membrane of Escherichiacoli.

Cathepsin G is a chymotrypsin-like protein-ase present in human polymorphonuclear leu-kocytes (31a), and the results of Odeberg et al.(26) indicate that its multiple electrophoreticforms are among the cationic bactericidal pro-teins described by Hirsch (15) and Zeya andSpitznagel (37). Those results are confirmed

INFECT. IMMUN.

and extended by the present direct demonstra-tion of bactericidal activity of purified cathepsinG. It is notable that a rather high concentrationof proteinase (24 ug/ml) was required to kill4.3 x 105 bacteria per ml (less than 1 gg [dryweight] per ml). This is similar, however, tothe amount of cationic protein needed to killE. coli and Proteus vulgaris (37). The ability ofcathepsin G to kill Acinetobacter 199A residesin its cationic nature and is not dependent onits proteolytic activity. Inactivation of the en-zyme with Dip-F does not interfere with itsbactericidal activity, and the same effect can beobtained with the nonlysosomal cationic pro-tein cytochrome c.Cathepsin D is also bactericidal to Acineto-

bacter. Here, however, the effect is dependenton the proteolytic activity of the enzyme. Par-tial or complete inhibition of cathepsin D bypepstatin correspondingly decreases the abilityof the enzyme to kill bacteria. Cathepsin D wasat least 10 times as active by weight as cathep-sin G.Each lysosomal proteinase seems to have its

own particular role in the defense against thegram-negative Acinetobacter 199A. CathepsinG is bactericidal, through its cationic proper-ties, cathepsin D has a bactericidal effect de-pendent on proteolysis, elastase hydrolyzes theouter membrane and is bacteriolytic, and cath-epsin B has a direct lytic effect. All of the fourlysosomal proteinases tested, together withnonlysosomal proteinases and peroxidase, po-tentiate lysis by lysozyme. Destruction of thebacterial cell envelope by the concerted attackof lysosomal proteinases and lysozyme can beseen in morphological studies with rabbit poly-morphonuclear leukocytes, which reveal thatearly steps in the attack produce fragmentationof the outer membrane and digestion of thepeptidoglycan.

ACKNOWLEDGMENTSWe acknowledge the support of the Medical Research

Council (to K.J.I.T. and R.C.O.). We are grateful to J. M.Lackie for the preparation of rabbit polymorphonuclear leu-kocytes containing phagocytized bacteria and to H. Ume-zawa for a gift of pepstatin. We thank R. A. Parker for theelectron micrographs.

LITERATURE CITED1. Baggiolini, M. 1972. The enzymes of the granules of

polymorphonuclear leukocytes and their functions.Enzyme 13:32-160.

2. Bainton, D. F. 1973. Sequential degranulation of thetwo types of polymorphonuclear leukocyte granulesduring phagocytosis of microorganisms. J. Cell Biol.58:249-264.

3. Barker, D. C., and K. J. I. Thorne. 1970. SpheroplastsofLactobacillus casei and the cellular distribution ofbactoprenol. J. Cell Sci. 7:755-785.

on April 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

LYSOSOMAL PROTEINASES 563

4. Barrett, A. J. 1967. Lysosomal acid proteinase of rabbitliver. Biochem. J. 104:601-608.

5. Barrett, A. J. 1972. Lysosomal enzymes, p. 46-135. In J.T. Dingle (ed.), Lysosomes. North-Holland Publish-ing Co., Amsterdam.

6. Barrett, A. J. 1973. Human cathepsin B. Purificationand some properties of the enzyme. Biochem. J.131:809-822.

7. Barrett, A. J., and J. T. Dingle. 1972. Inhibition oftissue acid proteinases by pepstatin. Biochem. J.127:439-441.

8. Becker, M. E., and S. E. Hartsell. 1954. Factors affect-ing bacteriolysis using lysozyme in dual enzyme sys-tems. Arch. Biochem. Biophys. 53:402-410.

9. Berlin, I., and H. Y. Neujahr. 1968. Studies of con-trolled lysis of washed cell suspensions ofLactobacil-lus fermentii and preparation of membrane-like frag-ments by a combined trypsin-lysozyme treatment.Acta Chem. Scand. 22:2972-2980.

10. Charney, J., and R. M. Tomarelli. 1947. A colorimetricmethod for the determination of the proteolytic activ-ity of duodenal juice. J. Biol. Chem. 171:501-505.

11. Cohn, Z. A. 1963. The fate of bacteria within phagocyticcells. I. The degradation of isotopically labeled bacte-ria by polymorphonuclear leukocytes and macro-phages. J. Exp. Med. 117:27-42.

12. Davies, P., G. A. Rita, K. Krakauer, and G. Weiss-mann. 1971. Characterisation of a neutral proteasefrom lysosomes of rabbit polymorphonuclear leuko-cytes. Biochem. J. 123:559-569.

13. Dean, R. T., and A. J. Barrett. 1976. Lysosomes. EssaysBiochem. 12:1-30.

14. Dewald, B., R. Rindler-Ludwig, U. Bretz, and M. Bag-giolini. 1975. Subcellular localisation and heteroge-neity of neutral proteases in neutrophilic polymor-phonuclear leukocytes. J. Exp. Med. 141:709-723.

15. Hirsch, J. G. 1956. Phagocytin: a bacterial substancefrom polymorphonuclear leukocytes. J. Exp. Med.103:589-611.

16. Ignarro, L. J. 1974. Regulation of lysosomal enzymesecretion: role in inflammation. Agents Actions4:241-258.

17. Inouye, M., and M. L. Yee. 1972. Specific removal ofproteins from the envelope ofEscherichia coli by pro-tease treatments. J. Bacteriol. 112:585-592.

18. Janoff, A., and J. Blondin. 1973. The effect of humangranulocyte elastase on bacterial suspensions. Lab.Invest. 29.454-457.

19. Jensen, M. S., and D. F. Bainton. 1973. Temporalchanges in pH within the pagocytic vacuole of thepolymorphonuclear leukocyte. J. Cell Biol. 56:379-388.

20. Klebanoff, S. J. 1971. Intraleukocytic microbicidal de-fects. Annu. Rev. Med. 22:39-62.

21. Klebanoff, S. J., and C. B. Hamon. 1972. Role ofmyelo-peroxidase-mediated antimicrobial systems in intactleukocytes. J. Reticuloendothel. Soc. 12:170-196.

22. Lackie, J. M. 1974. The aggregation of rabbit polymor-phonuclear leukocytes: effect of antimitotic agents,cyclic nucleotides and methyl xanthines. J. Cell Sci.16:167-180.

23. Miles, A. A., and S. S. Misra. 1938. The estimation ofthe bactericidal power of the blood. J. Hyg. 38:732-749.

24. Neville, D. M. 1971. Molecular weight determination ofprotein-dodecyl sulphate complexes by gel electropho-resis in a discontinuous buffer system. J. Biol. Chem.246:6328-6334.

25. Noller, E. C., and S. E. Hartsell. 1961. Bacteriolysis ofEnterobacteriaceae. I. Lysis by four lytic systems us-ing lysozyme. J. Bacteriol. 79:697-706.

26. Odeberg, H., I. Olsson, and P. Venge. 1975. Cationicproteins of human granulocytes. IV. Esterase activ-ity. Lab. Invest. 32:86-90.

27. Ogino, K., and K. Nakashima. 1974. Purification ofrabbit liver cathepsin B1. J. Biochem. 75:723-730.

28. Repaske, R. 1958. Lysis of gram-negative organismsand the role of versene. Biochim. Biophys. Acta30:.225-232.

29. Robinson, J. P., R. D. Robinson, and J. H. Hash. 1974.Electron microscopy of Staphylococcus aureus cellsand cell walls after treatment with lysozyme Chalar-opsis. J. Bacteriol. 117:900-903.

30. Salton, M. R. J. 1953. Cell structure and the enzymiclysis of bacteria. J. Gen. Microbiol. 9:512-523.

31. Shohet, S. B., J. Pitt, R. L. Baehner, and D. G. Po-plack. 1974. Lipid peroxidation in the killing of phag-ocytized pneumococci. Infect. Immun. 10:1321-1328.

31a. Starkey, P. M., and A. J. Barrett. 1976. Human ca-thepsin G catalytic and immunological properties.Biochem. J. 155:273-278.

31b. Starkey, P. M., and A. J. Barrett. 1976. Lysosomalelastase. Catalytic and immunological properties.Biochem. J. 155:265-271.

31c. Starkey, P. M., and A. J. Barrett. 1976. Neutral pro-teinases of human spleen: purification and criteriafor homogeneity of elastase and cathepsin G. Bio-chem. J. 155:255-263.

32. Thorne, K. J. I., and D. C. Barker. 1972. The occur-rence of bactoprenol in the mesosome and plasmamembranes of Lactobacillus casei and L. plantarum.J. Gen. Microbiol. 70:.87-98.

33. Thorne, K. J. I., M. J. Thornley, P. Naisbitt, and A. M.Glauert. 1975. The nature of the attachment of aregularly arranged surface protein to the outer mem-brane of an Acinetobacter sp. Biochim. Biophys. Acta389:97-116.

34. Thornley, M. J., A. M. Glauert, and U. B. Sleytr. 1973.Isolation of outer membranes with an ordered arrayof surface subunits from Acinetobacter. J. Bacteriol.114:1294-1308.

35. Thornley, M. J., K. J. I. Thorne, and A. M. Glauert.1974. Detachment and chemical characterization ofthe regularly arranged subunits from the surface ofan Acinetobacter sp. J. Bacteriol. 118:654-662.

36. Warren, G. H., J. Gray, and J. A. Yurchenko. 1957.Effect of polymyxin on the lysis of Neisseria catar-rhalis. J. Bacteriol. 74:788-793.

37. Zeya, H. I., and J. K. Spitznagel. 1968. Arginine-richproteins of polymorphonuclear leukocytes. J. Exp.Med. 103:589-611.

VOL. 14, 1976

on April 30, 2020 by guest

http://iai.asm.org/

Dow

nloaded from