A 41.7 kDa serine protease from Clostridium perfringens type a: Degradation of purified human serum proteins

Zbl. Bakt. 277, 145-160 (1992) © Gustav Fischer Verlag, StuttgartlNew York

A 41. 7 kDa Serine Protease from Clostridium perfringens Type A: Degradation of Purified Human Serum Proteins URSULA WOLF, DIERK BAUER, and WALTER H. TRAUB* Institut fur Medizinische Mikrobiologie und Hygiene, Universitat des Saarlandes, 6650 Homburg/Saar, Germany

With 7 Figures· Received January 2, 1992 . Accepted February 24, 1992

Summary Two clinical isolates of Clostridium perfringens type A produced a novel caseinolytic serine protease. Both enzymes had a molecular weight of 41.7 kilodaltons and an isoelectric point of 9.1. The two enzymes were immunogenic for rabbits and closely related serologically. Both enzymes partially degraded the heavy chains of human immunoglobulins (Ig) G and IgM, but not IgA. Purified human complement (C) components C3, C5, C8, and C9 were attacked; Clq was refractory. Both enzymes were active against human transferrin, alphajantitrypsin, alphaj-macroglobulin, haptoglobin, type III fibrinogen, and fibronectin. Creactive protein was refractory.

Zusammenfassung Zwei klinisch isolierte Stamme von Clostridium perfringens Typ A produzierten eine caseinolytische Serinprotease gekennzeichnet durch ein Molekulargewicht von 41.7 Kilodalton und einen isoelektrischen Punkt von 9,1. Beide Enzyme erwiesen sich als immunogen fur Kaninchen und serologisch nahe verwandt. Beide Proteasen degradierten partiell die schweren Ketten der Immunglobuline (Ig)G und IgM, jedoch nicht IgA. Die Humankomplement (C)-Komponenten C3, C5, C8 und C9, aber nicht Clq, wurden attackiert; ferner degradierten beide Enzyme Transferrin, alpha--Antitrypsin, alphaj-Makroglobulin, Haptoglobin, Typ III Fibrinogen und Fibronectin. C-reaktives Protein war refraktar,

Introduction

Clostridium perfringens type A, the most common causative agent of human gas gangrene (3, 11,29,30), had long been known to produce a great variety of exotoxins (12), their principal representatives being alpha-toxin (phospholipase C), theta-toxin (perfringolysin 0), and the 80 kilodalton (kDa) kappa-toxin (collagenase). In the past,

* Corresponding author 10 Zbl. Bakt. 277/2

146

U. Wolf, D.Bauer, and W.H. Traub

most workers concentrated on alpha-toxin since this exoenzyme proved to be a protective antigen (5,12). Sato et al. (19) showed a strain of C. perfringens to produce at least three distinct proteases, one of which proved to be a metalloprotease, another was a thiol protease, whereas the third protease, which had the largest molecular size, was not characterized further. Walter (28) demonstrated the 80 kDa collagenase of C. perfringens to be chemotactic for human neutrophil leukocytes. Fujiyama et al. (7) recently observed a lecithinase-negative, unidentified Clostridium species (strain M.O.6) to produce an IgA protease. We were interested to find out whether isolates of C. perfringens type A produced proteases capable of degrading purified human serum proteins analogous to metalloproteases of Serratia marcescens (25). This communication serves to present preliminary data with respect to a novel serine protease produced by two clinical isolates of C. perfringens type A that displayed human serum proteolytic activity.

Materials and Methods Bacteria. A total of 27 C. perfringens type A strains (11 clinical isolates, 15 faecal isolates, and control strain ATCC 13124) were screened for azocasein hydrolysis (see below). The strains had been identified as previously described (1, 14,23,24,26). Two of the isolates, C. perfringens 4-Cli (source: patient M.R., wound swab, 15.10.84) and 6-Cli (source: patient 1.M., wound swab, 31. 12.84) were chosen for further study. Media. Brucella agar, Chopped Meat broth (CMB), NIH Thioglycolate broth, and Proteose Peptone were purchased from Difco Laboratories, Detroit, Michigan, USA. The medium of Murata et al. (13) served for large-scale cultures of protease producer strains. Reagents. All chemical reagents, including sorbitol and those for phosphate-buffered saline (PBS), pH 7.5, were of analytical grade (E. Merck, Darmstadt). The serine protease trypsin (1: 250, from bovine pancreas; 4 U/mg; batch H) was purchased from Serva Biochemica GmbH, Heidelberg; a stock solution of 1 mg/ml was prepared in 1 mM HCl which was diluted to 50ftg/ml for azocoll hydrolysis assays. The thiol protease ficin (100 mg/10 ml in 10 mM sodium acetate buffer, pH 4.5; lot No. 12674520-16) was procured from Boehringer Mannheim GmbH, Mannheim, and utilized at a final concentration of 25 ug/ml. The Serratia marcescens SF 178 metalloprotease (25) was used at 2 mg/ml of 0.1 M sodium phosphate buffer, pH 7.0. The following protease inhibitors were used: Ethylenediaminetetraacetate (EDTA), disodium salt (lot C9; Serva; stock solution = 0.5 M in H 2 0 , pH 8.0); -l-p-aminobenzamidine-Z HCl (lot No. 07060; Serva; stock solution = 100 mM in 0.1 M Tris-HCl, pH 7.5); phenylmethylsulphonylfluoride (PMSF; lot No. 23040; stock solution = 100 mM in 100% (v/v) ethanol); N-(N-(L-3-transcarboxyoxiram-2-carbamyl)-L-leucyl)-agmatin (E-64; lot No. 12764721-15; Boehringer Mannheim; stock solution = 20 mg/ml in 50% (v/v) aqueous ethanol); 4-(2-aminoethyl)-benzenesulphonylfluoride hydrochloride (Pefabloc SC; lot No. 125 K 16201439; E. Merck; stock solution = 100 mM in 0.1 Tris-HCl, pH 7.5). The proteinase inhibitors were utilized experimentally in accordance with the following investigators (names in brackets): PMSF (Richardson et aI., 17), p-arninobenzamidine (Sugimura and Nishihara, 21), EDTA (Endo et aI., 6; Richardson et aI., 17). E-64 and Pefabloc SC were employed precisely in accordance with the instructions of the respective manufacturer. 1,4-dithiothreitol (lot No. G9, Serva), L-cysteine (lot No. 54F-0366; Sigma Chemie GmbH, Deisenhofen), and 2-mercaptoethanol (lot no. M 3357; BioRad Laboratories GmbH, Miinchen) were employed according to the methods of Young and Broadbent (32) and Endo et al. (6), respectively. Stock solutions of 10, 20, and 20 mM, respectively, were prepared in PBS, pH 7.5. Azocasein (sodium salt; batch D8) and isoelectric focusing reagents were obtained from Serva. Polyacrylamide gel electrophoresis reagents, including 8 molecular weight protein standards (range 14.4 - 200 kDa) and Coomassie Blue R-250, were purchased from Bio-Rad. Purified human immunoglobulin

C. perfringens Serine Proteases

147

(Ig)A (from colostrum), IgG, IgM, type III fibrinogen, and fibronectin were received from Sigma. Azocoll (100-250 mesh; lot No . 8010052) and the following purified human serum proteins were purchased from Calbiochem GmbH, Frankfurt: complement (C) components C1q, C3, C5, C8, and C9; alphaj-antitrypsin, and alphaj-macroglobulin. Transferrin was procured from Behringwerke AG, Marburg. All human serum proteins were stored at -65°C until further use. Type III acid-soluble collagen from calf skin and insoluble type I collagen from bovine Achilles tendon had been purchased from Sigma. Collagenase of C. histolyticum (0.9 Ulmg; lot 230 18) was procured from Serva. Horseradish peroxidase (HRP)-conjugated swine ant i-rabbit immunoglobulin (batch 030A) and alkaline phosphatase-conjugated swine anti-rabbit immunoglobulin (lot B 108) were procured from Dakopatts GmbH , Hamburg. Pelikan "Found India" @ink was purchased from Pelikan AG, Hannover. Tests for proteolytic activity. The method of Sato et al. (18, 19) was used to examine C. perfringens strains and prote ases for hydrolysis of azocasein ( ~40 nm). Protease activity was expressed as azocasein hydrolyzing units (ACU) per ml relative to mg protein/ml. Assays for azocoll hydrolysis (A S20 nm) were performed precisely according to the instructions of Calbiochem. Azocoll hydrolysis assays involving various prot ease inhibitors (Tables 3 and 4) were carried out as follows: 1 ml of inhibitor was combined with 50 111 of protease and incubated at room temperature for 60 min; 10mg of azocoll were added to each tube and the suspensions were agitated vigorously at 37 °C for 20 min, following which the suspensions were centrifuged (10000 x g, 10 min). The supernatant fluids were examined for A520 nm (Uvikon 860; Kontron Instruments GmbH, Miinchen). Assays for phospholipase C activity . The method of Kurioka and Matsuda (9) employing the chromogenic substra te, p-nitrophenylphosphorylcholine (10 mM NPPC; Sigma) together with sorbitol (60% (w/v)) in 0.25 M Tr is-HCI, pH 7.2, was used to examine C. perfringens strains (culture supernatants and fractions collected during various purification steps) as well as commercial phospholipase C (160 units/ml; Sigma; diluted 1 : 0-1 : 1024) for phospholipase C activity (A4 1O nm). Purification of C. perfringens proteases. Through trial and error, the following procedure was developed: Starter cultures consisting of 100 ml of NIH thioglycolate broth were inoculated with C. perfringens and incubated under station ary conditions at 35 °C for 8 h. The entire 100 ml of outgrowth were transferred to 2000 ml of fresh Mur ata medium and incubated at 35 °C for 16 h, following which the cell suspensions were centrifuged at 19700 x g, 10 °C, for 20 min. The supernatant fluids were membrane filter-sterilized (0.45 11m; Pressure Filtration Unit model SM 16274 ; Sartorius GmbH, Gottingen), The filtrat es were concentrated 10 times (Sartorius Cross-Flow System, Sartocon Type 175 21 ). The concentrated material was washed with 2000 ml of demineralized water in the Cross-Flow System, lyoph ilized, and stored at - 65 °C. Next, 200 mg of lyophilized retentate were dissolved in 50 ml of 20 mM sodium phosphate buffer, pH 7.5, and cooled to -4 c. Two volumes of methanol (precooled to - 65 °C) were added under constant stirring and held at -20 °C for 1 h. The suspension was centrifuged at 27000 x g, 4 °e, for 15 min. The precipitate (sediment) was dissolved in 10 ml of 0.02 M sodium phosphate buffer, pH 7.5, and dialyzed at room temperatu re against 121 of demineralized water for 1 h, after which the retentate was lyophilized and stored at -65 °C. The precipitate was subjected to Sephadex DEAE-A50 ion exchange chromatography (Deutsche Pharmacia GmbH, Freiburg), 20 mg of precipitate were dissolved in 1 ml of 50 mM Tr is-HCI buffer, pH 8.0, and placed on columms which measured 55 x 14 mm. The flow rate was adjusted to 30 mllh, and 1.45 ml fractions were collected with an Ultrorac 7000 fraction collector (LKB Instrument GmbH , Grafelfing), Elution was carried out with 3 ml of the same Tris buffer as above. The fractions were tested for azocasein hydrolysis. All positive fractions were pooled and subjected to ultrafiltration (Sartorius Centrisart I, SM 13249E; nominal exclusion = 20000 dalton s) following precisely the manufacturer's instructions, after which the materials were lyophilized and stored at - 65 °C. High pressure liquid chromatography (H PLC) . Alternatively, the two C. perfringens protease s could be purified with the aid of analytical HPLC. The hardware consisted of a 0

148

U. Wolf, D.Bauer, and W.H. Traub

Merck-Hitachi (E. Merck) liquid chromatograph, a proportioning valve, a variable wavelength UV monitor, and the processor 17. The column was a Merck Superformance'P 50-10 basic unit with glass cartridge. Lichrospherv 1000 S03- (5 urn) was the ion exchanger. Twenty to 30 mg of ethanol-precipitated protease material were dissolved per ml of elution buffer (50 mM sodium phosphate, pH. 5.0, plus 0.02% (w/v) of sodium azide); the NaCI gradient was 0-1 M. The flow rate was adjusted to 1 mllmin. Fractions were assayed for azocasein hydrolysis; positive fractions were lyophilized. Sodium dodecyl-sulphate-polyacrylamide electrophoresis (SDS-PAGE). This procedure was carried out according to the method of Laemmli (10). The wells received 20 ul of sample or standards. The stacking gels (0.125 M Tris, pH 6.8; 4% acrylamide) were electrophoresed at 15 rnA/gel and 10 °C; the separating gels (0.375 M Tris, pH 8.8; 12 or 7.5% acrylamide) were electrophoresed at 30 rnA/gel and 10°C. The gels were fixed, stained, destained, and dried as specified previously (25), utilizing BioRad equipment. In addition, SDS-PAGEgels of the two C. perfringens proteases (20 mg/ml; diluted 1 : 2 in nonreducing buffer; 40!t1 per slot) were shaken gently in 0.1 M TRIS-HCI, pH 7.5, with 0.02% (w/v) sodium azide (Merck) added, for 30 min, following which they were deposited on Skim Milk agar (140 mm diameter Petri dishes), the composition of which was based on the formula of Sokol et al. (20): 3% (w/v) Skim Milk (Difeo) and 1.5% (w/v) Bacto Agar (Difco) in 0.1 M Tris-HCI, pH 7.5. The plates were incubated at 35°C for two days and examined for clearing of the turbid medium. Isoelectric focusing. This procedure was performed using the Servalyt®PreNetsv (lot No. 06060) and protein test mixture 9 (lot No. 25081C) from Serva while strictly adhering to the supplier's specifications. An LKB model 2297 Macrodrive 5 served as power supply, and an LKB model 2117 Multiphor II electrophoresis unit was used for the separation of proteins. The pH gradient was 6-11. Production of anti-protease rabbit immune sera (RIS). New Zealand white rabbits (SAVO mediz. Versuchstierzuchten GmbH, Kisslegg/Allgau) Nos. 356 and 357 were immunized against C. perfringens proteases 4-Cli and 6-Cli, respectively, as follows: 2 mg of purified protease, dissolved in 100 ul of PBS, pH 7.5, were incorporated into 1 ml of Freunds' complete adjuvant (Behringwerke; batch 377004 A) and inoculated subcutaneously (s.c.); 4 weeks later, the animals received identical booster injections by the s.c, route. The rabbits were bled 3 weeks later through cardiac puncture under general anesthesia (sodium thiopental; Trapanalv; Byk-Gulden, Konstanz). The separated sera were stored at -65°C. Rabbit No. 146 served as a source of normal rabbit serum (NRS). Enzyme-linked immunosorbent assay (ELISA). The microtiter ELISA procedure employed has been described before (27). The rabbit immune and control sera were tested in triplicate. Alkaline phosphatase-conjugated swine anti-rabbit immunoglobulin (Dakopatts), diluted 1: 500, was used as the second antibody. The phosphatase substrate was Sigma 104® (Sigma). The titres of the rabbit sera were interpreted as the highest dilution of serum that yielded an A4 0 5 nm of 2: 0.200 relative to appropriate controls (substrate control = antigen + enzyme substrate; conjugate control = antigen + conjugate + substrate; and antiserum control = antigen + antiserum + substrate). Immunoblotting (Western blots). The method of Towbin et al. (22) served to examine RIS Nos. 356 and 357, diluted 1 : 100, for immunoreactivity against the two proteases of C. perfringens. SDS-PAGE gels were blotted onto 0.45 urn BA-S 85 nitrocellulose membranes (Schleicher and Schuell, Dassel) with the aid of the Multiphor II Novablot Electrophoretic Transfer System (Pharmacia LKB GmbH, Freiburg). The second antibody was HRP-conjugated pig anti-rabbit immunoglobulin, diluted 1 : 500, and the substrate was HRP Color Development Reagent, 4-chloro-l-naphthol (Bio-Rad). Exposure of purified human serum proteins to C. perfringens proteases. The human serum proteins were used at different concentrations (Table 5). The proteins were exposed to fresh and heat-inactivated (60°C, 15 min) proteases, respectively, and incubated at 35°C for 22h (IgA, IgG, and IgM) or at 35°C for 5 h (all other serum proteins), following which aliquots were examined with SDS-PAGE electrophoresis.

C. perfringens Serine Proteases

149

Results The prot eases of the C. perfringens strains 4-Cli and 6-Cli revealed azocasein hydroly tic activities of 89 ACU/mg lyophilisate (corresponding to 5562 ACU/mg protein ) and of 78 ACU/mg lyophilisate (corresponding to 5305 ACU/mg protein), respectively (Table 1). Both final enzy.me preparations lacked NPPC-hy drolytic activity . Th e molecular weights of both enzymes were 41.7 kDa (Fig. l) and their isoelectric point was 9.1. The enyzmes displayed caseinolytic activity after SDS-PAGE gels had been overlaid on Skim Milk agar (Fig. 2). The two enzymes were immunogenic for rab bits and were serologically related in terms of ELISA (Tab le 2) and immu noblot reactivity (Fig. 3). Both enzymes proved to be serine proteases (Table 3h their activities were enhanced markedly by dithiothreitol, L-cysteine, and 2-mercaptoethanol (Table 4). Coincubation of selected purified human serum proteins (Table 5) with the C. perfringens proteases yielded the following results: both proteases degraded C compo nents C3, C5, C8, and C9, but not Clq; however, commercial C. histolyticum collagenase degra ded Cl q (data not shown), The two enzymes attacked the heavy chain s of IgG and IgM, but not that of IgA; the L chains of all immunoglobulins were not affected . Furthermore, both enzymes partially degraded transferrin, alphaj-antitrypsin, alphaj-macroglobulin, haptoglobin, type III fibrinogen, and fibronectin. However, CRP proved to be refractory. Representative SDS-PAGE electropherograms are shown in Fig.4-6. The human serum proteolytic activities of the two proteases are summarized in Table 6.

Table 1. Summary of activities and yields of C. perfringens serine proteases during successive purification steps

C. perfringens strain

Preparation

4-Cli

Culture supernatant fluid (2 1), filtrate

Yield

Protein content

ACU

ACU/mg protein

244.1 ug/ml

16/ml

65.5

132.4 ug/mg

S2/mg

393

127/mg

1,624

Rerentate following wash (lyophilisare)

1,010 mg

Methanol precipitate

407 mg

78.2 ug/mg

38.7 mg

16 ug/mg

89/mg

5,562

217.4 ug/ml

13/ml

59.8

117 ug/mg

45/mg

384

( Iyop hili ~ate )

Sephadex DEAE-A5 0 eluate (lyophilisare) 6-Cli

Culture supernatant fluid (2 1), filtrate Retentate following wash (Iyophilisare)

1,100 mg

Methanol precipitate

390 mg

69.2 ug/mg

109/mg

1,575

31.9 mg

14.7 ug/rng

78/mg

5,305

(lyo philisa re)

Sephadex DEAE-A50 eluate (lyophilisate) a

One azocasein unit (1 ACU) was defined as the amount of enzyme produ cing an absorbance increase (A440nm) of 0,01 under the experimental conditions employed.

150

U. Wolf, D. Bauer, and W. H. Traub

1

2

4

3

7

6

5

8

9 KDa- 200 116,3 - - 92,5

662

45

31

21,5 Fig. l a. SDS-PAGE eleetropherogram of successive purification steps of C. perfringens proteases 4-Cli and 6-Cli. Lane 1 - Protease 4-Cli after pressure filtration (retentate) 2 - Protease 4-Cli after methanol precipitation 3 - Protease 4-Cli after DEAE-A50 ion exchange chromatography 4 - Protease 4-Cli following HPLC chromatography 5 - Protease 6-Cli after pressure filtration (rerenrate) 6 - Protease 6-Cli after methano l precipitation 7 - Protease 6-Cli after DEAE-A50 ion exchange chromatography 8 - Protease 6-Cli after HPLC chromatography 9 - Molecular weight protein standards (from top to bottom): Myosin, ~-galactosid ­ ase, phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, and soybean trypsin inhibitor.

1 2

3

4

5

6

7

B '9

-

-

--

200

1~,~ , 66,2

-

--

---

45

31

- - - - - ----

21,5

KDa

C. perfringens Serine Proteases

151

4

21,

14,." "----" Fig. 2. SDS-PAGE gel ove rla y on Skim M ilk aga r to demonstrate caseinolytic activities of two purified C. perfringens proteases. Lanes 1 and 2: Fount Indiaf ink stained blot s of molecular weight protein standards (see Fig. 1, lane 9; plu s lysozyme , 14.4 kD a (bottom)) placed adjacent to SDS-PAGE gel immediately pri or to photography. Lane 3: Protease 4-Cli Lane 4: Pro tease 6-Cli.

~ Fig. lb. SDS-PAGE elecrrophero gram of pu rified C. perfringens pr oteases 4-Cli and 6-Cli following DEAE-A50 ion excha nge chro matog ra phy. Lane 1 Protease 4-Cli 2 Protease 6-C1i 3 M olecular weig ht protein sta ndards 4 Prot ease 4-Cli 5 Protease 6-Cli 6 Molecular weight protein standards 7 - Protease 4- Cli 8 Protease 6-Cli 9 M olecular weight protein sta ndards (see Fig. l a, lane 9).

152

U. Wolf, D. Bauer, and W. H. Traub

Table 2. Reciprocal ELISA titres of two rabbit immune sera against two purified proteases of C. perfringens Titre of rabbit serum No. Antigen (protease)

356 (antiprotease 4-Cli)

357 (antiprotease 6-Cli)

146 (normal rabbit serum)

4-Cli 6-Cli

40.960 40.960

163.840 163.840

<40 <40

1

2

4

5 KD

I

200 ,116,3

' 92,5

' 662 ,

--

--

45

.... 31

-r' i •

21,5 : _ . 14.4

Fig. 3. Western blots (immunoblots) of rabbit immune sera Nos. 356 and 357, diluted 1: 100, against two purified C. perfringens proteases; slots received 20 ul of 20 mg/ml of enzyme, respectively. HRP-conjugated pig anti-rabbit immunoglobulin (diluted 1 : 500) was the second antibody. Lane 1: RIS No. 356 against protease 4-Cli Lane 2: RIS No. 356 against protease 6-Cli Lane 4: RIS No. 357 against protease 4-Cli Lane 5: RIS No. 357 against protease 6-Cli Lanes 3 and 6: molecular weight protein standards (as in Fig. 1, lane 9, except that lysozyme (14.4 kDa) was the eighth marker (bottom)). The blotted control proteins had been stained with Pelikan Fount India'" ink.

a

0.812

100 %

0.970

1.145

0.000

0% 116 %

0.000

0.000

1.050

0%

0%

100%

100 %

109 % 0.900

1.150

0.000

0.D18

0% 0%

0.014

0.900

Tr ypsin

0%

100%

C. perfringen s 6-Cli

100%

127%

0%

2%

1.5%

100%

2.927

0.000

0.755

0.750

0.4 31

0.750

Ficin

390 %

0%

100 %

100 %

5 7.5 %

100 %

0.000

0.765

0.76 1

0%

100 %

100 %

S.marcescens SF 178 rnetalloprotease

Azocoll hydrolysis (AS20nm) ' The proteases were empl oyed at the following concent rations: C. per fringens 4-Cli and 6-Cli = 20 mg/ml PBS, pH 7.5; trypsin = 50 ug/ml; ficin = 25 ug/ml, S. marcescens SF 178 metalloprotease = 2 mg/m!.

10mM

EDTA

0.000

ImM

0.941

0.000

lOmM

10 mM

0.000

0.811

C. perfrin gens 4-cli

ImM

-

Final concentration employed

E-64

Phenylmethylsulph onylfluoride (PMSF) - - p-aminobenzamidin e -pefabloc SC

None

Protea se inhibitor

Activity (AS20nm )a per cent (% ) of protease following exposure

Table 3. Effects of vario us inhibitors again st two C. perfringens serine proteases

W

VI

......

"'"'"

I»

as

n>

S'

V"l

:::

" '" ...n>

OQ

S'

o ...n> :::' '0

154

U. Wo lf, D . Bauer, and W. H . Tra ub

Table 4 . Enhance ment of activities of two C. perfringens serine proteases by thiol protease in hi bito rs Protease inhi bitor

Fin al co nce nt ra tion employed

Activity (% ) of C. perfringens protease 4-Cli

6- Cl i

100

None

( A S 20 n m

1,4-dith ioth reitol L-cystein e 2- merca ptoetha nol

5 mM 10 mM lOmM

100 = 0.750 )

3 70 353 3 76

(A 52 0 nm

= 0 .9 70)

356 35 7 3 76

T a ble 5. List of pu rified human seru m p roteins (concentrat ions, sol vents) em p loy ed for C. perfringens protease ex peri ments" Human serum p rotein

Solvent

Concentrati on

19A

0. 145 M N a CI 0. 145 M NaCI 0.05 TR IS-H C I 0. 15 M NaCl, 0.01 M glycine, pH 7.4

1.1 mg/ml 1.1 mg/ml 1.1 mg/mI

C1q C3 C5 C8 C9

PBS, pH pH pH pH pH

25 0 25 0 25 0 200 2 00

Alp ha I-an titrypsi n Alphaj-rnacroglo bulin Fib rinogen ty pe III Fib ronecti on T ransferri n H ap to globin C-react ive protein

PBS, p H 7.5 pH 7.5 p H 7 .5 p H 7.5 pH 7 .5 p H 7.5 p H 7.5

IgG IgM

a

7.5 7. 5 7. 5 7.5 7.5

ug/ml ug/rnl ug/ml ug/rnl ug/ml

1.25 mg/ml 25 0 ug/ml 4 mg/m l 1 mg/ml 2 mg/m l 25 0 ug/rnl 25 0 ug/rnl

The protea ses we re em ployed a r 20 mg Iyo philisat e/ml of PBS, pH 7.5 ; th e ac tivity of pr ot ease 4 -Cli was 58 .2 A CU/ m l lyoph ilisare a nd th at of protease 6-Cli was 4 4 .5 ACUI mg lyophilisare. For coincu ba ti on , 50 ILl of seru m protein were co mbined with 50 ILl of enzyme ; incubatio n was at 35°C for 22 h (im m unoglo bulins) and at 35 °C for 5 h (all other serum pr oteins),

Fig. 5 . SDS-PAGE elec trop he rog ram of human co mp leme nt compo ne nt s C3 a nd C5 foll ow - ~ ing ex posure to C. perfrin gens proteases 4-Cl i a nd 6-Cli. Lan e 1 C3 , contro l 2 C3 + protea se 4- Cli 3 C3 + protease 6-Cli 4 C5 + co nt ro l 5 C5 + protease 4-C li 6 C5 + p rotease 6- Cli 7 Pro tease 4- Cli, control 8 Pro tea se 6- Cl i, control 9 M ol ecular weight protein stand ards (see Fig. 1).

C. perfringens Serine Proteases

1

2

3

4

155

6

5

200

KDa

116,3 92,5 66,2

45

-

31

Fig. 4. SDS·PAGE electropherogram of human IgG following exposure to C. perfringens proteases 4-CIi and 6-C1i. Lane 1 - IgG, control 2 - IgG + protease 4-Cli, fresh 3 - IgG + protease 4-C1i (60 °C, 15 min) 4 - IgG + protease 6-C1i, fresh 5 - IgG + protease 6-C1i (60 °C, 15 min) 6 - Molecular weight proteion standards (see Fig. 1).

1

2

3

4

5

6

7

9

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U. Wolf, D. Bauer, and W. H. Traub

1

3

2

4

5

6

7· 8

9

Fig. 6. SDS-PAGEelectropherogram of human complement components C8 and C9 following exposure to C. perfringens proteases 4-Cli and 6-Cli. Lane t - C8, control 2 - C8 + protea se 4-Cli 3 - C8 + protease 6-Cli 4 - C9, control 5 - C9 + protease 4-Cli 6 - C9 + protease 6-Cli 7 - Protease 4-Cli, control 8 - Protease 6-Cli, control 9 - Molecular weight protein standards (see Fig. 1).

1

2

3

4

5

6

7

8

9

C. perfringens Serine Proteases

157

Discussion The two C. perfringens proteases appeared to be pure as revealed by SDS-PAGE electropherograms and immunoblots. Both enzymes proved to be immunogenic for rabbits and were essentially identical in terms of reciprocal ELISAand immunoblotting cross-reactivity. The activities of both enzymes were antagonized only by inhibitors of serine proteases, but stimulated markedly in the presence of dithiothreitol, Lcysteine, and 2-mercaptoethanol. Therefore, these two enzymes were categorized as serine proteases, with a possible thiol group in the active centre. To the best of our knowledge, the 41.7 kDa proteases produced by two representative clinical isolates of C. perfringens type A are novel enzymes with demonstrable proteolytic activity against various purified human serum proteins, including IgG and IgM, various "acute phase"-proteins, and several components of the complement system. The two proteases failed to degrade C component C1q; furthermore, both enzymes lacked activity against several chromogenic collagenase substrates (Wolf, U. et al., unpublished observations). In addition, the molecular weights of these proteases differed from that of the classical kappa toxin (80 kDa), a genuine collagenase (2, 8, 12, 15, 16, 19). With the aid of Sephadex G-150 gel filtration, a strain of C. perfringens type A (strain PB6K N5) had been shown by Sato et al. (19) to produce at least three proteases, which were presumptively characterized as an EDTA-sensitive metalloprotease, a thiol protease, which could be reactivated with cysteine, and an unknown protease of larger molecular size, which proved to be insensitive to iodoacetamide and EDTA, respectively; however, the authors did not state the molecular weights of any of these three proteases. Furthermore, C. perfringens type B, E, and D, but not A, strains are known to produce lambda toxin, a non-collagenolytic gelatinase, which, however, hydrolyzes various protein substrates, including azocoll, casein, and hemoglobin (12, 16). Nevertheless, it is felt that the two 41.7 kDa proteas described in this preliminary report represent novel exoenzymes. It is not yet known whether these proteases are produced by C. perfringens type A in vivo as well and might contribute to the pathogenesis of myonecrosis, i.e., clinical gas gangrene, with concomitant lack of generation of chemoattractants for polymorphonuclear leukocytes (11). Therefore, in the absence of relevant experimental animal data, the potential pathobiological significance of this multifacetted, albeit nonspecific putative bacterial aggressin currently remains conjectural.

Acknowledgments. We thank Ms. Reate Ebel for the preparation of this manuscript.

~ Fig.7. SDS-PAGE electropherogramof human alphaj-antitrypsin and alphaj-macroglobulin following exposure to C. perfringens proteases 4-Cli and 6-Cli. Lane 1 - Alpha--antitrypsin, control 2 - Alphaj-antitrypsin + protease 4-Cli 3 - Alphaj-antitrypsin + protease 6-Cli 4 - Alphaj-macroglobulin, control 5 - Alphaj-macroglobulin + protease 4-Cli 6 - Alphaj-macroglobulin + protease 6-Cli 7 - Protease 4-Cli, control 8 - Protease 6-Cli, control 9 - Molecular weight protein standards (see Fig.1).

H-chain: 53 L-chain: 25.1 H-chain: 70.8 L-chain: 25.1

IgG

58 166 major components: 57.5, 52.2 200 44 76.5

Alpha I-antitrypsin Alphay-macroglobulin Fibrinogen, type III Fibronection Haptoglobin Transferrin

algA, C1q, and CRP resisted the proteases.

C8 C9

144.5, 138, 79.4 138 79.4 72.4 79.4

C3 C5

IgM

Molecular weight (kDa) of protein/components

Human serum protein

56.2 kDa fragment generated Total degradation Total degradation Total degradation Total degradation 60, 52, 43, and 39 kDa fragments generated

Total degradation Faint 30.2 kDa fragment generated Total degradation Total degradation Total degradation

33.3 kDA fragment generated No change 32 kDa fragment generated No change

4-Cli

56.2 kDa generated 89.1 kDa fragment generated Total degradation 83.2 kDa fragment generated 43.6 kDa fragment generated 60, 52, 43, and 39 kDa fragments generated

Total degradation Faint 30.2 kDa fragment generated Unaffected 56.2 kDa fragment generated 52.5 kDa fragment generated

33.3 kDa fragment generated No change 32 kDa fragment generated No change

6-Cli

Degradation of protein/component by C. perfringens protease

Table 6. Summary of human serum proteolytic activities (SDS-PAGE electropherograms) of two C. perfringens proteases"

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C. perfringens Serine Protea ses

159

References

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Prof. Dr. Walter H. Traub, Institut fur Medizinische Mikrobiologie und Hygiene, Haus 43, D-6650 Homburg/Saar, Germany