Collagenase of Clostridium perfringens Type A: Degradation of Human Complement Component C1q

Zbl. Bakt. 276, 27-35 (1991) © Gustav Fischer Verlag, StuttgartlNew York

Collagenase of Clostridium perfringens Type A: Degradation of Human Complement Component Clq URSULA WOLF, DIERK BAUER, and WALTER H. TRAUB* Institut ftir Medizinische Mikrobiologie und Hygiene, Universitat des Saarlandes, D-6650 Homburg/Saar

With 2 Figures· Received May 15, 1991 . Accepted in revised form July 9, 1991

Summary The semipurified collagenases from Clostridium perfringens type A strains 2-Cli and ATCC 13124, both characterized by molecular weights of 79.4 kilodaltons, partially degraded purified human complement (C) component C1q. The following purified human serum proteins were refractory: C components C3, C4, C5, C6, C7, C8, and C9; immunoglobulin (Ig)A (from colostrum), IgG, and IgM; alpharmacroglobulin, haptoglobin, and Creactive protein. Zusammenfassung Zwei semigereinigte Collagenasen der Clostridium perfringens Typ A Stamme 2-Cli and ATCC 13124, gekennzeichnet durch ein Molekulargewicht von jeweils 79.4 Kilodaltonen, degradierten partiell die gereinigte Human-Komplement (C) Komponente Clq. Folgende gereinigte Humanserum-Proteine erwiesen sich als resistent; die C-Komponenten C3, C4, C5, C6, C7, C8 und C9; Immunglobulin (Ig)A (aus Colostrum), IgG and IgM; ferner alphar Makroglobulin, Haptoglobin und C-reactives Protein. Introduction

Clostridium perfringens type A, the most common causative agent of human gas gangrene, i.e., myonecrosis (2, 9, 32, 33), had long been known to produce various exotoxins (11). The most important exoenzyme was found to be alpha-toxin, a genuine phospholipase C, which proved a protective antigen in experimental animals (4). Additional exotoxins were kappa toxin, a collagenase (14) characterized by a molecular weight of 80 kilodaltons (kDa), and theta toxin (or perfringolysin 0), a 51 kDa thiolor SH-activated cytolysin (11). Both kappa and theta toxin were lethal for mice after

* Corresponding author

28

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

intravenous injection; and kappa toxin revealed local dermonecrotic activity in rabbits following intracutaneous administration (11). Very recently, Walter (31) demonstrated the 80 kDa collagenase of C. perfringens to be a chemoattractant for human polymorphonuclear leukocytes. We decided to purify kappa toxin from two selected strains of C. perfringens type A and to examine the enzymes for proteolytic activity against various purified human serum proteins, analogous to experiments performed previously with regard to two novel purified proteases of this microorganism (34).

Materials and Methods

Bacteria. C. perfringens strain 2-Cli (protocol no. Va 915; source: patient M. H., post partum lochiaj 26.1. 83) had been identified according to conventional criteria (1, 13,32, 33). C. perfringens strain ATCC 13124 served for control purposes. Media. NIH-Thioglycolate (NIH-THIO) broth and Protease Peptone were purchased from Difco Laboratories, Detroit, Michigan. Vitamins, thioglycolic acid, and purine bases were obtained from Sigma Chemie GmbH, Deisenhofen, and were used for the medium of Murata et al. (12) which was utilized for large batch cultures. The bacteria were maintained as previously described (27, 29, 34). Reagents. All chemical reagents, including sorbitol and those for phosphate-buffered saline (PBS), pH 7.5, were of analytical grade (E. Merck, Darmstadt). Azocasein (sodium saltj batch D8), acid-soluble collagen from calf skin and insoluble type I collagen from bovine Achilles tendon, type XII C. perfringens phospholipase C (EC 3.1.4.3j activity: 300 units/mg protein), and p-nitrophenylphosphorylcholine (NPPC) were purchased from Sigma. Collagenase of C. histolyticum (EC 3.4.24.3j activity 0.9 U/mgj lot 23018), the collagenase substrate Z-Gly-Pro-Leu-Gly-Pro (batch B8), and ninhydrin were procured from Serva Feinbiochemica GmbH, Heidelberg. Polyacrylamide gel electrophoresis reagents, including 8 molecular weight protein standards (range 14.4 - 200 kDa) and Coomassie Blue R-250, were obtained from Bio-Rad Laboratories GmbH, Miinchen, as were horseradish peroxidase (HRP) and 4-chloro-1-naphthol. Purified immunoglobulin (Ig)A (from colostrum), IgG, and IgM were supplied by Sigma. Azocoll and the following purified human serum proteins were purchased from Calbiochem GmbH, Frankfurt: complement (C) components C1q, C3, C4, C5, C6, C7, C8, and C9j alpharmacroglobulin, haptoglobin, and Creactive protein (CRP). All human serum proteins were stored at -65 ec until further use. HRP-conjugated pig anti-rabbit immunoglobulin (batch 095) was supplied by Dakopatts GmbH, Hamburg. Sheep and horse erythrocytes were purchased from Gesellschaft fiir Mikrobiologische Nahrmedien mbH, Walldorf. Purification of C. perfringens collagenases. The C. perfringens strains were inoculated into 25 ml of NIH-THIO broth and incubated at 35 ec for 5 h, after which the entire 25 ml were transferred into 2.5 liters of Murata medium. After incubation at 35 ec for 18 h, the cultures were centrifuged at 12,000 x g, 4 ec, for 20 min. The supernatant fluids were membrane-filtered (0.45 f-lmj Pressure Filtration Unit model 16274, Sartorius GmbH, Gottingen). The filtrate was concentrated tenfold (Mini Cross-Flow System, Sartorius) and subsequently dialyzed against 5 liters of 0.05 M phosphate buffer, pH 7.2. Next, 400 g of ammonium sulfate were dissolved in 1 liter of filtrate under constant stirringj the suspension was held at 4 ec overnight. The precipitate was sedimented (12,000 x g, 4 ec, 20 min), dialyzed, lyophilized (model GT2, Leybold-Heraeus GmbH, Koln-Bayental) and stored at -65 ec. For chromatography, 50 mg of Iyophilisate were dissolved in 3 ml of elution buffer (0.05 M Tris-HCI, 0.005 M CaCh, 0.02% (w/v) NaN3 , pH 7.5) and applied to 2.6 x 10 em columns of Sephacryl S-200 HR (Pharmacia Biotechnology, Uppsala, Sweden). The flow rate was adjusted to 15 mllh; 2 ml fractions were collected. The fractions were examined for protein content at 280 nm (model 2138 Uvicord S, LKB Instrument GmbH, Griifelfing) and for collagenase activity (see below). Fractions with demonstrable collagenolytic activity

Collagenase of Clostridium perfringens Type A

29

were combined, ultrafiltered (CX-30 Ultrafilter, Millipore GmbH, Eschborn), lyophilized, and stored at -65°C. Assays for proteolytic and collagenolytic activity. The method of Yagisawa et al. (34) was used to detect collagenolytic activity. To 200111 of the synthetic peptide Z-Gly-Pro-Leu-GlyPro (0.02 M in 0.02 M Veronal-HCl buffer, pH 8.2) were added 100 111 of enzyme suspension (3 mg/ml 0.033 M phosphate buffer, pH 7.4) and 10 111 of CaCh (2 mM in distilled water). The mixture was incubated at 30°C for 18 h. The liberated amino groups were determined with the colorimetric ninhydrin method of Rosen (19); a calibration curve was constructed with different concentrations of glycylproline (AS70nm). Azocoll hydrolysis was determined in accordance with the supplier's specifications. Briefly, 10 mg of azocoll were suspended in 1 ml of 0.1 M potassium phosphate buffer, pH 7.0; 100111 of sample (3 mg lyophilisate/ml of 0.033 M phosphate buffer, pH 7.4) were added. The mixture was incubated under constant stirring at 37"C for 30 min. Undegraded azocoll was removed by centrifugation. Absorbance of the supernatant fluids was measured at A S20nm ' A calibration curve was established with the commercial C. histolyticum collagenase (5 mg/ml of 0.05 M Tris buffer, pH 7.4, with added 0.005 M CaCI2). The method of Mandl et al. (10) was employed to detect hydrolysis of insoluble collagen; 25 mg of type I collagen were suspended in 4.9 ml of 0.033 M phosphate buffer, pH 7.4, following which 100111 of 0.1 % (w/v) enzyme solution (same buffer) were added. The mixture was incubated under constant stirring at 37°C for 18 h. The quantity of solubilizd collagen was determined with the colorimetric ninhydrin method of Rosen (19; see above). A calibration curve was constructed employing different concentrations of leucine (AS70nm). Assay for protein content. The BioRad Protein Standard Assay kit was employed under strict adherence to the manufacturer's instructions. Tests for phospholipase C activity. The semipurified collagenases (100 l1g/ml) were examined for phospholipase C activity according to the method of Kurioka and Matsuda (7), using 10 mM NPPC together with 60% (w/v) sorbitol in 0.25 M Tris-HCl, pH 7.2, as specified previously (28). The commercial phospholipase C (160 units/ml) and C. histolyticum collagenase (l00 l1g/ml) served as controls. Absorbance was measured at 410 nm. Tests for hemolytic activity. The semipurified collagenases (100 l1g/ml) were tested for hemolytic activity against 2% (v/v) sheep and horse erythrocytes using a microtiter method as described before (29). The commercial phospholipase C was utilized for control purposes. Exposure of human serum proteins to C. perfringens collagenases. Twentyfive 111 of serum protein (250 l1g/ml PBS, pH. 7.5; C1q, C3, C4, C5, C6, C7; alpha I-antitrypsin, alpha2-macroglobulin; 100 l1g/ml PBS, pH 7.5: C8, C9; 1.1 mg!ml of 0.145 M NaCl: IgA, IgG, and IgM) were combined with 25 111 of 0.5 mg!ml of collagenase 2-Cli and with 1 mg! ml of collagenase ATCC 13124, respectively. Control proteins received 25 111 of PBS, pH 7.5. The immunoglobulins were exposed additionally to heat-inactivated collagenases (60°C, 15 min). All mixtures were incubated at 35°C for 24 h, following which they were examined with the SDS-PAGE procedure. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE procedure of Laemmli (8) served to determine the molecular weights of the two collagenases and of the human serum proteins after enzymatic exposure. Wells received 20 111 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% acarylamide) were electrophoresed at 30 rnA/gel and 10°C. The gels were fixed, stained, destained, and dried as specified previously (28). lsoelectric focusing. This procedure was performed using the Servalyt® PreNets® kit (lot no. 06060) and protein test mixture 9 (lot no. 25081) from Serva under strict adherence 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 separation of proteins. The pH gradient was 3-10. Production of anti-collagenase rabbit immune serum (RIS). New Zealand White rabbit (SAVA mediz. Versuchstierzuchten GmbH, Kisslegg/Allgau) no. 263 was immunized as

30

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

follows: 0.5 mg of lyophilized, semipurified collagenase of C. perfringens strain ATCC 131-24 was dissolved in 1 ml of PBS, pH 7.5, and incorporated into 1 ml of Freund's complete adjuvant (Behringwerke AG, Marburg). The rabbit was administered the suspension subcutaneously (s.c.); 21 days later, the animal received an identical booster injection s.c. The rabbit was exsanguinated 4 weeks later through cardiac puncture under general anesthesia (Evipan®, Bayer AG, Leverkusen). The separated RIS was stored at -65°C, Rabbit no. 52 served as a source of normal rabbit serum (NRS). Enzyme-linked immunosorbent assay (ELISA). The microtiter ELISA procedure employed was the same as that specified previously (30). The sera were tested in quadruplicate. Alkaline phosphatase-conjugated swine anti-rabbit immunoglobulin (lot 108; Dakopatts GmbH, Hamburg), diluted 1: 500, was the second antibody. The phosphatase substrate was Sigma 104® (Sigma). The titres of the rabbit sera were interpreted as the highest dilution that yielded an ~05 nm of ~ 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. (26), as described previously (28), was utilized to examine RIS no. 263, diluted 1: 100, for immunoreactivity against the two collagenases of C. perfringens. The second antibody was HRP-conjugated pig anti-rabbit Ig, diluted 1 : 500. Results The collagenases of C. perfringens strains 2-Cli and ATCC 13124 revealed rather similar activity during the successive purification steps (Table 1). The collagenase from strain 2-Cli (100 f.l.g/ml) lacked phospholipase C activity in terms of NPPC-hydrolysis (A410nm = 0.029); however, the collagenase from strain ATCC 13124 (100 f.l.g/ml) yielded an ~10nm of 0.525. For comparison, the commercial phospholipase C (160 units/ml) yielded an A410nm of 4.0 at 1: 0, 1.34 at 1: 4, 0.46 at 1: 16, 0.22 at 1: 64, 0.14 at 1 : 256, and of 0.1 at a dilution of 1 : 1024, and the C. histolyticum collagenase (100 f.l.g/ml) had an A410nm of 0.016 (the ~10nm of the buffer blank control was 0.071). Furthermore, whereas the 2-CIi collagenase (100 f.l.g/ml) was free of hemolytic activity against sheep and horse erythrocytes, the ATCC 13124 collagenase (100 f.l.g/ml) hemolyzed both sheep and horse erythrocytes at a dilution of 1: 16; the commercial phospholipase C (160 units/ml) also hemolyzed both sheep and horse erythrocytes at a dilution of 1: 16. The hemolytic activities of the collagenase ATCC 13124 and of the commercial phospholipase C were abolished by heat treatment at 60°C for 1 h, but not by 50°C for 1 h (data not shown). Both enzymes were characterized by a molecular weight of 79.4 kDa and by an isoelectric point (IEP) of 4.5. RIS no. 263 reacted with both collagenases at a dilution of 1 : 163,840 (ELISA procedure); NRS no. 52 lacked detectable antibodies (titer = < 1: 40). Western blots with RIS no. 263, diluted 1 : 100, against the two semipurified collagenases (30 f.I.l of 20 f.l.g/ml of enzyme per slot) yielded one immunoreactive polypeptide with an apparent molecular weight of about 80 kDa, respectively (Fig. 1). The two enzymes degraded C1q, specifically the A, B, and C chains of C1q, which had apparent molecular weights of 33.1,31.6, and 28.2 kDa, respectively (Fig. 2). However, all of the other examined human serum proteins resisted collagenolytic attack.

C

b

a

92.3 mgl2.5 I

15.7 mg

Ammonium sulfate precipitate

Sephacryl S-200 HR eluate

Culture supernatant fluid

26 mg

Sephacryl S-200 HR eluate

Corresponding to C. histolyticum collagenase units. /lg glycylproline from synthetic substrate. /lM leucine (= units).

ATCC 13124

107.3 mgl2.5 I

Culture supernatant fluid

2-Cli

Yield

Ammonium sulfate precipitate

Preparation

C. perfringens strain

0.068

0.41 mglmg lyophilisate

0.47 mglmg lyophilisate

0.25 mglmg lyophilisate

43.8 mgl2.5 L

0.038

0.068

0.004

0.038

0.018

110 mgl2.5 L

0.43 mglmg lyophilisate

Azocoll hydrolysis a

Protein content

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

113

564.0

327.7

91.5

112

119

495.4

541.2

30.5

113

103

118

Activity Insoluble Hydrolysis of Z-Gly-Pro-Leu- collagen C Gly-Pro b

n

§::

w ......

";...

"0

~

'"

"::I

()Q

S·

"..,r:t'

"0

8

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00

"::II» '" "..... 0

()Q

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32

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

1 2

--

Fig. 1. Western blot (immunoblot) of rabbit immune serum no. 263, diluted 1 : 100, against collagenase 2-Cli (lane 1) and collagenase ATCC 13124 (lane 2); slots received 30 I-tl of 20 I-tg/ml of enzyme, respectively. HRP-conjugated pig antirabbit immunoglobulin (diluted 1 : 500) was the second antibody. Lane 3 = molecular weight protein standards (s. Fig. 2, lane 6), blotted, and stained with Coomassie Blue R-2S0. Discussion The molecular weights of both C. perfringens collagenases amounted to 79.4 kDa and their IEPs were 4.5, values in agreement with those derived by previous authors (3, 6, 20, 21, 23). However, the collagenase from C. perfringens strain ATCC 13124 apparently still was contaminated by alpha toxin (i.e., NPPC-hydrolysis) and by perfringolysin 0 (theta toxin) because the preparation hemolyzed both sheep and horse erythrocytes. Very recently, McDanel (11) again had cautioned investigators against contamination of allegedly purified exotoxins of C. perfringens by other toxins of this microorganism. This is why the two collagenases were designated as "semipurified", even though SDS-PAGE electropherograms and the immunoblots with the singular rabbit anti-collagenase (ATCC 13124) serum revealed apparent purity. Under our experimental conditions, the two collagenases were active only against C component Clq among the limited number of human serum proteins examined. Previously, C component Clq had been shown to be a complex protein characterized by a molecular

Collagenase of Clostridium perfringens Type A

1 2

3

33

4 5

Fig. 2. SDS-PAGE electropherogram of human complement Clq following exposure to collagenases of C. perfringens. lane 1 - Clq + collagenase 2-CIi lane 2 - Clq + collagenase ATCC 13124 lane 3 - Clq, control lane 4 - collagenase 2-CIi, control lane 5 - collagenase ATCC 13124, control lane 6 - molecular weight protein standards (from top to bottom: myosin, ~-galactosidase, phosphorylase B, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, lysozyme). weight of 459.3 kDa and composed of 6 A, 6 B, and 6 C chains, each of them with molecular weights of 23.8-27.55 kDa, respectively (15, 16, 17,22). Furthermore, it is known that the A, B, and C chains of C component Clq contain regions of 78-81 amino acid residues of collagen-like repeating sequence (18). In addition, Heinz et al. (5) demonstrated that Clq shared epitopes with type II collagen. Therefore, the SDSPAGE electropherograms were interpreted as supportive evidence in as much as it had been shown by Reid and coworkers that Clq was susceptible to proteolytic attack by the collagenase of C. histolyticum. It should be stressed that the separting gels utilized for SDS-PAGE electrophoresis did not permit resolution of large molecular weight proteins, such as alphaz-macroglobulin, which, however, yielded an apparent molecular weight of 166 kDa in our 3 Zbl. Bakt. 27611

34

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

experiments. It was not possible to determine whether the collagenases occupied the "bait region" (24) of alpharmacroglobulin and resulted in specific limited proteolysis in this region, as had been reported by Sottrup-Jensen and Birkedal-Hansen (25) for human fibroblast collagenase. In summary, the two collagenases under study attacked and degraded the A, B, and C chains of human C component Clq; whether the two enzymes had interacted with human alpharmacroglobulin is uncertain at this time. Our finding (34) that two novel proteases of C. perfringens were active against various human complement components (but not Clq), the heavy chains of IgG and IgM, as well as transferrin, alpharmacroglobulin, alphal-antitrypsin, haptoglobin, type III fibrinogen, and fibronectin, but not C-reactive protein, plus the observations of this study further attested to the enormous exoenzyme repertoire of C. perfringens. Whether the proteases of this microorganism might contribute to the typical scarcity of polymorphonuclear leukocytes in myonecrotic lesions (9), given their effects against various components of the complement system, various members of the group of "acute phase" proteins, and the heavy chains of IgG and IgM, with subsequent lack of generation of chemoattractants for neutrophil granulocytes, currently is unknown. Acknowledgments. We thank Ms. Beate Ebel for the preparation of this manuscript.

References 1. Allen, S. D.: Clostridium. In: Manual of Clinical Microbiology, 4, edition, pp. 434-444, E. H. Lennette, A. Balows, W. ]. Hausler jr., and H. ]. Shadomy (eds.). American Society for Microbiology, WashingtonIDC (1985) 2. Anonymous: Remembering gas gangrene. Lancet ii (1984) 851-852 3. Barrett, A. ]., c. G. Knight, M. A. Brown, and U. Tisljar: A continuous fluorimetric assay for clostridial collagenase and Pz-peptidase activity. Biochem. J. 260 (1989) 259-263 4. Boyd, N. A., R. o. Thomson, and P. D. Walker: The prevention of experimental Clostridium novyi and CI. perfringens gas gangrene in high-velocity missile wounds by active immunization. J. Med. Microbiol. 5 (1972) 467-472 5. Heinz, H.-P., K. Rubin, A.-B. Laurell, and M. Loos: Common epitopes in C1q and collagen type II. Behring Institute Mitteilungen Nr.84 (1989) 48 6. Kameyama, S. and K. Akama: Purification and some properties of kappa toxin of Clostridium perfringens. Jap. J. Med. Sci. BioI. 24 (1971) 9-23 7. Kurioka, S. and M. Matsuda: Phospholipase C assay using p-nitrophenylphosphorycholine together with sorbitol and its application to studying the metal and detergent requirements of the enzyme. Analyt. Biochem. 75 (1976) 281-289 8. Laemmli, U. K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 277 (1970) 680-685 9. MacLennan, ]. D.: The histotoxic clostridial infections of man. Bact. Rev. 26 (1962) 177-276 10. Mandl,!., H. Zipper, and L. T. Ferguson: Clostridium histolyticum collagenase: Its purification and properties. Arch. Biochem. Biophys. 74 (1958) 465-475 11. McDonel,]. L.: Toxins of Clostridium perfringens types A, B, C, D and E. Chapter 22, pp. 477-517. In: Pharmacology of Bacterial Toxins, Section 119, F. Dorner and]. Drews (eds.). Pergamon Press, Oxford (1986) 12. Murata, R., A. Yamamoto, S. Soda, and A. Ito: Nutritional requirements of Clostridium perfringens PB6K for alpha toxin production. Jap. J. Med. Sci. BioI. 18 (1965) 189-202 13. Nagler, F. P.O.: Observations on a reaction between the toxin of Cl. welchii (type A) and human serum. Brit. J. Exp. Path. 20 (1939) 473-485

Collagenase of Clostridium perfringens Type A

35

14. Oakley, C. L. K., G. H. Warrack, and W. E. van Heyningen: The collagenase (kappa toxin) of Cl. welchii type A. J. Path. Bact. 58 (1946) 229-235 15. Reid, K. B. M.: Chemistry and molecular genetics of C1q. Behring Institute Mitteilungen Nr. 84 (1989) 8-19 16. Reid, K. B. M.: Application of molecular cloning to studies on the complement system. Immunology 55 (1985) 185-196 17. Reid, K. B. M.: Proteins involved in the activation and control of the two pathways of human complement. Biochem. Soc. Transact. 11 (1983) 1-12 18. Reid, K. B. M. and A. J. Day: Structure-function relationships of the complement components. Immunol. Today 10 (1989) 177-180 19. Rosen, H.: A modified ninhydrin colorimetric analysis for amino acids. Arch. Biochem. Biophys. 67 (1957) 10-15 20. Sato, H., Y. Yamakawa, A. Ito, and R. Murata: Effect of protease on the production of Clostridium perfringens alpha toxin. Jap. J. Med. Sci. BioI. 30 (1977) 44-46 21. Sato, H., Y. Yamakawa, A. Ito, and R. Murata: Effect of zinc and calcium ions on the production of alpha-toxin and proteases by Clostridium perfringens. Infect. Immun. 20 (1978) 325-333 22. Schumaker, V. N., P. Zavodszky, and P. H. Poon: Activation of the first component of complement. Ann. Rev. Immunol. 5 (1987) 21-42 23. Smyth, C. J. and J. P. Arbuthnott: Properties of Clostridium perfringens (welchii) type-A a-toxin (phospholipase C) purified by electrofocusing. J. Med. Microbiol. 7 (1974) 41-66 24. Sottrup-Jensen, L.: a-Macroglobulins: Structure, shape and mechanism of proteinase complex formation. J. BioI. Chern. 264 (1989) 11539-11542 25. Sottrup-Jensen, L. and H. Birkedal-Hansen: Human fibroblast collagenase-a-macroglobulin interactions. Localization of cleavage sites in the bait regions of five mammalian amacroglobulins. J. BioI. Chern. 264 (1989) 393-401 26. Towbin, H., T. Staehelin, and J. Gordon: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Procedure and some applications. Proc. Nat. Acad. Sci. USA 76 (1976) 4350-4354 27. Traub, W. H.: Clostridium perfringens type A. Comparison of in vitro and in vivo activity of twelve antimicrobial drugs. Chemotherapy 32 (1986) 59-67 28. Traub, W. H. and D. Bauer: Interactions of purified Serrratia marcescens proteases with fresh human serum and with purified human serum proteins. Zbl. Bakt. Hyg. A 263 (1987) 561-571 29. Traub, W. H., D. Bauer, and U. Wolf: Virulene of clinical and faecal isolates of Clostridium perfringens type A for outbred NMRI mice. Chemotherapy, in press (1991) 30. Traub, W. H., M. Spohr, and D. Bauer: Active immunization of NMRI mice against Serratia marcescens. I. Phenol-water lipopolysaccharide fractions and purified metalloproteases. Zbl. Bakt. Hyg. A 265 (1987) 182-196 31. Walter, R. J.: Clostridial collagenase. A chemoattractant for human neutrophils. Inflammation 10 (1986) 347-361 32. Werner, H.: Anerobier-Infektionen. Pathogenese, Klinik, Therapie, Diagnostik, 2. Aufl. Georg Thieme Verlag, Stuttgart (1985) 33. Willis, A. T.: Clostridium: the spore-bearing anerobes. In: Topley and Wilson's Principles of Bacteriology, Virology and Immunity, 7. edition, vol 2, pp. 442-475, M. T. Parker (ed.). Edward Arnold, London (1983) 34. Wolf, U., D. Bauer, and W. H. Traub: A 44.7 kDa protease from Clostridium perfringens type A: Degradation of human serum proteins. Zbl. Bakt. (Submitted) 35. Yagisawa, S., F. Morita, Y. Nagai, H. Noda, and Y. Ogura: Kinetic studies on the action of collagenase. J. Biochem. 58 (1965) 407-416

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