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Fibrinolytic activity of purified serratia marcescens metalloproteases
Zbl. Bakt. Hyg. A 260, 35-40 (1985)
Fibrinolytic Activity of Purified Serratia marcescens Metalloproteases WALTER H. TRAUB and DIERK BAUER Institut fur Medizinische Mikrobiologie und Hygiene, Universitat des Saarlandes, 6650 Homburg/Saar, Federal Republic of Germany
With 3 Figures ' Received January 8, 1985 . Accepted February 20, 1985
Abstract Two purified rnetalloproteases of Serratia marcescens revealed fibrinolytic activity, i.e., degraded human fibrin in agar as well as commercially available human fibrinogen and fibrinogen in fresh human plasma.
Zusammenfassung Zwei gereinigte Metalloproteasen von Serratia marcescens erwiesen sich als Fibrinolysine. Beide Enzyme degradierten Human-Fibrin in Agar sowie kommerziell erhaltliches Human-Fibrinogen und Fibrinogen in frisch em Human-Plasma.
Introduction During the past two decades, Serratia marcescens emerged as an opportumsnc pathogenic, nosocomially significant microorganism (17). However, relatively little is known about the virulence determinants of this bacterial species (12). It was demonstrated that S. marcescens strains carrying lipopolysaccharide a-antigens 06 and/or 014 were somewhat more virulent for outbred NMRI mice than isolates characterized by other serogroup antigens (14). Clinical isolates of S. marcescens were shown previously to produce various exoenzymes (16). Of these, a purified metalloprotease, derived from S. marcescens strain BG, was demonstrated as a genuine bacterial aggressin with regard to experimental pneumonia in laboratory animals by Kreger and associates (9). We had previously noted that several clinical isolates of S. marcescens altered diverse proteins of fresh human serum (5). Subsequently, we purified and characterized representative metalloproteases derived from two nosocomially significant strains of S. marcescens (6). This study served to determine whether these two exoenzymes displayed fibrinolytic activity.
36
W. H. Traub and D. Bauer Materials and Methods
Bacteria. S. marcescens isolates SF 178 and SH 186 were recovered from patients with septicemia and post-operative wound infection in San Francisco, California, and Homburg/ Saar, respectively. Both isolates were of serotype 061014: H12 and bacteriocin type 18, and were maintained as previously described (15). Media. Tryptic Soy broth, Brain Heart Infusion broth, Nutrient broth, Bacto Agar, and Skim Milk (source of casein) were procured from Difco Laboratories, Detroit, Michigan. Skim Milk agar contained 3 % Skim Milk and 1.5 % Bacto-Agar. Fibrin agar was prepared as follows: 10 ml of a 1 % human fibrinogen (Behringwerke AG, Marburg) solution were added to 90 ml of Nutrient broth containing 1.5 % Bacto-Agar, mixed, and held at 55 °C for 10 min, after which 1 ml of fresh human serum (donor T.) was added (3). Reagents. Human type III fibrinogen (Fraction I, lot no. 13F-9330; 79 % protein content) was purchased from Sigma Chemie GmbH, Taufkirchen. The fibrinogen stock solution contained 4 mg per ml of phosphate-buffered saline, pH 7.5, which was subdivided into small aliquots, frozen and kept stored at -65°C until further use. Human test fibrinogen (lot no. 504 317 B, 910 mg dry substance) was obtained from Behringwerke AG, Marburg, and rehydrated with 25 ml of sterile demineralized water; this stock solution contained 1 % fibrinogen, i.e., 10 mg per ml of 0.1 M TRIS buffer, pH 8.0. Rabbit anti-human fibrinogen immune serum (lot no. 10541 B) was obtained from Behringwerke AG. Two healthy adults (B. and T.) repeatedly donated 20 ml blood samples which were heparinized; plasma was separated by centrifugation at 2500 x g for 10 min, 22°C, and utilized immediately. Purification of metalloproteases. The two S. marcescens metalloproteases were purified and lyophilized as described previously (6). The activities of the enzymes were monitored (440 nm) through hydrolysis of azocasein (lot no. 09061; Serva Feinbiochemica GmbH, Heidelberg); a 10 mg/ml solution of trypsin 1 : 250 (Serva; lot H; activity = ca. 4 U/mg) served as a control. Immunization of rabbits. New Zealand White rabbits (Medizinische Versuchstierzucht Nickisch, Osthofen-Neuteich) were immunized as follows: 1 ml of 200 j.tg of purified metalloprotease per ml of PBS was incorporated into 1 ml of Freunds' complete adjuvant (batch 32; Behringwerke AG). The rabbits received 1 ml of suspension subcutaneously; 4 weeks later, each animal received an identical booster injection (15). The animals were bled 4 weeks after the second injection. The rabbit immune sera (RIS) were processed and stored as 1 ml aliquots at -65 DC. Antibody titers of the anti-protease RIS were determined with the aid of a previously published enzyme-linked immunosorbent assay procedure (15). Exposure of fibrinogen and plasma to S. marcescens metalloproteases. Five mg of purified, lyophilized metalloprotease SF 178 and 10 mg of protease SH 186 were dissolved in 1 ml of the 4 mg/ml fibrinogen stock solution, 1 ml of B. plasma, and 1 ml of T. plasma, respectively. Control polystyrene tubes (Sarstedt, Niimbrecht) received 1 ml of either substrate alone. The tubes were incubated at 37°C and 200 rpm for 3 h in a New Brunswick G24 Environmental Incubation Shaker, after which the tube contents were subdivided and frozen immediately (-65°C) . Electroimmunoassays. These tests were performed according to the method of Laurell (8), utilizing 0.025 M Tricine buffer IV, pH 8.6, ionic strength 0.02 (Bio-Rad Laboratories GmbH, Miinchen), 1 % agarose Mr-0.25 (Serva), Bio-Rad equipment (Power Supply no. 500; Electrophoresis Cell no. 1415 ), and polyester agarose support films (50 em"; Serva). A total of 50 or 100 ul of rabbit anti-human fibrinogen immune serum was incorporated into 7.5 ml of 1 % agarose (height of the gel = 1.5 mm). Antigen wells (2.5 mm diameter) received 5 j.tl of undiluted and 1 : 4 diluted fibrinogen stock solution (protease-exposed and control solution) and undiluted, protease-exposed and control B. and T. plasma, respectively, at 2 V/cm. Electrophoresis was carried out at 85 V and 10 °C for 18 h. The agarose support films were pressed, washed, stained with Coomassie Blue R-250 (Serva), and destained according to the instructions of Axelsen (1). The rockets (2) were compared for peak areas (= height x 1/2 width) in order to demonstrate semiquantitatively any decrease in
Fibrinolytic Activity of S. marcescens
37
fibrinogen concentrations relative to controls. With protease-exposed human plasma, a record was made of the distances that the antigen migrated as compared with the homologous control. Two-dimensional immunoelectrophoresis. The tests were performed according to Axelsen (1), utilizing agarose ME (Miles Laboratories, Elkhart, Indiana), 0.02 M Tris-barbiturate buffer, pH 8.6, and 50 cm 2 polyester agarose films (see above). Antigen wells (2.5 mm diameter) received 5 ul of protease solution (5 or 10 mg/ml). Electrophoresis was carried out in the first dimension at 10 V/cm at 10 °C for 1 h employing the same equipment as above. A total of 100 and 150 f!1 of RIS no. 118 and 120, respectively, were incorporated into the 1 % agarose gels, and electrophoresis in the second dimension was carried out at 2 V/c'm at lO OC for 18 h. The agarose support films were processed as above.
Results The two purified S. marcescens rneralloproteases SF 178 and SH 186 were immunogenic for NZW rabbits; the ELISA-titres of RIS no. 118 against proteases SF 178 and SH 186 were 1: 1280, respectively, whereas RIS no. 120 revealed ELISA-titres of 1 : 5120 against both proteases. Both proteases were immunologically pure and closely related in terms of cross-reacting epitopes as determined with the aid of two-dimensional immunoelectrophoresis, i.e., following examination of RIS no. 118 and 120 against both enzymes, respectively. Preliminary experiments established that S. marcescens isolates SF 178 and SH 186 (needle stab-inocula) lysed fibrin in human fibrin agar following overnight incubation at 35 °C. Spot drop inocula (appro ximately 0.05 ml)
-
.. 1 2 3
5 6
Fig. 1. Degradation of human control fibrinogen by S. marcescens metalloproteases. Electroimmunoassay. Anode == top. Well 1: control fibrinogen (4 mg/ml), undiluted 2: control fibrinogen, diluted 1: 4 3: fibrinogen (4 mg/ml), undiluted + protease SF 178 4: fibrinogen, diluted 1 : 4 + protease SF 178 5: fibrinogen (4 mg/ml), undiluted + protease SH 186 6: fibrinogen, diluted 1 : 4 + protease SH 186
38
W. H. Traub and D. Bauer
o
1 2 3 4 5 6 Fig. 2. Degradation of human plasma fibrinogen by S. marcescens metalloprotease SF 178. Electroimmunoassay. Anode = top. Well 1: control fibrinogen (4 mg/rnl) 2: fibrinogen (4 mg/ml) + protease SF 178 3: control B. plasma 4: B. plasma + protease SF 178 5: control T. plasma 6: T. plasma + protease SF 178
1 ,2 3
5
Fig. 3. Degradation of human plasma fibrinogen by S. marcescens metalloprotease SH 186. Eleetroimmunoassay. Anode = top. Well 1: control fibrinogen (4 mg/ml) 2: fibrinogen (4 rng/ml) + prote ase SH 186 3: control B. plasma 4: B. plasma + protease SH 186 5: control T. plasma 6: T. plasma + protease SH 186
Fibrinolytic Activity of S. marcescens
39
of both purified metalloproteases (10 mg/ml) displayed potent (complete clearing) fibrinolytic activity on human fibrin agar within 2.5 h at 35°C. The two proteases reacted with human control type III fibrinogen (Fig. 1). Both proteases attacked fibrinogen in fresh human plasma as well (Fig. 2. and 3); the antigen revealed altered electrophoretic mobility following proteolytic exposure.
Discussion
It was of interest to note that both purified, immunologically pure metalloproteases of S. marcescens interacted with commercially available human fibrinogen to a greater extent than with fibrinogen in fresh human plasma. It is possible that.one or more of several proteinase inhibitors in plasma, such as alphaj-proteinase inhibitor, alphajmacroglobulin, alpha-trypsin inhibitor, alpha.vchymotrypsin inhibitor, anti-thrombin III, Cl-inactivator, alphaj-plasma inhibitor, or one of the recently described, slowly migrating (~r and y -globulin fractions) proteinase inhibitors (11) inhibited the two metalloproteases in fresh human plasma as opposed to a laboratory solution of fibrinogen. Previously, Miyata et al. (10) had submitted evidence indicating that human plasma alphaj-macroglobulin sterically inhibited a S. marcescens protease; the protease in turn cleaved alphaj-macroglobulin at the midregion with resultant conformational changes. Fibrinogen figures prominently among so-called acute-phase proteins (4, 7). Conceivably, the fibrinolytic activity of both metalloproteases might constitute an additional aggressin (13) of S. marcescens in the setting of human invasive nosocomial infections with subsequent spread and bloodstream invasion. Acknowledgment. This investigation was supported in part by a grant from the Deutsche Forschungsgemeinschaft (Tr 136/3).
References
1. Axelsen, N. H.: Handbook of irnrnunoprecipitation-in-gel techniques. Reprinted from Scand. J. Immunol. 17, Suppl. 10, by Blackwell Scientific Publications, Oxford (1983) 2. Bjerrum, 0.].: Electroimmunoprecipitation artefacts. Scand. J. Immunol. 17, Suppl. 10 (1983) 333-342 3. Deuriese, L. A. and A. van de Kerckhove: A comparison of methods used for testing staphylokinase (fibrinolysin) production in Staphylococcus strains. Antonie v. Leeuwenhoek 46 (1980) 457-465 4. Dinarello, C. A.: Interleukin-1 and the pathogenesis of the acute-phase response. New Engl. J. Med. 311 (1984) 1413-1418 5. Doerr, M. and W. H. Traub: Proteolytic activity of selected clinical isolates of Serratia marcescens against human serum proteins. Zbl. Bakt. Hyg., 1. Abt. Orig. A 252 (1982) 196-207 6. Doerr, M. and W. H. Traub: Purification and characterization of two Serratia marcescens proteases. Zbl. Bakt. Hyg. A 257 (1984) 6-19 7. Kushner, I., H. Geu/urz; and M. D. Benson: C-reactive protein and the acute-phase response. J. Lab. Clin. Med. 97 (1981) 739-749 8. Laurel!, C.-B.: Electroimmunoassay. Scand. J. Clin. Lab. Inv. 29, Suppl. 124 (1972) 21-37
40
W. H. Traub and D. Bauer
9. Lyerly, D. M. and A. S. Kreger: Importance of Serratia protease in the pathogenesis of experimental Serratia marcescens pneumonia. Infect. Immun. 40 (1983) 113-119 10. Miyata, K., M. Nakamura, and K. Tomoda: Interaction between Serratia protease and human plasma alphaz-macroglobulin.]. Biochem. 89 (1981) 1231-1237 11. 0dum, L.: Slow migrating proteinase inhibitors in human serum. Hoppe-Seyler's Z. Physiol. Chern. 365 (1984) 567-570 12. Simberkoff, M. S.: Experimental Serratia marcescens infection and defense mechanisms, pp. 157-164, in: A. von Graevenitz and S. J. Rubin (eds.), The Genus Serratia. CRC Press, Inc., Boca Raton Florida (1980) 13. Smith, H.: The elusive determinants of bacterial interference with non-specific host defences. Phil. Trans. roy. Soc. (Lond.) B 303 (1983) 99-113 14. Traub,W. H.: Virulence of nosocomial isolates of Serratia marcescens for NMRI mice: Correlation with O-antigens 06 and 014. Zbl. Bakt. Hyg., I. Abt. Orig. A 252 (1982) 360-369 15. Traub, W. H.: Passive protection of NMRI mice against Serratia marcescens: Comparative efficacy of commercial human IgG immunoglobulin preparations and rabbit anti-O, -H, -K, -live cell and -protease immune sera. Zbl. Bakt. Hyg., I. Abt. Orig. A 254 (1983) 480-488 16. Traub, W. H. and 1. Kleber: Detection of fibrinolytic and elastase activity among clinical isolates of Serratia marcescens. Path. Microbiol. 41 (1974) 334-340 17. von Graeuenitz, A.: Infection and colonization with Serratia, pp. 167-186, in: A. von Graevenitz and S. J. Rubin (eds.), The Genus Serratia. CRC Press, Inc., Boca Raton Florida (1980)
Professor Dr. Walter H. Traub, Institut fur Hygiene und Mikrobiologie, Universitat des Saariandes, Haus 43, 0-6650 Homburg(Saar)
Fibrinolytic Activity of Purified Serratia marcescens Metalloproteases WALTER H. TRAUB and DIERK BAUER Institut fur Medizinische Mikrobiologie und Hygiene, Universitat des Saarlandes, 6650 Homburg/Saar, Federal Republic of Germany
With 3 Figures ' Received January 8, 1985 . Accepted February 20, 1985
Abstract Two purified rnetalloproteases of Serratia marcescens revealed fibrinolytic activity, i.e., degraded human fibrin in agar as well as commercially available human fibrinogen and fibrinogen in fresh human plasma.
Zusammenfassung Zwei gereinigte Metalloproteasen von Serratia marcescens erwiesen sich als Fibrinolysine. Beide Enzyme degradierten Human-Fibrin in Agar sowie kommerziell erhaltliches Human-Fibrinogen und Fibrinogen in frisch em Human-Plasma.
Introduction During the past two decades, Serratia marcescens emerged as an opportumsnc pathogenic, nosocomially significant microorganism (17). However, relatively little is known about the virulence determinants of this bacterial species (12). It was demonstrated that S. marcescens strains carrying lipopolysaccharide a-antigens 06 and/or 014 were somewhat more virulent for outbred NMRI mice than isolates characterized by other serogroup antigens (14). Clinical isolates of S. marcescens were shown previously to produce various exoenzymes (16). Of these, a purified metalloprotease, derived from S. marcescens strain BG, was demonstrated as a genuine bacterial aggressin with regard to experimental pneumonia in laboratory animals by Kreger and associates (9). We had previously noted that several clinical isolates of S. marcescens altered diverse proteins of fresh human serum (5). Subsequently, we purified and characterized representative metalloproteases derived from two nosocomially significant strains of S. marcescens (6). This study served to determine whether these two exoenzymes displayed fibrinolytic activity.
36
W. H. Traub and D. Bauer Materials and Methods
Bacteria. S. marcescens isolates SF 178 and SH 186 were recovered from patients with septicemia and post-operative wound infection in San Francisco, California, and Homburg/ Saar, respectively. Both isolates were of serotype 061014: H12 and bacteriocin type 18, and were maintained as previously described (15). Media. Tryptic Soy broth, Brain Heart Infusion broth, Nutrient broth, Bacto Agar, and Skim Milk (source of casein) were procured from Difco Laboratories, Detroit, Michigan. Skim Milk agar contained 3 % Skim Milk and 1.5 % Bacto-Agar. Fibrin agar was prepared as follows: 10 ml of a 1 % human fibrinogen (Behringwerke AG, Marburg) solution were added to 90 ml of Nutrient broth containing 1.5 % Bacto-Agar, mixed, and held at 55 °C for 10 min, after which 1 ml of fresh human serum (donor T.) was added (3). Reagents. Human type III fibrinogen (Fraction I, lot no. 13F-9330; 79 % protein content) was purchased from Sigma Chemie GmbH, Taufkirchen. The fibrinogen stock solution contained 4 mg per ml of phosphate-buffered saline, pH 7.5, which was subdivided into small aliquots, frozen and kept stored at -65°C until further use. Human test fibrinogen (lot no. 504 317 B, 910 mg dry substance) was obtained from Behringwerke AG, Marburg, and rehydrated with 25 ml of sterile demineralized water; this stock solution contained 1 % fibrinogen, i.e., 10 mg per ml of 0.1 M TRIS buffer, pH 8.0. Rabbit anti-human fibrinogen immune serum (lot no. 10541 B) was obtained from Behringwerke AG. Two healthy adults (B. and T.) repeatedly donated 20 ml blood samples which were heparinized; plasma was separated by centrifugation at 2500 x g for 10 min, 22°C, and utilized immediately. Purification of metalloproteases. The two S. marcescens metalloproteases were purified and lyophilized as described previously (6). The activities of the enzymes were monitored (440 nm) through hydrolysis of azocasein (lot no. 09061; Serva Feinbiochemica GmbH, Heidelberg); a 10 mg/ml solution of trypsin 1 : 250 (Serva; lot H; activity = ca. 4 U/mg) served as a control. Immunization of rabbits. New Zealand White rabbits (Medizinische Versuchstierzucht Nickisch, Osthofen-Neuteich) were immunized as follows: 1 ml of 200 j.tg of purified metalloprotease per ml of PBS was incorporated into 1 ml of Freunds' complete adjuvant (batch 32; Behringwerke AG). The rabbits received 1 ml of suspension subcutaneously; 4 weeks later, each animal received an identical booster injection (15). The animals were bled 4 weeks after the second injection. The rabbit immune sera (RIS) were processed and stored as 1 ml aliquots at -65 DC. Antibody titers of the anti-protease RIS were determined with the aid of a previously published enzyme-linked immunosorbent assay procedure (15). Exposure of fibrinogen and plasma to S. marcescens metalloproteases. Five mg of purified, lyophilized metalloprotease SF 178 and 10 mg of protease SH 186 were dissolved in 1 ml of the 4 mg/ml fibrinogen stock solution, 1 ml of B. plasma, and 1 ml of T. plasma, respectively. Control polystyrene tubes (Sarstedt, Niimbrecht) received 1 ml of either substrate alone. The tubes were incubated at 37°C and 200 rpm for 3 h in a New Brunswick G24 Environmental Incubation Shaker, after which the tube contents were subdivided and frozen immediately (-65°C) . Electroimmunoassays. These tests were performed according to the method of Laurell (8), utilizing 0.025 M Tricine buffer IV, pH 8.6, ionic strength 0.02 (Bio-Rad Laboratories GmbH, Miinchen), 1 % agarose Mr-0.25 (Serva), Bio-Rad equipment (Power Supply no. 500; Electrophoresis Cell no. 1415 ), and polyester agarose support films (50 em"; Serva). A total of 50 or 100 ul of rabbit anti-human fibrinogen immune serum was incorporated into 7.5 ml of 1 % agarose (height of the gel = 1.5 mm). Antigen wells (2.5 mm diameter) received 5 j.tl of undiluted and 1 : 4 diluted fibrinogen stock solution (protease-exposed and control solution) and undiluted, protease-exposed and control B. and T. plasma, respectively, at 2 V/cm. Electrophoresis was carried out at 85 V and 10 °C for 18 h. The agarose support films were pressed, washed, stained with Coomassie Blue R-250 (Serva), and destained according to the instructions of Axelsen (1). The rockets (2) were compared for peak areas (= height x 1/2 width) in order to demonstrate semiquantitatively any decrease in
Fibrinolytic Activity of S. marcescens
37
fibrinogen concentrations relative to controls. With protease-exposed human plasma, a record was made of the distances that the antigen migrated as compared with the homologous control. Two-dimensional immunoelectrophoresis. The tests were performed according to Axelsen (1), utilizing agarose ME (Miles Laboratories, Elkhart, Indiana), 0.02 M Tris-barbiturate buffer, pH 8.6, and 50 cm 2 polyester agarose films (see above). Antigen wells (2.5 mm diameter) received 5 ul of protease solution (5 or 10 mg/ml). Electrophoresis was carried out in the first dimension at 10 V/cm at 10 °C for 1 h employing the same equipment as above. A total of 100 and 150 f!1 of RIS no. 118 and 120, respectively, were incorporated into the 1 % agarose gels, and electrophoresis in the second dimension was carried out at 2 V/c'm at lO OC for 18 h. The agarose support films were processed as above.
Results The two purified S. marcescens rneralloproteases SF 178 and SH 186 were immunogenic for NZW rabbits; the ELISA-titres of RIS no. 118 against proteases SF 178 and SH 186 were 1: 1280, respectively, whereas RIS no. 120 revealed ELISA-titres of 1 : 5120 against both proteases. Both proteases were immunologically pure and closely related in terms of cross-reacting epitopes as determined with the aid of two-dimensional immunoelectrophoresis, i.e., following examination of RIS no. 118 and 120 against both enzymes, respectively. Preliminary experiments established that S. marcescens isolates SF 178 and SH 186 (needle stab-inocula) lysed fibrin in human fibrin agar following overnight incubation at 35 °C. Spot drop inocula (appro ximately 0.05 ml)
-
.. 1 2 3
5 6
Fig. 1. Degradation of human control fibrinogen by S. marcescens metalloproteases. Electroimmunoassay. Anode == top. Well 1: control fibrinogen (4 mg/ml), undiluted 2: control fibrinogen, diluted 1: 4 3: fibrinogen (4 mg/ml), undiluted + protease SF 178 4: fibrinogen, diluted 1 : 4 + protease SF 178 5: fibrinogen (4 mg/ml), undiluted + protease SH 186 6: fibrinogen, diluted 1 : 4 + protease SH 186
38
W. H. Traub and D. Bauer
o
1 2 3 4 5 6 Fig. 2. Degradation of human plasma fibrinogen by S. marcescens metalloprotease SF 178. Electroimmunoassay. Anode = top. Well 1: control fibrinogen (4 mg/rnl) 2: fibrinogen (4 mg/ml) + protease SF 178 3: control B. plasma 4: B. plasma + protease SF 178 5: control T. plasma 6: T. plasma + protease SF 178
1 ,2 3
5
Fig. 3. Degradation of human plasma fibrinogen by S. marcescens metalloprotease SH 186. Eleetroimmunoassay. Anode = top. Well 1: control fibrinogen (4 mg/ml) 2: fibrinogen (4 rng/ml) + prote ase SH 186 3: control B. plasma 4: B. plasma + protease SH 186 5: control T. plasma 6: T. plasma + protease SH 186
Fibrinolytic Activity of S. marcescens
39
of both purified metalloproteases (10 mg/ml) displayed potent (complete clearing) fibrinolytic activity on human fibrin agar within 2.5 h at 35°C. The two proteases reacted with human control type III fibrinogen (Fig. 1). Both proteases attacked fibrinogen in fresh human plasma as well (Fig. 2. and 3); the antigen revealed altered electrophoretic mobility following proteolytic exposure.
Discussion
It was of interest to note that both purified, immunologically pure metalloproteases of S. marcescens interacted with commercially available human fibrinogen to a greater extent than with fibrinogen in fresh human plasma. It is possible that.one or more of several proteinase inhibitors in plasma, such as alphaj-proteinase inhibitor, alphajmacroglobulin, alpha-trypsin inhibitor, alpha.vchymotrypsin inhibitor, anti-thrombin III, Cl-inactivator, alphaj-plasma inhibitor, or one of the recently described, slowly migrating (~r and y -globulin fractions) proteinase inhibitors (11) inhibited the two metalloproteases in fresh human plasma as opposed to a laboratory solution of fibrinogen. Previously, Miyata et al. (10) had submitted evidence indicating that human plasma alphaj-macroglobulin sterically inhibited a S. marcescens protease; the protease in turn cleaved alphaj-macroglobulin at the midregion with resultant conformational changes. Fibrinogen figures prominently among so-called acute-phase proteins (4, 7). Conceivably, the fibrinolytic activity of both metalloproteases might constitute an additional aggressin (13) of S. marcescens in the setting of human invasive nosocomial infections with subsequent spread and bloodstream invasion. Acknowledgment. This investigation was supported in part by a grant from the Deutsche Forschungsgemeinschaft (Tr 136/3).
References
1. Axelsen, N. H.: Handbook of irnrnunoprecipitation-in-gel techniques. Reprinted from Scand. J. Immunol. 17, Suppl. 10, by Blackwell Scientific Publications, Oxford (1983) 2. Bjerrum, 0.].: Electroimmunoprecipitation artefacts. Scand. J. Immunol. 17, Suppl. 10 (1983) 333-342 3. Deuriese, L. A. and A. van de Kerckhove: A comparison of methods used for testing staphylokinase (fibrinolysin) production in Staphylococcus strains. Antonie v. Leeuwenhoek 46 (1980) 457-465 4. Dinarello, C. A.: Interleukin-1 and the pathogenesis of the acute-phase response. New Engl. J. Med. 311 (1984) 1413-1418 5. Doerr, M. and W. H. Traub: Proteolytic activity of selected clinical isolates of Serratia marcescens against human serum proteins. Zbl. Bakt. Hyg., 1. Abt. Orig. A 252 (1982) 196-207 6. Doerr, M. and W. H. Traub: Purification and characterization of two Serratia marcescens proteases. Zbl. Bakt. Hyg. A 257 (1984) 6-19 7. Kushner, I., H. Geu/urz; and M. D. Benson: C-reactive protein and the acute-phase response. J. Lab. Clin. Med. 97 (1981) 739-749 8. Laurel!, C.-B.: Electroimmunoassay. Scand. J. Clin. Lab. Inv. 29, Suppl. 124 (1972) 21-37
40
W. H. Traub and D. Bauer
9. Lyerly, D. M. and A. S. Kreger: Importance of Serratia protease in the pathogenesis of experimental Serratia marcescens pneumonia. Infect. Immun. 40 (1983) 113-119 10. Miyata, K., M. Nakamura, and K. Tomoda: Interaction between Serratia protease and human plasma alphaz-macroglobulin.]. Biochem. 89 (1981) 1231-1237 11. 0dum, L.: Slow migrating proteinase inhibitors in human serum. Hoppe-Seyler's Z. Physiol. Chern. 365 (1984) 567-570 12. Simberkoff, M. S.: Experimental Serratia marcescens infection and defense mechanisms, pp. 157-164, in: A. von Graevenitz and S. J. Rubin (eds.), The Genus Serratia. CRC Press, Inc., Boca Raton Florida (1980) 13. Smith, H.: The elusive determinants of bacterial interference with non-specific host defences. Phil. Trans. roy. Soc. (Lond.) B 303 (1983) 99-113 14. Traub,W. H.: Virulence of nosocomial isolates of Serratia marcescens for NMRI mice: Correlation with O-antigens 06 and 014. Zbl. Bakt. Hyg., I. Abt. Orig. A 252 (1982) 360-369 15. Traub, W. H.: Passive protection of NMRI mice against Serratia marcescens: Comparative efficacy of commercial human IgG immunoglobulin preparations and rabbit anti-O, -H, -K, -live cell and -protease immune sera. Zbl. Bakt. Hyg., I. Abt. Orig. A 254 (1983) 480-488 16. Traub, W. H. and 1. Kleber: Detection of fibrinolytic and elastase activity among clinical isolates of Serratia marcescens. Path. Microbiol. 41 (1974) 334-340 17. von Graeuenitz, A.: Infection and colonization with Serratia, pp. 167-186, in: A. von Graevenitz and S. J. Rubin (eds.), The Genus Serratia. CRC Press, Inc., Boca Raton Florida (1980)
Professor Dr. Walter H. Traub, Institut fur Hygiene und Mikrobiologie, Universitat des Saariandes, Haus 43, 0-6650 Homburg(Saar)