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Collagenase and protease activity in Bacteroides melaninogenicus Gisslow, Mary Therese 1975

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Collagenase and Protease A c t i v i t y i n Bacteroides melaninogenicus by Mary Therese Gisslow B.Sc, University of Santa Clara, 1970 Thesis submitted i n p a r t i a l fulfilment of the requirements for the degree of Master of Science i n the Department of Microbiology We accept t h i s thesis as conforming to the require_d standard The University of B r i t i s h Columbia A p r i l , 1975 In present ing t h i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or pub l i ca t ion of th is thes is fo r f inanc ia l gain shal l not be allowed without my wri t ten permission. Department of Microbiology The Un ivers i ty of B r i t i s h Columbia Vancouver 8, Canada Date A p r i l 2, 1975 i i ABSTRACT A rapid sensitive collagenase assay has been developed using "^C-acetyl-ated collagen as the substrate. Acid-soluble c a l f s k i n collagen was labelled 14 with acetic-1- C-anhydride at pH 8. The radioactively l a b e l l e d collagen remained susceptible to Clostridium histolyticum collagenase but resistant to nonspecific proteolysis indicating that denaturation of the collagen had not occurred as a result of the acetylation procedure. The rate of 14 release of C from labelled collagen by £. histolyticum collagenase was 14 proportional to enzyme and substrate concentrations. The results of the C assay correlated with those of other collagenase assays tested. Collagenolytic a c t i v i t y of Bacteroides melaninogenicus was examined both i n v i t r o and the guinea pig model system using known collagenase-produc-ing strains and recent i s o l a t e s . In contrast to the reported r e s u l t s of pre-vious workers, only two of the t h i r t y melaninogenicus strains isolated were found to have collagenase a c t i v i t y . Collagenase was found i n washed c e l l s of positive strains of 15. melaninogenicus as early as day one. No a c t i v i t y was ever observed i n culture supernatants. Studies of material aspirated from guinea pigs infected with pathogenic B^. melaninogenicus demonstrated the presence of collagenase i n the infectious exudates. The enzyme was associated with the b a c t e r i a l c e l l s , was dependent on reducing conditions and was stimulated by a factor i n f i l t e r e d exudate. P a r t i a l characterization of c e l l - f r e e protease a c t i v i t y i n B^. melanino- genicus has shown the protease to be dependent on reducing agents, requiring -3 5 x 10 M cysteine for a c t i v i t y . Other reducing agents were also e f f e c t i v e i n stimulating protease a c t i v i t y . The a c t i v i t y was stable at 4°C, resistant -3 to autodigestion, and sensitive to EDTA at concentrations of 10 M and above. The pH optimum for a c t i v i t y against Azocoll was 8.0. i i i TABLE OF CONTENTS Page I. INTRODUCTION 1 I I . MATERIALS AND METHODS 7 A. Organisms 7 B. Growth of organisms 7 C. Chemicals and chromatographic materials 8 D. Preparation of b a c t e r i a l c e l l s for enzyme assays 8 E. Preparation of culture supernatants for enzyme assays 8 F. Extraction of collagen 8 G. Acetylation of collagen 10 H. Assays for collagenase a c t i v i t y 10 I. Guinea pig infections 11 J. Assays for proteolytic a c t i v i t y 13 K. Polyacrylamide gel electrophoresis 13 L. Hydroxyproline assay 14 M. Protein determination :~. 14 I I I . RESULTS 15 A. Development of a new collagenase assay 15 1. Analysis of the citrate-extracted collagen., 15 a. Hydroxyproline 15 b. Polyacrylamide gel electrophoresis...- 15 c. Viscosity assays: enzyme s u s c e p t i b i l i t y 15 d. Gel formation at 37°C 15 2. Acetylation of collagen 16 a. Results of a t y p i c a l acetylation 16 b. Effect of pH on the eff i c i e n c y of acetylation 16 i v 3. Analysis of the acetylated collagen 16 a. P r e c i p i t a t i o n of collagen 16 b. S u s c e p t i b i l i t y of l^C-collagen to collagenase and other proteolytic enzymes. . 20 c. Protease-treated collagen 20 4. Kinetic studies Sf the collagenase assay 23 a. Release of r a d i o a c t i v i t y from labelled collagen with time by c l o s t r i d i a l collagenase. 23 b. Effect of substrate concentration 23 c. Effect of enzyme concentration 23 B. Collagenase i n Bacteroides melaninogenicus: i n v i t r o studies 30 1. Attempts to i s o l a t e collagenase-producing strains 30 2. Attempts to increase collagenase a c t i v i t y . 30 3. Collagenolytic a c t i v i t y i n collagenase-producing strains CR2A and K110 32 a. Viscosity assays. 32 b. !4 C assays 32 c. Study of collagenase production by K110 as a function of culture age 32 C. Collagenase i n B^. melaninogenicus infectious exudates 32 1. Studies of the in f e c t i o n 32 a. Establishment of a mixed i n f e c t i o n . . . 37 b. Organisms from the infectious mixture 37 c. Recombination of collagenase-producing strains with GP25..38 d. Production of a transmissible i n f e c t i o n with a single s t r a i n of 13. melaninogenicus 38 e. Characterization of the in f e c t i o n produced by s t r a i n 2D...40 2. Studies of collagenase a c t i v i t y i n material aspirated from infected animals 42 a. Demonstration fo collagenolytic a c t i v i t y i n exudate from an infected animal 42 b. Association of collagenolytic a c t i v i t y with b a c t e r i a l c e l l s i n exudate 42 c. Characterization of the stimulatory factor i n the supernatant 47 (1) Effect of u l t r a f i l t r a t i o n on the supernatant 47 (2) S t a b i l i t y of the f i l t r a t e 47 V E. Proteolytic a c t i v i t y i n B^. melaninogenicus culture supernatants...51 1. Demonstration of an ex t r a c e l l u l a r protease 51 2. Protease a c t i v i t y as a function of culture age 51 3. P u r i f i c a t i o n of the protease 51 4. P a r t i a l characterization of protease a c t i v i t y 61 a. S t a b i l i t y of the protease 61 b. Effect of cysteine concentration on protease a c t i v i t y 61 c. Effect of other reducing agents on protease a c t i v i t y 61 d. Effect of pH on protease a c t i v i t y 64 e. Effect of EDTA on protease a c t i v i t y 64 IV. DISCUSSION 70 V. LITERATURE CITED 74 v i LIST OF TABLES Page I. Properties of 13. melaninbgeriicus Strains 9 I I . Protocol for Collagenase Assay 12 I I I . Acetylation of Collagen 17 IV. Effect of pH on Efficiency of Acetylation 18 V. Non-precipitable "*"^C 19 14 „, VI. C Solubilized by Proteolytic Enzymes 21 14 14 VII. Release of C from Untreated and Pre-digested C-collagen by S u b t i l i s i n and Collagenase 22 VII I . Collagenase A c t i v i t y i n Thirty I3_. melaninogenicus Isolates 31 IX. Collagenase A c t i v i t y i n 13. melaninogenicus CR2A and K110 35 X. Collagenase A c t i v i t y as a Function of Culture Age 36 XI. Infections Produced by Recombination of 13. melaninogenicus with a Mixed Culture ....39 XII. Collagenolytic A c t i v i t y i n Fractionated Guinea Pig Exudate 44 XII I . F i l t r a t i o n of High Speed Supernatant 48 XIV. S t a b i l i t y of UM-10 F i l t r a t e . . . 49 XV. Ashing 50 XVI. Resistance to Autodigestion 62 XVII. Effect of Cysteine Concentration 63 XVIII. Effect of Reducing Agents 65 v i i LIST OF FIGURES Page 14 1. Release of C from acetylated collagen by (J. histolyticum collagenase 25 14 2. Release of C from acetylated collagen with increasing substrate concentration 27 14 3. Release of C from acetylated collagen as a function of collagenase concentration 29 4. Viscometric assay of collagenolytic a c t i v i t y 34 14 5. C released from collagen by whole exudate 41 6. Stimulation of collagenase a c t i v i t y by high speed supernatant 46 7. Protease a c t i v i t y i n CR2A and 2D supernatants 53 8. Proteolytic a c t i v i t y i n 2D supernatants 55 9. Protease a c t i v i t y as a function of culture age 57 10. Effect of BSA on chromatography of supernatant on G-75 60 11. Effect of pH ; 67 12. Effect of EDTA • 69 v i i i ACKNOWLEDGEMENTS To Dr. Barry C. McBride, for his encouragement, c r i t i c i s m , guidance and understanding, I wish to'extend my deepest thanks. I hope that my future work w i l l r e f l e c t not only the high standard of his s c i e n t i f i c research but also some of the c r e a t i v i t y and open-mindedness of his approach to science. I wish to thank Tom Edwards and Denis Mayrand for helpful comments, suggestions and s c i e n t i f i c discussion. Thanks are due also to several members of the department whose names are not mentioned here, for helpful suggestions and moral support. 1 I. INTRODUCTION Infections characterized by mixed populations of nonsporulating anaerobes have been known for some time (1,5,30). Examples include brain abscesses, lung abscesses, appendicitis, human bi t e wounds and periodontal disease. A char a c t e r i s t i c mixed b a c t e r i a l population, the so-called "fusospirochaetal complex" was observed i n these infections. Experimental mixed infections containing the same "fusospirochaetal complex" could be produced by subcutan-eous inoculation of experimental animals with human gingival debris or mater-i a l from a variety of abscesses (21,46,49). Rosebury and co-workers (40), i n a study of experimental mixed infections produced with gingival debris, found that the mixture retained i t s i n f e c t i v i t y through several animal passages or through several transfers as a mixed culture i n v i t r o , but that recombination of pure cultures isolated from the mixture f a i l e d to produce the " t y p i c a l " fusospirochaetal i n f e c t i o n . The c r i t e r i a l u s e d for defining a t y p i c a l transmissible i n f e c t i o n as used by these workers were summarized by Socransky and Gibbons (47); a t y p i c a l i n f e c t i o n i s described by Macdonald et a l . (28). Experimental mixed infections i n the guinea pig model system, as described by these workers, can be of two types. One i s a walled-off l o c a l i z e d abscess containing foul-smelling exudate which can be used to transmit the in f e c t i o n to a second animal. The other i s a rapidly spreading necrotic i n f e c t i o n which perforates the abdominal wall or the skin. The animal loses hair and necrosis of the skin occurs i n the abdominal region. The exudate readily infects other animals. Positive i n f e c t i v i t y i s indicated by death, development of spreading necrotic lesions, or development of a lo c a l i z e d transmissible abscess. Hard, nodular, nontrans-missible caseous abscesses or mild inflammation indicate no n i n f e c t i v i t y . Thus a t y p i c a l transmissible i n f e c t i o n i s one that (a) i s i n f e c t i v e according to 2 the above description and (b) can be passaged v i a the exudate to another ani-mal . Macdonald et a l . (28) succeeded i n producing t y p i c a l transmissible mixed infections i n guinea pigs when seventeen pure cultures isolated from an i n -fection were recombined and injected into an animal. By successively elimin-ating various organisms from the o r i g i n a l combination of seventeen, he was able to reproduce the t y p i c a l "fusospirochaetal" i n f e c t i o n with a combination of four organisms, none of which were fusiforms or spirochaetes (29). Inoc-ulation of an animal with the "pathogenic quartet," which included two strains of Bacteroides, a motile gram-negative rod and a fa c u l t a t i v e diphtheroid, always produced the t y p i c a l transmissible l e s i o n . One of the Bacteroides species was i d e n t i f i e d as melaninogenicus, and further studies on the mixed i n f e c t i o n (27) showed that t h i s organism was an essential component of the system. In the study of one mixed anaerobic i n f e c t i o n , i t was found that no i n f e c t i o n occurred when 15. melaninogenicus was omitted from the recombina-tion mixture. Other members of the i n f e c t i v e combination could be replaced or omitted depending on the s t r a i n of B_. melaninogenicus used (48). In the four-organism system the role of the diphtheroid was found to be the production of a naphthoquinone compound required by the jB. melaninogeni- cus s t r a i n . Substitution of a naphthoquinone-independent jB. melaninogenicus s t r a i n for the o r i g i n a l s t r a i n used showed that the diphtheroid was then no longer required for i n f e c t i v i t y and was i n fact eliminated from the mixture (27) . Recombination of B_. melaninogenicus with populations of organisms c-from which i t had been eliminated restored i n f e c t i v i t y to a number of systems (48). Evidence thus implicated 15. melaninogenicus as the primary pathogen i n mixed in f e c t i o n s , and the organism closest to being an overt pathogen of a l l the normal inhabitants of the o r a l cavity (27,48). On the assumption that elucidation of the pathogenic mechanisms involved i n experimental mixed infections might provide some understanding of the mechanisms involved i n the pathogenicity of c l i n i c a l anaerobic infections, investigators became concerned with the production of p o t e n t i a l l y damaging metabolites, toxins, or other factors which would explain how 13. melaninogeni- cus and associated organisms were able to cause i n f e c t i o n . Although Macdonald had suggested e a r l i e r that perhaps mixed infections were b a c t e r i a l l y nonspecif-i c but biochemically s p e c i f i c i n terms of toxins, l y t i c enzymes and other u damaging factors produced by the mixed population (26,29), the demonstration of the essential role of 15. melaninogenicus i n the experimental system sugges-ted that the "nonspecific" infections were i n fact dependent on the presence of )}_. melaninogenicus and that the role of other organisms was one of sup-porting and enhancing the i n vivo growth of the primary pathogen (27,48). Consequently J3. melaninogenicus was examined for pathogenic properties. 13. melaninogenicus i s a gram-negative, nonmotile, nonsperulating anaer-obic rod. The species i s somewhat loosely defined, comprising those organ-isms producing black colonies when incubated anaerobically on blood agar. The organism can isolated from the o r a l cavity, feces, and a number of naturally occurring c l i n i c a l infections (5,23,51,53). Most strains require hemin and many require vitamin K or a related naphthoquinone for growth (12,13). Although s i g n i f i c a n t differences have been found among strains i n carbohydrate fermentation patterns^ menadione requirement and colony morphology (42), i n -vestigators have been unable to f i n d any useful patterns dividing them into taxonomic groups and find the "species" to represent a biochemically and immunologically diverse group of organisms (6). Macdonald and Gibbons '(27) had shown that the organism produces H^S, DNase, indole, and NH^, and i t was known that the organism contained endo-toxins (31) . IS. melaninogenicus was found to be active against a number of protein substrates (12,27,42). A l l strains tested were able to substitute 4 for the original strain of 15. melaninogenicus in the mixed infection (27). While differences were found between strains with regard to protease, colla-genase, DNase, RNase, NH^ , indole, H^ S and fermentation patterns, no correla-tion between any of the above and pathogenicity could be found (27) u n t i l Kestenbaum (20) demonstrated a positive correlation between collagenase ac-t i v i t y and infectivity for four 15. melaninogenicus strains i n a guinea pig system. Collagen degradation i s a feature of periodontal disease (24,46,47), and although 15. melaninogenicus i s the only organism indigenous to the oral cavity known to produce a collagenase (12), the relationship between collagen-ase production and the pathogenicity of the organism either in oral lesions or in other mixed anaerobic infections remains unclear. (Whether factors other than collagenase were involved in Kestenbaum's system is not known.) Enhancement of a fusobacterial infection in rabbits by simultaneous injection of a crude cell-free preparation of I5_. melaninogenicus collagenase was demon-strated by Kaufman (19). Thus there i s evidence that 15. melaninogenicus collagenase plays a role in the organism's pathogenicity. The unique properties of collagen and the collagenolytic enzymes have resulted i n the formulation of specific c r i t e r i a for the definition of collagenases. A thorough discussion of the collagen molecule has been edited by Ramachandran (37), and a recent review of collagen biosynthesis has been written by Bornstein (4); while excellent reviews of the collagenolytic enzymes have been written by Seifter and Harper (44,45); Eisen and co-workers (9) and Nordwig (34). The collagen molecule is characterized by i t s sensi- •, t i v i t y to collagenases but resistance to other proteolytic enzymes. The basic unit or tropocollagen molecule consists of three polypeptide chains intertwined into a ri g i d h e l i c a l structure, the unusual nature of which i s 5 partly due to i t s amino acid composition. In most of the polypeptide chain every t h i r d amino acid i s glycine, and the molecule contains a large amount of proline and hydroxyproline. Because collagen i s one of the few proteins containing appreciable amounts of hydroxyproline, the purity of a collagen preparation can be determined by assaying the amount of t h i s amino acid pre?-sent (12,14). Solutions of native collagen have high v i s c o s i t i e s and form a r i g i d , opaque gel when incubated at 37°C. Both properties are absent i n g e l a t i n , the denatured form of collagen. Gelatin i s also susceptible to a number of proteases which are incapable of attacking native collagen. Native collagen can thus be distinguished from i t s denatured state by physical properties as well as enzyme s u s c e p t i b i l i t y . The collagenases constitute a class of unique proteases capable of attacking native collagen i n the h e l i c a l portion of the molecule. Because of the s u s c e p t i b i l i t y of ge l a t i n to a number of proteases, v a l i d assays for collagenase a c t i v i t y must use undenatured collagen as the substrate and must be performed under non-denaturing conditions. Many assays for collagen-o l y t i c a c t i v i t y are based on the measurement of physical changes i n collagen preparations, such as a decrease i n the v i s c o s i t y of collagen solutions (43,44). or the dissolution of collagen fibers i n suspensions or gels (3, 15,44). These procedures require a considerable amount of substrate and are d i f f i c u l t - t o quantitate. Synthetic peptides with sequences i d e n t i c a l to portions of the collagen molecule have been used with Clostridium histolyticum collagenase but are unsatisfactory for collagenases of different s p e c i f i c i t i e s or for enzymes of questionable p u r i t y , as a number of peptidases incapable of attack-ing, native collagen can cleave the synthetic peptides, and a number of true 14 collagenases have no a c t i v i t y against the peptide substrates (16). A C i / 14 assay based on the release of -^C-glycine-containing peptides from C l a b e l -led guinea pig skin collagen was developed by Gross and co-workers (32) and 6 14 3 i s widely used for tissue collagenases (7,10,39). C-proline and H-proline have been used to l a b e l chick embryo collagen (35,41). In a l l of these sys-tems the radioactive collagen i s prepared by in j e c t i n g the animal with the 14 C-amino acid shortly before extracting collagen from the tissue. Although small amounts of enzyme can be assayed quantitatively, the s p e c i f i c a c t i v i t y 14 of the collagen preparations i s variable, and the purity of the C-protein must be determined after l a b e l l i n g . The collagenase of JB. melaninogenicus d i f f e r s from other microbial c o l l a -genases i n that i t i s cell-bound and requires cysteine or some other reducing agent for a c t i v i t y (12). The enzyme i s released when the c e l l s lyse. Haus-man and Kaufman (17) succeeded i n i s o l a t i n g collagenase a c t i v i t y i n a p a r t i -culate f r a c t i o n obtained by centrifuging the autolysate supernatant at 100,000 x for one hour. Further attempts to purify the collagenase from the pa r t i c u l a t e f r a c t i o n , or to separate i t from the caseinolytic a c t i v i t y with which i t i s associated have been unsuccessful. This thesis i s concerned with the study of collagenase a c t i v i t y i n ]J. melaninogenicus both: i n v i t r o and i n the guinea pig model system. P a r t i a l characterization of protease a c t i v i t y i n 13. vmelaninogenicus culture super-natants w i l l also be discussed. Specific points to be covered include: (1) Development of a rapid and sensitive collagenase assay based 14 on the release of C-peptides from labelled collagen. (2) Studies on collagenase a c t i v i t y i n I3_. melaninogenicus c e l l s (a) i n v i t r o (b) i n the guinea pig model system. (3) Studies on protease a c t i v i t y i n melaninogenicus culture supernatants. 7 I I . MATERIALS AND METHODS A. Organisms. Bacteroides melaninogenicus strains K110 and CR2A, obtained from Dr. P.A. Mashimo, were collagenolytic strains o r i g i n a l l y isolated by Macdonald and co-workers (27,28). Other strains were isolated i n the laboratory from gingival scrapings taken from persons i n varying states of ora l health. The organismso were isolated from cultures grown on blood agar plates (Difco blood agar base to which was added 5% laked human blood). The plates were incubated either i n anaerobic jars evacuated and flushed with E^:CO^ (95:5) (Matheson) or i n an anaerobic glove box (Coy Mfg., Ann Arbor, Michigan) containing an atmosphere of N^^rCO,^ (85:10:5). After 4-7 days incubation at 37°C, black colonies were picked from the plates and subcultured on the same medium u n t i l pure cultures were obtained. Staphylococcus aureus was a laboratory stock s t r a i n . Cultures of both organisms were maintained by weekly transfer or l y o p h i l i z a t i o n . B. Growth of organisms. Liquid cultures of melaninogenicus were maintained i n the trypticase-yeast extract medium described by Gibbons and Macdonald (12). Glucose was routinely omitted from the medium as were menadione and NaHCO^. Liquid c u l -tures were incubated at 37°C either i n the anaerobic chamber or i n stoppered tubes gassed with H 2:C0 2 (95:5). Since neither vitamin nor menadione appeared to enhance the growth'of vitamin K-requiring strains when added to the medium, vitamin K requirers were grown on blood agar plates with a streak of S^. aureus. Liquid medium for vitamin K-requiring strains was prepared as follows: f i l t e r - s t e r i l i z e d vitamin K^ (0.5% i n 95% ethanol) was added to s t e r i l e 2% agar which had been cooled to 50°C. The mixture was dispensed into tubes i n 1 ml quantities and 8 allowed to s o l i d i f y . Ten ml of the standard l i q u i d medium then was added. Thioglycolate, was not included i n the medium as vitamin i s inactivated by reducing compounds. Table I l i s t s the vitamin K requirements and i n f e c t i v i t y of the 13. melan- inogenicus strains most frequently used. Strains not l i s t e d i n the table did not require vitamin K and were noninfective. C. Chemicals and chromatographic .materials. 14 Acetic-1- C anhydride was obtained from New England Nuclear. Azocoll was purchased from Calbiochem; Clostridium histolyticum collagenase, trypsin, s u b t i l i s i n , subtilopeptidase, pronase, vitamin K^, bovine serum albumin (BSA) and hydroxyproline were obtained from Sigma. Chymotrypsin was obtained from Worthington. Bio-Gel P-60 was supplied by Bio-Rad laboratories, and Sephadex G-.75.' was purchased from Pharmacia. D. Preparation of b a c t e r i a l c e l l s for enzyme assays. Cells for enzyme assays were harvested by centrifugation and resuspended i n the assay buffer at a concentration lOx that of the o r i g i n a l .culture. Cells used for v i s c o s i t y assays were dialyzed against Tris-HCl buffer (0.1M, pH 7.2) containing 10 cysteine for 4 hours before the assay. E. Preparation of culture supernatants for enzyme assays and for column chro-: t". jmatography. Cells were sedimented from 48 hour cultures by centrifugation. The supernatant was f i l t e r e d through a 0.45 u f i l t e r ( M illipore Corp.) to remove any remaining b a c t e r i a l c e l l s and was then concentrated to 1/10-1/20 the o r i g i n a l volume using an u l t r a f i l t r a t i o n apparatus (Amicon) f i t t e d with a PM-10 membrane. The concentrated supernatant was diluted to the o r i g i n a l volume with d i s t i l l e d water and re-concentrated twice before use. F. Extraction of collagen. Neutral salt-soluble collagen was extracted from guinea pig skin accord-9 Table I Properties of 13. melaninogenicus Strains s t r a i n vitamin K collagenase i n f e c t i v i t y pure culture mixed culture CR2A - + K110 + ? *2D - + + + GP14 + + + GP2 MA-R -ing to the method of Gross (14). Acid-soluble collagen was extracted from fresh f e t a l c a l f s k i n as described by Gallop and Se i f t e r (11), except that ultracentrifugation of the pooled c i t r a t e supernatants was replaced by f i l -t r a t i o n through fine sintered glass to remove particulate matter. Lyophilized collagen was stored i n stoppered flasks at -20°C. G. Acetylation of collagen. Lyophilized acid-soluble collagen was s o l u b i l i z e d i n 0.01% cold acetic acid by s t i r r i n g overnight at 4°C. The concentration of collagen was 2 mg/ml. Immediately p r i o r to the addition of the acetylating agent the pH of the collagen solution was brought to 8.0 by the addition of 1M K^ HPO^ ,. The acety-14 l a t i n g agent, acetic-1- C anhydride i n benzene, was added dropwise over a period of 2 hours. A t y p i c a l preparation consisted of 250 mg of collagen and 10.2 mg of anhydride (100 jiCi/mg). The temperature of the reaction mixture was maintained at 10°C i n a water-ice bath, and the mixture was s t i r r e d con-tinuously on a magnetic s t i r r e r . The pH was monitored by a pH meter and re-adjusted to 8.0 i f necessary by the addition of IN NaOH. When a l l of the an-hydride had been added, s t i r r i n g of the mixture was continued for another hour i n the cold. The mixture was a c i d i f i e d with g l a c i a l acetic acid to s o l u b i l i z e any collagen which had gelled and to f a c i l i t a t e removal of the benzene. The acetylated collagen was dialyzed against 15 1 of d i s t i l l e d water at 4°C to 14 remove C-acetic acid, a by-product of the reaction. D i a l y s i s was continued 14 u n t i l no further C was detected i n the dialysate, which usually required 5 days with frequent changes of d i s t i l l e d water. The acetylated collagen was lyop h i l i z e d and stored at -20°C. H. Assays for collagenase a c t i v i t y . 14 The C-collagen substrate was prepared by s o l u b i l i z i r i g l y o p h i l i z e d , acetylated collagen i n 0.01% acetic at a concentration of 1 mg/ml by s t i r r i n g overnight at 4°C, The resulting clear, viscous solution was stored at 4°C for periods of up to two months. Typical reaction mixtures for both C_. h i s t o - lyticum collagenase and 15. melaninogenicus collagenase are shown i n Table I I . Commercial enzyme from fj. histolyticum was used i n the assay buffer at a con-centration of 30 units/ml. The reducing agent employed with the B_. melanino- genicus enzyme was cysteine hydrochloride, made up to a concentration of 5 x 10 i n the assay buffer and neutralized by adding 5N NaOH before use. The cysteine solution was always prepared immediately p r i o r to use. The assay components were incubated for f i f t e e n minutes before the addition,-of substrate and incubation was continued at room temperature. The reaction was stopped and unreacted collagen precipitated by the addition of 0.04N phospho-tungstic acid (PTA) and 2N HC1 to f i n a l concentrations of 0.01N and 0.5N respectively. A 0.1 ml aliquot of the reaction mixture was added to a "micro-fuge" tube (Beckman) containing 50 u l each of PTA and HC1. The samples were l e f t at room temperature for ten minutes before centrifugation for f i v e minutes i n a Beckman-Spinco 152 microfuge (Beckman Instruments). 100 Ail of the supernatant was counted. The control sample for each assay contained buffer i n place of enzyme; Viscosity assays were performed as described by Se i f t e r and Gallop (43) using a viscometer with a flow rate of 80-100 seconds for d i s t i l l e d water. When I*, melaninogenicus c e l l s were used as the source of enzyme, the dialyzed -2 c e l l suspensions were preincubated with 5 x 10 M cysteine for 15 minutes at room temperature. I. Guinea pig infections. Cells for inoculation of animals were harvested from blood agar plates or from broth cultures. Colonies from plates were resuspended i n approxi-mately 1 ml of phosphate-buffered saline (PBS), pH 7.0, and a portion of the suspension was injected into the animal. Cells from broth cultures were harvested by centrifugation and resuspended i n the desired volume of PBS 12 Table I I Protocol for Collagenase Assay component volume (ml) !B. melaninogenicus histolyticum C-collagen (0.1%) Tris-HCl buffer (.05M, pH 7.2) with CaCl 2 (.005M) cysteine (.005M) collagenase (30 units/ml) c e l l suspension (lOx) 0.2 0.2 0.1 0.2 0.1 0.1 0.1 unless otherwise stated. Guinea pigs used for the studies were 200-250 grams i n weight. The animals were shaved and inoculatred subcutaneously i n the groin area. They were observed for up to s i x weeks. Exudate was aspirated from infected animals using a s t e r i l e disposable syringe while the animal was under l i g h t ether anesthesia. The exudate was examined for microbial purity by plating on blood agar. J. Assays for proteolytic a c t i v i t y . P roteolytic a c t i v i t y was determined by measuring a c t i v i t y against Azocoll or casein. The reaction mixture for the Azocoll assay contained: Tris-HCl buffer (0.05M, pH 7.2), 4.8 ml; neutralized cysteine hydrochloride i n the same buffer (0.05M), 1.2 ml; and enzyme, 0.5 ml. The reaction components were incubated at 37°C for 15 minutes before the addition of Azocoll (20 mg),fand incubation was continued at the same temperature i n a shaking water bath. Two-ml samples were removed at desired time i n t e r v a l s , c h i l l e d immediately i n ic e , and centrifuged or f i l t e r e d to remove insoluble material. The amount of dye released was determined by measuring the absorbance of the supernatant at 520 nm. The substrate for the casein assay was prepared by dissolving 1 g of f; .. casein (Hammarsten) i n 100 ml of phosphate buffer (pH 7.4), and heating 15 minutes i n a b o i l i n g water bath. The reaction mixture contained 2.5 ml casein, -2 1.0 ml of neutralized cysteine hydrochloride (5 x 10 M) when required, 0.5-1.0 ml of enzyme, and buffer to 5.0 ml. The mixture was incubated at 37°C and the reaction terminated by the addition of 5.0 ml of 10% t r i c h l o r o a c e t i c acid. After 30 minutes at room temperature the contents of the tubes were f i l t e r e d and the A o o n of the f i l t r a t e s determined. ZoU K. Polyacrylamide gel electrophoresis. Gel electrophoresis was performed using the method of Nagai et a l (31). Samples were denatured prior to electrophoresis by heating to 50°C for 15 min-14 utes. L. Hydroxyproline assay. Hydroxyproline was assayed by the method of Leach (22). Citrate-extract-ed collagen (5 mg/ml) was hydrolyzed i n vacuo i n 6N HC1 by heating to 108°C for 18 hours. The hydrolyzed samples were neutralized with NaOHy diluted to 100 ug protein/ml and f i l t e r e d p r i o r to assaying for hydroxyproline. M. Protein determination. Protein was determined by the method of Lowry et a l . (25) using BSA as a standard. 15 I I I . RESULTS A. Development of a new collagenase assay. The main c r i t e r i o n for any v a l i d collagenase assay i s the use of unde- -natured collagen as the substrate. Acetylation of the epsilon amino;groups of the lysine residues of collagen was the l a b e l l i n g procedure chosen because the reaction can be carried out under mild conditions minimizing the possi-14 b i l i t y of denaturation of the collagen molecule. The v a l i d i t y of the C assay was examined by determining (1) the purity and properties of the c i t r a t e extracted collagen, (2) the properties of the acetylated collagen and (3) the s e n s i t i v i t y of the detection method to changes i n enzyme and substrate concen-t r a t i o n . 1. Analysis of the citrate-extracted collagen. a. Hydroxyproline. The purity of the citrate-extracted collagen was determined by measuring the amount of hydroxyproline present i n a weighed sample. A value of 12 ug/100 ug protein, or 12%, was obtained when the citrate-extracted collagen was analyzed for hydroxyproline. This agrees with the value of 13% given for the hydroxyproline content of c a l f s k i n collagen (8). b. Polyacrylamide gel electrophoresis. When subjected to gel elec-trophoresis according to the method of Nagai (32), the heat-denatured collagen stained as three bands corresponding to the (X-, j5-t and ^-components. This i s i n agreement with the expected r e s u l t s . c. Viscosity assays; enzyme s u s c e p t i b i l i t y . Citrate-extracted collagen was susceptible to collagenase but resistant to trypsin when assayed viscometrically. The v i s c o s i t y of the collagen preparation was evidence that the collagen was not denatured. d. Gel formation at 37°C. Formation of the t y p i c a l r i g i d , opaque gel was observed when collagen was prepared according to the method of Bennick and Hunt (2). 2. Acetylation of collagen. a. Results of a t y p i c a l acetylation. Table I I I shows the results of a t y p i c a l acetylation of acid-soluble c a l f s k i n collagen. The amount of lab e l incorporated ranged from 4-7%. The proportion of lysine residues l a -belled was calculated using a value of 4.0% for the lysine content of collagen (8) and assuming that the epsilon amino group of lysine was the p r i n c i p a l group labelled under these conditions (38). b. Effect of pH on the e f f i c i e n c y of acetylation. The acetylation procedure was carried out i n phosphate buffer at pH values of 7.0, 7.5, and 8.0. The substrate, neutral salt-soluble collagen at a concentration of 0.2%, was prepared by s t i r r i n g overnight at 4°C i n the appropriate buffer. Equal amounts of the acetylating agent were added to each sample. The acetylated samples were dialyzed against 0.1M phosphate buffer, pH 7.0, for three days before analysis. As shown i n Table IV, the acetylation procedure was most effective at pH 8.0 of the pH values tested. 3. Analysis of acetylated collagen. a. P r e c i p i t a t i o n of collagen. Collagen was precipitated i n i n i t i a l experiments by adding ethanol to a f i n a l concentration of 50% (43). Since 8-10% of the r a d i o a c t i v i t y remained i n the supernatant after ethanol treat-ment, a number of common protein precipitants were tested i n an attempt to reduce background counts. Two types of collagen preparation were used i n these experiments. "Insoluble" collagen was a fine suspension of collagen 14 o fibers prepared by s t i r r i n g the C-collagen overnight at 4 C i n Tris buffer (.05M, pH 7.0), with added CaCl 2 (.005M); "soluble" collagen was prepared by s o l u b i l i z i n g the acetylated collagen i n 0.01% acetic acid, then neutralizing the solution. "Soluble" collagen was a more viscous, g e l - l i k e preparation than was "insoluble" collagen. Table V summarizes the results obtained with 17 Table I I I Acetylation of Collagen protein added (mg) 250 protein recovered (mg) 211 s p e c i f i c a c t i v i t y (dpm/mg collagen) 6.25 x 10^ lab e l incorporated (%) 7.0 lysine residues labelled (%) 20.2 14 C i n solution after addition of PTA and HC1 (%) 1.4 Table IV Effect of pH on Effic i e n c y of Acetylation pH s p e c i f i c a c t i v i t y (cpm x 10 /^mg) 7.0 1.5 7.5 0.45 8.0 5.9 Each reaction mixture contained r ^ c o l l a g e n , 5 mg; phosphate buffer, pH as indicated, 1 m mole; acetic-1- C-anhydride (5 AiCi//umole) , 5 /umoles. Total volume 2.5 ml, temperature 10°C, reaction time 1 hr. Table V 14 Non-precipltable C 19-collagen preparation p r e c i p i t a t i n g reagent concentration soluble 1 4C (%) insoluble acetone 30% 10.3 II ii it 50% 6 6~ 2 II BSA EtOH 2 mg/ml 50% 9.9 II ZnCl 1.0% EtOH 50% 9.1 II polyethylene gl y c o l EtOH 15% 50% 9.2 II EtOH 50% 8.7 ti PTA .01N . 6.0 HC1 .5N ii none — 40.5 soluble none 90.0 II PTA HC1 .01N .5N 0 20 several of the pr e c i p i t a t i n g reagents tested. The pr e c i p i t a t i n g agents were added to the collagen samples. After 15 minutes at ice bath temperature the samples were centrifuged 5 minutes i n the Microfuge. Control samples were centrifuged 5 minutes without addition of any p r e c i p i t a t i n g agent. Since the lowest backgrounds were obtained using "soluble" collagen and a combina-14 t i o n of PTA and HC1 (44), C-collagen was always prepared for assays by s o l u b i l i z a t i o n i n 0.01% acetic acid. Even i n l a t e r experiments when collagen of higher s p e c i f i c a c t i v i t y was obtained, background counts remained around 1-2% of the t o t a l (counfts.. 14 b. S u s c e p t i b i l i t y of C-collagen to collagenase and other proteo- l y t i c enzymes. Table VI shows the percentage of r a d i o a c t i v i t y released from 14 C-collagen by collagenase and a number of proteolytic enzymes. The sub-strate remains susceptible to the action of both C^. histolyticum and 13. melan- inogenicus collagenases but resistant to attack by a number of common pro-14 teases. Some release of C due to nonspecific proteolytic attack on the non-h e l i c a l portions of the molecule would be expected (36) . The release of a small percentage (10%) of counts by trypsin and other proteases was presumed to r e f l e c t this and i s i n agreement with the results of others (38). 14 c. Protease-treated collagen. C-acetylated collagen i n 0.01% acetic acid (1 mg/ml) was incubated with s u b t i l i s i n . The reaction mixture contained 5 mg collagen, 37 units of enzyme, and Tris-HCl buffer (0.05M, pH 8, with .005M CaC^) to a f i n a l volume of 10 ml. Incubation was continued for 6 hr at room temperature, after which the reaction mixture was dialyzed over-night against d i s t i l l e d water at 4°C to precipitate the collagen. The pre-c i p i t a t e d collagen was centrifuged and washed with d i s t i l l e d water u n t i l no protease a c t i v i t y , (as detected by measuring dye release from Azocoll) re-mained i n the p e l l e t . The p e l l e t was s o l u b i l i z e d i n 0.01% acetic acid. Pre-14 liminary incubation of the C-collagen with s u b t i l i s i n produced a substrate Table VI C Solubilized by Proteolytic Enzymes enzyme concentration (mg/ml) 1 4C s o l u b i l i z e d (%)* collagenase 0.2 75.0 trypsin 0.1 9.7 s u b t i l i s i n 17.5 3.2 subtilopeptidase 17.5 7.1 pronase 1.25 1.0 chymotrypsin 6.25 1.3 B. melaninogenicus 20.0 46.6 s t r a i n CR2A * Incubation time 2 hr. for CR2A, 1 hr. for other enzymes. 22 Table VII 14 14 Release of C from Untreated and Pre-digested C-collagen by S u b t i l i s i n and Collagenase 14 collagen enzyme soluble C untreated collagenase _C. histolyticum 61.0 J3. melaninogenicus 46.5 s u b t i l i s i n 7.4 pre-digested collagenase C_. histolyticum 44.0 13. melaninogenicus 47.5 s u b t i l i s i n 0.95 which remained susceptible to attack by both c l o s t r i d i a l and 13. melaninogeni- cus collagenases but was less susceptible to attack by proteases (Table V I I ) . This indicated that acetylation had not denatured the main h e l i c a l portion of the molecule. The results obtained also indicate that the "^ C has actually labelled the h e l i c a l , collagenase-susceptible portion of the molecule rather than only the non-helical, protease-susceptible ends of the collagen molecule. 4. Kinetic studies of the collagenase assay. a. Release of r a d i o a c t i v i t y from la b e l l e d collagen by c l o s t r i d i a l collagenase. The time course of release of r a d i o a c t i v i t y from ^C-collagen by histolvticum collagenase i s plotted i n Figure 1. The rate of release 14 of C was linea r during the early part of the reaction. Two rates of reac-t i o n were observed when low concentrations of collagenase were used, in d i c a -ting that a number of less reactive s i t e s are available to the enzyme after the i n i t i a l cleavage of the molecule. b. Effect of substrate concentration. The release of from l a -belled collagen when substrate concentration was varied followed a t y p i c a l saturation k i n e t i c s pattern as shown i n Figure 2. When higher concentrations of substrate were used, however, accurate sampling was made impossible by the high v i s c o s i t y of the reaction mixture. c. Effect of enzyme concentration. The rate of release of radioac-14 t i v i t y from C-collagen during the e a r l i e s t part of the reaction varies dir-r e c t l y with enzyme concentration, as shown i n Figure 3. In this case a con-centration of 1.1 ug/ml of C_. histolyticum collagenase produced s i g n i f i c a n t 14 release of C. Smaller amounts of enzyme could be detected i f the reaction mixture was incubated for longer periods of time. 24 Figure 1. Release of C from acetylated collagen by C. histolyticum-collagenase. 1A 5 The reaction mixture contained: C-collagen, 1.9 x 10 cpm/mg, 200 ug; Tris buffer, pH 7.2, 160 umoles; C a C l 2 > 4 umoles; C. histolyticum collagenase, 1.5 units. Total volume, 1.05 ml; temperature, 37°C. 25 Figure 2. Release of C from acetylated collagen with increasing substrate concentration. Each reaction mixture contained: X HC-collagen, 1.9 x 10D cpm/mg, as indicated; Tris buffer, pH 7.2, 480 umoles; CaCl 2, 12 pmoles; C. histolyticum collagenase, 0.375 units. Total volume 2.45 ml, tem-perature 37°C, reaction time 30 minutes. Figure 3. Release of C from acetylated collagen as a fungtion of c o l -lagenase concentration. Each reaction mixture contained: "^C-collagen, 1.9 x 10"* cpm/mg. 200 ug; Tris buffer, pH 7.2, 160 umoles; CaCl^, 4 umoles; enzyme as i n d i -cated. Total volume 1.05 ml, temperature 37°C. 29 0.5 1.0 1.5 Collagenase (units) 30 B. Collagenase i n 13. melaninogenicus; i n v i t r o studies. 1. Attempts to i s o l a t e collagenase-producing s t r a i n s . Thirty strains of J3. melaninogenicus isolated from different gingival samples were tested for 14 a c t i v i t y against C-collagen. The results are shown i n Table V I I I . Since the experiments were done on separate occasions the incubation times and ages of the c e l l s vary somewhat. From the data i t can be seen, however, that only 14 two of the t h i r t y strains were active against C-collagen. No a c t i v i t y was found i n any of the supernatants of theij.samples tested. Assays for collagenase a c t i v i t y using the viscometric method gave results i d e n t i c a l to those obtain-14 ed with the C assay. The lack of collagenase a c t i v i t y i n the majority of strains tested contrasts with the findings of others (12,42) who detected collagenase a c t i v i t y i n a l l strains tested. 2. Attempts to increase collagenase a c t i v i t y . Macdonald et a l . (27) found that collagenolytic a c t i v i t y was apparently increased i n J3. melaninogen- icus c e l l s grown i n d i l u t e medium, which suggested that the enzyme might be repressed i n the normal growth medium. In an e f f o r t to either de-repress or to induce collagenase, a number of 13. melaninogenicus strains from the above group of t h i r t y were grown i n medium containing 1/10 the normal amount of trypticase and supplemented with insoluble collagen. Growth i n collagen-supplemented medium was about 1/10 that i n normal medium, as estimated by 14 absorbance at 660hm, and counts released from C-collagen remained at backr,; :•: ground levels for organisms grown both i n standard and i n collagen-supplemen-ted media. Substitution of acid-soluble collagen for the insoluble collagen had no ef f e c t . Because cobalt ions have been found to stimulate JC. histolyticum collagen-I | ase and Zn i s an i n t r i n s i c part of the enzyme structure (44,45), both ions were tested on 13. melaninogenicus c e l l s . Neither ion had any activating effect when added to the reaction mixture. 31 Table VIII Collagenase A c t i v i t y i n Thirty B. melaninogenicus Isolates i s o l a t e culture age incubation time UC released collagenase (days) (hrs) (%) a c t i v i t y 2 2 1 4.4 -13 2 1 2.6 4 8 2 1 0.7 -12 2 1 6.9 -15 2 1 3.0 -9 2 1 9.8 ? 6 2 1 4.9 -14 2 1 0.25 11 2 1 2.1 — 10 2 1 1.6 -16 7 2 4.3 -17 7 2 3.7 -DG 7 2 3.7 -TP 7 2 4.5 -BW 7 2 3.5 -PN 7 2 4.3 -MA-R 7 2 4.6 -H-l 7 2 3.9 -DW 7 2 5.3 — R6 7 2 3.9 -3D 3 2 0.25 -2D , 3 2 23.0 + AF 3 2 1.9 — FC-1 3 2 1.3 -CP 3 2 0.13 -JG 3 2 0.38 -GP2 5 2 4.7 — GP14 3 3 44.0 + FC 3 2 1.2 -TN 3 2 0.13 32 3. Collagenolytic a c t i v i t y i n collagenase producing strains CR2A and K110. a. Viscosity assays. Figure 4 i l l u s t r a t e s the reduction i n viscos- — • i t y of a collagen solution when incubated with c e l l s of 1$. melaninogenicus -:: strains CR2A and K110. BothK^B. melaninogenicus strains were active against collagen when assayed by t h i s method. 14 14 b. C assays. Both strains were active against C-collagen as il-lustrated i n Table IX. A l l a c t i v i t y was found i n the c e l l suspensions rather than i n the supernatants. c. Study of collagenase production by K110 as a function of culture 14 age. K110 c e l l s and supernatants were assayed for a c t i v i t y against C-colla-gen over a period of 10 days. Cells were grown i n standard trypticase-yeast extract medium, centrifuged and resuspended i n buffer at 20x th e i r o r i g i n a l concentration. Collagenase a c t i v i t y was detectable as early as day one and was found exclusively i n the c e l l s (Table X). CR2A produced sim i l a r r e s u l t s . Assays for collagenase a c t i v i t y using the viscometric method also gave posi-tiv e results at day one. This contrasts with the finding of Gibbons and Mac-donald (12), who found that collagenolytic a c t i v i t y was not evident u n t i l the culture began to lyse (72 hrs). However, their assay method, based on the release of hydroxyproline from collagen gels, may not have been sensitive enough to detect a c t i v i t y during e a r l i e r stages of growth. C. Collagenase i n B_. melaninogenicus infectious exudates. 1. Studies of the i n f e c t i o n . Although Macdonald and co-workers (27) established 15. melaninogenicus as an essential component of the mixed anaero-bic i n f e c t i o n i n the guinea pig system, and Kestenbaum demonstrated a correla-tio n between collagenase a c t i v i t y and i n f e c t i v i t y (20), l i t t l e else i s known about the relationship of 15. melaninogenicus: collagenase to pathogenicity. Some of the objectives i n attempting to establish the i n f e c t i v e system i n guinea pigs were to (1) determine whether collagenase a c t i v i t y of the organisms 33 Figure 4. Viscometric assay of collagenolytic a c t i v i t y . The reaction mixtures contained: collagen, 8 mg; Tris buffer, 9 pH 7.0, 320 /umoles; CaC^, 2 ;umoles,; cysteine 72 Limoles; 10 b a c t e r i a l c e l l s . Total volume, 5.2 ml, temperature 25°C. AT i s the elapsed time (flow time) for each reading. The midpoint i s defined as the time elapsed from the beginning of the assay to the beginning of the p a r t i c u l a r reading, plus 1/2 the elapsed time for that reading (1/2 A T). 35 Table IX Collagenase A c t i v i t y i n .B. melaninogenicus CR2A and K110 addition soluble C (cpm) % t o t a l 1 4C none 0 0 K110 c e l l s 5074 56.5 supernatant 262 2.9 CR2A c e l l s 3411 38.0 supernatant 248 2.8 Corrected for background Incubation time 3 hr. Table X Collagenase A c t i v i t y as a Function of Culture Age age of culture absorbance 1 4 c s o l u b i l i z e d (%)* (days) (660 nm) c e l l s supernatant 1 0.95 50 0 2 1.2 64 0.6 3 1.2 60 3.0 4 1.5 49 0 7 0.85 96 1.5 8 0.62 36 0.6 9 0.45 33 3.7 10 0.37 11 0.7 * Incubation time 5 hr. correlated with inf e c t i v i t y . , (2) determine whether collagenase could be i n -duced by animal passage, and (3) determine whether collagenase was produced i n the i n f e c t i o n i t s e l f . a. Establishment of a mixed i n f e c t i o n . I n i t i a l attempts to estab-l i s h a transmissible mixed i n f e c t i o n i n guinea pigs were unsuccessful. Gin-g i v a l scrapings from a number of persons i n varying states of o r a l health were cultured on blood agar plates for one week, following which the colonies were scraped from the plates, resuspended i n s t e r i l e saline and injected into animals. The response was at best a small nodular les i o n confined to the s i t e of i n j e c t i o n , from which a small amount of thick pustular material could sometimes be aspirated. In no instance could an i n f e c t i o n be produced i n a second animal by i n j e c t i o n of the exudate. A transmissible mixed i n f e c t i o n was f i n a l l y established with a mixed culture obtained from a periodontal patient. The day following the i n j e c t i o n the animal developed an abscess containing watery, dark coloured, foul-smell-ing exudate which contained large numbers of c o c c o b a c i l l i , spirochaetes, long rods, and motile organisms. Inoculation of a second animal with 0.1 ml of ex-udate produced a rapidly spreading i n f e c t i o n . Autopsy of the infected animals showed marked tissue necrosis i n the area of i n j e c t i o n , with perforation of the peritoneal wall i n one animal. The characteristics of the i n f e c t i o n were simi l a r to those described by Macdonald (28) for very acute mixed anaerobic infections. b. Organisms from the infectious mixture. A s e r i a l d i l u t i o n of ex-udate from an infected animal was prepared and samples were plated on blood agar. Saline suspensions of colonies scraped from the plates were injected into animals with the purpose of establishing a t y p i c a l i n f e c t i o n with a minimum number of species. Organisms removed from the 10 ^ d i l u t i o n of exudate were able to produce the t y p i c a l i n f e c t i o n ; the 13. melaninogenicus s t r a i n isolated from t h i s mixture was designated GP 14. The in f e c t i v e mixture was maintained as a mixed culture on blood agar and was also transferred several times on trypticase soy agar to eliminate the 15. melaninogenicus from the mix-ture. The mixed culture without 13. melaninogenicus was designated GP 25 and was maintained as a mixed culture on blood agar. The mixed culture alone was not i n f e c t i v e , but i n f e c t i v i t y was restored when GP 14 was added back to the . culture. Strain GP 14 was not infectious by i t s e l f . c. Recombination of collagenase-producing strains with GP 25. Four known collagenase-producing strains of IS_. melaninogenicus were recombined with the noninfectious "helper organisms" of the GP 25 mixture i n an attempt to produce i n f e c t i o n . The 15. melaninogenicus strains used were GP 14, o r i g i n a l l y isolated from the mixture; 2D, which was isolated from a periodontal patient; and K110 andCCR2A,which were collagenase producing strains o r i g i n a l l y isolated by Gibbons and Macdonald (27,28). The 13. melaninogenicus strains were grown with the mixture on blood agar plates p r i o r to i n j e c t i o n into animals. Guinea pigs weighing 200 g were injected with 0.6 ml of a 2 ml saline suspension of organisms scraped from the plates. The results i n Table XI show that infec-tions were produced by both GP 14 and 2D but not by CR2A and K110. Strain K110 has subsequently been fount} to be i n f e c t i v e when inoculated i n combination with one organism from the GP 25 mixture. d. Production of a transmissible i n f e c t i o n with a single s t r a i n of j3. melaninogenicus. 15. melaninogenicus s t r a i n 2D was tested for i t s a b i l i t y to produce an in f e c t i o n without the support of other organisms. Cells from a l i q u i d culture were washed, resuspended i n s t e r i l e PBS and injected into a guinea pig. Within 18 hours the animal had developed symptoms of a rapidly spreading i n f e c t i o n : marked edema i n the thoracic area, darkening of skin and loss of hair i n the thoracic area, and considerable weight loss. Material aspirated from the animal was dark i n colour, watery and foul-smelling, and Table XI Infections Produced by Recombination of melaninogenicus with a Mixed Culture jB. melaninogenicus s t r a i n i n f e c t i o n t r a n s m i s s i b i l i t y added to GP25 (5 days) K110 l o c a l i z e d response CR2A l o c a l i z e d response (slight) GP14 rapidly spreading abscess, burst to outside + 2D" very rapidly spreading i n f e c t i o n ; animal dead by day 5 + when examined by phase-contrast microscopy appeared to contain a pure culture of 13. melaninogenicus along with red blood c e l l s , white blood c e l l s and pus c e l l s . Fatty acid analysis revealed that acetic, propionic, isobutyric, bu-t y r i c and i s o v a l e r i c acids were present. These acids are produced by 13. mel- aninogenicus i n i n v i t r o culture. Inoculation of the exudate onto blood agar showed that i t contained a pure culture of JB. melaninogenicus. Strain 2D was thus one of the few 13. melaninogenicus strains capable of producing an in f e c t i o n without the support of other organisms (27,49,50). The i n f e c t i o n produced symptoms simi l a r to those described i n the l i t e r a t u r e for infections produced by CR2A (27) . However, CR2A was never found to produce an i n f e c t i o n , either alone or i n combination with other organisms. Thus i t would appear to have l o s t some factor required for i n f e c t i v i t y . e. Attempts to characterize the in f e c t i o n produced by 2D. Since 2D was in f e c t i v e by i t s e l f , the 2D i n f e c t i o n was chosen as the simplest system to study i n an attempt to delineate some of the requirements for i n f e c t i v i t y . However, because of the v a r i a b i l i t y among animals and the fact that 2D lo s t i n f e c t i v i t y with continued culture, the results of this study can only be noted as general trends. The following observations were made: (1) Guinea pigs weighing less than 250 g were more susceptible to in f e c t i o n than larger guinea pigs. 9 (2) The production of a successful i n f e c t i o n usually required 10 c e l l s as determined by direct count. However, since viable counts (as de-termined by plate count and Most Probable .Number methods) were oftenjless than 10% of the t o t a l counts, the number of viable c e l l s required to produce an i n ^ 9 fection may have been much less than 10 . (3) 48-hour c e l l s produced successful infections i n a greater per-centage of cases (66%) than did 24-hour c e l l s (30%). 72-hour c e l l s were as effective as 48-hour c e l l s . 40 «• 14 Figure 5. C released from collagen by whole exudate. 14 5 Each reaction mixture contained: C-collagen, 4.5 x 10 cpm/mg, 200 jig; Tris buffer, pH 7.0, 20 umoles; CaCl 2, 1 umolet; cysteine where indicated, 10 umoles; exudate, aspirated 48 hrs. after i n f e c t i o n , 1.5 x 10 b a c t e r i a l c e l l s . Total reaction volume, 0.5 ml,temperature, 25°C. 41 42 (4) Successful infections could usually be produced with c e l l s from . lyop h i l i z e d cultures when the cultures maintained i n the laboratory did not in f e c t . This supports the idea that the s t r a i n l o s t some property required f o r i n f e c t i v i t y . 2. Studies of collagenase a c t i v i t y i n material aspirated from infected  animals. Material aspirated from guinea pigs infected with 2D was studied to determine i f collagenase was,:' (1) present i n the i n f e c t i o n , (2) c e l l - a s s o c i a -ted or free, and (3) dependent on reducing conditions. Since there was con-siderable f l u i d accumulation i n infected animals (5-10% of the animal's body weight could often be aspirated), 10-15 ml of exudate could be aspirated from an infected animal without d i f f i c u l t y . a. Demonstration of collagenolytic a c t i v i t y i n exudate from an 14 infected animal. Figure 5 shows the release of counts from C-collagen by a sample of exudate taken from an infected animal. The exudate contained 1.5 x 1 0 ^ c e i l s / m l by direct count. Cysteine stimulated collagenase a c t i v i t y i n the exudate. b. Association of collagenase a c t i v i t y with b a c t e r i a l c e l l s i n  exudate. In order to determine whether the collagenase a c t i v i t y observed i n the guinea pig exudate was associated with the b a c t e r i a l c e l l s , the following experiment was done. Exudate was fractionated according to the following scheme: Exudate > Fraction A 270 x £ 15 min Low speed p e l l e t Low speed supernatant-•Fraction C 17,000 x £ 15 min resuspend i n Tris I Fraction B High speed p e l l e t ^ "V^High speed | supernatant resuspend I Fraction D Fraction E The following fractions were assayed i n the presence and absence of cysteine: (a) whole exudate, (b) low speed p e l l e t , (c) low speed supernatant, (d) high speed p e l l e t , and (e) high speed supernatant. A l l fractions were adjusted to their o r i g i n a l volumes. As shown i n Table XII, collagenase a c t i v i t y was found i n fractions A, B, C, and D ( s l i g h t ) . No a c t i v i t y was apparent i n the c e l l -free supernatant. Definite dependence on cysteine was observed i n the active fractions. • Since a c t i v i t y i n the low speed supernatant was much higher than that of the b a c t e r i a l c e l l s (high speed p e l l e t ) resuspended inlibuffer, i t was f e l t that some factor i n the high speed supernatant was required for or stimu-lated collagenase a c t i v i t y i n the b a c t e r i a l c e l l s . In an ef f o r t to demonstrate a c t i v i t y i n the b a c t e r i a l c e l l s , a sample of exudate was fractionated accord-ing to the above scheme and the high speed p e l l e t resuspended either i n buf-fer or i n high speed supernatant prior t<3 assay. Figure 6 shows that a c t i -v i t y was much greater i n the c e l l s resuspended i n supernatant than i n those resuspended i n buffer. No a c t i v i t y was apparent i n the supernatant alone. In a l l cases collagenolytic a c t i v i t y was dependent on cysteine. A c t i v i t y was 1, higher i n c e l l s resuspended i n high speed supernatant than i n the whole exu-date, which suggests that the whole exudate contains an i n h i b i t o r which i s Table XII Collagenolytic A c t i v i t y i n Fractionated Guinea Pig Exudate c 14 * tr a c t i o n cysteine C released (cpm x 10""^ ) A - 1.8 + 2.6 B - .16 + 1.7 C - .95 + 2.6 D - .14 + .76 E - .15 + .25 Incubation time 3 hr. A l l values corrected for background. 45 Figure 6. Stimulation of collagenase a c t i v i t y by high speed supernatant. Each reaction mixture contained: C-collagen, 4.5 x 10 cpm/mg, 200 pg; T r i s buffer, pH 7.0, 100 umoles; cysteine, 10 jumoles; CaCl 2 > 1 umole; 2D c e l l s resuspended as indicated. Total volume, 0.5 ml; temperature, 25 C. 46 removed by low speed centrifugation. c. Characterization of the stimulatory factor. Collagenase a c t i v i t y i n exudates from guinea pig infections was now known to be (1) associated with the b a c t e r i a l c e l l s , (2) dependent on cysteine and (3) stimulated by a factor i n the high speed supernatant. Stimulation might be due to a factor such as a protease, which would act on p a r t i a l l y digested collagen to increase the rate of release of counts, ,,or to some other molecule or ion which stimulated collagenase a c t i v i t y . (1) Effect of u l t r a f i l t r a t i o n on the high speed supernatant. In order to roughly determine the size of the stimulatory factor i n the high speed supernatant, a sample of supernatant was f i l t e r e d using an Amicon u l t r a -f i l t r a t i o n apparatus f i t t e d with UM-10 and UM-05 membranes, which r e t a i n glo-bular molecules of 10,000 and 500 molecular weight, respectively. B a c t e r i a l c e l l s from a sample of exudate were harvested as previously described and resuspended either i n buffer, high speed supernatant, UM-10 f i l t e r e d superna - i tant or UM-05 f i l t e r e d supernatant. As i s shown i n Table X I I I , very l i t t l e difference was found between the whole supernatant and the f i l t e r e d superna-tants, indicating that the stimulatory factor was a small molecule. (2) S t a b i l i t y of the f i l t r a t e . B a c t e r i a l c e l l s from a sample of exudate, collected as previously described, were resuspended i n (1) UM-10 f i l t e r e d supernatant, (2) f i l t e r e d supernatant which had been placed at 100°C for 15 minutes, (3) f i l t e r e d supernatant which had been stored at -20°C for three weeks, and (4) sample #3 which had been placed at 100°C for 15 minutes. No s i g n i f i c a n t differences were observed as shown i n Table XIV. To determine whether the stimulatory factor was a small organic molecule or a metal ion, a sample of the UM-10 f i l t r a t e was ashed by placing i t at 550°C for 3 hours. The ashed sample was resuspended i n the o r i g i n a l volume. As shown i n Table XV, ashing the f i l t r a t e had no effect on i t s 48 Table XIII F i l t r a t i o n of High Speed Supernatant resuspension treatment Y^C released (cpm x 10 /10 8cells/30 min) buffer supernatant UM-10 f i l t r a t e UM-05 f i l t r a t e 0.81 2.1 . 1.8 2.5 49 Table XIV S t a b i l i t y of UM-10 F i l t r a t e resuspension treatment •^ C released (cpm x 10" 3/10 8cells/30 min) UM-10 f i l t r a t e boiled f i l t r a t e frozen f i l t r a t e , boiled 1.8 2.1 1.7 2.2-50 Table XV Ashing resuspension treatment _C released (cpm x 1 0 / 1 0 8 ce l l s / h r ) buffer UM-10 f i l t r a t e ashed f i l t r a t e 1.9 4.2 5.7 stimulatory a c t i v i t y , which suggested that the stimulatory factor was probab-I | _^ l y an ion. Addition of Ca or Na to the reaction mixture had no stimulatory effect. D. Proteolytic a c t i v i t y i n 13. melaninogenicus culture supernatants. 1. Demonstration of an e x t r a c e l l u l a r protease. Proteolytic a c t i v i t y against Azocoll i n the culture supernatants of I3_. melaninogenicus strains 2D and CR2A i s shown i n Figure 7. A d e f i n i t e increase i n the rate of dye release occurs when 0.01M cysteine i s included i n the reaction mixture. A lag was observed before dye release could be measured; this may have been due to the fact that the Azocoll had to be reduced before enzymic attack would occur. A c t i v i t y against Azocoll was lo s t when the supernatant was bubbled with a i r for 15 minutes (Figure 8), but was restored with cysteine. The concentrated supernatant was active against casein but showed very l i t t l e a c t i v i t y against 14 BSA. No a c t i v i t y could be demonstrated against C-collagen. The proteoly-t i c a c t i v i t y was retained by an Amicon PM-30 membrane which retains globular molecules of 30,000 MW. Most of the a c t i v i t y was retained by an XM-50 mem-brane (50,000 MW retention), suggesting that the protease was either about 50,000 MW or associated with a large molecule or c e l l fragment. 2. Protease a c t i v i t y as a function of culture age. Protease a c t i v i t y was assayed i n culture supernatants of 2D prepared at various stages i n growth. Supernatants were assayed without concentration. Protease a c t i v i t y against Azocoll, shown i n Figure 9, could be measured a f t e r 10 hours of growth and increased with increasing c e l l numbers u n t i l 48 hours when a further increase i n protease a c t i v i t y was noted. Similar experiments also demonstrated an increase i n proteolytic a c t i v i t y i n mid- to late stationary phase which contin-ued as the c e l l s began to lyse. 3. P u r i f i c a t i o n of the protease. Attempts at fractionation of the concentrated supernatant with the object of purifying the protease produced Figure 7. Protease a c t i v i t y i n CR2A and 2D supernatants. Each reaction mixture contained: T r i s buffer, pH 7.2, 300 umol cysteine, where indicated, 100 umoles; supernatant, 2.5 mg protein; Azocoll, 30 mg. Total volume 10 ml, temperature 37°C. 53 Figure 8. Proteolytic a c t i v i t y i n 2D supernatants. Each reaction mixture contained: Tris buffer, pH 7.2, 300 pmoles cysteine, where indicated, 100 jumoles; supernatant, 2.4 mg "protein; Azocoll, 30 mg. Total volume 10 ml, temperature 37°C. 55 Minutes Figure 9. Protease a c t i v i t y as a function of culture age. Each reaction mixture contained: Tris-HCl buffer, pH 7.2, 240 pmoles; cysteine, 80 jumoles; supernatant, 9.3 mg protein; Azocoll, 25 mg. Total volume 8.0 ml, temperature 37°C, reaction time 60 minutes. variable r e s u l t s . Supernatant from a 2D culture was concentrated and low molecular weight molecules removed as described i n Methods. Chromatography on Bio-Gel P-60 resulted i n the recovery of only one th i r d of the material applied to the column (as estimated by A 0 0 _ ) . A c t i v i t y against Azocoll was z o U not detected i n the effluent. When a sample of.supernatant was chromatograph-ed on a Sephadex G-75 column (100 cm x 2.5 cm), only about one half of the ma-t e r i a l applied to the column was recovered, and a c t i v i t y against Azocoll was detected i n many fractions, suggesting that the protease a c t i v i t y might be as-sociated with another constituent of the supernatant. Since the protease a c t i v i t y appeared to be either s t i c k i n g nonspecifically to the longer column or to associate with some other component of the concen-trated supernatant, i t was f e l t that chromatography of the supernatant i n the presence of a large c a r r i e r protein such as BSA might allow the protease to complex with the c a r r i e r and elute with i t . Figure 10 shows the elution pro-f i l e s of concentrated supernatant, BSA, and a combination of supernatant and and BSA from a G-75 column. A complex has apparently formed between the BSA and some constituent of the supernatant, as the BSA peak has been shifted to the l e f t , indicating a larger molecule. Protease a c t i v i t y eluted with the complex. Rechromatography of the complex, however, produced a pattern simi-l a r to the o r i g i n a l e lution p r o f i l e of the concentrated supernatant i n F i -gure 10. Considerable v a r i a t i o n was encountered among the elution patterns of d ifferent samples or of the same sample chromatographed at different times. The reason for the va r i a t i o n i s not known although i t may have been due to breakdown of some of the components of the supernatant with storage, or to proteolytic action on some of the protein material. Another p o s s i b i l i t y i s that the protease may have been associated with a component of the c e l l w a l l , which would result i n the association of proteolytic a c t i v i t y with random fragments when the c e l l s lysed. 59 Figure 10. Effect of BSA on chromatography of supernatant on G-75. Column dimensions, 46.6 x 1.2 cm; flow rate, 30 ml/hr; f r a c t i o n s i z e , 1.5 ml; eluant, Tris-HCl, 0.05M, pH 7.0. Samples applied: (1) concentrated supernatant (2.5 mg protein/ml), 1.0 ml, > 2 O.D./cm, • •. (2) BSA (1 mg/ml) , 1.0 ml, 0.6 O.D./cm, • - -•• (3) BSA + concentrated supernatant (1:1, v:v), 1.5 ml, 1.9 O.D./cm, • — • . Azocoll a c t i v i t y , . 61 4. P a r t i a l characterization of protease a c t i v i t y . a. S t a b i l i t y of the protease. Aliquots of a concentrated supernatant of 2D were placed i n tubes. Cysteine ( f i n a l concentration 10 M) was added to half the samples. The samples were stored at 4°C and at -20°C; those con-taining cysteine were under an atmosphere of 1^:002 (80:20). Samples were re-moved and assayed at int e r v a l s over a period of seven weeks. The results i n -dicated that proteolytic a c t i v i t y against Azocoll i s stable under these con-ditions for at least seven weeks although a s l i g h t amount of a c t i v i t y may be los t on storage at 4°C without cysteine. The protease was inactivated by b o i l i n g , a temperature of 100°C for fi v e minutes reducing the rate of dye release to 1/3 that of the control. No a c t i v i t y was present after treatment at 100°C for ten minutes. Resistance of the protease to autodigestion was indicated by the following experiment. Samples of the concentrated supernatant were incubated at room temperature with and without cysteine. Aliquots were assayed at i n -tervals for protease a c t i v i t y . As i l l u s t r a t e d i n Table XVI, no decrease i n protease a c t i v i t y was apparent i n any of the samples, indicating that the pro-tease i s resistant to autodigestion for at least a period of four hours. b. Effect of cysteine concentration on protease a c t i v i t y . In order to determine the concentration of cysteine required for protease a c t i v i t y , cysteine concentrations {of the reaction mixture were varied from 10 to -1 -3 10 M. A concentration of at least 5 x 10 M cysteine was required for ac-t i v i t y (Table XVII). With lower concentrations of cysteine the rate of dye release never exceeded that of the control. Increasing the cysteine concen-r. tr a t i o n resulted only i n a s l i g h t increase in.protease a c t i v i t y . c". Effect of other reducing agents on protease a c t i v i t y . Mer-captoethanol, d i t h i o t h r e i t o l and sodium thioglycolate were tested for their effectiveness i n stimulating protease a c t i v i t y . The effectiveness of the 62 Table XVI Resistance to Autodigestion time at 25°C cysteine a c t i v i t y (brs) (10-2M1 ( A 5 2 0/15') 0 - 0.13 + 1 - 0.13 + 0.17-2 - 0.18 + 0.14 A - 0.15 + 0.15 Table XVII Effect of Cysteine Concentration cysteine added f i n a l concentration- a c t i v i t y (/jmoles) (M) ( A.../10') none 0 .03 .065 lO " 5 .01 .65 l O " 4 .02 6.5 l O " 3 .04 32.5 5 x 1 0 - 3 .38 65.0 l O " 2 .32 325.0 5 x 1 0 - 2 .49 650.0 10" 1 .44 reducing agents was greater at pH 8.0 than at pH 7.0. D i t h i o t h r e i t o l was the most effective of those tested (Table XVIII). d. Effect of pH on protease a c t i v i t y . Protease a c t i v i t y was measured at pH values form 4.0 to 9.0. Acetate buffers were used from pH 4.0 to 5.5, phosphate buffers from pH 6.0 to 7.5, and Tris-HCl buffers from pH 7.0 to 9.0. The results shown i n Figure 11 indicate that the optimum pH for ac-t i v i t y against Azocoll i s 8.0. Good a c t i v i t y was always obtained at pH values between 7.0 and 8.5. e. Effect of EDTA on protease a c t i v i t y . EDTA was tested for i t s effect on protease a c t i v i t y by adding i t to the reaction mixture to give f i n a l -2 -5 concentrations ranging from 10 M to 10 M. The protease showed some sensi--2 -3 t i v i t y to EDTA at concentrations of 10 M and 10 M, which reduced the rate -4 of dye release to about 40% that of the control. EDTA at 10 M had no effect and 10 "*M EDTA was i f anything s l i g h t l y stimulatory (Figure 12) . Table XVIII Effect of Reducing Agents reducing agent Limoles f i n a l concentration (M) pH activity ( A 5 2 ( J / 1 0 ' ) -none 0 0 8.0 .21 dithiothreitol 650 1 0 " 1 II .56 65 lO" 2 II .53 6.5 lO" 3 II .41 mercaptoethanol 650 1 0 " 1 II .59 65 lO" 2 II .33 6.5 i o " 3 II .17 thioglycolate 650 lO" 1 • II .44 65 i o " 2 it .25 6.5 i o " 3 it .17 none 0 0 7.0 .02 cysteine 65 i o " 2 ii .17 dithiothreitol 65 i o ~ 2 II .12 v mercaptoethanol 65 i o " 2 ti .10 thioglycolate 65 i o - 2 II .02 Figure 11. Effect of pH. Each reaction mixture contained: buffers, pH as indicated, acetate 300 umoles, phosphate, 300 umoles, Tris 300 umoles; cysteine, 100 umoles supernatant 1.25 mg protein; Azocoll, 20 mg. Temperature, 37°C, pre-incubation 10 minutes. Total volume, 6.5 ml. 67 Figure 12. Effect of EDTA. Each reaction mixture contained: Tris buffer (pH 8), 300 jomoles; cysteine, 60 /jmoles; EDTA, as indicated; supernatant, 1.25 mg protein; Azocoll, 20 mg. Total volume 6.5 ml, temperature 37°C, preincubation 10 min. 69 Minutes IV. DISCUSSION The development of a rapid and sensitive assay for collagenolytic a c t i -v i t y should f a c i l i t a t e collagenase studies not only i n b a c t e r i a l systems but 14 m mammalian systems as w e l l . The advantage of using C-acetylated collagen would appear to be the speed of the assay r e l a t i v e to the viscometric method and the ease of preparation of large amounts of substrate of high s p e c i f i c 14 a c t i v i t y r e l a t i v e to;/the C-glycine method. A possible disadvantage of 14 using acetylated collagen rather than C-glycine labelled collagen i s the fact that the radioactive label i n the former i s located i n a functional group which has been chemically attached to the collagen molecule, whereas i n the l a t t e r the l a b e l occurs i n an amino acid i n t r i n s i c to the collagen molecule. Studies done on the assay, however, have not revealed any differences between the behaviour of the labelled and the unlabelled substrates. The acetylated collagen does not appear to have been denatured by the l a b e l l i n g procedure, as i s evidenced by i t s continued resistance to nonspecific proteolytic attack, and appears to be a suitable assay substrate as evidenced by treatment with collagenase.., To date, perfect correlation has been obtained between the 14 results 6f the C-collagen assay and those of other collagenase assays used. In contrast to the findings of other workers (12,42), studies i n t h i s laboratory have shown that only a small percentage of human ora l isolates of 15. melaninogenicus are collagenolytic. In addition, the presence of collagenolytic a c t i v i t y i n 15. melaninogenicus strains studied so far has been found to correlate with the presence of other characteristics i n the organism including a s p e c i f i c f a t t y acid p r o f i l e and hemagglutinating a c t i v i t y (T. Ed-wards, personal communication). The collagenase of I5_. melaninogenicus was found to be associated with the c e l l s and dependent on reducing conditions for a c t i v i t y , as reported by others (12). Gibbons and Macdonald (12) found that degradation of collagen gels by J3. melaninogenicus cultures was not evident u n t i l culture autolysis began at 72 hours, which suggested to them that the enzyme was i n t r a c e l l u l a r and was released with l y s i s of the c e l l s . S i m i l a r l y , Hausman and Kaufman found no evidence of collagenase a c t i v i t y i n culture supernatants u n t i l autolysis began (17). In contrast to the findings of these workers, c o l l a -genase a c t i v i t y was found i n washed c e l l s of J3. melaninogenicus as early as day 1. No a c t i v i t y was ever detected i n the culture supernatants even after autolysis had occurred. The characteristics of the transmissible mixed i n f e c t i o n produced by the i n j e c t i o n of guinea pigs with organisms cultured from gingival material were sim i l a r to those of the rapidly spreading infections described by Macdonald and co-workers (28). Results obtained when the J3. melaninogenicus s t r a i n was eliminated from and subsequently recombined with the mixture were i n agree-] ment with those reported by- others. However, i n contrast to previous reports that a number of JB. melaninogenicus strains could replace the o r i g i n a l i n the infectious mixture, t y p i c a l infections were obtained only when certain of the collagenase producing strains were recombined with the mixture.• This may be due to the fact that the 13. melaninogenicus strains used by previous workers were a l l collagenolytic. Kestenbaum (20) found a positive correlation between collagenase a c t i v i t y of the 13. melaninogenicus strains used and the severity of the i n f e c t i o n , whether or not the B^. melaninogenicus strains differed i n other respects such as protease production was not established, thus i t cannot be said that the differences i n the severity of the infections were dependent solely on differences i n collagenase a c t i v i t y . The data ob-tained using the experimental mixed i n f e c t i o n system support the idea of a correlation between collagenase a c t i v i t y and i n f e c t i v i t y insofar as successful infections were produced with collagenolytic ]3. melaninogenicus strains and noncollagenolytic strains were never i n f e c t i v e . The determination of whether collagenase a c t i v i t y i s essential for a successful i n f e c t i o n w i l l require a mutant of aii i n f e c t i v e , collagenase-producing s t r a i n d i f f e r i n g from the par-ent s t r a i n only i n the absence of collagenase a c t i v i t y . I t evident, however, that collagenase a c t i v i t y alone i s not s u f f i c i e n t for i n f e c t i v i t y , as one collagenase-positive s t r a i n (CR2A) was never found to produce any i n f e c t i o n either'alone or i n combination with other orgafrisms. Assuming that i t i s the same organism described by Macdonald (27), i t can be presumed that CR2A has l o s t some " i n f e c t i v i t y factor" required for pathogenicity. Of interest also i s the observation that 2D, which o r i g i n a l l y produced infections si m i l a r to those described for CR2A, appeared also to be losing an " i n f e c t i v i t y factor" and becoming noninfective both«.in pure and i n mixed culture. Both s t r a i n s , however, remained strongly collagenolytic. The observation that infections were produced more frequently with older c e l l s of 2D indicates that a factor required for i n f e c t i v i t y i s produced during the stationary phase of growth. Studies of material aspirated] from guinea pig infections have shown that 2D elaborates collagenase i n the i n f e c t i o n as well as i n v i t r o . The a c t i v i t y i s d e f i n i t e l y associated with the b a c t e r i a l c e l l s . The nature of the stimula-tory factor i n f i l t e r e d exudate remains unclear; however, due to the fact that i t was not destroyed by ashing i t i s probably a metal ion. 13. melaninogenicus has been shown to possess proteolytic a c t i v i t y but the a c t i v i t y has not been characterized. An organism dependent on peptides for growth (52) would be expected to be ac t i v e l y p r o t e o l y t i c . The organism may elaborate more than one protease, as a c t i v i t y has been demonstrated i n the washed c e l l s as well as i n the supernatant, and Hausman and Kaufman have found caseinolytic a c t i v i t y associated with a particulate f r a c t i o n from the autoly-sate supernatant (17). The role of the protease i f any i n i n f e c t i o n cannot be established yet; conceivably proteolytic a c t i v i t y could enhance collagenolytic 73 a c t i v i t y to promote the spread of i n f e c t i o n as well as being of n u t r i t i o n a l significance to the organism. Preliminary studies i n this laboratory indicate that dermonecrotic a c t i v i t y i n 15. melaninogenicus supernatants may be associa-ted with protease a c t i v i t y . The role of collagenase i n I5_. melaninogenicus infections and i t s r e l a -tionship to pathogenicity remains unclear. I5_. melaninogenicus has been i s o l a -ted for years from a number of c l i n i c a l infections, i s known to comprise 5% of the cultivable f l o r a of the gin g i v a l crevice, and i t i s known that some strains produce a collagenolytic enzyme. However, the common speculation that collagenase production by 15. melaninogenicus i s largely responsible for the pathogenesis of periodontal disease and other infections may be an oversimpli* f i c a t i o n i n view of the finding that considerable differences exist between strains with regard to collagenase a c t i v i t y and i n f e c t i v i t y . In experimental infections produced with collagenolytic strains of the organism, 13. melanino- genicus has been established as the primary pathogen and has been demonstrated to produce collagenase i n s i t u . However, i n naturally occurring infections the r e l a t i v e proportions of "pathogenic"and "nonpathogenic" 13. melaninogenicus strains i s not known. Collagenase-positive organisms obviously exist i n such situations as they have been isolated from infections, but t h e i r contribution:-to the t o t a l i n f e c t i v i t y of the b a c t e r i a l population i s unknown. The finding that the majority of human o r a l i s o l a t e s of 13. melaninogenicus are noninfec-tiv e and collagenase-negative suggests that i f 15. melaninogenicus collagenase i s to be implicated i n the pathogenesis of periodontal disease i t must be postulated either that the non-collagenolytic organisms are induced to pro-duce collagenase i n the disease state or that collagenolytic 13. melaninogenicus strains are introduced into the population. LITERATURE CITED Altemeier, W.A. 1942. The pathogenicity of appendicitis p e r i t o n i t i s . Surgery 11: 374-384. Bennick, A. and A.M. Hunt. 1967. Collagenolytic a c t i v i t y i n o r a l tissues. Arch. Oral B i o l . 12: 1-9. Berman, M. B., R. Manabe and P.F. Davison. 1973. Tissue collagenase: a simple, semiquantitative enzyme assay. Anal. Biochem. 54_: 522-534. Bornstein, P. 1974. The biosynthesis of collagen. Annu. Rev. Biochem. 43: 567-603. Burdon, K.L. 1928. Bacterium melaninogenicum from normal and pathologic tissues. J. Infec. Dis. 42_: 161-171. Courant, P.R. and R.J. Gibbons. 1967. Biochemical and immunological heterogeneity of Bacteroides melaninogenicus. Arch. Oral B i o l . 12: 1605-1613. Donoff, R.B., J.E. McLennan and H.C. G r i l l o . 1971. Preparation and pro-perties of collagenases from epithelium and mesenchyme of healing mammalian wounds. Biochim. Biophys. Acta 227: 639-653. Eastoe, J.E. 1967. Composition of collagen and a l l i e d proteins, p. 1-72. In G.N. Ramachandran (ed.), Treatise on collagen, v o l . 1. Academic Press, Inc., New York. Eisen, A.Z., E.A. Bauer and J . J . Jeffrey. 1970. Animal and human c o l l a -genases. J. Invest. Dermatol. 55_: 359-373. Eisen, A.Z., E.A. Bauer and J . J . Jeffrey. 1971. Human skin collagenase. The role of serum alpha-globulins i n the control of a c t i v i t y i n vivo and i n v i t r o . Proc. Nat. Acad. S c i . U.S. 68: 248-251. Gallop, P.M. and Sam S e i f t e r . 1963. Soluble collagens, p. 635-641. In S.P. Colowick and N.O. Kaplan (ed.), Methods i n enzymology, v o l . 6. Academic Press, Inc., New York. Gibbons, R.J. and J.B. Macdonald. 1961. Degradation of collagenous sub-strates by Bacteroides melaninogenicus. J. B a c t e r i o l . 81_: 614-621. Gibbons, R.J. and J.B. Macdonald. 1960. Hemin and vitamin K compounds as required factors for the c u l t i v a t i o n of certain strains of Bacteroides  melaninogenicus. J. B a c t e r i o l . 80: 164-170. Gross, Jerome. 1958. Studies on the formation of collagen. T.'.. Proper-ti e s and fractionation of neutral s a l t extracts of normal guinea pig connective tissue. J. Exp. Med. 107: 247-263. 75 15. Gross, Jerome, and C.M. Lapiere. 1962. Collagenolytic a c t i v i t y i n am-phibian tissues: a tissue culture assay. Proc. Nat. Acad. S c i . U.S. 48: 1014-1022. 16. Harper,E., and J.Gross. 1970. Separation of collagenase and peptidase a c t i v i t i e s of tadpole tissues i n culture. Biochim. Biophys. Acta 198: 286-292. 17. Hausman, Ernest, amd E l i a s Kaufman. 1969. Collagenase a c t i v i t y i n a particulate f r a c t i o n from Bacteroides melariiriogeriicus. Biochim.iBiophys. Acta 194: 612-615. 18. Hite, K.E., M. Locke and H.C. Hesseltine. 1949. Synergism i n experi-mental -.infections with nonsporulating anaerobic bacteria. J. Infec. Dis. 84: 1-9. 19. Kaufman, E.J., P.A. Mashimo, E. Hausman, C.T. Hanks and S.A. E l l i s o n . 1972. Fusobacterial i n f e c t i o n : enhancement by c e l l free extracts of Bacteroides melaninogenicus possessing collagenolytic a c t i v i t y . Arch. Oral B i o l . 17: 577-580. 20. Kestenbaum, R.C., J. Massing and S. Weiss. 1964. The role of collagen-ase i n mixed infections^containing Bacteroides melariiriogeriicus. IADR abstract. 21. K l i n e , B.S. 1923. Experimental gangrene. J. Infec. Dis. 32: 481-483. 22. Leach, A.A. 1960. Notes on a modification of the Neuman and Logan method for the determination of hydroxyproline. Biochem. J. 7_4: 70-71. 23. Loesche, W.J., R.N. Hockett and S.A. Syed. 1972. The predominant cultivab l e f l o r a of tooth surface plaque removed from i n s t i t u t i o n a l i z e d subjects. Arch. Oral B i o l . 17_: 1311-1326. 24. Loesche, W.J., K.U. Paunio, M.P. Wooldolk and R.N. Hockett. 1974. Collagenolytic a c t i v i t y of dental plaque associated with periodontal pathology. Infec. Immunity £: 329-336. 25. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall 1951. Protein measurement with the F o l i n phenol reagent. J. B i o l . Chem. 193: 265-275. 26. Macdonald, J.B., R.J. Gibbons and S.S. Socransky. 1960. B a c t e r i a l meehanismsr. i n periodontal disease. Ann. N.Y. Acad. S c i . 85_: 467-478. 27. Macdonald, J.B., S.S. Socransky and R.J. Gibbons. 1963. Aspects of the pathogenesis of mixed anaerobic infections of mucous membranes. J. of Dent. Res. 42: 529-544. 28. Macdonald, J.B., R.M. Sutton and M.L. K n o l l . 1954. The production of fuspspirochaetal infections ingguinea,.pigs, withrrecdmbined, pure cultures. J. Infec. Dis. 95: 275-284. 76 29. Macdonald, J.B., R.M. Sutton, M.L. K n o l l , E.M. Madlener and R. M. Grains ger. 1956. The pathogenic components of an experimental fusospiro-chetal i n f e c t i o n . J. Infec. Dis. 98: 15-20. 30. Meleney, F.L. 1931. Bacteri a l synergism i n disease process with a confirmation of the synergistic b a c t e r i a l etiology of a certain type of progressive gangrene of the stomach w a l l . Ann. Surg. 19: 961-981. 31. Mergenhagen, S.E. and H.W. Scherp. 1960. Lysis of reconstituted c o l l a -gen and catabolism of products of collagenolysis by the o r a l micro-biota. Arch. Oral B i o l . 1: 333-338. 32. Nagai, Y., J. Gross and K.A. Piez. 1964. Disc electrophoresis of collagen components. Ann. N.Y. Acad. S c i . 121: 494-500. 33. Nagai, Y., C.M. Lapiere and J. Gross. 1966. Tadpole collagenase. Preparation and p u r i f i c a t i o n . Biochemistry 5_: 3123-3130.. 34. Nordwig, A. 1971. Collagenolytic enzymes. Advances i n enzymology 34: 155-205. 35. Peterkofsky, B., and R. Diegelmann. 1971. Use of a mixture of protein-ase-free collagenases for the s p e c i f i c assay of radioactive collagen i n the presence of other proteins. Biochemistry 6_: 988-994. 36. Piez, K.A., 1967. Soluble collagen and the components resulting from i t s denaturation, p. 207-252. In G.N. Ramachandran (ed.), Treatise on collagen, v o l . 1. Academic Press, Inc., New York. 37. Ramachandran, G.N., ed. 1967. Treatise on collagen. Academic Press, Isic/., New York. 38. Riordan, J.F. and B.L. Vallee. 1972. Acetylation, p. 494-499, In S.P. Colowick and N.O. Kaplan (ed.), Methods i n enzymology, v o l 25. Academic Press, Inc., New York. 39. Robertson, P.B., R.E. Taylor and H.M. Fullmer. 1972. A reproducible quantitative collagenase r a d i o f i b r i l assay. C l i n . Chim. Acta hl\ 43-45. 40. Rosebury, T., J.B. Macdonald and A.R. Clark. 1950. Bacteriologic survey of gingi v a l scrapings from periodontal infections by direct examination, guinea pig inoculation and anaerobic c u l t i v a t i o n . J. of Dent. Res. 29: 718-731. 41. Sakamoto, S., P. Goldhaber and M.J. Glimcher. 1972. A new method for the assay of tissue collagenase. Proc. Soc. Exp. B i o l . Med. 139: 1057-1059. 42. Sawyer, S.J., J.B. Macdonald and R.J. Gibbons. 1962. Biochemical characteristics of Bacteroides melaninogenicus. Arch. Oral B i o l . _7: 685-691. 77 43. S e i f t e r , S. and P.M. Gallop. 1962. Collagenase from Clostridium histolyticum, p. 659-665. In S.P. Colowick and N.O. Kaplan (ed.), Methods i n enzymology, v o l . 5. Academic Press, Inc., New York. 44. S e i f t e r , S S . and E. Harper. 1970. Collagenases, p. 613-635. In S.P. Colowick and N.O. Kaplan (ed.), Methods i n enzymology, v o l . 19. Academic Press, Inc., New York. 45. S e i f t e r , S. and E. Harper. 1971. The collagenases, p. 649-697. In P.D. Boyer (ed.), The enzymes, v o l . 3. Academic Press, Inc., New York. 46. Smith D.T. 1930. Fusospirochetal disease of the lungs produced with cultures from Vincent's angina. J. Infec. Dis. 4j>: 303-310. 47. Socransky, S.S. 1970. Relationship of bacteria to the etiology of periodontal disease. J. of Dent. Res. 49_: 203-222. 48. Socransky, S.S. and R.J. Gibbons. 1965. Required role of Bacteroides melaninogenicus i n mixed anaerobic infecti o n s . J. Infec. Dis. 115: 243-247. 49. Takazoe, I. and T. Nakamura. 1971. Experimental mixed i n f e c t i o n by human gingiv a l crevice material. B u l l . Tokyo Dent. C o l l . 12: 85-93. 50. Takazoe, I . , M. Tanaka and T. Homma. 1971. A pathogenic s t r a i n of Bacteroides melaninogenicus. Arch. Oral B i o l . 16: 817-822. 51. Takeuchi, H., M. Sumitani, K. Tsubakimoto and M. Tsutsui. 1974. Oral microorganisms i n the"gingiva of individuals with periodontal desease. J. of Dent. Res. 53: 132-136. 52. Wahren,AA. and R.J. Gibbons. 1970. Amino acid fermentation by Bacter-oides melaninogenicus. Antonie van Leewenhoek _36_: 149-159. 53. Weiss, C. 1943. The pathogenicity of Bacteroides melaninogenicus and i t s importance i n surgical infections. Surgery 13: 683-691. 

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