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Isolation of two trypsin-like proteases associated with the outer membranes of Bacteroides gingivalis Scott, Helen G. 1988

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Isolation of two trypsin-like proteases associated with the outer membranes of Bacteroides gingivalis  By  HELEN  G. SCOTT  D.D.S., University of Alberta, 1976  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  In  THE FACULTY OF GRADUATE STUDIES THE FACULTY OF DENTISTRY DEPARTMENT OF CLINICAL DENTAL SCIENCES  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA  November  1988  © Helen G. Scott, 1988  In  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  partial  fulfilment  University  of  British  Columbia,  for  this or  thesis  reference  thesis by  this  for  his  and  scholarly  or  thesis  study.  her  for  of  gain  shall  permission.  Department  of  n i n i r a l  The University of British 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6(3/81)  September  2,  n p n f a l  Columbia  1988  S r i p n p P R  that  agree  may  representatives.  financial  requirements  I agree  I further  purposes  the  be  It not  is  that  the  Library  permission  granted  by  understood be  for  allowed  an  advanced  shall for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  ABSTRACT  Two proteases, P-1 and P-4, present in the outer membranes of  gingivalis  Bacteroides  A T C C 33277, were isolated and partially purified by S D S - P A G E .  The purification procedures resulted in a specific activity 45-fold (P-1) and 40-fold (P-4) greater than that of the crude fractions. of proteolytically  active  P-1  produced three  bands  Electrophoresis  corresponding  to  molecular weights of 235, 220 and 200 kD whereas electrophoresis of proteolytically active P-4 produced one major band corresponding to 74 kD. The optimum temperature and pH for activity were determined using N-5benzoyl-D-arginine-p-nitroanilide as substrate. The  proteases were most  active at 37 °C and at pH values between 6.0 and 6.5.  Both proteases required a reducing agent for activity and were inhibited by a variety of serine and thiol protease inhibitors.  Arginine-containing peptides  were readily hydrolyzed whereas the proteases were less active against glycine-containing peptides.  The proteases hydrolyzed IgA, IgG, gelatin,  azocoll and azoalbumin; acid-soluble collagen was not degraded.  The results of this investigation suggest that these are trypsin-like proteases which have a thiol component as part of the active site.  ii  TABLE OF CONTENTS  Abstract  ii  List of tables  v  List of figures  vi  Acknowledgements  vii  Introduction  1  Classification of black-pigmented Bacteroides  1  Antigenic and serologic characteristics of B. gingivalis  3  Ultrastructural studies  3  Ecology of Bacteroides gingivalis  4  Growth and nutrition  4  Metabolic end-products of Bacteroides gingivalis  7  Formation and characterization of vesicles  7  Adherence properties of Bacteroides gingivalis  8  Fimbriae  9  Surface-binding proteins  9  Hemagglutination properties  10  Experimental infections involving B P B s  11  Periodontal diseases  12  Virulence factors of Bacteroides  gingivalis  14  Adherence properties  14  Capsule  15  Outer membrane vesicles  16  Lipopolysaccharide  17  Metabolic end-products  17  Enzyme activities  18  Types of proteases  21  Proteases of Bacteroides gingivalis  22  Protelytic activity of other pathogenic organisms Aim of the investigation  27 29  iii  Material and methods Bacterial strain and culture conditions Isolation and purification of proteases Preparation of outer membranes Preparative polyacrylamide gel electrophoresis Protein determination Protein profiles of OM-1 and OM-2 Protein profiles of P-1 and P-4 Proteolytic activity of OM-1, OM-2, P-1, P-4 Peptidase activity Optimum pH Optimum temperature and heat stability Inhibition of protease activity Hydrolysis of protein substrates Immunoreactivity Chemicals  Results Purification Peptidase activity Optimum pH Optimum temperature and heat stability Inhibition of enzyme activity Activity against protein substrates Immunoreactivity  Discussion  Bibliography  iv  LIST OF TABLES  TABLE  PAGE  1  Purification of  2  Peptidase activity of P-1 and P-4  52  3  Effect of protease inhibitors and metal ions  59  4  Activity against protein substrates  60  v  P-1 and P-4  47  LIST OF FIGURES  FIGURE  >  PAGE  1  S D S - P A G E of outer membranes-1  43  2  S D S - P A G E of outer membranes-2  44  3  Zymogram of proteases after preparative S D S - P A G E  45  4  S D S - P A G E of P-1  49  5  S D S - P A G E of P-4  50  6  S D S - P A G E showing proteolytic purity of OM-2, P-1, P-4  51  7  Optimum pH for the hydrolysis of BAPNA by P-1  55  8  Optimum pH for the hydrolysis of BAPNA by P-4  56  9  Optimum temperature for the hydrolysis of BAPNA  57  10  Heat stability of P-4  58  1 1  Hydrolysis of IgA and IgG by P-1 and P-4  62  12  Hydrolysis of gelatin and collagen by P-1 and P-4  63  13  Hydrolysis of collagen by trypsin and P-1  64  vi  I  ACKNOWLEDGEMENTS  I am deeply grateful to Dr. B. C. McBride for his understanding and guidance, and for his continued support and encouragement of this project, and for the financial support of the University of British Columbia. My sincere thanks to Drs. John Silver and Jukka Uitto for serving on my committee and for their guidance and support, and I gratefully acknowledge the constant encouragement of the members of the Division of Periodontics. I wish to give special thanks to Dr. Daniel Grenier who so generously shared his knowledge and experience with me, and who was always a source of encouragement. I give my sincere thanks to Meja Song, Heather Merilees and Pauline Hannam who asssisted me by sharing their technical skills and experience, and for their constant support and encouragement. I extend to my fellow students, Angela Joe, Nadarajah Ganeshkumar, Umadatt Singh and Blair Heffelfinger, my sincere gratitude for consistently sharing their experience and knowledge, and for their fellowship and humour. I also wish to thank Bruce McCaughey and Harold Traeger for their photography in the preparation of this work. I take this opportunity to express my gratitude to my family and friends their unfailing support and confidence.  vii  for  INTRODUCTION  Bacteroides findings  gingivalis  is c o n s i d e r e d to be a periodontal pathogen b a s e d upon the  of n u m e r o u s  investigations  which  have  identified this o r g a n i s m  in  association with a variety of periodontal d i s e a s e s (reviewed by v a n Winkelhoff et al, 1988).  Furthermore, B. gingivalis  p o s s e s s e s a number of  virulence factors  which may be potentially d a m a g i n g to the periodontal t i s s u e s .  T h e periodontal  pocket p r o v i d e s an e c o l o g i c a l environment w h i c h is ideal for c o l o n i z a t i o n by these  organisms.  Classification of black-pigmented Black-pigmented  Bacteroides  Bacteroides  ( B P B ) are G r a m - n e g a t i v e , obligately  anaerobic,  non-motile, n o n s p o r e f o r m i n g , r o d - s h a p e d o r g a n i s m s which p r o d u c e b r o w n to black c o l o n i e s o n solid m e d i a containing blood.  T h i s group of m i c r o - o r g a n i s m s  w a s first d e s c r i b e d by O l i v e r a n d W h e r r y (1921) w h o n a m e d the Bacterium  b e c a u s e they b e l i e v e d the dark pigment to be  melaninogenicum  melanin.  T h e s e bacteria were later a s s i g n e d to the genus Bacteroides  Bacteroides  and  Courant  and named  (Roy and Kelly, 1939).  melaninogenicus  T h i s w a s the only B P B s p e c i e s r e c o g n i z e d until 1970, (1962)  and  Gibbons  (1967)  had  although S a w y e r et al  shown  biochemical  i m m u n o l o g i c a l heterogeneity a m o n g strains of B. melaninogenicus. and M o o r e (1970) divided B. melaninogenicus their f e r m e n t a t i v e (strongly  abilities:  fermentative),  fermentative)  and  B.  organism  B.  B.  subsp.  melaninogenicus  s u b s p . intermedius subsp.  melaninogenicus  Holdeman  into three s u b s p e c i e s b a s e d upon  melaninogenicus  melaninogenicus  and  (weakly (non-  asaccharolyticus  fermentative).  Sufficient  biochemical  and  genetic  differences  were  found  between  saccharolytic and asaccharolytic s u b s p e c i e s to elevate B. melaninogenicus asaccharolyticus  to the s p e c i e s level ([B.  1  asaccharolyticus],  the  subsp.  Finegold  and  B a r n e s , 1977).  Further research  (Coykendall et al, 1980;  v a n S t e e n b e r g e n et  al, 1979) indicated that the D N A of asaccharolytic B P B strains isolated from the h u m a n oral cavity c o n t a i n e d b e t w e e n 4 7 a n d 4 9 mol% g u a n i n e plus  cytosine  (G+C) w h e r e a s the D N A isolated from non-oral strains contained between 52 a n d 53 mol% (G+C).  Furthermore, no D N A homology w a s found between the D N A s of  the oral a n d the non-oral strains. asaccharolytic  species,  It w a s therefore p r o p o s e d , that a s e c o n d  Bacteroides  be recognized  gingivalis,  designate oral strains (Coykendall et al, 1980;  which  would  v a n S t e e n b e r g e n et al, 1982).  A n e w s p e c i e s of asaccharolytic Bacteroides, which c a n b e d i s t i n g u i s h e d B.  a n d from B.  gingivalis  S t e e n b e r g e n et al (1984):  asaccharolyticus,  Bacteroides  has been  endodontalis.  proposed  from  by v a n  This proposal c a m e after  the evaluation of the two asaccharolytic B P B strains by V a n S t e e n b e r g e n et al, (1982).  T h e s e two strains were originally isolated by Sundqvist ( G . Sundqvist,  P h . D. thesis, University of U m e a , U m e a , S w e d e n , 1976, a s reviewed by M a y r a n d &  Holt,  1988).  B.  endodontalis  c a n be  differentiated  from  the  other  asaccharolytic s p e c i e s by its lower G + C content a n d by the a b s e n c e of antigens c o m m o n to other a s a c c h a r o l y t i c B P B s . identified by their differing polyacrylamide endodontalis  Furthermore, all three s p e c i e s c a n b e  protein profiles  gel electrophoresis  following s o d i u m d o d e c y l  (van S t e e n b e r g e n  sulfate-  et a l , 1 9 8 4 ) .  B.  h a s b e e n isolated from mixed oral infections, predominantly from  p y o g e n i c infections of odontogenic origin (van Winkelhoff et al, 1985) a n d from root c a n a l infections ( H a a p a s a l o et al, 1986).  Bacteroides  gingivalis  periodontal p o c k e t  is isolated primarily from the h u m a n gingival s u l c u s or  a n d differs from the non-oral  B.  asaccharolyticus  production of p h e n y l a c e t i c acid ( K a c z m a r e k a n d C o y k e n d a l l , 1 9 8 0 ; 1979),  by  Mayrand,  hemagglutination of s h e e p erythrocytes (Slots a n d G e n c o , 1979), by  a trypsin-like e n z y m e activity (Slots, 1981), a n d by serology 1978;  by the  M o u t o n et a l , 1981).  2  ( M a n s h e i m et al,  Antigenic and serologic characteristics of Bacteroides gingivalis Reed et al (1980) demonstrated the antigenic specificity of B. and B. gingivalis  asaccharolyticus  using Immunoelectrophoresis and immunodiffusion techniques.  The results of their work showed that none of the strains from non-oral sites was antigenically similar to B.  gingivalis  and furthermore, no common  antigens  were found between the asaccharolytic and saccharolytic BPB strains.  Parent et al (1986) observed that B.  gingivalis  consisted of at least two  serogroups, and found that the human biotype exhibited 25 surface antigens, two of which were specific for the biotype. serogroups of B. gingivalis  Fisher et al also demonstrated two  (J.G. Fisher, J.J. Zambon, P. Chen and R.J. Genco,  J . Dent. Res. 65:816, abstr. 817, 1986) and found that these groups were associated with the virulence of the strain. major protein band at 54 kD which was  The virulent strains produced a absent  in the avirulent  strains.  Furthermore the electrophoretic profile produced by cell envelope proteins of virulent  and  avirulent  strains differed  (Bramanti  and  Holt, J . Dent.  Res.  66:223, abstr. 930, 1987), and this difference was apparently represented in the pathogenicity of the organism.  Ultrastructural  studies  Electron microscopy observations (Takazoe et al, 1971) revealed the presence of an external capsule and fimbria-like structures in a pathogenic strain of  gingivalis.  B.  The electron microscopy studies of Mansheim and Kasper (1977),  revealed a typical Gram-negative morphology:  the cells had an inner and outer  cell membrane separated by a thin peptidoglycan layer.  Furthermore, the oral  strain, but not the non-oral strain, was found to have capsular material external to, and associated with, the outer membrane.  A recent electron microscopic  survey by Handley and Tipler (1986) also revealed the presence of fimbriae and of a layer of material external to the outer membrane.  In addition, B. gingivalis  produces outer membrane vesicles (blebs) which are  3  formed by pinching or budding of the outer membrane (Grenier 1987;  Listgarten and Lai, 1979;  Woo et al, 1979).  and Mayrand,  The number of vesicles  released may be related to growth conditions (McKee et al, 1986). The vesicles have  been  shown to be proteolytic and collagenolytic, to hemagglutinate  erythrocytes and to mediate attachment between noncoaggregating  bacterial  species (Grenier and Mayrand, 1987).  of Bacteroides  Ecology  Bacteroides pocket  gingivalis  gingivalis  colonizes the human gingival sulcus and periodontal  (Slots and Genco, 1979) and has occasionally been isolated from oral  mucosal surfaces  (van Winkelhoff, 1986b;  Zambon et al, 1981).  The organism  is usually absent from a healthy gingival sulcus, however, it may constitute <15% of the cultivable subgingival flora from individuals with gingivitis (Slots, 1982;  Slots et al, 1978; White and Mayrand, 1981; Zambon et al, 1981), and  the proportion of B. gingivalis  increases  significantly  periodontitis (Loesche et al 1985; Slots, 1977b; al, 1979;  White and Mayrand, 1981).  in cases  of  adult  Spiegel et al, 1979; Tanner et  In addition, this organism is routinely  found in those sites which are deemed to be actively progressing (Slots et al, 1986;  Growth  Tanner et al 1984).  and  nutrition  Morphologically, B P B are uniform or pleomorphic rods; cells in broth culture are coccobacillary, 0.5 urn wide by 1-2 urn long. Growth on blood agar produces colonies which are 1-2 mm in diameter, convex, and form black pigment in 710 days. This black pigmentation, originally thought to be melanin by Oliver and Wherry (1921), was found by Shah et al (1979) to be protohemin, a watersoluble,  intracellular or cell-associated derivative of hemoglobin  (Duerdan,  1975).  Bacteroides  gingivalis will grow on solid medium or in broth culture containing  small peptides plus hemin and vitamin K as growth factors (Gibbons and MacDonald, 1960; Wahren and Gibbons, 1970).  4  Vitamin K can be replaced by  menadione (Gibbons and MacDonald, 1960), and succinate can take the place of hemin as a growth factor (Mayrand and McBride, 1980). proposed a selective medium for B.  gingivalis  Hunt et al (1986)  {Bacteroides  gingivalis  agar,  BGA) containing bacitracin, colistin and nalidixic acid as the selective agents. They found that this medium could be used to differentiate between B. and B. asaccharolyticus  and could isolate B. gingivalis  gingivalis  from other oral bacteria.  Colistin and nalidixic acid inhibit aerobic and facultatively anaerobic, Gramnegative bacteria, and bacitracin inhibits Gram-positive bacteria and is active against many other oral species.  Seddon et al (1988) described chemically defined and minimal media for the growth of Bacteroides  gingivalis.  The chemically defined medium  contained only  twelve components and was able to support the growth of cells in culture which were morphologically identical to those grown in other media. metabolic end-products  Furthermore, the  of cells grown in this medium were reproducible and  yielded patterns similar to those produced in complex media.  The growth rates  were approximately 50% slower than those of cells grown in a complex medium, however,  when the defined medium was supplemented with protein hydrolysates,  the rate of cell growth was increased.  The  effect  B. gingivalis  of hemin concentration  upon the physiology  strain W50 was studied by McKee et al (1986).  and virulence  of  They found that  no growth occurred in the absence of hemin, although a complex proteinaceous medium supplemented with vitamin  was used. Cells grown under conditions of  hemin limitation produced few fimbriae,  but large numbers of extracellular  vesicles were observed surrounding the cells and free within the medium. Conversely, under conditions of hemin excess, the cells were heavily fimbriated and few vesicles were noted.  Furthermore, cells grown in medium lacking hemin  were avirulent when injected into mice whereas the injection of cells grown in medium supplemented with hemin produced up to 50% mortality in mice. conclusion was that  Their  the hemin concentration of the medium modulated not only  5  cell growth, but also the virulence of the organism.  Based upon this apparently  obligate requirement for hemin, it is not surprising that B. gingivalis  is highly  effective in degrading heme-containing plasma proteins (Carlsson et al, 1984) or that there is a proportionate increase in the number of Bacteroides  species  when bleeding develops as a result of disease progression (Loesche and Syed, 1978).  Shah and Williams (1987) found that B. gingivalis  grew best in medium in  which glucose was absent, and if glucose was present, little was metabolized (3%).  Prolific growth was produced in medium containing protein hydrolysates.  Nutritional relationships can exist between certain bacteria the result of which is an enhancement of growth for one or both organisms.  For example, BPB  species exhibit enhanced growth in the presence of naphthoquinone, a vitamin Krelated compound (MacDonald et al, 1963) and the hemin required by  gingivalis  B.  can be replaced by the succinate produced by facultative organisms  which ferment glucose (Mayrand & McBride, 1980) or by capnophilic bacteria (Grenier & Mayrand, 1985).  More recently, Grenier & Mayrand (1986) found  that protoheme, produced by W. recta in coculture with B. gingivalis, the growth of this Bacteroides  gingivalis  species.  enhanced  Furthermore, once established,  B.  can promote its own growth by suppression of other bacterial species  by the production of hematin or bacteriocin (Takazoe et al, 1984).  The growth of a pathogenic strain of B. gingivalis  (W50) under conditions of  varying pH was examined by McDermid et al (1988).  Using chemostat  conditions, stable growth occurred over the pH range 6.7 to 8.3, but could not be maintained at pH 6.5 or pH 8.5.  A maximum yield of cells was obtained when the  cultures were maintained between pH values of 7.0 and 8.0.  The enzymatic  activity of the cells was also found to vary according to the growth pH.  Trypsin-  like activity increased with the growth pH and was maximal at pH 8.0, whereas the specific enzyme activity of collagenase and hyaluronidase was greatest at, or below a neutral growth pH.  6  Metabolic  end-products  of  Bacteroides  gingivalis  Some products resulting from the metabolism of nutrients by B. gingivalis  are  considered to be virulence factors of the organism (van Steenbergen et al, 1982).  These include organic acids:  isovaleric (Lambe et al, 1982;  acetic, proprionic, butyric, isobutyric,  Reed et al, 1980; Shah et al, 1976) which are  produced in common with other Bacteroides is produced only by B. gingivalis  species. However, phenylacetic acid  (Kaczmarek and Coykendall, 1980;  Mayrand,  1979) and was found to be directly proportional to the Trypticase content of the medium;  L-phenylalanine and peptides containing this amino acid also enhanced  the production of phenylacetic acid  (Bourgeau and Mayrand, 1983).  Ammonia and indole (MacDonald and Gibbons, 1962; van Steenbergen et al, 1986b) and volatile sulphur compounds, including hydrogen sulphide, dimethyl sulphide and methylmercaptan (Tonzetich and McBride, 1981) are produced by  B. gingivalis  and are considered to be virulence factors detrimental to the host.  The large quantities of ammonia produced (Shah et al, 1987) contribute to the rise in pH during growth of B.  gingivalis  in broth culture (McDermid, 1988)  and may have a similar effect within the periodontal pocket where the pH has been shown to increase with an increase in pocket depth and with the severity of the host inflammatory response (Bickel and Cimasoni, 1985).  Formation  and  characterization  of  extracellular  vesicles  The formation of extracellular vesicles by B. gingivalis  has been reported by  Williams and Holt (1985) and by McKee et al (1986).  Williams and Holt  (1985) recovered outer membrane fragments (vesicles) by ultracentrifugation of culture supernatant.  They found that although there was a distribution of  sizes, vesicles of approximately 50 nm predominated.  The polypeptide profile of  the vesicles was similar, but not identical, to that of the outer membranes, as analyzed by S D S - P A G E . There were at least two polypeptide bands, at 16 and 18 kD,  found in the outer membrane fraction of B. gingivalis  detectable in the vesicle fraction.  which were  not  In addition, there were minor differences  7  between the fractions  in the intensity of the protein staining of some of the  bands.  Grenier and Mayrand (1987) characterized some of the biological activities of extracellular vesicles isolated from B. gingivalis.  They, too, found a variation  in vesicle size from 20-150 nm, but a predominance of vesicles of 50 nm diameter.  However, in contrast to the study of Williams and Holt (1985),  Grenier and Mayrand (1987) found two supplementary  polypeptide  bands  between 45 and 66 kD in the vesicle fraction which were not apparent in the polypeptide profile of the outer membranes.  This difference between the two  studies could be the result of having used two different strains of B. for comparison of the polypeptide patterns;  gingivalis  gingivalis  Williams and Holt (1985) used B.  strain W whereas Grenier and Mayrand (1987) used a nonpathogenic  strain, A T C C  33277.  The vesicles were found to be both proteolytic and  collagenolytic and were able to agglutinate sheep erythrocytes, activities which are identical with those of the whole cell.  Furthermore, the vesicles mediated  coaggregation between bacterial cells of two species, Eubacterium and Capnocytophaga  ochracea,  saburreum  by forming a physical link or bridge between the  cells, as seen by electron microscopy.  This coaggregation was stable over a pH  range from 4.5 to 8.5 (Grenier & Mayrand, 1987).  Adherence properties of  Bacteroides  gingivalis  A primary requirement of an organism, in order to colonize and grow within a host, is an ability to attach to host surfaces.  Specific adhesins on the surface of  bacterial cells are responsible for the attachment to specific host receptors (Holt, 1982).  The adhesins on the cell surface of Gram-negative  bacteria  include type-specific pili or fimbriae, hemagglutinins and other surface-binding proteins  (Ofek and Perry,  1985).  colonize the dentition at discrete sites.  It is characteristic  of B.  gingivalis  to  Attachment potential, host and bacterial  interactions, nutritional, physical and chemical factors are determinants which will affect the colonization pattern.  8  1.  Fimbriae  Fimbriae from B. gingivalis Yoshimura et al, (1984) and that the fimbriae from B.  have been isolated, purified and characterized by Yoshimura et al, (1985). are  gingivalis  thin, curly,  approximately 5 nm long and with a diameter of 4 nm.  Their studies indicate heat-stable  filaments  The component subunit,  fimbrillin, has an apparent molecular weight of 43 kD and a primary structure different from that of other Gram-negative bacteria (Yoshimura et al, 1985). Furthermore,  native  fimbriae  and  denatured  fimbrillin  have  differing  immunological characteristics and show little cross-reactivity.  Work done by Okuda & Takazoe (1974) seemed to indicate that the fimbriae conferred  hemagglutinating  (HA)  activity,  however,  a pure  preparation of  fimbriae (Yoshimura et al, 1984) showed neither HA activity, nor inhibition of HA activity.  Slots and Gibbons (1978) reported that B. gingivalis  was able to  attach to both buccal and crevicular epithelial cells, and to the surfaces of Grampositive bacteria.  However, the attachment of B. gingivalis  to epithelial cells  and to erythrocytes was inhibited by both saliva and serum. These fluids did not inhibit the attachment to Gram-positive bacteria.  What this suggests is that  there may be two types of adhesins on the surface of B. gingivalis.  This  hypothesis is supported by the fact that both those strains which have HA activity,  and those which  do not, exhibit fimbriae on their cell surface.  Furthermore, heating for 15 minutes at 60 ° C abolished the HA activity of a partially purified preparation of fimbriae (Slots and Gibbons, 1978).  2.  Surface-binding  properties  The  ability to bind to specific  host proteins  may  assist the organism in  colonization, in protection from host defences or by providing key nutrients.  B.  gingivalis  has been found to bind fibrinogen (Lantz et al, 1986).  binding was rapid, highly specific and saturable. indicated that B. gingivalis  The  Furthermore, this group  possessed a cell-associated thiol protease which  9  could degrade fibrinogen.  Winkler et al (1987) investigated the ability of some Gram-positive and Gramnegative  oral organisms,  membrane-like  matrix  including B.  and  to  gingivalis,  purified  fibronectin, laminin and Type IV collagen.  to attach to a basement-  basement  membrane  proteins:  Their results showed that, generally,  the Gram-negative organisms bound in greater numbers to the intact matrix, whereas the Gram-positive organisms attached preferentially to the isolated proteins.  Of all the Gram-negative organisms tested, B. gingivalis  intact matrix in the greatest numbers and although it did not  bound to the  bind to a great  degree to the isolated proteins, it attached in greater numbers to the isolated Type IV collagen than did either of the other Gram-negative organisms or the Grampositive organisms.  The apparent affinity of B. gingivalis  for the intact matrix,  rather than for the isolated proteins, may be related to involvement of a threedimensional structure of the matrix in adherence.  The importance of the binding of B. gingivalis  to the isolated Type IV collagen  should not be overlooked in that the basement membrane, which is composed of Type IV collagen, offers the last potential barrier to bacterial translocation from the pocket to the underlying connective tissues.  3.  Hemagglutination  properties  The ability of B. gingivalis  to hemagglutinate erythrocytes (Boyd and McBride,  1984; Slots and Genco, 1979) differentiate  B. gingivalis  is an important taxonomic character which can  from other Bacteroides  species.  However,  receptor responsible for HA activity has not been completely elucidated. components (capsular polysaccharide and lipopolysaccharide [LPS]) from B, gingivalis extracted  from  the  Surface extracted  neither exhibited nor inhibited HA activity, however, pili  B.  gingivalis  Although, as mentioned earlier,  did posess  HA ability (Okuda et al, 1981).  others have had difficulty reproducing this  work.  10  Boyd and McBride (1984) showed that the HA activity was associated with lowmolecular-weight LPS, protein, and loosely bound lipid of the outer membrane and furthermore, that procedures which removed fimbriae from the surface of B.  had no effect  gingivalis  upon the ability of the whole cells to cause  hemagglutination. In their study, the hemagglutinating activity and the bacterial aggregating activity associated with a crude outer membrane fraction were separated.  The fraction responsible for bacterial aggregation comprised a large  quantity of protein and carbohydrate with little lipid material, whereas the fraction  responsible  for  hemagglutination  contained  loosely-bound  lipid,  carbohydrate and only a small amount of protein.  The work of Yoshimura et al (1984) also showed that a preparation of purified fimbriae from B. gingivalis  had neither a positive correlation with HA activity  nor did it inhibit HA activity. factor of B.  Further investigation into the hemagglutination  by Okuda et al (1986) resulted in the isolation and  gingivalis  characterization of a hemagglutination factor from culture supernatant of strain 381.  gingivalis mainly  of  protein  B.  Their results indicated that the factor was composed  (73%)  with  smaller  quantities  of  sugar  (12%)  and  phosphorous (6%). The apparent molecular weight was 40 kD, as determined by SDS-PAGE.  Experimental  infections  involving  asaccharolytic  BPBs  Asaccharolytic BPB species have often been cited as pathogens in experimental infections (Grenier & Mayrand,1985; Mayrand & McBride, 1980; Gibbons, 1965).  Socransky &  In these studies, combinations of bacteria were isolated from  various oral sources and were tested for their ability to induce abscess formation and transmissible infections when inoculated subcutaneously into guinea pigs. The general consensus from these studies was that individual bacterial species or isolates were not able to induce an infection, however, mixtures of two or more bacteria produced a pathogenic effect.  A common finding of these studies was that when BPBs were included in the  11  infectious mixture, a transmissible infection could be produced. the BPB species, and especially asaccharolytic BPBs, mixture, transmissibility was not exhibited.  However, when  were deleted from the  It is possible that the synergistic  infective mechanism is related to growth factors produced by the associated bacteria.  However, a recent study by Grenier & Mayrand (1987) has shown that  six out of fourteen strains of Bacteroides pathogenic in pure culture.  gingivalis  tested for virulence were  These pathogenic strains tended to be highly  collagenolytic and proteolytic whereas strains which were not pathogenic had lower collagenolytic activity but possessed a high proteolytic activity.  Periodontal  diseases  Specific periodontal diseases have been described and are distinguished by such factors as the age of the patient, the type and distribution of bone and attachment loss and by the species of bacteria associated with the disease (Page & Schroeder, 1982).  For example, adult periodontitis has been associated with BPBs  intermedius  and B.  gingivalis)  Slots,  1986a;  (Slots,1979; Zambon,  1985; Zambon  organisms  (Listgarten  Capnocytophaga  and Actinobacillus Slots  &  &  actinomycetemcomitans,  Listgarten,  et al, 1981),  and  1988;  spirochetes  Hellden,1978).  and Actinobacillus  (B.  Socransky,  1977;  and other  motile  Bacteroides  actinomycetemcomitans  gingivalis, have been isolated  from patients diagnosed as having juvenile periodontitis (Kornman & Robertson, 1985; Liljenberg & Lindhe, 1980; Moore et al 1985; Newman et al, 1976; Slots, 1976).  At  least 300  different  periodontal pocket. sulcus  are  bacterial  species  have  been  identified  within  the  In health, bacteria comprising the microflora of the gingival  primarily  Gram-positive,  facultative  organisms  (Slots,  1977a),  whereas in adults, a proportionate increase in the number of Gram-negative, anaerobic species occurs as the disease increases in severity (Slots, 1977b; Slots et al, 1978).  This is the case as well for juvenile periodonditis where the  subgingival microflora has been found to comprise Gram-negative facultative or  12  anaerobic rods  (Baehni et al, 1979; Kornman & Robertson, 1985; Moore et al,  1985; Slots, 1976;  Tanner et al, 1979).  Bacteroides gingivalis has been repeatedly implicated in the establishment and progression of periodontal diseases.  Many recent studies have confirmed the  finding of Burdon (1928) that Bacteroides species were present in disease sites (reviewed by Slots, 1982; Slots & Listgarten, 1988;  van Winkelhoff et al,  1988). B. gingivalis has been recovered not only from sites in adults diagnosed as having generalized advanced periodontitis, (Loesche et al, 1985; Slots, 1977b; Spiegel et al, 1979; Tanner et al, 1979; White & Mayrand, 1981) but also from sites in adult patients which were classified as actively progressing (Slots et al, 1986; Tanner et al, 1984).  That periodontal diseases are the result of mixed infections has been the indication from all of the investigations periodontal health and disease.  into the microflora related to both  The composition of the subgingival flora found in  health varies significantly from that found in the various forms of periodontal disease.  In a state of health, 95% of the total microflora, as determined by phase  contrast or darkfield microscopy, is composed of nonmotile rods and cocci (Listgarten and Hellden, 1978).  A s disease progressses, there is a marked  decrease in the numbers of cocci and a proportionate increase in the number of motile rods and spirochetes (Lindhe et al, 1980; Temporo et al 1983).  Di Murro et al (1987), in a study evaluating the subgingival microflora isolated from patients diagnosed as having rapidly progressive periodontitis, found B. gingivalis to be consistently involved.  Furthermore, a recent report by van  Dyke et al (1988), indicated that B. gingivalis was an etiologic agent in severe, recurrent adult periodontitis.  B. gingivalis has been demonstrated in both generalized juvenile periodontitis (Loesche  et al, 1985;  Wilson  et al , 1985)  and in localized  periodontitis (Kornman & Robertson,1985; Moore et al, 1985).  13  juvenile  Additional  evidence  periodontal  for the role of B.  diseases  investigators  agree  comes  that  from  in patients  in the various types of  gingivalis  immunological diagnosed  studies.  Virtually  with generalized  periodontitis or adult periodontitis the serum antibody levels to B. are higher than in other groups of individuals ( Altman et al, 1982; al,  1982; Ebersole  juvenile gingivalis  Ebersole et  et al, 1986; Farida et al, 1986; Mouton et al, 1981;  Taubman et al, 1982; Vincent et al, 1985).  Elevated levels of antibody to B.  are also noted in gingival crevicular fluid as compared to serum  gingivalis  levels (Ebersole et al, 1985; Schonfeld & Kagan, 1982;  Tew et al, 1985b).  A recent study by Holt et al (1988) indicated that B. successfully  implanted  into  the  periodontal  could be  gingivalis  microbiota  of  monkeys.  B.  strain 3079.03 was isolated from a ligature-induced periodontal site  gingivalis  in a cynomolgus gingivalis  all  strain.  monkey and was used to derive a rifampin-resistant Subsequent to the implantation of the  had serum antibody levels to the B. gingivalis  B.  derived strain, not only  strain increased as compared to  controls, but in addition, significant bone loss was observed radiographically at implanted sites, ostensibly due to a burst of activity.  Thus,  this work appears  to demonstrate a direct connection between the implantation of B. gingivalis  and  clinical alterations in serum antibody levels and in crestal bone levels.  Virulence factors  of Bacteroides  gingivalis  Potentially pathogenic organisms require a combination of properties in order to manifest their effect. B. gingivalis  1.  Much research has been directed toward the properties of  which contribute to its virulence.  Adherence  properties  One of the first requirements of an organism, in order to initiate disease, is an ability to attach to its host, for only in so doing will the organism be able to colonize and multiply within the host.  The features of B. gingivalis  which effect  adherence have been discussed previously under adherence properties and include  14  fimbriae, hemagglutinins and surface-binding proteins.  The recent work by Ohmori et al (1987) and Hanazawa et al (1988) helps to elucidate the virulence properties of the fimbriae of B. gingivalis. (1987)  reported  that  human  gingival  fibroblasts  spontaneously  thymocyte-activating factor (FTAF), which in turn stimulates thymoctye proliferation. The importance of  Ohmori et al produce  mitogen-induced  F T A F is that it may function to  activate the immune response, which has a potentially damaging effect upon the periodontal tissues of the host.  In the follow-up study, Hanazawa et al (1988)  examined culture supernatants from gingival fibroblasts for their ability to stimulate thymocyte proliferation. purified fimbriae on F T A F  Once this was established, the effect of  production was determined by culturing gingival  fibroblasts with various doses of purified fimbriae.  Lipopolysaccharide was not  detected on a silver-stained gel following S D S - P A G E of the fimbriae. results indicated that the fimbriae stimulated F T A F dependent manner.  Their  production in a dose-  Thus fimbriae may have a duel role in the pathogensis of  disease: attachment and the activation of factors which elicit a response from the host defense system.  2.  C a p s u l e of  Bacteroides  The capsule of B. gingivalis  gingivalis  has a number of  functions:  as a physicochemical  barrier between the cell and the external environment, as protection against dessication by binding water molecules, as a defense against the host immune system by avoiding the phagocytic action of polymorphonuclear  leukocytes  (PMNLs), and if entrapment by PMNLs does occur, the capsule aids in preventing hydrolytic degradation of the organism.  The capsule consists of a layer of  electron-dense material, external to the outer membrane, approximately 15 nm thick (Handley & Tipler, 1986; Listgarten & Lai, 1979; Woo et al, 1979) and is composed of a polysaccharide heteropolymer (Mayrand & Holt , 1988).  Studies relating virulence to the presence of a capsule have shown that those strains which are encapsulated have greater resistance to phagocytosis (Okuda &  15  Takazoe, 1973).  Furthermore, when capsular material  species was added to a system utilizing Staphylococcus  extracted from a BPB  aureus, phagocytosis was  inhibited.  Mayrand & Holt  (1988), reviewing the work of van Steenbergen et al (in  publication), noted that virulent strains of B. gingivalis killing by human serum and by PMNLs plus serum. organisms did not autoagglutinate, hydrophilic than less virulent strains. that  were more resistant to Furthermore, the virulent  had a thicker capsule and were  more  The conclusion from their results was  observed differences in virulence can be attributed, at least in part, to  differences in capsular structure.  3. The  Outer outer  membrane membrane  vesicles vesicles  (previously  described)  identical, to the outer membrane of B. gingivalis.  are  similar,  if not  Their virulence is related  to  proteolytic and collagenolytic activities, as well as to an ability to hemagglutinate erythrocytes and to promote adherence between noncoaggregating  species  (Grenier & Mayrand, 1987).  Although their role in the pathogenesis of disease is unclear, it is possible that, because of their small size, the vesicles could easily cross epithelial barriers which would be impermeable to whole cells.  In this way, the lytic capacity of the  cell could be expanded, allowing it to obtain nutrients from a larger area, and creating a pathway for invasion of the whole cell into the tissues.  Furthermore,  the vesicles could compete for antibodies of the host defence system, thereby impeding the specific antibacterial immune defense (Grenier & Mayrand, 1987).  In a study by Smalley & Birss (1987), vesicles from B. were found to have tryspin-like enzyme  gingivalis  activity, supporting the finding of  Grenier & Mayrand (1987) who found not only a trypsin-like, collagenolytic acitvity, associated with the extracellular vesicles.  16  strain W50  but also  a  In a more  recent study by Smalley, Birss & Shuttleworth (1988), a trypsin-like enzyme was purified from a cell- and particle-free culture supernatant of B. gingivalis. The activity was associated with both a 58 kD peptide (by S D S - P A G E ) and a higher molecular-weight complex.  Both prepared fractions had the ability to  degrade human plasma fibronectin in the presence and the absence of a reducing agent  4.  (dithiothreitol).  L i p o p o l y s a c c h a r i d e of Bacteroides  gingivalis  The lipopolysaccharide (LPS) of B. gingivalis is associated with the outer leaflet of the outer membrane and comprises three covalently-linked components:  a  polysaccharide O antigen which extends into the surrounding medium from the surface of the outer membrane, a core polysaccharide found at the surface of the outer membrane, and the lipid A moiety which is embedded in the outer membrane.  The studies of Mansheim & Kasper (1977) and Mansheim et al  (1978) indicated that the L P S of BPB species was biochemically different  from  that of other Gram-negative bacteria in that it lacked heptose and 2-keto-3deoxyoctonate.  The absence of these compounds in lipopolysaccharide from BPB  species has been disputed (B. Johne, I Olsen, & K. Bryn, J . Dent. Res. abstr.  67:368,  2046,1988).  The virulence of L P S from B. gingivalis is manifested in a number of ways: (1) incubation of  gingival fibroblasts in culture with L P S inhibits growth of the  fibroblasts (Larjava et al, 1987; Layman & Diedrich, 1987),  (2) L P S has  been shown to cause bone resorption in vitro ( Millar et al, 1986; Nair et al, 1983)  and is able to inhibit bone collagen formation (Millar et al, 1986), (3)  through induction of interleukin-1, L P S is able to activate the inflammatory response which is postulated to have a role in disease pathogenesis (Hanazawa et al,  1985).  5.  Metabolic  end-products  of Bacteroides  gingivalis  Other substances which may contribute to the virulence of B. gingivalis the characteristic end-products of asaccharolytic metabolism. 17  include  For example,  butyric and proprionic acid have been shown to have a cytotoxic effect upon various human or animal cells in culture (Goldstein et al, 1984; Grenier & Mayrand, 1985; Singer & Buchner, 1981; Touw et al, 1982; van Steenbergen et al,  1982).  Volatile  sulphur  compounds,  methylmercaptan and dimethyl disulphide,  including  hydrogen  sulphide,  produced by B. gingivalis  (Tonzetich  & McBride, 1981) have a potentially damaging effect upon tissues of the host because they can influence the permeability of the oral mucosal tissues (Ng & Tonzetich, 1983) and can reduce collagen synthesis (Tonzetich & McBride, 1981).  Indole and ammonia (MacDonald & Gibbons, 1961; van Steenbergen et  al, 1986b) are also potentially toxic metabolic products of B.  6.  Enzyme  activities  a.  Perturbation  of  o f Bacteroides host  defense  gingivalis.  gingivalis mechanisms  is able to elaborate specific enzymes which may contribute to its  B. gingivalis  virulence by destroying or impeding the defensive response of the host immune system. to  Carlsson et al (1984a) found the presence of enzymes  degrade  the  macroglobulin.  plasma  proteinase  In vivo, these  inhibitors,  proteinase  which were able  3-1-antitrypsin  inhibitors  probably  and  3-2-  function to  regulate the activity of proteases released from PMNLs (Starkey & Barrett, 1977), however, the destruction of such inhibitors by B. gingivalis  could favor  greater tissue degradation and more rapid disease progression.  Enzymes  which  can  specifically  degrade  the  plasma  proteins  albumin,  haemopexin, haptoglobulin and transferrin have been reported (Carlsson et al, 1984).  The degradation of the iron-transporting  requirement  of B. gingivalis  for hemin.  proteins may reflect the  Nilsson et al (1985) also found a  virulent strain of B. gingivalis  (W30) able to recognize and inactivate other  important plasma proteinase  inhibitors including complement  (Cl)  inhibitor  which is a key modulator of both the classical and alternative pathways of complement activation.  18  Furthermore,  antithrombin,  plasminogen,  complex, clotting factor X and most of the eliminated  after 30 minutes  of incubation  prekallikrein,  prothrombinase  2-antiplasmin were functionally with the bacterial  suspension.  Bacteroides gingivalis is able to alter the host defense system by degrading IgG, IgM and complement factors C3 and C5 (Sundqvist et al, 1985) and secretory IgA (Sato  et al, 1987).  immunoglobulins  Kilian  (1981)  demonstrated  the degradation of  A-), A2 and G and in a follow-up investigation (Mortensen 81  Kilian, 1984) purified and characterized an immunoglobulin A1 protease from a B P B species, B. melaninogenicus subsp. melaninogenicus.  It may not be  possible to compare this work directly with that of either Sundqvist et al (1985) or Sato et al (1987) because the bacterial strain used in the study of Mortensen & Kilian (1984) was a sacchrolytic strain whereas the studies of Sundqvist strains  b. The  et al (1985) and Sato et al (1987) utilized oral  asaccharolytic  (B. gingivalis).  Provision  of nutrients  extensive proteolytic enzyme system of B. gingivalis is able to degrade a  wide variety of proteinaceous substances.  The large number of small peptides  produced by protein hydrolysis may then be transported into the cell and used to satisfy its nutritional requirements.  c.  Destruction of host  tissues  A strong fibrinolytic activity is evidenced by B. gingivalis (P.A. Mashimo & J . Slots, J . Dent. Res. 62: 663, abstr. 123, 1983; Lantz et al, 1986; Nitzan et al, 1978; Wikstrdm et al, 1983) which provides it with the potential for tissue invasion.  Other enzymatic activity may be responsible for the destruction of tissue matrix. The early studies by Courant et al (1965) and Socransky (1970), in which whole plaque samples were analyzed, indicated that the plaque contained lytic enzymes capable of destroying the major components of the gingival connective tissue.  More recently, Grenier & Mayrand (1983) investigated the production  19  of enzymes  by B. gingivalis  which  had the potential  components of the gingival ground substance. elaborated  gingivalis  gelatinase,  to degrade  various  Their findings showed that B.  chondroitin  deoxyribonuclease and fibrinolysis all of which  sulfatase,  hyaluronidase,  may have an effect upon tissue  destruction in the pathogenesis of periodontal disease.  Gibbons & MacDonald (1961) and Robertson et al (1982) noted the cellassociated collagenase activity  of B. gingivalis.  This organism has subsequently  been shown to be the only BPB species having specific collagenase activity which will degrade native Type 1 collagen (Mayrand & Grenier, 1985; 1987;  van Steenbergen & de Graaff, 1986).  Sundqvist et al,  This type of collagen is the major  constituent of gingival connective tissue (From & Schultz-Haudt,  1963) and  although it is resistant to many proteolytic enzymes, it can be degraded by both bacterial and mammalian collagenase (Loesche et al, 1982).  B.  gingivalis  Slots, 1981;  also possesses  a trypsin-like  van Winkelhoff et al, 1986c)  activity (Laughon et al, 1982;  which appears to be an important  part of collagen degradation.  The trypsin-like activity of B. gingivalis in cultures  grown  at increasingly  (McDermid et al, 1988).  strain W50 was found to be enhanced higher pH  values,  maximal  at pH  8.0  Furthermore, the finding of McDermid et al (1988)  that collagen degradation required the action of collagenase and a trypsin-like activity emphasizes the importance of these two enzymes in tissue degradation.  The trypsin-like activity of B. gingivalis van Winkelhoff  (Laughon et al, 1982;  Slots, 1981;  et al, 1986c) may also contribute to the virulence of this  organism by the conversion of latent host collagenase into active collagenase (Golub et al, 1985) and by the activation of the complement system, thereby stimulating prostaglandin-mediated osteoclastic bone resorption 1982).  (Schenkein,  Unlike eucaryotic collagenases, which cleave native collagen at a single  site, collagenase  from B. gingivalis  hydrolyzes collagen into small peptides  20  (Robertson et al, 1982; Toda et al, 1984), perhaps making the fragments more susceptible to degradation by other, nonspecific proteases.  The trypsin-like activity may also contribute to the virulence of B.  gingivalis  by the induction and activation of host procollagenase (H. Birkedal-Hansen, J . Dent. Res. 62: 101, abstr. S51, 1987).  T y p e s of  proteases  Proteases may be characterized as one or more of the following: 1. serine protease: the active site.  characterized by the presence of a unique serine residue at  This group includes mammalian trypsin, chymotrypsin and  elastase and several bacterial proteases.  The usual reaction catalyzed by these  enzymes is the hydrolysis of peptide bonds in proteins and peptides.  Trypsin  cleaves bonds only after lysine and arginine whereas chymotrypsin cleaves bonds only after large hydrophobic residues, eg. tyrosine. protease which is able to degrade elastin.  Elastase is a specific  Trypsin and chymotrypsin can be  inhibited by specific inhibitors which bind to the active site.  2. thiol protease: characterized by the presence of a cysteine side  chain at the  active site, comparable to the serine residue of a serine protease.  Papain is an  example of a thiol protease. These proteases can be inhibited by reagents which react with thiol groups or by a variety of metal ions which form complexes with thiol groups.  3.  carboxyl (acidic) protease:  characterized by the presence of two aspartic  acid residues at the active site. These proteases are active at acid pH.  Pepsin is  a representative of this group and will hydrolyze peptides with hydrophobic residues on either side of the scissile bond.  Pepsin can be inhibited by the  specific inhibitor, pepstatin.  4.  metalloprotease:  characterized by the presence of a bound metal (usually  21  zinc) at the active site.  These proteases require divalent metal ions for activity.  This group of enzymes includes carboxy peptidases A and B (exopeptidases) and thermolysin  (endopeptidase).  Inhibitors  of  these  proteases  include  ethylenediaminetetracetic acid and ascorbic acid.  Destruction of both hard and soft tissues can occur as a result of the enzyme activities of Bacteroides  gingivalis.  The major component of the tissues of the  periodontium is Type I collagen which, in disease, can be rapidly destroyed (Page & Schroeder,  1976) possibly by both specific collagenase and nonspecific  protease activity.  The proteases of Bacteroides gingivalis have been the subject  of much research.  Proteases  of  Bacteroides  gingivalis  Uitto & Raeste (1978) determined collagenase activity against Type I collagen using gingival fluid from both healthy and disease sites.  This activity was found  to be 7 times greater in inflamed versus healthy tissue, however in both groups, collagenolysis  was increased by brief exposure to trypsin, suggesting the  presence of latent collagenase which could be activated by neutral protease. Collagenase activity was enhanced in both groups by bacterial plaque and by incubation of leukocytes with bacterial plaque extract.  In a later study, Uitto (1983) examined the effect upon basement membrane collagen (Type bacterial plaque.  IV) using proteinases from human gingiva, leukocytes and As determined by the release of hydroxyproline, the highest  activity was found in leukocyte and plaque extracts. Type IV collagen, because of discontinuities  in the  triple  helical  structure  (Schuppan  et  al,1980)  is  susceptible to degradation by nonspecific proteases, for example, gelatinase and elastase-like enzymes.  Robertson et al (1982) assessed the collagenolytic activity of a number of oral organisms,  including B P B species, Actinobacillus  (A.a.), Fusobacterium,  Capnocytophaga,  22  actinomycetemcomitans  and Selenomonas.  Degradation of  collagen was determined by S D S - P A G E following incubation of collagen in solution and collagen fibrils with cell sonicates and culture supernatants.  These  investigators found that the BPB species had a cell-associated collagenolytic activity, whereas collagenolytic activity was observed in both the cell sonicates and media preparations of A.a.  Enhancement of the proteolytic activity was found  when the cells were grown in peptide-depleted medium.  Toda et al (1984), however, did find some collagenolytic activity supernatant from B. gingivalis.  in culture  The activity was enhanced by the addition of  reducing agents such as dithiothreitol (DTT) and L-cysteine, suggesting the presence of a thiol-dependent collagenolytic activity, the production of which increased with bacterial growth (up to 40 hours).  The origin of this activity  was not precisely defined.  The work of Mayrand & Grenier (1985) further elucidated the ability of oral organisms to degrade collagen. including six BPB  BPB species, were evaluated.  examined, B. gingivalis  previously  In this study, twelve species of oral bacteria, Their results indicated that,  of the  was the only species which could degrade collagen  sterilized with ethylene oxide.  A rapid collagenolysis occurred which  was determined to be the result of the combined activity of a specific collagenase and nonspecific proteases.  Although other strains were capable of degrading  collagen, the hydrolysis was much slower (24 vs 2 hours of incubation) and appeared to be the result of the activity of nonspecific proteases only.  The  findings of Sundqvist et al (1987) and van Steenbergen & de Graaf (1987) support the results of Mayrand & Grenier (1985). gingivalis  These studies also found B.  to be the only BPB species, of those examined, to demonstrate specific  collagenolytic activity.  The results of Sundqvist et al (1987) indicated that the  activity was cell-associated.  As well as having a specific collagenolytic activity, B. gingivalis  has been shown  to possess other proteolytic activities, both cell-free and cell-associated, which  23  may be important in the pathogenesis of disease. These nonspecific proteases may produce  destruction of tissue directly or may induce factors of the host defense  system which can have a deleterious effect upon the periodontal tissues.  Fujimura & Nakamura (1981) characterized two partially purified proteases from cell extracts of a strain of B. melaninogenicus.  Since the supernatant from  an ultracentrifugation following cell sonication was used as the starting material, it is unlikely that the proteases isolated were cell-bound.  These proteases were  inhibited by thiol compounds, but not by diisopropylfluorophosphate (DFP), an inhibitor of serine proteases.  Neither collagenase nor elastase activity was  detected.  A partially purified protease from B. gingivalis strain 381 was characterized by Yoshimura et al (1984) using the chromogenic synthetic substrate, benzoylD-L-arginine-p-nitroanilide preparation  containing  (BAPNA). both  The activity was found in a whole cell  the inner  and outer  membranes  and the  peptidoglycan, therefore the exact localization of the enzyme was not possible, although it was classified as being membrane-bound. The trypsin-like activity was inhibited by thiol protease inhibitors and stimulated by reducing agents such as DTT and L-cysteine.  Ono  et al (1987) also found a trypsin-like  protease  from  culture  supernatant  of  B.  activity gingivalis  in a partially  purified  strain 381.  These  investigators suggested that the activity was due to the same enzyme as previously studied by Laughon et al (1982) and Yoshimura et al (1984).  The  work by Ono et al (1987) extended the characterization of this protease by determination of the apparent molecular weight by S D S - P A G E to be 49,000 D. It should be noted, however, that the protease studied by Yoshimura et al (1984) was partially purified from a whole cell envelope fraction, whereas Ono et al (1987) used culture supernatant as a starting material.  Tsutsui et al (1987) used the supernatant from the ultracentrifugation of  24  sonicated B. gingivalis  cells.  A protease was isolated and purified by ammonium  sulphate precipitaiton and sequential column chromatography. molecular weight by S D S - P A G E was found to be 50,000 D.  The apparent  This membrane-free  protease was inhibited by serine and trypsin inhibitors and was most active at an alkaline pH (8.5).  The activity was enhanced by the reducing agents L-cysteine  and 2-mercaptoethanol.  Although there are similarities to the trypsin-like  activity found by Yoshimura et al (1984), this protease was slightly inhibited by M g Mg  + +  + +  and EDTA, whereas significant inhibition from EDTA and activation by  was found by Yoshimura et al (1984).  In addition, the activity found by  Tsutsui et al (1987) was not significantly affected by p-chloromercuribenzoic acid (PCMB), although it did affect the activity of the protease reported by Yoshimura et al (1984).  In a later study, Fujimura & Nakamura (1987) examined the characteristics of a trypsin-like  protease  isolated from a Triton X-100  soluble extract of sonicated cells of B. gingivalis sequential column chromatography.  (nonionic  detergent)  strain 33277 and purified by  This enzyme was active at neutral pH (7.5),  inhibited by thiol inhibitors and EDTA, with an apparent molecular weight of 65,000 D as determined by S D S - P A G E .  The properties of this enzyme are  similar to those of the enzyme reported by Yoshimura et al (1984), however, different strains of B. gingivalis  were used in each of these studies.  Sorsa et al (1987) found both a trypsin-like protease and collagenase in a supernatant fraction resulting from a low speed centrifugation of sonicated cells of B. gingivalis  strain 33277.  The trypsin-iike protease was able to degrade  native Type IV collagen and denatured Type I collagen whereas the collagenase cleaved native Type I collagen at multiple cleavage sites over time.  The trypsin-  like protease was inhibited by serine protease inhibitors, EDTA, and ascorbic acid;  activity was enhanced by reducing agents. The importance of this is that  basement membrane is composed of Type IV collagen, thus such a nonspecific, trypsin-like activity could facilitate the penetration of other bacterial toxins and  25  enzymes from the periodontal pocket into the underlying connective tissue.  Grenier & McBride (1987) were able to isolate, purify and characterize glycylprolyl protease from the  outer membranes of B. gingivalis  a  strain 33277.  The purification was completed by preparative S D S - P A G E and showed a single band with an apparent molecular weight of 29,000 D for the active form of the protease.  This protease was inhibited by reducing agents, in contrast to other  proteases isolated from B. gingivalis.  Proteolytic activity was exhibited against  both a synthetic peptide containing the glycylprolyl peptide and denatured collagen.  The protease was found in all B. gingivalis  strains tested, but not in  other BPB species examined.  Two groups have investigated the effect of specific peptidases Abiko  et  al  (1985)  characterized  a  partially  of B.  purified  gingivalis.  glycylprolyl  dipeptidylaminopeptidase isolated from spent culture medium, supernatant from washed cells and supernatant from cell extracts of B. gingivalis  strain 381.  The  enzyme was found to enhance the hydrolysis of partially degraded Type I collagen.  Suido et al (1987) isolated two peptidases from the culture supernatant of B. gingivalis  strain 381.  One, a glycylprolyl peptidase thought to be the same as  that found by Abiko et al (1985), was inhibited by serine protease inhibitors. The other enzyme, N-CBz-glycyl-glycyl-arginyl peptidase,  was first associated  with the cell fraction, but after 48 hours in culture, could be found in the supernatant. This enzyme was characterized as a thiol protease, however, it was not determined whether or not it represented any of the previously described thiol proteases.  Finally, Otsuka et al (1987) isolated a protease(s) from culture supernatant from B.  gingivalis  strain  381.  This  protease  was  separated  into  three  isoenzymes, all of which were dependent upon thiol group reagents for activity (DTT).  The  previous studies of Yoshimura et al (1984), Robertson et al  (1982) and Toda et al (1984) suggest the presence of a thiol-dependent  26  trypsin-like protease associated with the proteolytic activity of B.  gingivalis.  On the basis of their results with specific substrates and inhibitors and the requirement for reducing agents for activity,  Otsuka et al (1987) suggest that  the designation of this enzyme as a trypsin-like protease be changed to one of a thiol protease.  The highly varied and extensive proteolytic nature of B. gingivalis shown in the work of Grenier, Chao, McBride (in press).  has  been  Eight proteases were  isolated by these researchers using BSA-polyacrylamide gel electrophoresis. The proteases were isolated, and subsequently characterized, from  culture  supernatants, cell extracts, purified outer membranes, and outer membrance vesicles.  Although only a portion of the proteolytic nature of B.  gingivalis  can be  appreciated from the literature cited, the importance of proteolysis in disease pathogenesis cannot be underestimated,  especially as the organism appears to  have a direct effect upon the periodontal tissues, and is able to not only  induce a  response from the host which can enhance disease progression, but can also inhibit the defense system of the host.  Proteolytic  activity  of  other  pathogenic  organisms  A number of organisms considered to be pathogenic have been shown to possess cell-associated  or cell-free  products  which  are  an  integral  virulence and contribute to their ability to cause disease.  Pseudomonas  aeruginosa,  part of  their  One such organism is  an opportunistic pathogen capable of producing a broad  spectrum of disease under a variety of conditions (reviewed by Pollack, 1984). More recently, Heck et al (1986) found Pseudomonas  elastase was capable of  degrading human collagen, Types III and IV, and that Type I collagen was degraded by both P. aeruginosa  elastase and an alkaline protease.  Degradation products  resulting from the incubation of collagen with the proteases at 25 ° C  were  visualized  Their  by staining with Coomassie  27  blue following  SDS-PAGE.  conclusions were that the tissue destruction observed in P. aeruginosa infections may be the result of collagen degradation by nonspecific extracellular proteases.  Collagenolytic activity was observed by Mufioz et al (1984) to be related to the virulence of Entamoeba  histolytica.  This group had previously shown that E.  histolytica produced collagenase which was specific for Type I collagen.  Their  subsequent study in 1984 examined pathogenic and nonpathogenic strains of E. histolytica.  The results  indicated  that collagenolytic  activity  correlated  positively with the virulence of the organism as determined by the ability of the organisms  to produce  relationship between  liver lesions  in hamsters.  This  evidence  for the  collagenolysis and virulence is similar to that documented  by Grenier & Mayrand (1987) in their study of pathogenic and nonpathogenic strains of Bacteroides  gingivalis.  The pathogenicity of Legionella pneumophila has been associated with a tissuedestructive protease (TDP) by Williams et al (1987) and  Conlan et al (1988).  Conlan et al (1988) was able to demonstrate the in vivo production  of T D P in  the lungs of infected guinea pigs, while the immunocytochemical techniques of Williams et al (1987) demonstrated the presence of T D P in situ. findings reveal an important role for  These  T D P in the pathogenesis of Legionnaires'  disease.  An extracellular protease produced by Aeromonas salmonicida is thought to be a virulence factor in the pathogenesis of fish furunculosis.  A study by Sakai  (1985) demonstrated the importance of the protease in disease by inducing a protease-deficient mutant which, when injected into salmon and trout, produced neither disease nor mortality.  Serratia  marcesens  produces a number of extracellular proteases which are  capable of degrading defense-oriented humoral proteins and tissue components. Molla et al (1986) determined that a virulent strain of Serratia produced an extracellular protease which cleaved IgG and lgA1.  28  The protease was not  inhibited by endogenous human proteinase  inhibitors, 3-1-protease  inhibitor  and 3-2-macroglobulin, and the inhibitors themselves were completely degraded. Extensive pulmonary edema and hemorrhage was noted by Lyerly & Keger (1983)  following  administration  of  a  purified  protease  from  Serratia  marcescens to the lungs of guinea pigs and mice. The tissue destruction observed was similar to that produced during acute Serratia pneumonia.  A i m s of the study All of the organisms described have in common a pathogenic role in specific diseases.  The ability of these organisms to produce disease appears to be the  result of the associated.  combined effects of proteolytic activity, either cell-free or cell-  The organism with which this study was concerned was Bacteroides  gingivalis and the specific aims of the investigation were to isolate, purify and characterize two membrane-associated proteases.  The importance of identifying and characterizing the proteolytic activity of B. gingivalis  relates to its presumed role in the pathogenesis and progression of  various periodontal diseases.  The findings of many investigators  potentially  of  destructive  effects  B. gingivalis  proteases  indicate  in the form of  perturbation of the host defense system and direct tissue destruction.  The  degradation of host proteins is a potential means by which the organism can satisfy  its metabolic requirements.  The clinical aspect of the identification of Bacteroides  gingivalis  proteases is  that it may be possible to use specific protease inhibitors in the prevention or treatment of periodontal diseases.  29  MATERIALS  and  METHODS  Bacterial strain and culture conditions 33277, was grown in Brain  Heart  Bacteroides  gingivalis,  Infusion broth (3.7%; Difco)  hemin (10 ug/ml; Sigma) and vitamin K (1 ug/ml; Sigma). incubated in an anaerobic chamber ( N - H - C 0 2  2  Isolation and purification of proteases  2  strain  containing  The cultures were  [85:10:5]) at 37 ° C .  Protease was prepared from the  two sources of outer membranes outlined below:  Preparation of outer membranes 1.  Outer membranes-1  (OM-1):  Outer  membranes  were  prepared  following  the method of Boyd and McBride (1984) and Grenier and McBride (1987). Bacterial cells from Bacteroides centrifugation at 8,000 x g  strain 33277, were harvested by  gingivalis,  from an early-stationary-phase culture.  The cells  were washed twice in 0.15 M NaCI and suspended in 50 mM phosphate buffer (pH 7.4) containing 0.15 M NaCI.  To remove the outer membranes,  the cells were  sheared through needles of progressively smaller gauge to a final needle size of 26 1/2 gauge. second  This was  periods.  followed by mixing in a Waring blender for seven  30-  The blender jar and contents were placed in an ice bath for 2  minutes following each 30-second mixing.  The mixture was centrifuged for 20  minutes  cells  at 8,000  supernatant  was  x g to remove centrifuged  whole  for 2 hours  and debris.  at 80,000  The  resulting  x g and the gel-like  translucent pellets of outer membranes were suspended in distilled water and lyophilized.  2.  Outer membranes-2 (OM-2): A cell extract was prepared according to the  method of Grenier, Chao, and McBride (in press).  Sixty grams (wet weight) of  cells treated as described in the previous section (OM-1) were (discontinous sonication, five 3-minute periods, with continuous  sonicated  cooling in an  ice bath, and 2 minute resting periods between each sonication; 30% duty cycle, output 5; Sonifier Cell Disrupter 350; Branson Sonic Power Co.).  30  The sonicated  mixture  was centrifuged twice  (20 minutes  at 8,000  x g)  to remove  cell  debris, and the resulting cell extract was concentrated to approximately 1/3 of its original volume by lyophilization.  The findings of Grenier, Chao, and McBride (in press) show that the proteolytic activity of the cell extract is identical to that of purified outer membranes and furthermore,  this  ultracentrifugation.  activity  can  be  removed  from  the  cell  extract  by  These findings are good evidence that the activity in the cell  extract is due to the presence of outer membrane fragments, and not to soluble proteases unique to this cellular fraction.  Preparative 1.  polyacrylamide  outer membranes-1:  of Laemmli proteases.  (1970),  gel  electrophoresis  (PAGE)  Preparative electrophoresis, with the buffer system  was used to accomplish the initial separation of the  One hundred mg of lyophilized outer membranes were suspended in  3.5 ml of 50 mM Tris hydrochloride buffer (pH 7.2) and sonicated ( 1 x 1 0 s). The membranes were then solubilized by adding 2.5 ml of sodium dodecyl sulphate  (SDS) solubilization  buffer (0.125 M Tris hydrochloride, 4% S D S ,  20% glycerol, 1% bromphenol blue) and incubating the mixture for 30 minutes at 37 ° C . onto  This concentration and volume of outer membrane solution was loaded  6 mm thick,  12% (wt/vol) polyacrylamide  resolving  gels  with  4.5%  (wt/vol) polyacrylamide stacking gels.  Electrophoresis was carried out at a constant current of 80 mA for two gels, with cooling, for 16 hours (overnight).  Following electrophoresis, a narrow vertical  strip (approximately 2 cm wide) was cut from one side of the gel and the remainder of the gel stored at 4 ° C . The strip was treated according to the method of Foltmann et al (1985 ) in order to detect proteolytic activity. was washed  The gel strip  with 2.5% Triton-X 100 in 0.3 M sodium acetate buffer (pH 5.3)  for 1 hour to remove S D S .  The strip was then equilibrated in the acetate buffer  without Triton X-100 for 1 hour.  31  Proteolytic activity was detected by placing the gel strip upon a previously prepared 1% agarose gel containing 1% skim milk  and 25 mM dithiothreitol  (DTT).  proteolytic  After incubation for 5-6 hours at 37 ° C ,  detected as white bands in a clear matrix.  activity  was  Two of these bands, representing  protease material, were selected for further purification and characterization (P-1 and P-4).  The designations P-1 and P-4 are taken from the work of  Grenier, Chao, and McBride (in press) which describes the proteolytic profile of Bacteroides  gingivalis.  The profiles of the proteolytic activities found in Bacteroides  culture  gingivalis  supernatants, outer membranes, vesicles and cell extracts were analyzed in SDS-polyacrylamide gels containing covalently bound bovine serum albumin. Proteolytic  activity  of  P-1  (M  r  200,000 D) and  P-4 ( M  r  80,000  D) was  found in culture supernatants, in cell extracts, in purified outer membranes and in the vesicle preparation of Bacteroides  The section of the gel  gingivalis  .  corresponding to proteolytic activity was excised from the  remaining gel slab, cut into small pieces and suspended hydrochloride buffer (pH 7.0). constant agitation.  in 10 mM Tris  The proteins were eluted overnight at 4 ° C with  The eluate from P-1 and from P-4 was dialyzed against  distilled water for 8 hours at 4 ° C and then lyophilized.  The lyophilized eluates  (P-1 and P-4) from the preparative electrophoresis procedure were suspended separately  in  1.0  ml  of  10  mM  Tris  hydrochloride  buffer  (pH  7.2).  Solubilization of the protein contained in the fractions was accomplished by adding 0.5 ml of SDS-solubilization buffer and incubating the solutions for 30 minutes at 37 ° C . (wt/vol)  The solutions were then loaded onto 1.5 mm thick, 10%  polyacrylamide  resolving  gels  with  4.5%  (wt/vol)  polyacrylamide  stacking gels and subjected to a second electrophoresis. The electrophoretic purification procedure was carried out at a constant current of 20 mA for two gels, with cooling, for 16 hours (overnight).  32  The areas of proteolytic activity  were detected and prepared as described above. The electrophoretic purification procedure was repeated once more using the lyophilized eluate from the previous run as the sample. The partially purified proteases were suspended in 50 mM Tris hydrochloride buffer (pH 7.0) and kept at -20 ° C .  2.  outer membranes 2:  Aliquots of 5 ml of the cell extract were incubated  with 2 ml of the SDS-solubilization buffer for 30 minutes at 37 ° C .  The  electrophoretic purification procedure was carried out on 6.0 mm gels and 1.5 mm gels as outlined above for outer membranes-1, and the purified protease material suspended and stored in the same manner as that purified from the outer membrane-1 material.  The proteases isolated and purified from both OM-1 and OM-2 were used in the characterization studies.  All of the results reported, except those for peptidase  activity, were obtained from proteases purified from OM-2.  Protein  determination  The amount of protein in the purified fractions and in the crude fractions of OM1 and OM-2 was determined according to the Bradford (Bio-Rad) assay for protein.  A standard curve was prepared using bovine serum albumin (BSA)  standard stock solution (100 mg/ml; Sigma) diluted with distilled water to 25 pg/ml.  Aliquots of this diluted standard solution, containing 0 to 20 u,g of  protein, were reacted with 0.2 ml of the Bio-Rad colour reagent and the absorbance measured at A g 5  Protein  profiles  of  OM-1  5  and  OM-2  Electrophoresis of OM-1 and OM-2 was carried out on mini-slab gels (0.075 cm thicknesss; Bio-Rad Laboratories, Richmond, Calif.)  containing 12% (wt/vol)  polyacrylamide in the resolving gel and 4.5% (wt/vol) polyacrylamide in the stacking gel. Two sets of samples were prepared: (i)  lyophilized OM-1  and OM-2 were incubated separately with the S D S -  solubilization buffer for 30 minutes at 37 ° C (nonboiled fractions), and  33  (ii)  lyophilized OM-1  and OM-2 were incubated separately with the  SDS-  solubilization buffer for 5 minutes at 100 ° C (boiled fractions).  Aliquots from both preparations, containing 30 u.g of protein, were loaded onto the gels and electrophoresis carried out at room temperature at a constant current of 200 volts (for two gels).  An aliquot containing standard molecular  weight proteins was loaded onto the gels as a reference. included serum  The reference proteins  myosin ([H chain], 200,000 D), phosphorylase b (97,000 D), bovine  albumin  (68,000  D), ovalbumin  (25,700 D) and /Mactoglobulin  (43,000  (18,400 D).  D),  <?-chymotrypsinogen  Following electrophoresis,  the  protein profiles were visualized by staining with silver nitrate following  a  modification of the procedure of Oakley et al (1980).  Protein  profiles  of  P-1  and  P-4  The molecular weight of the proteases was determined by S D S - P A G E using 12% (wt/vol) polyacrylamide mini-slab gels.  The migration of the proteases was  compared to the migration of known protein standards (as described above). Aliquots of each of the proteases were incubated with SDS-solubilization buffer for either 5 minutes at 100 °C, or for 30 minutes at 37 ° C prior to loading onto the gels.  Following electrophoresis, the gels were stained with silver nitrate  (Oakley et al, 1980).  Lipopolysaccharide (LPS) was determined according to the  method of Tsai and Frasch (1980).  Proteolytic  activity  of  OM-1,  OM-2,  P-1  and  P-4  In addition to the silver stained samples, OM-1 and OM-2 were compared for proteolytic activity, along with an outer membrane vesicle preparation (kindly provided  by  Dr. Daniel Grenier,  University  of British  Columbia), and the  partially purified protease fractions, using the method of Kelleher and Juliano (1984) and D. Grenier & B.C. 1988).  Bovine  serum  polyacrylamide gels to  McBride (J. Dent. Res.67:368, abstr. 2045,  albumin  (BSA)  was  incorporated  provide the substrate for proteolysis.  34  into  the  SDS-  The BSA-acrylamide conjugate was prepared according to the method of Kelleher and Juliano (1984).  Four hundred mg of linear polyacrylamide were dissolved  in 20 ml of 0.2 M sodium phosphate buffer (PBS), pH 6.8, and then mixed with 8 ml of 25% gluteraldehyde (in water).  The mixture was incubated for 24  hours at 37 °C. The non-conjugated gluteraldehyde was removed by dialyzing the mixture against 10 L of distilled water at 4 ° C for four days with three changes of water per day. To the dialyzed mixture 2 ml of 10% BSA (0.2 M PBS, pH 7.2) were added and the mixture allowed to react for 24 hours at 25 °C.  Following  incubation, the reaction was halted by adding 250 mg of glycine and 3 mg of sodium azide.  The mixture was then incubated for 24 hours at 4 ° C and again  dialyzed against 10 L of distilled water containing 0.05% sodium azide at 4 ° C for 2 days. The BSA-acrylamide conjugate was stored at 4 °C.  Proteolytic  activity  was  determined  by  analyzing  the samples  in  SDS-  polyacrylamide gels containing covalently bound bovine serum albumin (BSA). Briefly, the electrophoresis was carried out as described above, using 10% (wt/vol)  polyacrylamide  polyacrylamide  was  in  the  mixed  with  resolving  gel.  BSA-acrylamide  Prior  to  casting,  conjugate  to  the  give  a  concentration of 200 ug of protein per ml of gel (5% [vol/vol] BSA-conjugate in the total casting volume).  The crude fractions and partially purified proteases  were prepared by incubation with the SDS-solubilization buffer for 30 minutes at 37 ° C .  Aliquots of the prepared samples were loaded onto the  BSA-  polyacrylamide mini-slab gels and subjected to electrophoresis at a constant current of 200 volts (for two gels).  Following electrophoresis, the gel was shaken gently for 30 minutes in 100 mM Tris hydrochloride buffer (pH 7.0)  containing 2% Triton X-100,  washed twice  in distilled water and then equilibrated in the Tris buffer without Triton X-100 for 30 minutes.  After equilibration, the gel was incubated for 2 hours at 37 ° C  in a development buffer of 100 mM Tris hydrochloride (pH 7.0) mM calcium chloride and 50 mM L-cysteine as reducing agent.  containing 2.5 The gel was  subsequently stained for protein with Coomassie brilliant blue R-250.  35  After  destaining, the proteolytic activity could be visualized as clear bands against a dark blue background.  Determination  of  peptidase  activity  Peptidase activity was determined according to the method of Berdal and Olsvik (1983).  Each of the proteases was incubated with each of the chromogenic  peptides in the same manner as outlined below for the BAPNA (N-<?-Benzoyl-Darginine-p-nitroanilide) assay:  Briefly, 75 pi of each of the proteases was incubated with 75 uJ of a 2 mM solution of each of the chromogenic peptides in a reaction mixture containing 225 u_l of 50 mM Tris hydrochloride buffer (pH7.5 at 37 °C) and 2 mM DTT . Incubation was carried out overnight at 37 ° C , following which, p-nitroaniline was measured after diazotization.  the release of  Trichloroacetic acid (40% in  water, 375 pi) was added to the mixture followed by 112.5 u.l of freshly prepared  sodium  nitrite  (0.1%  in  water).  temperature, 112.5 pi of sodium sulphamate  After  5  minutes  at  room  (0.5% in water) was added and the  reaction mixture incubated for 5 minutes at room temperature.  Lastly, 112.5  uJ of freshly prepared N-1-naphthylethylene diamine dihydrochloride (0.1% in water) was added and following 5 minutes incubation at room temperature, the absorbance at A  5  4  5  was recorded.  The diazotization procedure alters the absorption maxima and increases the sensitivity of the test.  Determination of peptidase activity was semi-quantitave  based upon the following scale:  no colour reaction (0), faint red colour (+/-),  clearly visible red colour (+), strong red colour (++).  Determination  of  optimum  pH  A reaction mixture was prepared containing 75 u.l of the partially  purified  proteases (P-1, 1.7 u,g total protein;  u.l of 2  mM  P-4, 1.1 pg total protein), 75  N-<?-Benzoyl-D-arginine-p-nitroanilide  36  (BAPNA) and 225 u.l of one of  the following buffers:  0.2 M acetate buffer, pH 4.8 and 5.4; 0.2 M phosphate  buffer, pH 5.6 to 8.1; 0.2 M Tris hydrochloride buffer, pH 7.0 to 8.6.  Each of  the buffers contained sufficient DTT such that the reaction mixture was 2 mM in DTT.  The reaction mixture was incubated overnight  at 37 ° C  and the release of p-  nitroaniline was measured after diazotization as outlined above for the BAPNA assay.  Determination of optimum temperature and heat stability Using the BAPNA assay outlined above, the optimum temperature for activity was determined by incubating the substrate overnight with the proteases in a sample mixture containing 50 mM Tris hydrochloride buffer, pH 7.5,  and 2 mM DTT.  The incubation was carried out at 25, 30, 37, 45 ° C .  The heat stability of the proteases was determined as above, except that the protease used (P-4)  was incubated at temperatures of 4, 37, 55, 80, and 100  ° C for 30 minutes, 2 hours or 24 hours prior to determination of enzyme activity using the BAPNA assay.  Inhibition of protease activity Protease activity was determined in the presence of various protease inhibitors and metal ions: EDTA, N-9-p-tosyl-L-lysine  phenylmethylsulfonyl fluoride (PMSF),  chloromethyl  ketone  tosylamine-2-phenyIethyIchloromethylketone p-chloromercuribenzoic  hydrochloride  (TLCK), L-1-  (TPCK), S D S , iodoacetate,  acid (PCMB), aprotinin, C a C I , HgCI , MgCI , Z n C I . 2  2  2  2  Each of the purified proteases was incubated with each of the inhibitors for 30 minutes at 37 ° C prior to determination of protease activity using the BAPNA assay .  A positive control containing protease, substrate and buffer with DTT,  but no inhibitor, was taken to be 100% reactive.  The relative reactivity of the  proteases following incubation with each of the selected inhibitors was calculated on this basis.  37  Hydrolysis  of  protein  substrates  by  P-1  and  P-4  Proteolytic activity against azocoll and azoalbumin was determined according to the method of Mayrand and McBride (1980). azocoll assay contained 15 u.l of purified  The reaction mixture for the  protease, 50 u.l of azocoll (30%),  450  pi of 50 mM Tris hydrochloride buffer (pH 7.5 at 37 °C) and 2 mM DTT.  The  mixture was incubated overnight at 37 °C, subsequently centrifuged at 12,000 x g for 3 minutes and the absorbance of the supernatant read at A  5 8 0  A sample  .  mixture containing azocoll, buffer and DTT served as the control.  The reaction mixture for the azoalbumin assay contained 100 u.l of purified protease, 500 u1 of azoalbumin (3% in 50 mM Tris hydrochloride buffer, 7.5 at 37 °C), and 25 mM DTT.  pH  The mixture was incubated overnight at 37 ° C .  Following the incubation, 500 u.1 of 20% trichloroacetic acid (TCA) was added to stop the reaction and the resulting 12,000 x g material) A  3 7 5  .  for 3  minutes.  To  precipitate was removed by centrifuging the  resulting  supernatant  at  (TCA-soluble  150 pi of 6 N NaOH was added, and the resulting absorbance read at  A sample mixture containing azoalbumin, buffer and DTT served as the  control.  Proteolysis of IgA, IgG, gelatin, and acid-soluble collagen was determined by assaying for lower-molecular-weight degradation products by S D S - P A G E .  The  proteins in question were incubated overnight at 37 ° C in a reaction mixture containing 25 uJ of partially purified protease, 25 u,l of protein substrate (1 mg/ml), 25 u.l of mM DTT.  50 mM Tris hydrochloride buffer (pH 7.5 at 37 °C), and 2  Subsequently , the samples were incubated for 5 minutes at 100 ° C  with 20 u.l of SDS-solubilization buffer. loaded  onto  mini-slab  gels  Aliquots of each of the samples were  containing  an  concentration to assay for degradation products. were stained with Coomassie prepared  appropriate  polyacrylamide  After electrophoresis,  brilliant blue R-250.  the gels  Control samples  were  containing the same reaction mixture but these were not incubated at  37 °C, instead solubilization buffer was immediately added to the samples and the  38  mixtures incubated for 5 minutes at 100 ° C .  Determination  of  immunoreactivity  The antibody used in this determination was prepared Dr. Daniel Grenier, University of British Columbia. membrane-associated  Bacteroides  gingivalis  and  kindly provided by  The antibody was against a  glycylprolyl protease  (Grenier  and McBride, 1987) and did not contain antibody against lipopolysaccharide. Cross-reactivity between the partially purified proteases (P-1 and P-4) and the prepared antibody was determined by Western blotting (Burnette, 1981). Electrophoresis  was carried out as described  above  on a 12% (wt/vol)  polyacrylamide resolving gel onto which had been loaded aliquots of P-1 (0.68 u.g of total protein), P-4 (0.45 u.g of total protein), crude OM-1 (20 u.g of total protein), crude OM-2 (20 u,g of total protein) and outer membrane vesicles (10 u.g of total protein).  After the run, electrophoretic blot transfer of material was  conducted according to the method of Burnette (1981). The gel was placed upon three layers of 3M Whatman filter paper buffer  (glycine  [14.4 g/L], Tris  previously wetted in the blotting  hydrochloride  ml/L], pH 8.3). Nitrocellulose paper, previously  [3.02 g/L], methanol [200 wetted in Tris buffered saline  (TBS; 20 mM Tris hydrochloride, 0.5 M NaCI; pH 7.5) was placed upon the gel and another three layers of wetted filter paper placed over the nitrocellulose paper.  The whole stack was placed into the holding cassette of the Bio-Rad Blot  cell (Bio-Rad Laboratories, Richmond, Calif.) and the cell filled with  blotting  buffer.  Electrophoretic transfer was carried out for 2 hours at room temperature with a constant current of 60 volts.  Following electrophoresis, the nitrocellulose was  first shaken gently with blocking buffer (3% BSA) for 1 hour, and then with the first antibody (anti-glycylproyl protease, 1:400 in 1% BSA) for 1 hour. incubation with the first antibody, the nitrocellulose was water followed by washing for 20 minutes  39  After  washed with distilled  in 2-100 ml changes  of T B S  containing  0.05% Tween-20 (TTBS).  Subsequently, the nitrocellulose was  shaken gently with the second antibody (goat anti-mouse IgG horseradish peroxidase conjugate [Bio-Rad], 1:3000 in 1% BSA)  for 1 hour, followed by  washing in distilled water and T T B S as outlined above.  Staining of the antigen-  antibody complexes with antibody raised in rabbit to the glycylprolyl protease was carried out with the Bio-Rad Immun Blot (GAR-HRP) assay kit.  Chemicals Synthetic peptides, IgG, IgA, azocoll, azoalbumin , acid-soluble collagen and the protease inhibitors P M S F , T P C K , TLCK, P C M B were purchased from Sigma. Electrophoresis  chemicals  (glycine,  SDS,  acrylamide,  bis-acrylamide,  ammonium persulfate and TEMED) were purchased from Bio-Rad.  40  RESULTS  Purification Comparison  of  OM-1  and  The yield of purified  OM-2.  outer membranes was 233 mg, dry weight, (7.8 mg per  liter of culture) in one preparation, from an original wet weight of cells of 104 gm (3.5 gm per liter of culture).  In a second preparation,  560 mg, dry weight,  (14.0 mg per liter of culture), of outer membranes were purified from an original wet weight of cells of 155 gm (3.9 gm per liter of culture).  The protein  content of the outer membrane fractions was determined to be 1.5 mg/ml;  the  cell extract contained 2.0 mg/ml of protein.  The protein profile of the outer membrane preparation (OM-1) obtained by silver staining (Fig. 1) was similar to that previously reported in Boyd & McBride (1984) and Grenier & McBride (1987).  The pattern was similar, but  not identical, to that of a cell extract prepared by sonication, as outlined in Materials and Methods for outer membranes-2 (OM-2, Fig. 2). bands revealed in the profile of OM-2 had the same M  r  The major  as those of OM-1, the  differences being in the number and intensity of staining of the minor bands.  An  apparent difference is the absence of several low molecular weight bands in the protein profile of  OM-2 (Fig. 2, lanes E, F, G, H ) as compared to the protein  profile of OM-1 (Fig. 1, lanes E, F, G, H).  The low molecular weight bands are  not visible in either the boiled or nonboiled samples of OM-2 (Fig. 2, lanes E, F, G, H).  A possible explanation is that the integrity of the proteins in OM-1 are  preserved whereas the proteins in  OM-2 are subjected to proteolytic activity  from proteases released during cell sonication.  Electrophoresis and silver  staining of boiled samples of both OM-1 and OM-2 produced major bands corresponding to M o f 68 kD , 45 kD and 40 kD. r  corresponding  to  M  r  A number of minor bands  both higher and lower than these major bands were  apparent.  41  Electrophoresis followed by silver staining of the nonboiled samples revealed a major band corresponding to an M o f 45 kD in the OM-1 preparation (Fig. 1, r  lanes B and F) and a similar major band corresponding to 40 kD in the OM-2 preparation (Fig. 2, lanes B and F).  These major bands were also found in the  respective protein profiles of the boiled samples (Figs. 1 and 2, lanes A and E).  In Figures 1 and 2, lanes C, D, G, and H show the protein profiles of the boiled and nonboiled samples following solubilization in buffer containing reducing agent (5-mercaptoethanol).  When compared with the protein profiles of the  boiled and nonboiled samples (Figs.1 and 2, lanes A, B, E, F) solubilized in buffer without reducing agent, it apparent that there are fewer bands in those lanes which contain samples solubilized in buffer containing reducing agent (Figs. 1 and 2, lanes C, D, G, H), most noticeable when comparing lanes F and H. It is likely that the reducing agent is able to protect the sulfhydryl bonds in the proteases,  thus resulting in fewer visible bands.  The skim milk zymogram obtained following preparative gel electrophoresis of OM-1  and OM-2 revealed three proteolytic bands with apparent  weights above 68,000 D (Fig. 3).  molecular  The bands were not similar in their intensity  of activity, the lower molecular weight band (P-4) and the intermediate band having more activity against the casein substrate than the higher molecular weight band (P-1).  The relative intensity of the bands was consistent from one  preparation to another.  P-1 and P-4 were selected for further purification and  characterization.  42  Figure  1.  SDS-PAGE  of outer membrane-1  preparation.  Lane A: boiled OM-1, 0.2 ug dry weight, SDS-sol buffer* Lane B: nonboiled OM-1, 0.2 ug dry weight, SDS-sol buffer Lane C:  boiled OM-1, 0.2 u.g dry weight, /J-ME-sol buffer**  Lane D: nonboiled OM-1, 0.2 ug, dry weight, /3-ME-sol buffer Lane E: boiled OM-1, 2.0 ug dry weight, SDS-sol buffer Lane F: nonboiled OM-1, 2.0 ug dry weight, SDS-sol buffer Lane G : boiled OM-1, 2.0 ug dry weight, tf-ME-sol buffer Lane H: nonboiled OM-1, 2.0 ug dry weight, /3-ME-sol buffer  * sodium dodecyl sulfate solubilization buffer **  fl-mercaptoethanol  solubilization  buffer  Arrows indicate molecular weight markers, from top to bottom, respectively: 97,000 D (phosphorylase b) 68,000 D (bovine serum albumin) 43,000  D (ovalbumin)  25,700 D (<9-chymotrypsin)  43  43a  Figure 2.  SDS-PAGE  of outer membrane-2 preparation.  Lane A: boiled OM-2, 0.1 x 10" ug total protein, SDS-sol buffer* 3  Lane B: nonboiled OM-2, 0.1 x 10' ug total protein, SDS-sol buffer** 3  Lane C: boiled OM-2, 0.1 x 10" ug total protein, 3  Lane D: nonboiled OM-2, 01. x 1 0  -3  fl-ME-sol  buffer  ug total protein, /?-ME-sol buffer  Lane E: boiled OM-2, 1.0 x 10~ ug totall protein, SDS-sol buffer 2  Lane F:  nonboiled OM-2, 1.0 x 1 0  -2  ug total protein, SDS-sol buffer  Lane G : boiled OM-2, 1.0 x 10" ug total protein, 2  Lane H: nonboiled OM-2, 1.0 x 1 0  -2  fl-ME-sol  u.g total protein,  buffer  tf-ME-sol  buffer  * sodium dodecyl sulfate solubilization buffer tf-mercaptoethanol  solubilization  buffer  Arrows indicate molecular weight markers, from top to bottom, respectively: 97,400 D (phosphorylase b) 68,000 D (bovine serum albumin) 43,000  D (ovalbumin)  25,700 D (<9-chymotrypsin)  44  44a  Figure 3. Zymogram of proteases after preparative SDS-PAGE.  45  45a  Proteolytic  activity  of  samples  against  bovine serum  albumin  (BSA)  The protocol outlined in Materials and Methods for the detection of proteolytic activity  on  BSA-polyacrylamide  gels was  selected following  a number of  experiments to determine the optimum length of time for treatment of the gels in Triton-X-100  and for the  incubation of the gels in the development buffer.  The  test gels were subjected to treatment times in Triton X-100 of 15, 30, 60 or 120 minutes whereas the incubation times in development buffer were from 0.5,1.5, 2.0 to 5.0 hours. volume  varied  The optimal concentration of BSA in the casting  was determined to be 5% after testing the protocol using gels containing  4, 5 or 15% BSA-conjugate (vol/vol) in the total casting volume.  The optimal  treatment time in Triton-X-100 was 30 minutes whereas the optimal incubation time in development buffer was 2 hours.  Determination  of  total  protein  After the third preparative  P A G E , the amount of protein contained in the  proteases purified from the OM-1 preparation was determined according to the Bradford (Bio-Rad) protein assay, and found to be 1.2 mg/ml (P-1) and 1.6 mg/ml (P-4), whereas the same proteases purified from the OM-2 preparation contained 67.5 u,g/ml (P-1) and 45.0 pg/ml (P-4). found in P-1 and P-4  The lower protein content  purified from the OM-2 preparation is not unexpected in  that the cells used to obtain the extract had been previously stripped, to a large degree, of the outer membranes with which the proteases are associated.  Specific  activity  of  P-1  and  P-4  The purification procedures resulted in an increase of specific activity and  40-fold  respectively  greater  than  that of the  (Table 1).  46  OM-2  preparation,  P-1  45-fold  and  P-4,  T A B L E 1.  Purification of P-1 and P-4 from the OM-2 preparation of Bacteroides gingivalis 33277  Fraction  Protein  Total  Sp act  Purifi-  Yield  (mg)  units*  U/mg of  cation  (%)  protein  OM-2  20  6.0  0.3  1  100  P-1  0.067  0.9  13.4  45  15 **  P-4  0.067  0.8  1 1.9  40  13 **  One unit of enzyme activity is defined as the amount of enzyme which released I (xmol of p-nitroaniline from 2 mM B A P N A in 120 minutes at 37 ° C . This is not necessarily the true yield since the original preparation contained a number of BAPNA-degrading proteases.  Following the second S D S - P A G E purification, the protein profile of boiled P-1 revealed one major band corresponding to an M  r  68 kD and six minor bands  corresponding to M f r o m approximately 50 to 18 kD (Fig. 4, lane E) whereas r  nonboiled P-1 revealed three major bands corresponding to M in the range of r  200 kD and one minor band with an M o f approximately 50 kD (Fig. 4, lane F). r  The 50 kD samples.  band was present in the profiles of both the nonboiled and boiled The  bands  were visualized  by silver staining  the gels  after  electrophoresis.  Following the third and final S D S - P A G E purification, electrophoresis of boiled P-1 produced five bands which corresponded to molecular weights ranging from 68 kD  (major band) to 18 kD  (Fig.4, lane G).  major bands following electrophoresis.  Nonboiled P-1 produced three  The bands obtained after silver staining  corresponded to molecular weights of 235 kD, 220 kD, and 200 kD (Fig. 4, lane H).  47  After the second S D S - P A G E purification, electrophoresis of boiled P-4 produced eight  bands  corresponding  to M  r  of 68 to 18 kD (Fig. 5, lane E) whereas  nonboiled P-4 revealed one major band corresponding to an M of 200 kD (Fig. r  5, lane F).  After the third and final S D S - P A G E purification, electrophoresis of boiled P-4 produced two major bands which corresponded to molecular weights of 68 and 50 kD, and a minor band corresponding to 40 kD  (Fig. 5, lane G).  Nonboiled P-4  produced one major band following electrophoresis which corresponded to a molecular weight of 74 kD (Fig. 5, lane H).  The three bands produced following  electrophoresis of the boiled P-4 (68, 50, 40 kD)  were also present in the  protein profile of boiled P-1 (Fig. 4, lane G).  The  M  r  of the  partially  purified proteases  determined  by  silver  staining  correlated well with the bands of proteolytic activity found in gels in which BSA had been incorporated as a substrate for the proteases (Fig. 6).  Proteolytic  activity can be visualized (Fig. 6, lanes C and D) in the area of 200 kD (P-1) and at 74 kD (P-4; Fig. 6, lane B).  Lane D contains protease  (P-1) eluted from  the upper one-half of a gel section excised from a preparative gel whereas Lane C contains protease (P-1) eluted from the lower one-half of the same preparative gel section.  If the two were combined, it would be equal to the whole P-1  protease as visualized following silver staining in Figure 4.  No bands were detected following electrophoresis of the purified proteases and subsequent staining of the gels for lipopolysaccharide.  48  Figure  4.  SDS-PAGE  of  P-1.  Lane A:  boiled OM-1, 2.0 ug dry weight, SDS-sol buffer*  Lane B:  nonboiled OM-1, 2.0 u.g dry weight, SDS-sol buffer  Lane C:  boiled P-1, after preparative S D S - P A G E , SDS-sol buffer  Lane D:  nonboiled P-1, after preparative S D S - P A G E , SDS-sol buffer  Lane E:  boiled P-1, after 2nd purification by S D S - P A G E , SDS-sol buffer  Lane F:  nonboiled P-1, after 2nd purification by S D - P A G E , SDS-sol buffer  Lane G :  boiled P-1, 0.2 u.g total protein, after 3rd purification by S D S - P A G E , SDS-sol buffer  Lane H:  nonboiled P-1, 0.2 u.g total protein, after 3rd purification by SDS-PAGE, SDS-sol buffer  sodium dodecyl sulfate solubilization buffer  Arrows indicate molecular weight markers, from top to bottom, respectively: 200,000 D (myosin, H chain) 97,000 D (phosphorylase b) 68,000 D (bovine serum albumin) 43,000 D (ovalbumin) 25,700 D (^-chymotrypsin) 18,400 D (/3-lactoglobulin)  49  A  B  C  D  E  F  HI  49a  G  H  jm  Figure 5.  SDS-PAGE of P-4.  Lane A: boiled OM-1, 2.0 u.g dry weight, SDS-sol buffer* Lane B: nonboiled OM-1, 2.0 u.g dry weight, SDS-sol buffer Lane C: boiled P-4, after preparative S D S - P A G E , SDS-sol buffer Lane D: nonboiled P-4, after preparative S D S - P A G E , SDS-sol buffer Lane E:  boiled P-4, after 2nd purification by S D S - P A G E , SDS-sol buffer  Lane F:  nonboiled P-4, after 2nd purification by S D S - P A G E , SDS-sol buffer  Lane G : boiled P-4, 0.2 ug total protein, after 3rd purification by S D S - P A G E , SDS-sol buffer Lane H:  nonboiled P-4, 0.2 u.g total protein, after 3rd purification by SDS-PAGE, SDS-sol buffer  * sodium dodecyl sulfate solubilization buffer  Arrows indicate molecular weight markers, from top to bottom, respectively: 200,000 D (myosin, H chain) 97,000 D (phosphorylase b) 68,000 D (bovine serum albumin) 43,000 D (ovalbumin) 25,700 D (<9-chymotrypsinogen) 18,400 D (/Mactoglobulin)  5 0  A  B  C  D  E  50a  F  G  H  Figure 6. Proteolytic purity of OM-2, P-1 and P-4.  Lane A:  nonboiled OM-2, 30 u.g total protein, SDS-sol buffer *  Lane B:  nonboiled P-4, 0.22 u.g total protein, SDS-sol buffer  Lane C:  nonboiled P-1 (lower 1/2), 0.45 ug  Lane D:  nonboiled P-1 (upper 1/2), 0.45 ug total protein, SDS-sol buffer  Lane E:  total protein, SDS-sol buffer  molecular weight markers, from top to bottom, respectively: 200,000 D (myosin H chain) 97,400 D (phosphorylase b) 68,000 D (bovine serum albumin) 43,000  D  25,700 D  (ovalbumin) (<9-chymotrypsinogen)  * sodium dodecyl solubilization buffer  51  A  B  C  51a  D  E  Peptidase  activity  Peptidase activity is shown in Table 2.  There was no  proteases regarding peptidase activity.  Both were able to readily hydrolyze  substrates containing benzoyl-arginine groups: arginine-p-NA  and  the  glutamylglycyl-L-arginine-p-NA. L-Pro-p-NA  the dipeptide, N-3-Benzoyl-D-  tetrapeptide,  N-Benzoyl-L-isoleucyl-L-  The two dipeptides containing glycine,  and Gly-L-Arg-p-NA  aminopeptide L-  difference between the  Gly-  , were less actively hydrolyzed, and the  -glutamyl-p-NA was only weakly degraded.  T A B L E 2.  P e p t i d a s e activity of P-1 a n d P-4  Substrate  P-1 Activity*  P-4  Activity*  L-Alanine-p-N A  0  0  L-Arginine-p-N A  0  0  L- o'-Glutamyl-p-N A  + / -  +/-  L-Leucine-p-N A  0  0  L-Lysine-p-N A  0  0  L-Methionine-p-N A  0  0  L-Proline-p-N A  0  0  L-Valine-p-N A  0  0  + +  + +  + / -  + / -  +  +  0  0  + +  + +  0  0  N-3-Benzoyl-D-arginine-p-N A Glycyl-L-proline-p- N A Glycyl-L-arginine-p-N A L-Alanyl-L-alanyl-Lphenylalanine-p-NA N - B e n z o y l - L - i s o l e u c y I-Lglutamylglycyl-L-arginine-p-N A Methoxysuccinyl-L-alanyl-L-alanylL- proline-L-valine-p-N A  * T h e d e t e r m i n a t i o n of p e p t i d a s e activity on s y n t h e t i c s u b s t r a t e s w a s s e m i - q u a n t i t a t i v e b a s e d u p o n t h e f o l l o w i n g s c a l e : no c o l o u r r e a c t i o n (0), faint r e d c o l o u r (+/-), c l e a r l y v i s i b l e red c o l o u r (+), strong red c o l o u r (++).  52  Optimum  pH  The optimum pH for activity of both proteases was found to be between pH 6.0 and 6.5, although there was significant activity over the pH range of 5.5 to 7.0 (Figs. 7 and 8).  Both proteases appeared to have significant activity in the acetate  buffer whereas  Optimum  activity appeared to be inhibited in Tris hydrochloride buffer.  temperature  As determined by the BAPNA assay, the optimum temperature for activity for both proteases was found to be 37 ° C (Fig. 9). Activity declined at temperatures either higher or lower than this value.  P-4 appeared to be much more sensitive  to changes in temperature than did P-1. 90%  At 45 °C, P-1 retained approximately  of the relative reactivity whereas P-4 had only 45% of the relative  reactivity at the same temperature. reactivity whereas  P-1  At 25 °C, P-4 exhibited 42% of the relative  retained 47% of the relative activity  at the  same  temperature.  Heat  stability  P-4 was used as the representative sample to determine the heat stability of the partially purified proteases, based upon its apparent sensitivity to changes in temperature.  The  proteolytic activity of P-4 was completely  incubation  at 80 ° C or after boiling for 5 minutes.  abolished after 2 hours of Incubation at temperatures  below 55 ° C for 30 minutes or for 2 hours had relatively little effect upon proteolytic activity and 41% of the relative activity was retained after 16 hours of incubation at 55 °C.  Electrophoresis of P-4 which had been incubated at 37 ° C for 48 hours revealed autodegradation.  This could be visualized, following silver staining, as a loss of  53  resolution of the protein bands when compared to those of the non-incubated control (Fig. 10).  The major band  (Fig. 10, lanes A, C, E) corresponds to the  74 kD band seen in Figure 5 (lane H) following electrophoresis of nonboiled P4.  The major bands (Fig. 10, lanes B, D, F) correspond to the 68, 50, and 40  kD bands seen in Figure 5 (lane G) following electrophoresis of boiled P-4. Comparison of the bands in Lanes B, D, and F (Fig. 10) reveals the loss of the 50 kD band in Lane D (boiled P-4, incubated 48 hours at 37 °C).  The loss of this  band indicates autodegradation of the P-4 protease after this incubation period.  The protease remained stable for at least 3 months at -20 ° C ; repeated freezing and thawing had no apparent effect upon proteolytic activity.  54  Figure 7.  Optimum pH for the hydrolysis  of B A P N A  by  P-1.  The reaction mixture contained 75 u,l of purified P-1, 75 u.l of 2 mM BAPNA, 225 U.I of buffer, and 2 mM DTT. buffer, pH 4.8 and 5.4;  The following buffers were used: 0.2 M acetate  0.2 M phosphate buffer, pH 5.6 to 8.1;  0.2 M Tris  hydrochloride buffer, pH 7.0 to 8.6.  The reaction mixture was incubated overnight at 37 ° C and the release of pnitroaniline was measured after diazotization as outlined for the BAPNA assay in Materials and Methods.  55  55a  Figure 8.  Optimum pH for the hydrolysis  of B A P N A  by  P-4.  The reaction mixture contained 75 u.1 of purified P-4, 75 u.1 of 2 mM BAPNA, 225 u.1 of buffer and 2 mM DTT. buffer,  pH 4.8 and 5.4;  The following buffers were used: 0.2 M acetate  0.2 phosphate buffer, pH 5.6 to 8.1;  0.2 Tris  hydrochloride buffer, pH 7.0 to 8.6.  The reaction mixture was incubated overnight at 37 ° C and the release of pnitroaniline was measured after diazotization, as outlined for the BAPNA assay in Materials and Methods.  56  56a  F i g u r e 9. O p t i m u m t e m p e r a t u r e for the h y d r o l y s i s of B A P N A b y P-1 a n d P-4.  The reaction mixtures contained 75 u.l of either P-1 or P-4, 75 u.l of 2 mM BAPNA, 225 u.1 of Tris hydrochloride buffer, pH 7.5 and 2 mM DTT. reaction mixtures were incubated overnight at the following temperatures:  The 25,  30, 37, and 45 ° C .  The release of p-nitroaniline was measured after diazotization as outlined for the BAPNA assay in Materials and Methods.  57  110 -i  Temp.  57a  Figure  10.  Heat  stability  of  P-4.  Lane A: nonboiled P-4, incubated 48 hours at 4 ° C Lane B: boiled P-4, incubated 48 hours at 4 ° C Lane C: nonboiled P-4, incubated 48 hours at 37 ° C Lane D: boiled P-4, incubated 48 hours at 37 °C Lane E:  nonboiled P-4, control maintained at -20 ° C  Lane F:  boiled P-4, control maintained at -20 ° C  Lane G: molecular weight markers: 97,400 D (phosphorylase b) 43,000  D  (ovalbumin)  25,700  D  (3-chymotrypsinogen)  58  58a  Inhibition  of  enzyme  activity  The effect of various inhibitors and metal ions on the hydrolysis of BAPNA is shown in Table 3. The proteases were strongly inhibited by the chymotrypsin inhibitor T P C K , and by the thiol inhibitors P C M B , iodoacetate and Hg ions. Inhibition occurred to a lesser extent from the serine protease inhibitor PMSF, from the trypsin inhibitor TLCK, from the anionic detergent S D S , and from Zn ions, which are inhibitory to thiol proteases.  The activity of the proteases  against BAPNA was enhanced by EDTA, aprotinin, and C a and Mg ions.  T A B L E 3. Compound  Effect of protease inhibitors a n d metal ions on e n z y m e activity Concn  P-1 % R e s i d u a l  (mM)  activity  None  P-4 % R e s i d u a l activity  100  100  EDTA  20  304  241  PMSF  4  44  85  TLCK  2  44  41  TPCK  2  8  7  20  50  29  PCMB  4  2  0  Iodoacetate  2  0  9  8 ]ig/ml  318  234  2  217  203  2  2  1  2  323  347  2  74  60  SDS  Aprotinin CaCI  2  HgCI  2  MgCI ZnCI  2  2  * Activity w a s m e a s u r e d by the B A P N A a s s a y a s outlined in Material and M e t h o d s .  59  Activity  against  protein  substrates  The activity of the proteases against protein substrates is shown in Table 4.  Both  of the proteases were able to degrade gelatin, IgA, IgG and the two azo substrates, azocoll and azoalbumin, under the conditions of the experiments.  Acid-soluble  collagen was not hydrolyzed by either protease during overnight incubation at 25 °C.  T A B L E 4. Substrate  Proteolytic activity of P-1 and P-4 P-1  Activity*  P-4  Activity*  Azocoll  +  +  Azoalbumin  +  +  Gelatin*  +  +  IgG*  +  +  IgA*  +  +  Acid-soluble collagen*  * T h e d e t e r m i n a t i o n of activity a g a i n s t p r o t e i n s w a s b a s e d u p o n the ability of the p r o t e a s e s to p r o d u c e fragments of lower molecular weight a s c o m p a r e d to the original protein profile s e e n in p o l y a c r y l a m i d e gels stained with C o o m a s s i e blue.  The degradation of IgA and IgG is apparent (Fig. 11) as a loss of bands following overnight incubation (37 ° C ) of the substrates with the proteases (Fig. 11, lanes B, D, F, H) as compared with the IgA and IgG samples which were not incubated with the proteases (Fig. 11, lanes A, C , E, G).  Gelatin was completely degraded by both P-1 and P-4 (Fig. 12). The degradation is indicated by the loss of bands (Fig. 12, lane B, gelatin with P-1 and lane F, gelatin with P-4) following overnight incubation (37 ° C ) of the substrate with the proteases as compared to the profiles seen in lanes A and E (Fig. 12) which contain samples of non-incubated substrate and protease. The 68 kD band seen in  60  lanes A, B, C, and D (Fig. 12) may be a contaminant related to the P-1 protease rather than to the substrate, since it appears to remain unaltered following the overnight incubation.  It was initially thought that P-1 had collagenolytic activity since acid-soluble collagen was completely  degraded following overnight incubation with the  protease at 37 ° C (Fig. 12, lane D). Therefore, a subsequent experiment, having a reaction mixture which comprised acid-soluble collagen, boiled P-1, a nonspecific protease (trypsin), Tris hydrochloride buffer and DTT, was completed following  identical  experimental  conditions.  After  electrophoresis  of the  samples,  it was evident that collagenolysis had occurred in the presence of  trypsin and boiled Fraction 1 (Fig. 13, lane B).  Since boiling was previously shown to completely abolish the proteolytic activity of the purified protease, the degradation which occurred could not be the result of proteolytic activity due to P-1.  The results suggest that the  non-specific  protease (trypsin) was responsible for the collagenolytic activity in concert with the disruption of the helical structure of collagen which occurs at 37 ° C (Fig. 13, lane B).  The collagenolytic activity of the nonboiled P-1 could be the  result of a similar effect upon acid-soluble collagen during incubation  at 37 ° C .  Alternatively, this could result from a small amount of residual S D S in the P-1 protease which may disrupt the tertiary structure of the collagen making it accessible to a non-specific protease.  61  molecule,  Figure  11.  Lane A:  Hydrolysis  of immunoglobulins  P-1 with IgA, T  A and G  by  P-1 and P - 4 .  0  Lane B: P-1 with IgA, T * 1 8  Lane C:  P-4 with IgA, T  0  Lane D: P-4 with IgA, T Lane E: P-1 with IgG, T  1 8  0  Lane F: P-1 with IgG, T Lane G: P-4 with IgG, T  1 8  0  Lane H: P-4 with IgG, T  1 8  * no incubation ** overnight incubation of protease fraction with substrate at 37 ° C  Arrows indicate molecular weight markers, from top to bottom, respectively: 97,400 D (phosphorylase b) 43,000  D  25,700 D  (ovalbumin) (^-chymotrypsin)  The reaction mixtures contained 25 pi protease, 25 pi IgA or IgG (1 mg/ml water), 25 u.1 Tris hydrochloride buffer, pH 7.5 at 37 ° C , 2mM DTT.  The  mixtures were either immediately boiled for 5 minutes with 20 u.1 of SDSsolubilization buffer, or incubated overnight at 37 ° C followed by boiling for 5 minutes with 20 u.1 of SDS-solubilization buffer.  Aliquots of 20 pi were loaded  onto a 12% (wt/vol) mini-slab gel. The gel was stained with Coomassie blue dye after electrophoresis.  62  A  B  C  E  D  62a  F  G  H  Figure  12.  Hydrolysis  of gelatin and collagen  Lane A:  P-1 with gelatin, T *  Lane B:  P-1 with gelatin, T *  by  P-1  and  P-4.  0  1 8  Lane C: P-1 with collagen, T  0  Lane D:  P-1 with collagen, T-|  Lane E:  P-4 with gelatin, T  Lane F:  P-4 with gelatin, T  8  0  1 8  Lane G: P-4 with collagen, T  0  Lane H: P-4 with collagen, T  1 8  no incubation overnight incubation of protease fraction and substrate at 37 ° C  Arrows indicate molecular weight markers, from top to bottom, respectively: 200,000 D (myosin H chain) 97,400 D (phosphorylase b) 68,000 D (bovine serum albumin) 43,000 D (ovalbumin)  The  reaction  (1 mg/ml)  mixtures  contained  25  u.l protease  fraction,  25  uJ gelatin  or acid-soluble collagen (1 mg/ml 0.1% acetic acid), 25 u.l Tris  hydrochloride buffer, pH 7.5 at 37 °C, 2 mM DTT. immediately boiled for 5 minutes  The mixtures were either  with 20 u,l of SDS-solubilization buffer, or  incubated overnight at 37 °C followed by boiling for 5 minutes with 20 ju.l of SDS-solubilization buffer.  Aliquots of 20 uJ were loaded onto a 7.5% (wt/vol)  polyacrylamide resolving mini-slab gel. The gel was stained with Coomassie blue dye after electrophoresis.  63  A  B  C  D  E  63a  F  G  H  Figure  Lane A:  13. H y d r o l y s i s  of  collagen  by  trypsin  and  P-1.  boiled P-1, trypsin and collagen, To*  Lane B: boiled P-1, trypsin and collagen, T18* Lane C: nonboiled P-1, trypsin and collagen, To Lane D: nonboiled P-1, trypsin and collagen, T18 Lane E: trypsin and collagen, To Lane F: trypsin and collagen, T18  * no incubation overnight incubation of P-1 and /or tryspin with  substrate at 37 ° C  Arrows indicate molecular weight markers, from top to bottom, respectively: 200,000 D (myosin H chain) 97,400 D (phosphorylase b) 68,000 D (bovine serum albumin) 43,000 D (ovalbumin)  Reaction mixture for Lanes A-D:  25 u.l boiled or nonboiled P-1, 25 u.l acid-  soluble collagen (1 mg/ml 0.1% acetic acid), 25 u.l trypsin (0.4 mg/ml water), 25 u.l Tris hydrochloride buffer (pH 7.5 at 37 °C), 2mM DTT. for Lanes  E and F:  Reaction mixture  25 uJ trypsin (0.4 mg/ml water), 25 u.1 acid-soluble  collagen (1 mg/ml 0.1% acetic acid), 25 u.l Tris hydrochloride buffer (pH 7.5 at 37 °C), 2 mM DTT.  The reactions mixtures were either boiled immediately  ( T ) with 20 u.l of the SDS-solubilization buffer for 5 minutes or incubated 0  overnight ( T ) at 37 ° C followed by boiling for 5 minutes with 20 u.l of the 1 8  SDS-solubilization buffer.  Aliquots of 20 u,l were loaded onto a 7.5% (wt/vol)  polyacrylamide resolving mini-slab gel. The gel was stained with Coomassie blue dye after electrophoresis.  64  A  B  C  D  64a  E  F  Reaction  with  antibody  No cross-reactivity  to g l y c y l p r o l y l  protease  was found when the partially purified proteases  reacted with antibody to a glycylprolyl protease.  were  This was determined by  Western blotting as outlined in Material and Methods. It is not surprising to find a lack of cross-reactivity since the glycylprolyl protease was deemed to be pure, based upon the electrophoretic pattern produced and subsequent staining with silver which is a very sensitive method for detection of protein (Oakley et al, 1 980)  65  DISCUSSION  The proteolytic activity of Bacteroides  gingivalis  gives this organism  many  potential advantages in terms of pathogenicity. A number of proteases have been characterized with regard to molecular weight, temperature and pH optima, specific inhibitors and ability to hydrolyze specific substrates.  The  proteases,  preparation membrane  P-1  and  P-4,  were  isolated  from the outer  membrane  (OM-1) obtained by shearing whole cells and from the outer preparation  (OM-2) derived from the sonication of whole cells  (Grenier & McBride, in press).  Since the shearing method for removal of the  outer membranes is very gentle, it is likely that little cell breakage occurred during this procedure.  However, sonication is a destructive process,  probably resulted in cell damage,  and  releasing intracellular proteins as well as the  outer membranes.  Grenier & McBride (in press) have described the proteolytic activity of culture supernatants, purified outer membranes, outer membrane vesicles and cell extracts  of  Bacteroides  electrophoresis.  using  gingivalis  BSA-polyacrylamide  gel  Their findings indicate that the proteolytic activity of the  purified outer membranes is identical to that of the cell extracts.  This activity  was found to be absent from the cell extracts following ultracentrifugation which would result in the removal of the outer membrane fragments from the cell extract.  The  loss  of  proteolytic  activity  from  the  cell  extract  after  ultracentifugation is confirmation that the proteolytic activity is associated with the outer membranes rather than with soluble proteases unique to the cell extract.  The BSA-polyacrylamide gel electrophoretic procedure proved to be a  rapid and very sensitive method for monitoring proteolytic activity during the purification of P-1 and P-4.  Gel electrophoresis was chosen as the method of purification because it is rapid, sensitive,  and  reproducible.  Furthermore,  66  SDS-polyacrylamide  gel  electrophoresis offers the ability to easily separate proteins according to their molecular weight.  Separation of high molecular weight proteins is often not  possible by more traditional methods because of the formation of protein aggregates.  The higher molecular weight protease (P-1) was fractionable to proteolytic purity,  as  determined  by  electrophoresis  conjugated to bovine serum albumin.  using  SDS-polyacrylamide  gels  The area of proteolysis was visible as a  single band on the stained gel which suggests that P-1 was pure in terms of its proteolytic activity.  Three narrow bands, corresponding to molecular weights of 235 kD, 220 kD and 200 kD, were visible following S D S - P A G E of nonboiled P-1 and subsequent silver staining for protein.  Several bands were visible following electrophoresis  and silver staining of boiled P-1 (68-18 kD).  These bands may be due to  denaturation of the protease after boiling with SDS-solubilization buffer. possible that at 37 ° C , in the presence of SDS, molecules migrate as one band.  It is  the protease and associated  However, during incubation at 100 ° C , in the  presence of SDS, the associated molecules may separate from the protease and each migrate as a separate band.  The outer membrane is associated with  insoluble hydrophobic molecules, protein and lipopolysaccharide.  It is very  likely that some of these molecules remain associated with the protease following purification procedures.  The position of P-1  protelytic activity visible on a BSA-polyacrylamide  gel  (corresponding to 200 kD) correlated well with the position of the three bands seen on a silver stained gel after electrophoresis of nonboiled P-1 kD).  (235-200  The presence of three bands visible after silver staining may simply  reflect the conformation of the protease, as suggested by Heukeshoven & Dernick (1985) and is not an indication that each band has individual activity.  proteolytic  The results of the study by Heukeshoven & Dernick (1985) indicated  67  that differences  in staining occurred  when  the protein  three-dimensional  structure was changed by treatment with detergents, e.g. SDS. three-dimensional  Furthermore, the  protein structure, and therefore the presentation of the  reactive moiety in space, appeared to be the single most important factor in stainability.  The appearance of a single band of proteolytic activity on the BSA-  polyacrylamide gel would tend to support this, and is additional proteolytic and protein purity of P-1.  evidence of the  Furthermore, the appearance of a single  major band, corresponding to 68 kD, after electrophoresis of boiled P-1  is  another indication that the three bands represent a single protease.  P-4,  fractionnated  to proteolytic  purity  (as above  for  P-1),  in its  active  (nonboiled) form, had a molecular weight corresponding to 74 kD whereas the boiled protease produced two major bands after S D S - P A G E corresponding to 68 and 50 kD.  It may be that these two bands again represent molecules associated  with the nonboiled protease and are produced as the protease  during treatment  a result of the denaturation of  with SDS-solubilization  buffer  at  100  °C.  Alternatively, the bands could be the result of a conformational change in the protease  following  denaturation  of  the  protease  by  boiling  with  SDS-  solubilization buffer.  The position of proteolytic activity (74 kD) on the BSA-  polyacrylamide  correlates  gel  very  well  with  the  single  major  band  corresponding to 74 kD, visualized by silver staining after S D S - P A G E  of  nonboiled P-4, and is an indication of a high degree of proteolytic and protein purity of the P-4 protease.  Lipopolysaccharide was not found in either P-1 or P-4, after S D S - P A G E and silver staining for lipopolysaccharide (results not shown).  Although the proteases had different molecular weights, they were identical in their activity.  The lower molecular weight protease (P-4) appeared to be more  reactive than the higher molecular weight protease (P-1).  The proteases were  identical in their requirement for reducing agent for activity and in pH and temperature optima.  The small difference in heat stability between P-1 and P-4 68  may be due to a protein complex which stabilizes P-1, making it less susceptible to changes in temperature.  The proteases were identical in their activity against  synthetic peptides and protein substrates.  Both proteases were especially active against synthetic peptides containing arginine which, on the basis of this substrate specificity, suggests that the proteases inhibition  are  trypsin-like  resulted  rather  than  chymotrypsin-like,  from the specific chymotrypsin  even  inhibitor, T P C K .  though It is  interesting that although the glycylprolyl dipeptide was slowly hydrolyzed, both denatured collagen and gelatin were readily degraded. the collagenolytic appears  activity of the  higher  molecular  Further investigation of weight  warranted, based upon the potential ability of  protease  (P-1)  P-1 to degrade acid-  soluble collagen at 37 ° C .  Both proteases completely degraded IgG and IgA. terms of virulence,  This ability is important in  giving the organism the potential to interfere with the host  defense system and thus enhance its own survival.  Previous studies (Mortensen  & Kilian, 1984; Sato et al, 1987; Sunqvist et al, 1985) have  reported similar  results.  The inhibition studies indicated that both P-1 and P-4 are serine proteases. Inhibition was produced by several serine protease inhibitors, including those active against both trypsin- (PMSF and TLCK) and chymotrypsin-like enzymes (TPCK).  In addition, the sensitivity of these proteases to thiol inhibitors  (PCMB, Hg and Zn ions) suggests that these groups are present at the active site. These findings are similar to those of other investigations describing trypsinlike proteases of B. gingivalis Sorsa et al, 1987; EDTA  (Fujimura & Nakamura, 1987;  Yoshimura et al, 1984).  Ono et al, 1987;  The enhancement of activity from  and aprotinin may be the result of chelation of an inhibitory cation  associated with the proteases.  Supportive evidence is the increase in activity  when cations, C a and Mg, are added to the reaction mixture.  69  The immunoreactivity studies indicate that the glycylprolyl protease purified from outer membranes of B. gingivalis different from the proteases  33277 (Grenier and McBride,1987) is  purified in this work.  The specificity of the  antibody to the glycylprolyl protease was reinforced by the fact that only the band representing the M  r  of the glycylprolyl protease (29,000 D) was detected  when the antibody was reacted with crude preparations of outer membrane, cell extract  and with the outer membrane vesicle  preparation,  as outlined in  Materials and Methods.  Whether these fractions represent a single protease or two different proteases is not entirely clear from this investigation, since in all characteristics molecular weight, the proteases were identical.  except  The initial detection of three  proteolytic bands on the skim milk zymogram could represent three different proteases or a single protease having three proteolytically active moieties. Further work using immunological and genetic techniques may be able to resolve this.  Preparation of specific antibody to each of the purified proteases, and  investigation of any cross-reactivity, should determine whether these are unique proteases  or  part  of  single  large  protein.  Furthermore,  isolation  and  purification of the intermediate protease seen on the zymogram would likely provide more information concerning this group of proteases.  It is quite likely, based upon the findings of this investigation, that the three bands apparent on the initial zymogram represent different forms of a single protease.  Evidence for this conclusion is the appearance of the major band  corresponding  to 68 kD which is common to both P-1  and P-4  following  electrophoresis of boiled P-1 and boiled P-4.  The trypsin-like protease described by Fujimura and Nakamura (1987) was also isolated from a cell extract of sonicated cells of B. gingivalis.  However,  their protease, soluble in Triton X-100, was found to be active at neutral pH (pH 7.5) and had a lower molecular weight by S D S - P A G E (65 kD) than either the P1 or P-4 protease in this study.  The earlier investigation of Fujimura and  70  Nakamura (1981) revealed the presence of two proteases, probably membranefree, which were also  inhibited by thiol inhibitors; however,  no  molecular  weights were determined.  Yoshimura et al (1984) and Ono et al (1987) described trypsin-like proteases with similar activities to the P-1 and P-4 proteases, however, the proteases were isolated from different crude fractions in each of these investigations.  The  protease isolated by Yoshimura et al (1984) could have been associated with either the inner or the outer membrane of the cell whereas Ono et al (1987) used culture supernatant as a starting material.  Yoshimura et al (1984) did not  determine the molecular weight of the partially purified protease whereas the M  r  of the protease purified by Ono et al (1987) was determined to be 49 kD by S D S - P A G E . Differences other than M exist among the proteases found in these r  two investigations and the proteases purified in this study.  The  maximum  activity of the protease was determined to be at pH 7.6 (Ono et al, 1987), however, information regarding pH optimum is not given by Yoshimura et al (1984).  In contrast, both P-1 and P-4 had their optimum activity between the  pH values between 6.0 and 6.5.  Furthermore, P-1 and P-4  appear to be  associated only with the outer membrane of the cell.  The trypsin-like protease isolated from culture supernatant by Sorsa et al (1987) appeared similar to P-1  and P-4 in activity. The  given for their protease was 35 kD (by gel filtration).  molecular weight Therefore, it is not  possible to make a direct comparison to the proteases isolated in this study.  Activity similar to that found for P-1 and P-4 was apparent in extracellular vesicles (Grenier & Mayrand, 1987).  The vesicle fraction was found to degrade  azocoll and to possess trypsin-like activity, evidenced by the hydrolysis of BAPNA.  The vesicles were also active against collagen.  Furthermore, the  polypeptide pattern (by SDS-PAGE) of the vesicle preparation was found to be similar, although not identical to that of an outer membrane fraction.  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