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The adherence properties of Bacteroides gingivalis Singh, Umadatt 1990

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The adherence properties of Bacteroides gingivalis  by  UMADATT SINGH  B.Sc,  Acadia University, 1980  M.Sc,  Acadia University,  1982  A THESIS SUBMITTED IN PARTIAL FULFILLMENT O F THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Department of Microbiology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA DECEMBER  1989  © Singh Umadatt, 1989  In presenting  this thesis in partial fulfilment of the  degree at the  and  study. I further agree that permission for extensive  copying of this thesis for scholarly purposes may or  by  his or  her  representatives.  be  permission.  Department The University of British Columbia Vancouver, Canada  (2/88)  granted by the head of  It is understood  publication of this thesis for financial gain shall not be  DE-6  advanced  University of British Columbia, I agree that the Library shall make it  freely available for reference department  requirements for an  that  copying  allowed without my  my or  written  Abstract A Bacteroides  gingivalis adhesin mediating attachment to red blood cells and buccal  epithelial cells was isolated, cloned and characterized. The isolation procedure involved gentle  stirring  of the cells followed  by ammonium  sulphate  precipitation,  exchange and gel chromatography. The native molecule had a M in excess of 1 0 r  ion6  kDa  and was made up of subunits with an M of 43 kDa. Antisera raised to the adhesin and r  its subunits reacted with antigens on the surface of B. gingivalis cells. No with fimbriae was  seen. The IgG fractions from these  reaction  antisera inhibited the adherence  of B. gingivalis to host tissue. Proteolytic enzymes destroyed binding capability of whole cells and of the purified adhesin but the molecular weight of the haemagglutinin was not altered.  A genomic library of B. gingivalis DNA was created in £ . coli JM83.  5500 colonies  were screened by a colony immunoassay with anti-S. gingivalis serum and by a direct haemagglutinating assay. clones,  1-3,and  1-49  337 clones tested positive by the immunoassay and two tested  positive  for  haemagglutinating  activity.  Both  haemagglutinating positive recombinants had inserts of 3.2 kb. One clone, 1-49 was chosen for further characterization.  E. coli 1-49 expressed a protein of 43 kDa that  was not present in E. coli JM 83 control as seen by S D S - P A G E and Western blot analysis.  Anti-1-49 serum inhibited the haemagglutinating  and E. coli 1-49. This serum  activity of  B. gingivalis  reacted with surface molecules on B. gingivalis and E.  coli 1-49 as seen by immunogold electron microscopy and immunofluorescence, and to the purified haemagglutinin by Western blot analysis. B. gingivalis,  the  haemagglutinating  activity  Like the haemagglutinin on  of E. coli 1-49  was destroyed by  heating and proteolytic enzymes but the apparent size of the molecule as determined by S D S - P A G E was not affected.  A  bacterial coaggregating adhesin from B. gingivalis was isolated and characterized.  ii  The isolation procedure involved adsorption of the solubilized adhesin on S . mitis followed  by elution with glycine buffer. S D S - P A G E of the boiled adhesin revealed a  protein with an M  r  of 46 kDa. Proteolytic digestion destroyed all bacterial aggregating  activity and hydrolysed the 46 kd protein. Antisera raised to the 46 kDa protein reacted with surface molecules on all strains of B. gingivalis tested. This antiserum inhibited the coaggregation reaction between B. gingivalis and other bacteria.  Vesicles produced by B. gingivalis were found to enhance the binding of S. sanguis to serum coated hydroxy apatite (SeHA). Maximum vesicle mediated binding took place at 37°C and was destroyed by heating.  The lipopolysaccharide from several black pigmented bacteroides were isolated and characterized physically, chemically and immunologically. All of the L P S were of the smooth type and contained the sugars rhamnose, glucose, galactose, glucosamine and galactosamine; no KDO or heptose were found.  iii  TABLE O F CONTENTS Abstract  ii  Table of contents  iv  List of tables  v  List of figures  x  Abbreviations.Nomenclature and Symbols  iii  XJJJ  Acknowledgement  X J V  Introduction  1  Specificity of adherence  1  Methods of study  3  Bacterial surface components involved in adherence  5  (1)  Firhbrial adhesins  5  (2)  Fibrils  7  (3)  Non-fimbrial proteinaceous  (4)  Carbohydrates  adhesins  8 8  Microbial adherence in the oral cavity  9  (a) Ecosystem  ,  9  (b)  Dental plaque  1 1  (c)  Adherence  1 2  Bacteroides gingivalis  14  (a)  Classification  15  (b)  Ecology  1 6  (c)  Serology  1 7  (d)  Pathogenicity  1 8  Virulence factors  21  (1) Adherence  21  (a) Haemagglutination  2 1  (b)  23  Bacterial coaggregation  iv  (c) Adherence to other substrates  24  (2)  Collagenase  25  (3)  Other proteolytic enzymes  2 6  (4)  Toxic products  26  (5)  Capsule  2 7  Vesicles  2 7  Lipopolysaccharides  30  Materials and Methods  3 6  Bacteria and cultivation  3 6  Isolation of haemagglutinin  3 7  Haemagglutination assay  3 8  Haemagglutination inhibition assays  3 8  Enzymatic treatment  3 9  Sodium periodate treatment  3 9  B-Mercaptoethanol  3 9  Electrophoretic techniques  4 0  Preparation of B. gingivalis chromosomal DNA  41  Construction of genomic library  41  Colony immunoassay  4 2  Immunological procedures  44  Binding to epithelial cells...  45  Isolation of bacterial aggregating factor  4 7  Bacterial aggregation assay  4 8  Bacterial aggregating inhibition  4 8  Modification of cell surface  4 9  Vesicles  5 0  Lipopolysaccharides  5 2  Results  5 6  V  Isolation of haemagglutinin  ,  5 6  Characterization of the haemagglutinin  6 0  Dissociation of the haemagglutinin  6 2  Chemical composition  6 2  Inhibition of haemagglutination  63  Immunogold labelling  6 3  Enzymatic treatment  6 7  Cloning of the haemagglutinin  7 3  Genomic library  7 7  Recombinant plasmids  7 7  Restriction analysis  7 7  Southern blot  77  Expression of cloned antigen  82  Prevalence of the 43 kDa protein  82  Immunogold electronmicroscopy  86  Inhibition by antisera  86  Binding to buccal epithelial cells  89  Characterization of the recombinant-epithelial cell reaction.  94  Bacterial coaggregating adhesin  98  Purification  98  Characterization of the solubilized BA  101  Enzymatic digestion of the BA  ;  105  Inhibition of bacterial aggregating activity by antisera..  105  Immunogold labelling  109  Vesicles  113  Isolation of vesicles  113  Effect of vesicles on attachment of S. sanguis to SeHA  11 3  Kinetics of the binding reaction  11 7  Spectrophotometry adherence assay  1 25  vi  Lipopolysaccharides  1 27  Analysis of L P S from Bacteroides  1 27  Chemical analysis of L P S  "129  Gas-liquid chromatography of sugars from L P S  131  Fatty acid composition  31  1  Western electroblotting analysis  134  Discussion  36  1  Bibliography  1  vii  4  7  LIST OF TABLES  TABLE  1  PAGE  Effect of a number of putative inhibitors on haemagglutinating activity  2  64  Effect of various surface modifying substances on the haemagglutinating activity of  3  B. gingivalis  Effect of immune sera on haemagglutinating activity of 66  B. gingivalis 4  Effect of enzyme treatment on haemagglutinating activity of intact cells and purified haemagglutinin of B. gingivalis....  5  activity of  purified B. gingivalis  haemagglutinin  7  74  Time course of HA inactivation by proteinase K  9 10  76  Effect of immune sera on haemagglutinating activity of E. coli 1-49  91  Adherence to buccal epithelial cells  92  Effect of protease, heat, and putative inhibitors on the attachment  to oral epithelial cells  11  Effect of immune IgG on binding to epithelial cells  12  Effect of various compounds on aggregation  13  75  Effect of P M S F on proteinase K digestion of the purified B. gingivalis haemagglutinin  8  71  Effect of increasing amounts of proteinase K on haemagglutinating  6  65  96 97 10 3  Effact of various substances on the bacterial aggregating activity of B. gingivalis  10 4  viii  14  Effect of enzymatic treatment on the bacterial aggregating activity of B. gingivalis and the purified BA  15  Effect of immune serum on bacterial aggregating activity  16  Effect of anti-46 kDa antiserum on the binding of B. gingivalis to immobilized S. mitis  17  Influence of vesicles on the attachment of S. sanguis 117  Langmuir isotherm parameters for the adsorption of S. sanguis to SeHA with and without the addition of vesicles  19  20 21  23  1 21  The effect of heat and putative receptor on the attachment of S. sanguis to SeHA beads  12 4  Visual adherence assay for the attachment of bacteria to SeHA  13 6  Chemical content of lipopolysaccharides of the black pigmented oral Bacteroides  22  108  110  to SeHA and SHA 18  106  13 0  Carbohydrate composition of lipopolysaccharides from BPB Fatty acid composition of L P S from B P B  ix  132 1 33  LIST O F FIGURES  FIGURES  1  PAGE  Elution of haemagglutinating activity and protein from a DEAE-Sepharose column  57  2  S D S - P A G E of fractions eluted from a DEAE-Sepharose column..  58  3  Chromatography of HA on Sephacryl S-1000  59  4  S D S - P A G E gradient gel analysis of fractions from Sephacryl S-1000 column  5  61  Cells of B. gingivalis reacted with anti-43kDa serum  68  6  Immunogold bead labelling of thin sections of B. gingivalis  69  7  Immuno gold bead labelling of thin sections of B. gingivalis  7 0  8 9 10 11  Colony immunoblot  78  Screening of recombinant clones for haemagglutinating activity... Restriction endonuclease analysis of the recombinant clones  81  Southern blot hybridization with the 3.2 kb Pst 1 restriction fragment of E. coli 1-49  13  83  Western blot of anti-1-49 antiserum against cell lysate of E. coin -49.  14  84  Western blot of the purified haemagglutinin from B. gingivalis and cell lysates of Bacteroides  15  80  Linear restriction map of the the cloned insert from recombinant E. coli 1-49  12  79  sp. using anti-1-49 serum  as the probe  85  Immuno gold bead labelling of E. coli 1-49  8 7  x  16  Immuno gold bead labelling of B. gingivalis  88  17  Immuno gold bead labelling of E. coli 1-49  90  18  Scanning electron micrograph of bacteria bound to buccal epithelial cells  19 20 21 22  93  Electron micrograph of bacteria attached to epithelial cells.. Adsorption of the BA by S. mitis  1 00  Western-blot analysis of the BA adsorption on S. mitis  1 02  S D S - P A G E analysis of the enzymatic digestion of the bacterial aggregating adhesin from B. gingivalis 33277  23  95  Immuno gold bead labelling of B. gingivalis  107 111  24  Immuno gold bead labelling of thin sections of B. gingivalis. .  112  25  Transmission electron micrograph of a vesicle preparation ...  114  26 27  B. gingivalis S D S - P A G E profile  11 5  Scanning electron micrograph of SeHA incubated with (A) B. gingivalis vesicles, (B) S.  sanguis,  and (C) B. gingivalis vesicles 28  ,  Adsorption isotherm of S. sanguis bound to SeHA with and without vesicles  29  11 9  Langmuir adsorption isotherm for the adherence of S. sanguis to SeHA, and SeHA pre-incubated with B. gingivalis vesicles....  30  118  1 20  Scatchard adsorption isotherm for the adherence of S. sanguis to SeHA pre-incubated with B. gingivalis vesicles  31  122  Adhesion of S. sanguis to SeHA, pre-incubated with increasing quantities of B. gingivalis vesicles  32  S D S - P A G E analysis of LPS from black pigmented Bacteroides..  xi  1 23 128  33  Reaction of L P S from B P B with antisera to B. gingivalis 33277 ..  xii  135  ABBREVIATIONS, NOMENCLATURE AND SYMBOLS  Anti-43  kDa  Antiserum raised against subunits of the haemagglutinin  Anti-HA  Antiserum raised against the native haemagglutinin  Anti-46 kDa  Antiserum raised against the bacterial aggregating adhesin  ATCC  American Type Culture Collection  BA  Bacterial  BPB  Black Pigmented Bacteroides  DNA  Deoxyribonucleic acid  EDTA  Ethylenediaminetetraacetic  HA  Haemagglutinin  HEPES  N-2-hydroxyethylpiperazine-N -2-ethanesulphonic  kb  1000 base pairs  kDa  1000  M  Relative molecular mass  r  agglutinin  acid  daltons  PAGE  Polyacrylamide gel electrophoresis  PBS  Phosphate buffered saline  PBS/T  Phosphate buffered saline/ Tween 20  SDS  Sodium dodecyl sulphate  SHA  Saliva-coated hydroxyapatite beads  SeHA  Serum-coated hydroxyapatite beads  SeHA-V  Vesicle covered serum-coated hydroxyapatite beads  TE  Tris/EDTA  xiii  acid  ACKNOWLEDGEMENTS  I sincerely acknowledge the financial, inspirational and technical support of Dr. B.C.  McBride during the course of this project.  My sincere thanks to my committee members Drs. R. A. J . Warren, R. E. W. Hancock and R. S . Molday for serving on my committee and their  advice  throughout the course of this work.  I wish to thank Andre Wong for his work with the electron microscope, and Bruce McCaughey for his photographic assistance.  Finally, I like to extend a sincere thank you to my wife Patricia for her encouragement and unfailing love during the course of this study. dedicated to my wife and my son Michael.  xiv  This thesis is  INTRODUCTION The  introductory section of this thesis will be a general discussion of the principles  governing  bacterial adherence to host tissue. This will be followed by a description of  adherence in the oral cavity and its importance in the pathogenesis of oral organisms. This latter section will include a detailed description of Bacteroides  gingivalis  including a discussion of potential pathogenic factors.  Adhesion or adherence can be defined as the irreversible attachment of an organism to a surface. In the oral cavity this surface can be another bacterium, soft tissue (epithelial cells) or hard tissue (teeth) which may be covered with an organic layer. Any molecule or structure responsible for adhesive activities will be called an adhesin. The entity on surfaces to which adhesins bind will be called the receptor. It has been postulated that when a bacterium approaches a putative receptor it binds via a reversible interaction (147)  and  other  interactions  stabilize  or  contribute  towards  the  irreversible  attachment (42). It is to be expected that molecules on the surface of the bacteria would be responsible for this attachment.  Specificity of adherence The question arises whether bacteria adhere to host tissue via specific surface molecules or via non-specific interactions. The specificity of the interaction of bacteria with host tissues was first suggested by data from in vivo experiments.  Gibbons et al (67) pointed  out the apparent preference of particular bacteria for certain tissues over other tissues (tissue tropism). For example Streptococcus  salivarius was found in large numbers on  the tongue and low numbers in dental plaque and subsequently it was shown to bind to epithelial cells.  This idea of the specificity of the interaction between bacteria and host  tissue is further supported by the species specificity of certain bacterial infections. For example, gonococcal infections are limited to humans, (as the bacteria responsible for the disease process is capable of attaching to human epithelial cells via their pili)  1  (104), group A streptococcal infection to humans (228) and diarrheagenic E.coli K 88 infection to pigs (106). Cheney et al (21) demonstrated convincingly the high degree of specificity between adherence and disease. They showed that certain diarrheagenic strain of E. coli which were capable of attaching to the gut epithelial cells of rabbit produced diarrhea in these animals, whereas these same strains of E. coli, unable to attach to the epithelial cells of guinea pigs and rats produced no disease in these animals.  Susceptibility to certain infections is a genetic trait related to the availability of appropriate receptors. For example it was demonstrated that certain pigs are genetically immune to E. coli K 88 infection. By crossing susceptible and resistant pigs the investigators showed that susceptibility is coded for by chromosomal dominant genes and moreover is mediated  by receptors for E. coli K 88 on the brush borders of the  intestinal epithelial cell (160, 207).  It should be emphasized however, that the colonization of a surface is a complex process which may involve factors such as bacterial motility, chemotaxis and the ability to reproduce  in that environment  (9). Various non-specific factors  also  influence  bacterial adherence. Healthy surfaces are constantly bathed by secretions laden with antibacterial  enzymes  and antibodies  which  are impedements  to colonization.  Additionally unattached bacteria are swept away by mechanical means such as coughing, sneezing and peristalsis, and the hydrodynamic forces exerted by fluid flowing over the mucosal surfaces, eg saliva and crevicular fluid.  Successful colonizers are those capable not only of attaching, but also of multiplying, this is particularly true on (94).  surfaces where host cells are continuously desquamated  In the oral cavity there are surfaces eg tooth, which circumvent problems of  shedding and thus allow a stable flora to develop.  Nevertheless, attachment to such  surfaces must be of such a nature to resist cleansing mechanisms such as mastication and salivary flow (8, 62).  2  Adherence is a complex process which may depend on several forces interacting at the same time. For instance it has been postulated that binding to oral surfaces electrostatic  (201),  lectin-like  (69,162)  and  hydrophobic  involves  interactions  (8).  Electrostatic and hydrophobic interactions may be less specific in nature but are important in stabilizing the specific lectin-like interaction.  Methods of study Several different approaches have been used in an attempt to gain a better insight as to how bacteria bind to a surface. The method chosen has depended on whether the objective was to (a) identify, isolate and characterize the adhesin or receptor, (b) characterize the adhesive interaction, (c) study the expression of genes involved, or (d) study the in vivo activity of the adhesin.  A number of adhesins have been isolated by conventional biochemical procedures including ultracentrifugation and column chromatography.  One of the best example is E .  coli where a variety of unique cell binding adhesins have been purified (46). In  oral  system adhesins have been purified from a number of organisms including S. salivarius and Actinomycetes  viscosus  (24,243). The  structural  characterization  of  these  adhesins has been determined by microscopic examination (55,256,257,260,).  An effective method for identifying adhesins has been to study adhesive negative mutants. In these studies spontaneous or chemically induced mutants were isolated following enrichment  by adsorption. A comparsion was then made  between  the cell wall  polypeptides of the mutant and wild type. For example, Weiss et al (246) coaggregating deficient mutant of Bacteroides  isolated a  loeschii which was found to  lack a  specific 75 kDa cell wall polypeptide. In other cases monospecific antisera was prepared by adsorption of the anti-wild type serum with the mutant, the adhesin  was then  identified by Western blot analysis. This monospecific antiserum was also used to inhibit adherence of the wild type cells (25,28) or to immunoprecipitate the adhesin (243).  3  A routine method of study for understanding the adherence reaction has been to chemically or enzymatically modify the cell surface of the bacteria or epithelial cell and to determine how adherence is affected.  McBride et al (152) showed that  Streptococcus  sanguis bound to sialic acid suggesting the presence of a sialic acid lectin. Weerkamp and McBride (242) found that treatment of S. salivarius with protease reduced to Fusobacterium (258)  binding  nucleatum thus implicating a protein as the adhesin. Yamazaki et al  showed that trypsin treatment of  Eikenella  corrodens  prevented  it from  attaching to buccal epithelial cells. They also found that neuraminidase treatment of the epithelial cell enhanced adherence, exposing a galactose residue thus implying that the adherence reaction involved a lectin-like protein on the bacterial surface.  The nature of the receptor has been studied by measuring the inhibitory activity of soluble  receptor  analogues. Well  studied  examples  are  the  mannose-sensitive  haemagglutinin (HA) of enterobacteriaceae (47) and the lactose sensitive A. viscosusS. sanguis interaction (119). Another example is the adherence of binding of Eikenella  corrodens to epithelial cells  Recently, recombinant DNA technology has been adhesion.  galactose sensitive  (258).  used to identify the genes involved in  The most thoroughly studied examples are  the E. coli adhesins which bind  the organism to gut epithelial cells. The number of genes involved in the adherence reaction was elucidated and the structural and regulatory sequences have been located (129).  Jacobs et al (101)  did site specific mutagenesis and localized the amino acids  involved in the adhesin-receptor reaction. This will be discussed in detail in subsequent paragraphs.  Bacterial surface components involved in adherence Bacteria have a complex cell surface comprised of proteins, carbohydrates  and  amphiphilic compounds such as LTA and LPS. Each of these components has the potential  4  to act as an adhesin. In some cases the adhesins are organized into polymerized structures such as fimbriae or capsule, which can be visualized by electron microscopy.  (1) Fimbrial adhesins (a)  Morphology  In general two classes of fimbrial adhesin exist, rigid and flexible. An example of former are the  type 1 fimbriae of E. coli which measures between  the  2 and 10 nm in  diameter and may be as long as 4 urn (181).  (b)  Composition  Fimbrial adhesins are proteinaceous polymers whose structural elements are primarily composed  of  identical  subunits  (186,197).  Since  the  subunits  are  disassociated by sodium dodecyl sulphate, (220), dilute  acid (157)  normally  or guanidine  hydrochloride (54), it is reasonable to believe that they are held together by hydrogen and hydrophobic bonds. The molecular weight of fimbrial subunits ranges kDa  (159,220).  Most fimbrial  adhesins are  negatively  charged  from 8 to 26  molecules with  isoelectric points in the range of 3.7 to 5.6 (183). Genetic and biochemical analysis have shown that there are a number of fimbriae associated proteins which are different from the fimbrial structural subunits and which actually form the example Lindberg et al (129)  adhesin.  For  showed that a distinction existed between the adhesin  molecule and the pap pili subunit of E. coli J-96. Clegg et al (17) demonstrated that mutants of Klebsiella pneumoniae can be constructed which possess intact fimbriae but lack the ability to agglutinate erythrocytes. This implies that adhesin responsible for recognition of receptors can be a moiety separate from that of the fimbrial structural element.  (c)  Role of fimbriae in adherence  Fimbriae have a well documented role in adherence. For example in E. coli the mannose sensitive (MR)  haemagglutinin was shown to be the typel fimbriae (45). Type 1 and  5  type 2 fimbriae of Actinomyces  viscosus have been shown to be involved in binding to  saliva coated hydroxyapatite and to S. sanguis respectively (42). The in vivo role of fimbriae in adherence has also been investigated. An example are the enterotoxigenic (ETEC) strains of E. coli which have lost their adherence mediating fimbriae and are no longer pathogenic (20,77). Similarly non-fimbriated gonococcus were found to be noninfective in human studies (104 ).  (d)  Genetics  Recently there have been a number of studies involved in the analysis of fimbrial genes. These studies have shown that there are multiple genes that are responsible for the production, export, assembly and anchorage of the fimbriae. These genes can be located on a plasmid as is the case with the E. coli K 88 fimbriae (180) or on the chromosome as found in E. coli J 96 (172).  For example, genes responsible for type 1 fimbriae  have been cloned from uropathogenic E. coli J 96 (148). The genes responsible for the formation of this fimbriae are organized into an operon in the order pil E , F, D, C , B, A.hyp. The pil B and pil C genes code for 30,000 and 86,000 M proteins respectively. r  The proteins  are involved in assembly and anchorage of the fimbriae.  The pil E gene  codes for a 31 kDa protein, mutagenesis in this gene results in a nonadhesive piliated bacteria. S o although type 1 pili are needed for receptor binding  sometime their  presence is not sufficient for binding. The receptor binding function requires in addition to the polymerized pili the product of the pil E gene (148). The pil F gene codes for an 18.2 kDa protein, mutation in this gene results in pilus of increased length. This led to the conclusion that the pil F gene product act as a competitive inhibitor of pilus polymerization.  The pap pili genes which are located on chromosomal DNA were cloned from E. coli J96 (172). This pili  is responsible for the MR haemagglutination  and uroepithelial  attachment of this bacteria. The pap genes are arranged in similar fashion to those of the type 1 fimbriae.  By mutant analysis coupled with immunoelectron microscopy it was  6  shown by Lindberg et al (129)  that the pap pili are heteropolymers composed of the  major pilin protein pap A, the minor pilin pap E and pap F, and the adhesin pap G . These latter three proteins are located at the tip of the pili.  It has become clear from studies of type 1 and pap pili that the receptor binding component is different from the fimbrial subunit and that fimbrial length and numbers are regulated by associated genes. A logical question ensues as to why the adhesin is associated only with the pilus tip.  An attractive possibility is that the pilus is needed to  place the adhesin outside the complete L P S O-chain found on E. coli. This suggestion is based on the finding that smooth strains of E. coli J 96 binds only if the pap A gene is intact, the pap A protein is not required in rough mutants which lack the O-side chain (129).  (2)  FibriHae  These are structures which were originally found  on the surface of gram-positive  bacteria where they formed part of the "fuzzy coat". Recently they have been seen on many gram-negative organisms (90).  They are different from fimbriae in that they  have  usually long and flexible.  no definite  diameter  and are  Fibrillae  may  be  peritrichous, polar or located in patches on the side of the bacteria. They vary in length from  70 nm to 200 nm. In S . salivarius  they have been divided into four distinct  groups depending on their adhesive properties, length and organization (243). A fibrillar haemagglutinin has been purified from Campylobacter  pylori, an organism  responsible for gastritis, peptic and duodenal ulcers in humans (55).  Adhesins with  fibrillar structures have also been isolated from several other bacteria including E. coli, Yersinia sp. (111), S. sanguis (128) and Bacteroides  (3)  loeschii (246).  Non-fimbrial proteinaceous adhesins  Not all proteins on the surface of bacterial cells are incorporated into structural  7  elements such as fimbriae and fibrils; they  can also exist as discrete elements on the  cell surface. For example E. coli (47) and Enterobacter fimbrial  non fibrillar  Mycoplasma  surface proteins that are responsible for  binds to sialic acid residues. A 300 nucleatum  (165). Capnocytophaga  (2) possess non haemagglutination.  attaches to epithelial cell via a 165 kd protein (7) which  pneumoniae  Fusobacterium  aerogenes  kDa protein  binds the  periodontopathogen  to buccal epithelial cell in a galactose sensitive reaction  ochracea  coaggregated with S. sanguis, A. naaslundii and A.  israelii via a cell surface protein in a  reaction that was inhibited by L-rhamnose and  D-fucose (245). Microscopic examination did not reveal any polymerised structures on the cell surface.  (4) Carbohydrates Other structures on bacterial cell surface have been known to have adhesive properties; these include  capsules and lipopolysaccharides (224,249). One of the most studied  example in which surface located carbohydrates is responsible for adherence is that of the  extracellular  glucan synthesized by S. mutans. This material is involved in  mediating the attachment of the organism to teeth and to other S. mutans (87).  S. sanguis 34 has carbohydrate receptors which mediate coaggregation with A. viscosus and A, naeslundii lectins. The receptor has been found to consist of repeating subunits of a hexasaccharide containing N-acetylglucosamine, galactose, glucose and rhamnose joined  by  phosphodiester  Corynebacterium, polysaccharides Agrobacterium  bonds  (52).  The  Erwinia, Pseudomonas which  are  believed  and to  plant  pathogens  Xanthomonas  bind  specifically  Agrobacterium,  produce to  the  extracellular host  (204).  tumifaciens is an invasive pathogen which initiates crown gall tumours  in a variety of plants. The adherence of virulent Agrobacterium  to a specific site on the  host cell wall exposed by wounding has been shown by Lippincott and Lippincott (130) to be due to the LPS O-antigen.  8  Microbial adherence in the oral cavity The mouth is a nutrient rich environment ideally suited for the growth of bacteria. Within the mouth there are several types of surfaces which lend themselves to colonization. These include the keratinized epithelium found in such places as the tongue, non-keratinized epithelium which lines the dentogingival sulcus,  the pellicle coating  the surface of the teeth, and other attached bacteria (66). These surfaces will be modified by the environment providing a multitude of potential receptors.  Oral surfaces are subjected to a number of modifying influences including the effect of saliva, crevicular fluid and ingested food. Saliva secreted by major and minor salivary glands continuously rinse oral surfaces to remove non-adherent organisms. Crevicular fluid plays a similar role in the gingival crevice. Both fluids supply nutrients to the indigenous microflora and remove waste products.  The food eaten can alter the pH and depending on its composition can favour the colonization of a particular group of organisms (85).  For example; fermentation of  carbohydrates leads to the production of acids and the selection of an acidogenic flora. The most profound effect is caused by sucrose which in addition to being fermented, is converted to high molecular weight glucans which bind large numbers of the strongly acidogenic S. mutans to the teeth (27).  (a) Ecosystem There are several environmental niches in the mouth created by anatomical diversity and a variety of allo-and autogenic factors. The complexity of the oral environment is reflected by diversity of the microbial flora which is estimated to consist of greater than three hundred species (161). These organisms are arranged into unique ecosystems. They modify their environment by consuming oxygen, altering pH, removing nutrients, releasing metabolic end products and elaborating enzymes which may change the nature of the bacterial receptors on the immobilized surfaces.  9  The most studied ecosystems of the oral cavity include the tongue, buccal surfaces, tooth and gingival crevice. The tongue is an aerobic environment which is thought to be the area of the most rapid cell proliferation (85). S. salivarius is found on the tongue in small numbers  on the tooth probably because of its ability to attach to the tongue  epithelium. The buccal surfaces are a sparsely populated aerobic ecosystem colonized by a limited number of organisms.  The area between the tooth and the gum, known as the gingival crevice is anaerobic in nature and supports the growth of a strict anaerobic population. This niche is relatively isolated from other ecosystems in the mouth (85). Organisms have the possibility of binding to non-keratinized epithelium or to the subgingival regions of the tooth. Food is supplied to the organisms by crevicular fluid which is a nutrient rich serum transudate which seeps into the crevice through the periodontium.  The type of bacteria isolated from this habitat reflects the state of health of the gingival tissues. In the healthy state most of the microflora are gram-positive facultative rods and cocci whereas in the diseased state there is a change to gram-negative anaerobic motile rods (221,222).  Another unique site in the oral cavity is the tooth. The exposed area of the tooth is enamel which is composed primarily of hydroxyapatite which has phosphate and calcium ions exposed on its surface (120). Since many more phosphate groups than calcium ions are exposed, hydroxyapatite  has a net negative charge. However, this surface is rarely  exposed directly to the bacteria, since the enamel is covered by a thin layer of organic material termed the acquired pellicle (140). This layer is normally less than a micron in thickness and consists mainly of salivary glycoproteins (4,30).  10  (b) Dental plaque Bacterial accumulations on the tooth surfaces are referred to as dental plaque. Plaque consists of bacteria embedded in an amorphous matrix of salivary, crevicular and bacterial polymers (66,68). Plaque develops rapidly on protected areas of the teeth but will eventually cover all smooth surfaces. The bacterial composition of the dental plaque differs with age of the plaque. When the plaque material is associated with the teeth above the gingival tissue it is designated as supragingival plaque, and when it is below the gingiva it is called subgingival plaque (66).  The development of dental plaque involves three types of adherence reactions. The first step is a selective adsorption to the pellicle. Subsequent accumulation of organisms involves  bacteria-bacteria binding.  By homotypic cell-cell interactions, organisms of  one species may accumulate and maintain  their position in the growing plaque.  Heterotypic cell-cell interactions leading to coaggregation between species are also of importance. Many in vitro studies have shown that oral bacteria can coaggregate when mixed together  (51,66,71,73,119,153,155). McBride et al (153)  demonstrated that  coaggregation supports the colonization of Veillonella in gnotobiotic rats infected with Streptococcus.  An unusual example of interbacterial aggregation in plaque are the so  called "corn-cob" formations in which coccal forms adhere via surface fibrils to a central filamentous organism (132).  The ability of salivary constituents to induce aggregation of a variety of oral streptococci is well documented and has been extensively reviewed by Bowden et al (10) and Gibbons (66).  Such interactions may promote homotypic bacterial aggregation in the plaque  matrix. Salivary  constituents bound to bacterial  cell surface  may  also  promote  heterotypic bacterial aggregation, thus contributing to the species diversity seen in plaque  (52).  11  (c) Adherence Adherence in the oral cavity follows the same principles as that of the adherence of microorganisms to any other surface of the body. The interactions between receptor and adhesin can be ionic, hydrophobic or lectin-like (225). The adhesins can be fimbriae, fibrillae or other proteins, carbohydrates and amphiphilic substances.  Adhesion to the tooth surface is studied in an in vitro adherence model which consists of adding bacteria to hydroxyapatite  beads treated with saliva to form the in vitro  equivalent of the acquired pellicle. These experimental pellicles have similar properties to a naturally radiolabeled  acquired pellicle (22,72). Adsorption can be quantified by use of bacteria  (128)  or by turbidimetric  (93)  cultural  (154)  or by visual  techniques (179).  The saliva coated hydroxyapatite (SHA) model system has been used to study the kinetics of binding. The binding isotherm is obtained by plotting the number of unbound cells (U) at equilibrium against the bound cells (B). Such adherence data can be fitted to a Langmuir adsorption isotherm (3, 72) U/B=K/N +1/NU. A plot of U/B vs U allows an estimation of the dissociation constant K and its reciprocal the affinity constant 1/K from the X intercept. The theoretical number of binding sites (N) on the S H A can be estimated from the slope of the graph.  Binding and Langmuir isotherms were used to compare the adherence of different species of oral bacteria to S H A and HA (26). Analysis of such isotherms indicates that the range of binding sites present for different species is much greater on S H A than on the untreated mineral (26). Binding to HA was found to be relatively non-specific whereas binding to S H A was a specific reaction. The salivary pellicle therefore imparts a degree of specificity to the adsorption process. Liljemark et al (128)  In a series of  showed that S. sanguis,  higher  competition experiments  S. mitis and S. salivarius  did not  compete with each other for binding sites on S H A . This data provided strong evidence that  12  bacterial adhesins were recognizing specific receptors on the salivary constituents adsorbed to HA and demonstrated that different bacterial species do have unique adhesins recognizing specific S H A receptors.  Molecular analysis has revealed that the binding of S. sanguis to S H A involves a number of different adhesins (42, 69, 162) which recognize different receptors on S H A . Some strains have different adhesins for the same receptor while other strains have multiple adhesins for the same receptor. Treatment of S H A with neuraminidase reduced its ability to bind S. sanguis  C 5 , did not affect binding of strain FC-1 (69)  and completely  eliminated binding of strain 12 (162).  Morris and McBride (162)  compared the adherence of S. sanguis 12 and S. sanguis 12  na (a variant which did not aggregate) to SHA. Their results showed that strain 12 bound to S H A via two types of salivary receptors. One receptor was sensitive to neuraminidase and the other was sensitive to preincubation at pH 5.0 ( 3 7 ° C ) . Strain 12 na has lost the adhesin which binds to the neuraminidase sensitive receptor, but retains the adhesin binding to the pH sensitive receptor. Based on this data a two site binding  model was  proposed (162).  This brief review of the literature clearly shows the complexity of adherence in the oral cavity. Oral organisms have the possibility of binding to host tissue or to other bacteria. The interaction between the microorganism and the surface to which it is attached can be attributed to host synthesized polymers (eg glucans). Regardless of the mechanism, the phenomenon of adherence is characterized by a high degree of specificity.  Bacteroides gingivalis The following section will encompass a detailed  discussion of Bacteroides  gingivalis  with emphasis on its adherence properties and other potential virulence factors.  13  B. gingivalis  is a gram-negative, non-motile, non-sporulating anaerobic rod. The  species is usually defined as short rods but often appears as rods with rounded ends which vary in size and shape from small coccoid forms to long filamentous organisms. When grown on blood agar plates, B. gingivalis and other asaccharolytic  Bacteroides  produce a black pigment. The chemistry and location of the black pigment gave rise to considerable speculation, but eventually it was shown that it was protohemin with traces of  protoporphyrin  (177,194,212)  Most strains of B. gingivalis require hemin or are stimulated by it and many require vitamin k (menadione) for growth (70,98). The roles of vitamin k and hemin for the growth of B. gingivalis are not fully understood. Gibbons and McDonald (70)  have  speculated that vitamin k acts as an electron carrier in an electron transport system. This proposal was later supported by Shah et al (210). Vitamin k has also been found to stimulate synthesis of phosphosphingolipids in the cell envelope suggesting a role in membrane permeability (126).  It has been suggested that the dark pigment produced by the Bacteroides is a mechanism for storage of hemin. This is consistent with the report of Rizza et al (194) who showed that when B. gingivalis was transferred from high concentrations of hemin to a medium lacking hemin that the cells divided about 8-10  times suggesting that heme had  accumulated within the cell.  (a) Classification Originally all oral anaerobes isolated from the oral cavity, urine and feces which produced a black pigment when grown on blood agar plates were classified as Bacteroides  melaninogenicum  new name Fusiformis nigrescens the 7  t h  (177). In 1947 Schwabacher et al ( 205 ) proposed a based on pigmentation but this name was rejected and  edition of B e r g e y s Manual (203)  14  retained  Bacteroides  melaninogenicus.  Although  Gibbons an  coworkers  (70)  in the  1960>s  showed  biochemical  and  immunological heterogeneity among strains of B. melaninogenicus, only one species of black pigmented Bacteroides (BPB) was recognized. An increased understanding of microbial physiology coupled with analysis of metabolic end products, it became evident that B. melaninogenicus  could be divided into several  fermentative activities. In 1970  subspecies" as a result of their  Holdeman and Moore (97)  described three different  subspecies of B. melaninogenicus on the basis of biochemical activity and quantitative and qualitative differences in the production of  volatile fatty acids. The saccharolytic  strains were divided into two subgroups: B. melaninogenicus which was strongly fermentative and B. melaninogenicus  subsp.  melaninogenicus  subsp. intermedius  which  was weakly fermentative. Assacharolytic strains, that is those which did not lower the pH of a glucose-based medium, were grouped in B.  melaninogenicus  subsp.  asaccharolyticus.  The studies of Finegold and Barnes (57) showed clearly that the biochemical and genetic characteristics  of  the  saccharolytic and  asaccharolytic strains  were  sufficiently  different to justify elevation of the asaccharolytic subspecies to species level. Further studies by Shah et al (209) and van Steenbergen (240) led to the separation of oral and non-oral B. melaninogenicus  species based on their genetic heterogenicity  particularly  among the asaccharolytic strains. This led Coykendall et al (31) to propose the new species  B. gingivalis  asaccharolyticus  for the asaccharolytic strains isolated from oral sites.  was retained for the non-fermentative  Bacteroides  sites. Results from deoxyribonucleic acid hybridization  B.  sp. from non-oral  experiments  indicated little  similarity between oral and non-oral types of asaccharolytic Bacteroides. The DNA base content  of B. gingivalis varied from 46.5  asaccharolyticus  to 48.4  mol % G+C, while that of  B.  varied between 49.2 and 53.6 mol % G+C.  Recently, van Steenbergen (240)  re-evaluated  two strains of B.  asaccharolyticus  isolated by Sunquist ( G . Sunquist Ph.D thesis, University of Umea. Umea Sweden 1976).  15  Though these strains were classified as B. asaccharolyticus, they had little or no DNA homology with either authentic B. asaccharolyticus  or B. gingivalis. A new name B.  endodontalis was proposed for these strains. Recently Shah and Collins (211) proposed a new genus Porphyromonas  for these closely related species of B. gingivalis, B.  endodontalis and B. asaccharolyticus.  There are a number of black pigmented bacteroides (BPB) which have been isolated from the mouth. These include Bacteroides  denticola, Bacteroides  loescheii,  Bacteroides  buccae and Bacteroides macacae.  (b)  Ecology  The principal habitat  of B P B is the oral cavity where they are found in the gingival  crevice and in the supragingival dental plaque. There are major differences in the frequency in which different B P B can be isolated from the oral cavity. In the healthy gingival sulcus less than 2 % of the cultivable microflora is made up of B P B (217).  Various B P B have been found to be associated with different forms of periodontal diseases.  B. gingivalis is found in low numbers if present at all, in healthy and  gingivitis sites in the oral cavity (44,209,216). In chronic gingivitis the predominant isolate is  B. intermedius  (216). In adult periodontitis the numbers of B.  gingivalis  increases significantly (218,250). This species may constitute in excess of 65 % of the flora in the deep periodontal pockets, with the severity of the disease correlating significantly with the percentage of B. gingivalis present (233).  (c) Serology Immunological analysis has proven to be an important tool in classifying closely related microorganisms (202).  In the B P B early serological work revealed the heterogeneity of  antigens in different strains that were classified as B. melaninogenicus  16  (29). This is  not unexpected since it was subsequently shown that they were different species.  The cross reaction of B. gingivalis with other B P B has been investigated by a number of groups. Mansheim et al (144) demonstrated that antisera to gingivalis intermedius  was  not  and B,  serologically cross-reactive  with similar material  They later showed  melaninogenicus.  immunofluoresence that there  capsular material from B. from  B.  by immunodiffusion and  was no serological cross-reactivity between  outer  membrane antigens from oral and non-oral strains of the asaccharolytic Bacteroides (146). The  antigenic  specificity of B.  demonstrated by Reed et al (192)  asaccharolyticus  and  B. gingivalis  was  using immunodiffusion and Immunoelectrophoresis.  In their study it was shown that none of the strains of B. from non-oral sites) was antigenically  similar to B.  asaccharolyticus  gingivalis.  (obtained  Furthermore,  no  common antigens were observed between the asaccharolytic and saccharolytic B P B strains.  These immunogenic differences between B. gingivalis and other oral Bacteroides were further revealed by the use of monoclonal antibodies to  B. gingivalis. Naito et al (169)  found that five monoclonal antibodies raised to surface antigens of B. gingivalis reacted with sonicated antigen from all strains of B. gingivalis tested. No serological crossreactivity was seen with antigens from nine other species of B P B tested. Hanazawa et al (88) showed that three of four monoclonal antibodies to B. gingivalis did not react with any other Bacteroides whereas one monoclonal antibody reacted with antigen from B. intermedius.  B.  gingivalis  can  be  divided  into  at  immunoelectrophoresis, Parent et al (184) B. gingivalis  least  two  serotypes.  Using  crossed-  compared the surface antigens of oral  from humans and animals. They identified serotype-specific and cross  reacting antigens.  The human biotype exhibited 25 surface antigens, 2 of which were  specific for the biotype. The animal biotype had 12 surface antigens and 2 were specific  17  for the biotype. Four common antigens were found in all strains of  B. gingivalis.  Fisher et al (58) demonstrated two serogroups within human strains of B. gingivalis and these groups appeared to be correlated with virulence. antigenic differences comes from  recent work of  Support for the concept of  Bramanti and Holt (14)  indicated that there was a relationship between the pathogenicity of B.  which  gingivalis  strains and the electrophoretic patterns of cell envelope proteins. Virulent strains (W 83, W 50) exhibited polypeptide bands at 56 and 49 kd, while avirulent strains showed bands at 72, 53 and 37 kDa.  (d) Pathogenicity There  is a large body of evidence which show that B. gingivalis is an important oral  pathogen. by  Experimental infections  Weiss (247)  who  with Bacteroides  sp. were first reported in 1937  demonstrated that two clinical isolates of B.  melaninogenicus  were pathogenic in dogs. Later, direct evidence was obtained which showed that Bacteroides  sp. played an important role in the pathogenesis of transmissible mixed  anaerobic infections in animal models (41,223,229).  Combinations of bacteria isolated  from various sources ( plaque, necrotic dental pulp and periodontal pockets) were tested for their capacity to induce abscess formation and transmissible infections when injected subcutaneously into guinea pigs. The results showed a synergistic effect; i.e individual bacterial species or some Bacteroides were not able to induce an infection whereas mixtures of two or more bacteria did produce an infection (83).  Most of these experimental infections have another common trait: when the BPB were present in the infections mixture, it was possible to produce a transmissible infection; when the B P B were deleted from the innocula a transmissible infection was not produced, indicating a key role for the Bacteroides  species (151).  Observations that the BPB can produce animal infections in pure culture have been infrequent. The first such observation was made by McDonald et al (139) who noted that  18  B. melaninogenicus  CR2A (B. gingivalis) could produce an infection in guinea pigs  when injected in pure culture. This infection was transmissible to other animals. It was also shown that this organism could produce an infection in conventional and germ free mice (224). More recently it has been shown that a number of strains of fl. gingivalis ( W 50, W 83, BH 18/10, 22B4 and RB46D-1) are capable of producing infections when  as pure cultures (82,232). Kastelein et al (114)  demonstrated  S.  gingivalis  W 83 virulence in guinea pigs and Swiss mice.  The guinea pig has been used extensively for the study of infection involving Bacteroides because it was thought that mice were less susceptible to these bacteria (223). However, recently Neiders et al (170) using a mice abscess model showed that eight B. gingivalis isolates from plaque associated with periodontal disease produced secondary lesions when injected intraperitoneally on the abdomen. Histological examination of the tissue showed evidence of invasion of the connective tissue.  (e) BPB and periodontal disease The association of any organism with a specific disease process is not easily proven. The first step in determining the etiological agents of different periodontal disease would be to define the microbiota present in sulci of healthy individuals and individuals with different clinical syndromes (36).  In the healthy sulci the majority of the organism  found are gram-positive rods and cocci. Tanner et al (233) found that for adults with periodontitis the microflora changed to one with a predominance of gram-negative rods. White and Mayrand (250) studied the flora of healthy gingival crevices and periodontal pockets to determine if a relationship exists between the presence of B. gingivalis and the severity of the disease. Samples from patients with severe inflamation showed a greater proportion of S. gingivalis. They found also that 6. gingivalis was not present in the healthy sulci.  Zambon et al (262)  in a similar type of study found a positive  correlation  the  B P B and  between  proportion  of  19  both  the  severity  of  gingival  inflammation  and periodontal pocket depth suggesting that these organism may  contribute to the pathogenesis of certain forms of periodontal diseases.  In conjunction with these studies, several researchers did comparative analysis on patients that were under remission from periodontitis after conventional antibiotic and other treatments.  Loesche and co-workers (134)  treated 5 advanced periodontitis  patients with metronidazole. They found that isolates of B. gingivalis comprised from 35-40 % of the subgingival microflora prior to treatment and decreased to 0 % of the flora immediately after treatment. At  6 months after the treatment, the proportion of  B. gingivalis had not returned to pretreatment level and neither had the disease.  Bacterial infections are frequently accompanied by an immune response that is specific for the pathogenic organism. Several studies have been carried out correlating the presence of serum antibodies to that of B P B especially B. gingivalis.  Using an Elisa  assay Mouton et al (164) measured levels of antibody specific for B.  asaccharolyticus  in serum samples obtained from new born infants, children, periodontal normal adults and endentulous adults, patients with adult periodontitis, juvenile periodontitis and acute necrotizing ulcerative gingivitis. They found elevated levels of serum IgG to B. gingivalis in patients with adult periodontitis.  Similar results were obtained by Patters and Korman (185) with  periodontitis  who found that subjects  had significantly greater anti-fi. gingivalis IgG and IgA than did  periodontal^ healthy subjects. In a more recent study Ebersole et al (50) human systemic antibody levels to  B. gingivalis and B. intermedius  juvenile periodontitis or adult periodontitis and in normal persons. gingivalis serum IgG  levels were seen in adult with periodontitis.  20  assessed  in subjects with Elevated anti-S.  Virulence factors It can be seen from this brief discussion that there is strong evidence implicating B. gingivalis as an important oral pathogen. The next obvious question was to define the determinants which make this organism a pathogen. Specific adhesins, proteases, endotoxins and capsules have all been looked at as potential virulence factors (142).  (1) Adherence  B.  gingivalis  grows in the sulcus where it is exposed to the cleansing action of  crevicular fluid and thus it must be able to adhere (9). The organism has been shown to possess  a  number  of  adhesive properties  including the  ability  to  agglutinate  erythrocytes, bind to epithelial cells and to coaggregate with a number of oral plaque organisms  (a)  (214).  HaemannltJtination  The haemagglutinating  activity  of the B P B was first studied  by Okuda and Takazoe  (175) who showed that the reaction was limited to B. gingivalis. They found that saliva and serum inhibited haemagglutinating activity of B . gingivalis.  Confusion  still  exists  as  to  the  nature  of  the  adhesin  responsible for  haemagglutinating activity of this bacteria. Okuda et al (175)  the  suggested that the  haemagglutinating activity was due to a fimbriae. They showed that fimbriated strains haemagglutinated whereas non-fimbriated strains did not and therefore they concluded that the adhesin was fimbrial. Slots  and Gibbons (214)  haemagglutinin  (HA)  haemagglutinating  showed that  positive  activity  and  strains  partially of  purified pili (fimbriae) from  B. asaccharolyticus  concluded that  fimbriae  were  6  possessed responsible for  haemagglutination. However, electron microscopic examination of the preparations revealed the presence of aggregated pili mixed with globular material. Attempts to eliminate the globular material were unsuccessful and thus it was questionable whether  21  the fimbriae were responsible for the haemagglutinating activity. polysaccharides did not exhibit haemagglutination  L P S and surface  nor did they inhibit the reaction  (174). Boyd and McBride (11) fractionated the outer membrane of B. gingivalis  and  found that the haemagglutinating activity was associated with a fraction containing low molecular weight L P S , protein and loosely bound lipid. They also demonstrated that there was no correlation between the presence of fimbriae and haemagglutination. This result was further reinforced by Suzuki et al (231) who  screened 53 isolates of B. gingivalis  for fimbriae by negative staining and immunological methods.They found that over 50 % of the isolates had no fimbriae but had haemagglutinating activity, thus casting further doubt on the hypothesis that fimbriae were the HA of B. gingivalis.  Recently fimbriae from B. gingivalis  have been isolated and their morphological,  biological, immunological and chemical properties characterized (260,261). These structures are heat stable, curly filaments approximately 5 nm in width which are devoid of haemagglutinating activity. The fimbrilin subunit which can be dissociated by boiling in S D S has an apparent molecular weight of 43 kDa.  Dickinson et al (40) cloned the gene encoding the fimbrial subunits into E. coli. They found that the gene was present in a single copy on the bacterial chromosome and that the predicted size of the mature protein was 36 kDa. The protein sequence had no marked similarity to other fimbrial sequences and no homologous sequences could be found in other B P B spp. suggesting that the fimbrillin represents a class of fimbrial subunit protein of limited distribution.  There have been several claims to the purification of the HA from B.  gingivalis.  Recently Inoshita et al (100) isolated an exohaemagglutinin from the culture medium of B.  gingivalis  chromatography.  by  ultracentrifugation  followed  by  gel  filtration  and  affinity  Biochemical analysis and S D S - P A G E revealed that the isolated HA  contained 3 major protein bands and no detectable L P S . Haemagglutination inhibition  22  studies showed that the HA was inhibited by L-arginine and arginine containing peptides suggesting that the arginine residues may be part of the ligand-binding sites on erythrocytes. Okuda et al (176) also isolated a HA case ammonium sulphate precipitation  from culture supernatant, in this  was followed by column chromatography with a  hydrophobic column, DEAE-Sephadex and Sephadex G-100 gel filtration columns. When the purified HA was analysed on S D S - P A G E several protein bands were present. In electron microphotographs of the preparation, the HA appeared to have a vesicle or tube like structure.  Haemagglutinating activity was destroyed by heating and inhibited by L-  arginine and L-lysine. They suggested that the guanido group on arginine may act as a contact residue between the adhesin and the receptor on erythrocytes. Monoclonal antibody against the HA bound to cell surface and inhibited haemagglutinating activity of both the cells and the purified HA.  Recently, Mouton et al (163) crossedimmunoaffinity  identified a cell bound HA of B. gingivalis by use of  electrophoresis. A  polyclonal antiserum  with  specificity  restricted to the HA was produced and on immunoblotting experiments two antigens with apparent molecular weights of 33 and 38 kDa were detected. The antiserum  inhibited  haemagglutinating activity.  (b) Bacterial coaggregation As indicated, B. gingivalis attaches to other bacteria associated with dental plaque. Slots and Gibbons (214) common  oral  showed that  B. gingivalis coaggregated with several species of  isolates including S. sanguis,  S. mitis, S. mutans, S. salivarius,  A.viscosus, A. israelii and A. naeslundii. This interaction unlike haemagglutination was not inhibited by either saliva or serum suggesting that the reaction was critical for the survival of B. gingivalis in the oral cavity. The inhibition data indicated that there was two types of adhesin on the surface of B. gingivalis.  Boyd and  McBride  (11)  demonstrated that the outer membrane contained the coaggregation activity . They were  23  able to separate the HA and the bacterial agglutinin (BA) adhesins. The coaggregating preparation contained a number of protein, carbohydrates and high molecular weight LPS but no fimbriae like structure.  Schwarz et al (206)  using  A. viscosus  attached to salivary coated  hydroxyapatite  (actinobeads) and ^H-labelled B. gingivalis carried out a quantitative evaluation of the cohesion of B. gingivalis and A. viscosus. They found that B. gingivalis  adhered  tenaciously to the actinobeads , thus showing that cohesion with bacteria in preformed plaque could fosters establishment of B. gingivalis on teeth.  With the exception of Boyd and McBride (11) no attempt has been made to identify or isolate the adhesin responsible for coaggregation of B. gingivalis.  (c) Adherence to other substrates In addition to haemagglutination and bacterial coaggregation, B. gingivalis has been shown to bind to a number of other substrates found in the oral cavity. Lantz and coworkers (123)  reported that strains of B. gingivalis specifically bind and degrade  fibrinogen. Binding was rapid, reversible and saturable. They speculate that interaction with fibrinogen may mediate colonization and establishment of periodontal  B. gingivalis in the  microbiota.  In another study, Winkler et al (253) demonstrated the attachment of B. gingivalis to an in vitro basement-membrane like matrix and to selected individual macromolecular constituents of this matrix. binding of B. gingivalis.  Preincubating the bacteria with fibronectin decreased the Similar results were obtained when  the  matrices were  preinubated with anti-fibronectin antibodies.  Recently  Naito  and Gibbons  (168)  studied the  attachment  of  B. gingivalis  to  hydroxyapatite beads treated with either human type 1 or type 1V collagen or to  24  particles of bovine collagen. They found that promoted attachment  collagen on hydroxyapatite  surfaces  . B. gingivalis also bound to particles of collagen. Heating of  bacteria abolished their adhesive properties. Human serum, fibronectin and protease inhibitors  were inhibitory  to the  reaction. Cimasoni et  al  (23)  showed that  B.  gingivalis can attach to serum, saliva and crevicular fluid coated hydroxyapatite beads.  (2)  Collaoenase  Some types of periodontal diseases are characterized by the loss of gingival connective tissue. Collagen is the major constituent of the gingival connective tissue and while this protein is resistant to a wide variety of proteolytic enzymes, it is degraded by bacterial and tissue collagenases (133).  For this reason the presence  of  collagenase  is an  important factor in the pathogenesis of any oral pathogen. Gibbons and McDonald (70) were the first to demonstrate the collagenolytic activity of B. gingivalis. was  The enzyme  found to be cell associated and to be stimulated by reducing agents such as  dithiothreitol and cysteine (149, 198). Lantz and her associates ( J . Dent. Res. 289, Abstr. 1076, 1983)  indicated that the enzyme may be within the periplasmic space.  Maximum activity was seen when the cells were grown in peptide deficient medium or when the culture reached stationary growth phase (150).  Roeterink et al (200) showed  that among the B P B , B. gingivalis produced the most damage to collagen tissue of the palate of mice.  Mayrand and Grenier (150) showed that B. gingivalis is the only BPB to produce a true collagenase. As opposed to eukaryotic collagenases which cleave undenatured collagen at a single site, the collagenase from B. gingivalis hydrolysis collagen into small peptides (149,  198).  B. gingivalis  also produces a factor which induces the production and  activation of mammalian procollagenase (H. Birkedal-Hansen, J . Periodontal Res. 62: 101 Abstr. 551, 1987).These results suggests that B. gingivalis has the potential to destroy periodontal connective tissue directly or indirectly.  25  (3)  Other proteolytic  activity  There are a number of reports of proteolytic enzymes found in B. gingivalis. Various amidopeptidases (1) thiol (178) and trypsin-like (230) proteases have been purified from this organism. polyacrylamide  Grenier et al (78) using bovine serum albumin conjugated to  demonstrated the presence of 8 proteases with different M . These f  enzymes have been shown to be active against fibrinogen (123) fibronectin (125) and other glycoproteins found on epithelial cell surfaces and in the connective tissue matrix. One of these was purified and proved to be a pro-gly specific enzyme of molecular weight 29,000  (78).  Some clinically important bacteria produce enzymes which  hydrolyse serum proteins  involved in host defense against microbial infections (110). Bacteroides  gingivalis is  capable of degrading immunoglobulins (237 ) as well as the C 3 and C 5 polypeptide of the complement system in vivo and in vitro (230). This organism is also capable of degrading  human  plasma proteinase  inhibitors  alpha-1-antitrypsin  and alpha-2-  macroglobulin (18). By degrading complement proteins and host proteinase inhibitors B. gingivalis can evade opsonization and phagocytosis by host cells and permits greater destruction of host tissue (227).  (4) Tissue toxicity Culture supernatant or whole cells of B. gingivalis are cytotoxic for monkey kidney cells and squamous epithelial cells B. gingivalis Other  (79).  Butyrate and propionate, two by products of  metabolism are potent inhibitors of several human cell lines (239).  potentially  toxic  factors  include  L P S , ammonia,  compounds ( hydrogen sulphide and methylmercaptan) been shown to increase cell permeability.  26  indole, volatile  sulphur  (235). The mercaptans have  (5) Capsule Electron microscopic observation revealed  a layer of ruthenium red  material 15 nm  thick covering the outer surface of several strains of B. gingivalis (90). Takazoe (173)  Okuda and  demonstrated that encapsulated strains of B. melaninogenicus  were  resistant to phagocytosis whereas non capsulated strains were not. These observations were later confirmed and expanded by van Steenbergen et al (238) chemiluminescence that virulent strains of B. gingivalis  who showed by  (W 83, W 50) were more  resistant to phagocytosis than non-virulent strains such as 376  and HG185.  The  virulent strains had a thicker capsule and a more hydrophilic surface than the avirulent strains. This observation indicates that a major difference between virulent and nonvirulent strains of B. gingivalis is the thickness of their capsular layer.  Vesicles This section will include a general description of vesicles with emphasis on the vesicles produced by Bacteroides gingivalis and their role as a potential pathogenic factor.  (1)  Formation  All gram-negative bacteria have the potential to produce vesicles. Vesicles are formed by a budding off of the released.  outer membrane and they can remain attached to the cell or be  The growth conditions appear to influence the quantity of vesicles formed.  McKee et al (156)  compared growth in hemin excess and hemin limitation and found  that B. gingivalis released  five times more vesicles when grown under hemin limiting  conditions. Knox et al (115) showed that B. coli, produced large quantities of vesicles with lysine as the  limiting  H ae m o p h i I u s (39)  factor.  along  (19,122,131,189,209,255) release  In the with  oral cavity  several  several genera  species  of  including Bacteroides  these vesicular structures. These vesicles are  similar to structures previously found in dental plaque (61,86).  27  (2) Composition Vesicles are derived from the outer membrane, and thus it is reasonable to assume that the chemical constituents of these two structures will (251)  compared  outer  membrane  be  similar.  William and Holt  and vesicles polypeptide profiles of several  Bacteroides and found that the major protein bands were similar but the vesicular preparation had more minor polypeptide bands. The LPS and carbohydrate composition of the outer membrane and vesicles have not been compared.  (3) Biological activities The surface of many bacteria plays a crucial role in pathogenicity. Several  determinants  of virulence are surface components and therefore vesicles by virtue of their mode of formation would be expected to retain these attributes. Vesicles have been shown to possess a number of biological activities, the following sections describe some of the biological functions of vesicles.  (a) Adherence It is not unreasonable to assume that  vesicles should possess the same adherence  properties as that of the outer membrane and a number of studies have shown that this is true.  Scanning electron microscopy revealed that like the whole cells of B. gingivalis  vesicles can attach to hydroxyapatite coated with saliva, serum and crevicular fluid (23).  Purified B. gingivalis  vesicles also binds to erythrocytes causing them to  agglutinate (80). Similarly Celesk and London (19) showed that Cytophaga DR 2002 attaches to S H A beads.  B. gingivalis  vesicles can act as a bridge to permit the  coaggregation of two bacteria which did not normally aggregate when mixed as is the case with Eubacterium  saburreum and Capnocytophaga  ochracea  (80). Treatment of the  vesicles with heat or proteolytic enzymes destroyed their ability to act as a bridge whereas similar treatment of the Eubacterium or Capnocytophaga did not affect attachment.  This would indicate  unimodal interaction  of a vesicular protein or  glycoprotein with a non-proteinaceous component on the bacterial cells.  28  The aggregative properties associated important  with  vesicles suggests that they may play an  role in the colonization of the periodontal pocket (118). The accumulation of  diverse sulcular flora could be mediated by intergeneric binding mediated by B. gingivalis vesicles. Vesicles would induce coaggregation  resulting in the formation of  microcolonies which could be resistant to phagocytosis by macrophage.  The examples cited above relate to vesicles with properties similar to that of outer membrane. There are also reports of vesicles with different adherence properties than that of the parent cell from which they were derived. For example Kagermeir et al (109)  studied the aggregation of Cytophaga with A. israelii. They found that vesicles  derived from Cytophaga did not bind to A. israelii but whole cells did. They suggested two possible explanation (1) the receptors on the outer membrane are located in areas which do not give rise to vesicles, (2) the receptors are anchored to the peptidoglycan or cytoplasmic membrane of the bacteria.  (b)  Enzymatic  activity  Enzymes are another biological activity associated with both outer membranes and vesicles.  Grenier and Mayrand (80) compared the proteolytic activities of vesicles and  whole cells of B. gingivalis and found them to be similar. In a recent study, Grenier et al (78) characterized the proteolytic activities of outer-membranes and vesicles of B. gingivalis  by S D S - P A G E using as a substrate bovine serum albumin covalently  conjugated to the polyacrylamide. A total of eight major proteolytic activities were present in both outer membranes and vesicles. shown  Pseudomonas  tragi vesicles have  been  to have proteolytic activity associated with them (48). Using immunological  procedures, Thompson et al (234) showed that a neutral protease was present on the cell surface and in the cell-bound vesicles.  A wide variety of other enzymes have been found to be present on vesicles. For example  29  the periodontal pathogen Capnocytophaga show an increase in the secretion of acid and alkaline phosphatases during the growth cycle (189). It has been estimated that over 50 % of these enzymes were released with the vesicles. The vesicles elaborated by Actinibacillus  actinomycetecomitans  contain a leucotoxin. Lai et al (122) observed that  leucotoxic strains of this organism produce numerous vesicles whereas non-leucotoxic strains released no vesicles.  It has been postulated that vesicles, due to their small size, may reach sites not available to the whole cell. An interesting  example of this is B. succinogenes  bacteria which produces large quantities of vesicles which contain the extracellular carboxymethyl cellulase activity (84).  a common rumen more than 50% of  These vesicles are located  between the adhering cells and cellulose fibres. The secreted vesicles are thought to facilitate hydrolysis of the substrate by reaching sites not available to the whole cells.  It may be that vesicles by virtue of their small size, adherence properties and enzymatic activities may play a role in the pathogenesis of bacteria. Vesicles may have been responsible for the immunofluorescence seen by Pekovic and Fillery (187)  at the  surface as well as in the interstitial spaces between epithelial cells in the oral cavity.  Lipopolysaccharides Lipopolysaccharides  are a heterogenous collection of molecules unique to the outer cell  envelope of gram-negative bacteria. This polymeric bacterial amphiphile is made up of three  major  constituents  which  are  linked  covalently  (56).  The  lipid A  portion  consisting of five or six fatty acids is attached to diglucosamine phosphate (254). Covalently attached to the lipid A is the rough core region of L P S consisting of 11 to 14 saccharides including unique octoses, heptoses and a variety of hexoses substituted with phosphate and ethanolamine. This rough core is often but not always capped with a repeating structure of saccharides of variable length which bears the name O-antigen and can constitute the major antigenic structure of gram-negative cells (59,188).  30  Lipopolysaccharides may be found in non-covalent association with envelope proteins (99,182). When  L P S is disassociated from  its surrounding molecules there is  disruption of the outer envelope which leads to a breakdown of the cell permeability barrier as measured by the penetration of antibiotics and other bactericidal agents (53).  Thus L P S is critical for the structural integrity of the cell and for changes in the  function of some of the L P S associated envelope proteins (38).  Lipid A structure The lipid A is usually covalently attached to the octose-2-keto-3-deoxyoctonate which forms the proximal part of the rough core. The large majority of lipid A's studied contain a 6 (1-6)  linked-D-glucosamine disaccharide that contains two phosphoryl group, one  bound as an ester and the other to the glycosidic hydroxyl group of the reducing glucosaminyl residue (6). hydrolysis.  Free lipid A can be prepared from L P S by mild acid  The lipid A of Salmonella  structure has been elucidated.  has been subjected to intense analysis and its  It contains straight chain and hydroxy fatty acids joined  by amide linkage to C-3 of glucosamine (254).  There is a high degree of chemical similarity between the lipid A's of bacteria belonging to the family Enteriobacteriaceae (13).  This is further emphasized by the fact that  antibody to E. coli lipid A cross reacts with the (182).  Mutharia et al (166)  Pseudomonas  aeruginosa  lipid A of other bacteria in this family  showed that monoclonal antibody to the lipid A from reacted with a variety of gram-negative species tested,  showing the conservation of the lipid A moiety.  However, there are lipid A which show  no serological cross reactivity. For example antiserum to lipid A of Salmonella does not react with lipid A of R. palustrus and R  vividus (135).  Biological effect of lipid A Lipid A is the endotoxic principle of gram-negative bacterial lipopolysaccharide, the polysaccharide portion serves as a solubilizing carrier for the  31  insoluble lipid A.  It has  been shown that the endotoxic activity of the soluble free lipid is comparable to that of intact L P S (64).  L P S and lipid A exhibit a high affinity for cell membranes, for other  lipids and for proteins. The lipid reacts with several different organs.  For example in  the liver, lipid A leads to the depletion of carbohydrate reserves, inhibition of enzyme induction and inhibition of glucogenesis (76).  Cell types sensitive to L P S include  macrophages, polymorphonuclear leukocytes, platelets, B-lymphocytes and fibroblasts (13).  Lymphocytes are induced to undergo mitogenesis by L P S and lipid A.  In addition L P S and lipid A activate a clotting enzyme on the standard lipid A assay, the Limulus amoebocyte assay (13).  Lipid A is also pyogenic and induces the Schwartzman  phenomenon and hemodynamic changes leading to shock and death (137,138). However, due to different chemical structures there is also a difference in levels of biological activity, for example Brucella  sp. lipid A has been found to exhibit very low toxicity  in experimental animals (120) whereas toxicity  in animals (65,105,193,  lipid A from Salmonella  sp. displayed  high  248).  Core The core of LPS usually contains D-glucosamine, D-glucose and D-galactose and an inner lipid A proximal region consisting of an oligosaccharide of core specific sugars, Lglycero-D-manno-heptose  (L-D-hep) and 2-keto-3-deoxy-D-manno-octonate  each forming a branched trisaccharide  (91).  (KDO)  Evaluation of the core structure of LPS  was greatly facilitated when so-called rough (R) mutants of Salmonella were found. Depending on the defect, these mutants synthesized complete or incomplete core structures linked to lipid A but were devoid of O-chain (137). The R mutants of Salmonella were found to be non-virulent, were phagocytized without opsonization by macrophage and were sensitive to toxic agents (195).  32  O-side chain The O-specific chains of LPS are made up of repeating units of identical oligosaccharides (196)  containing a number of different sugars. However, in some cases an oligomer of  a single sugar type is found as in the case of E. coli 09 (190, 137).  The number of  repeating units usually varies and this gives rise to the typical ladder like profile of L P S when  analysed on sodium dodecyl sulphate  polyacrylamide gel electrophoresis  (136,213).  Antigenicity The O-chain contains the immunodominant structures against which anti-LPS antibodies are directed during infection or on immunization  (63,64,136).  It has been found that  active vaccination with E. coli J-5 L P S and passive immunization with antiserum to J 5  protects  against bacteremia from a variety of gram-negative species (15). It was  found that immunization with LPS and the polysaccharide side chain, promoted clearance of P. aeruginosa (32,89).  Virulence The role of L P S in the virulence of P. aeruginosa  was studied in mice (33).  The  virulence of several strains was found to be related to the state of the O-polysaccharide side chain.  Strains with complete side chains were far more virulent than those with  abbreviated structures. Strains without O-side chains were avirulent and were cleared rapidly.  These  strains were  also serum  sensitive  indicating that  L P S O-chain  polysaccharide acts to mask the complement binding site that initiates cell lysis (33).  B. gingivalis L P S (1)  Composition  The LPS from B P B have not been subjected to the same extensive analysis as has the LPS from Salmonella lacks  and Escherichia  coli. It has been reported that the L P S from B P B  2-keto-3-deoxyoctonate (KDO), heptose and B-hydroxy myristic acid which are  33  common to enterobacterial LPS (95,113). and  gas-liquid-chromotographic  However, recently using mass spectroscopic  analysis (103)  revealed  the  presence  of  small  quantities of these compounds in L P S from certain strains of B. gingivalis and B. intermedius. Chemical analysis of the O-side chain showed the presence of both neutral and amino sugars with rhamanose, mannose, galactose and glucose the most predominant neutral sugars and glucosamine the predominant amino sugar (102,117,145).  The lipid A portion was shown to contain over 20 fatty acids (103) with a of  palmitic acid, hexadecanoic acid and stearic acids (117,145).  could  not detect  6-OH-myristic  acid in some preparations  predominance  Some investigators (140)  while  others  reported the presence of hydroxy fatty acids (167).  (2)  Mitooenicitv  The mitogenic effects of L P S from B P B were studied and compared to that of E. coli (117). It was found that the two LPS'S affected the LPS-nonresponsive C3H/HeJ spleen cells equally.  This response was specific to B cells . This may have biological  significance since it has been reported that established lesions of chronic inflammatory periodontal disease in humans are primarily infiltrated with B-cell (49,141,208).  (3)  Bone resorption  The correlation between the incidence of B. gingivalis and alveolar bone loss is well documented (215,218). Millar et al (158), using a low and high molecular weight fraction of L P S in a bone resorption assay found that both fractions release of  4  5  stimulated the  C a from prelabelled fetal rat bones. More importantly they  found that  collagen formation was reduced by as much as 40% . These two results suggest that LPS may play an important role in disease. Koga et al (117)  alveolar bone loss that is  found that B.  gingivalis  production from macrophage to activate thymocytes. activities  including enhancement  of immune  34  associated with periodontal  L P S stimulated IL-1  interleukin-1  is known to have many  responses, stimulation  of  thymocyte  proliferation, activation of B-cells and stimulation of bone resorption. These findings show that one of the mediators of alveolar bone loss in human periodontitis could be IL-1 stimulated by B. gingivalis LPS.  (4)  Endotoxic effects  The biological potency of LPS from B. gingivalis and other BPB is low in comparison to L P S from aerobic gram-negative organisms when assessed by pyrogenicity, chick embryo lethality and the Schwartzmann test (167,124).  Mansheim et al (145)  found  that LPS preparations gave no positive skin reactions in rabbit in doses up to 1mg, whereas  12.5 u.g oiSalmonella  typhi endotoxin gave a positive reaction. Limulus lysate  gelation  and chick embryo lethality occurred at doses 30-fold or more greater than that  in Salmonella endotoxin controls.  (5)  Serology  Dahlen and Baltzer (34) found  that antibody to lipid A of B. gingivalis did not cross  react with lipid A from any of the oral anaerobes they studied. Enterobacteriaceae  Anti-lipid A from  did not cross react with lipid A from any of the B P B . These results  confirm that Bacteroides species have a unique lipopolysaccharide structure (96).  The studies to be described in this thesis were undertaken with the objective of identifying, isolating and characterizing the adhesins used by B. gingivalis in its attachment to host tissue and to other bacteria in dental plaque. In addition the LPS of a number of the BPB was studied.  35  Material and methods  Bacteria and cultivation Bacteroides  33277,  gingivalis  B.  levii  29147,  B.  33185,  denticola  B.  25260 were obtained from the American Type Culture Collection  asaccharolyticus  (Rockville, Md.). B. gingivalis W 12, W 50 and W 83 were obtained from G . Bowden, University of Winnipeg, Manitoba, Canada. B. intermedius BMA and 2332H and B. gingivalis  2D were isolated and characterized in our laboratory. Escherichia  coli JM  83 [ara A (lac-pro AB) rspL (=strA) 0 80 lacZAM15] and J M 101 [supE thi A (lacproAB) {F'traD36 proABIaclq lacZ AM15}] were used as recipients (241)  for the  plasmid vector p U C 18 [Ap ] (259). Streptococcus mitis, S. sanguis, S mutans and S. r  salivarius glycerol at Bacteroides  were human oral isolates. All bacterial cultures were stored in 10 -70°C.  E a c h experiment  strains  were  grown  was in  Microbiology systems, Cockeyville, MD.)  started  Brain-Heart  from  a frozen  Infusion  stock  broth  culture.  (BHI)  supplemented with hemin (5  %  ug/ml)  (BBL and  menadione (0.5 ug/ml) at 3 7 ° C in an anaerobic chamber (Coy Manufacturing, Ann Arbor, Michigan) containing an atmosphere of N : H : C 0 2  2  2  (85: 10: 5) for 48 h. When  solid medium was needed, agar (1.5 %) was added to BHI broth together with whole blood at a final concentration of 5 % . S. mitis and other streptococci were grown at 3 7 ° C in trypticase soy broth (TSB, BBL Microbiology systems, Cockeysville, MD.) supplemented with yeast extract  (3 g/liter).  E. coli J M 83 and J M 101  were grown in Luria-  Bertani broth (10 g tryptose, 5 g NaCl, 5 g yeast extract per liter, pH 7.5) medium at 37°C.  When solid medium was needed, agar (1.5 %) was added to LB broth. Ampicillin,  100 ug/ml, (Sigma) was added to the medium when required.  For adherence assays, S. sanguis 12 was grown overnight in presence of 1.5 u.Ci of [ H]-methyl-thymidine 3  per  ml of culture  medium. The  centrifugation, washed three times in 50 mM H E P E S  cells were  harvested by  buffer (pH 7.2), sonicated for 5  sec (30 % duty cycle, output 5, Sonifier Cell Disrupter 350), washed twice more in  36  H E P E S buffer and finally resuspended to an absorbance of 4.0, as measured at 580 nm.  Isolation of the haemagglutinin B. gingivalis was grown for 48 h after which the cells were harvested by centrifugation at 10,000 x g for 10 min. Cells were washed twice in 0.05 M phosphate buffer (pH 7.4)  and  resuspended to  1/10  the  original  culture  volume  in  20  mM  Tris-  hydrochloride (pH 7.4) containing 0.15 M NaCl and 10 mM MgCI - The suspension was 2  stirred for 15 min at room temperature on a magnetic stirrer.  The cells were pelleted  by centrifugation and the proteins in the supernatant were precipitated by the addition of ammonium sulphate to 40 % . After stirring at 4 ° C overnight, the precipitated proteins were collected by centrifugation and buffer (pH 8.0).  resuspended in a small volume of 20 mM Tris-HCl  This material was dialysed extensively against 3 mM Tris-HCl (pH  8.0). The dialysate was clarified by centrifugation at 10,000 x g for 20 min.  The dialysate was then subjected to ion-exchange chromatography, on a column (16x1.5 cm) of DEAE-Sepharose which was equilibrated with 20 mM Tris-HCl (pH 8.0).  After  loading the sample, the column was washed with 100 ml of 20 mM Tris-HCl (pH 8.0), eluted with a linear gradient of 0-0.3  M NaCl in 20mM Tris-HCl, (pH 8.0). Fractions  of 2 ml were collected and screened individually for haemagglutinating activity and analysed on S D S - P A G E .  Fractions  which exhibited haemagglutinating activity and  showed a single silver staining band at 43 kDa on 12 % S D S - P A G E were pooled and lyophilized.  This lyophilized sample was resuspended in 20 mM Tris-HCl (pH 8.0) and applied to a Sephacryl  S-1000 (Pharmacia Fine Chemicals Uppsala, Sweden) column (80 x 1.5  cm) equilibrated with 20 mM Tris-HCl buffer (pH 8.0). 20 mM Tris-HCl (pH 8.0) containing 50 mM NaCl.  The column was eluted with  Fractions of 3 ml were collected and  were screened individually for haemagglutinating activity and analysed on S D S - P A G E .  37  Haemagglutination assay Haemagglutinating activity was assessed in U-bottom polystyrene microtitre plates (Dynatech  Corp.) with formalized sheep or human erythrocytes resuspended in  phosphate buffered saline (PBS) pH 7.2 at a concentration of 2 % (v/v). and recombinant clones were washed twice in P B S and resuspended to an A  B. 6  6  0  gingivalis of 1 and 3  respectively. Equal volumes (100 u.l) of erythrocytes and bacterial cells or fractionated samples were mixed together and the plates shaken for 20 min after which they were allowed to sit for 1h. Results were scored on the basis of the settling pattern of the erythrocytes. To establish aggregation titres, the bacterial cells or fractionated samples were serially diluted in P B S (pH 7.2) and the last well containing visibly detectable aggregated erythrocytes was taken as the end point of the reaction.  Haemagglutination inhibition assays (a) A number of substances were tested for their ability to block the haemagglutinating activity of B. gingivalis. The materials to be tested were dissolved in P B S (pH 7.2) to the concentration indicated, serially diluted in 50 u.l of the same buffer  and mixed with  an equal volume of bacterial cells or purified haemagglutinin (HA). The concentration of HA or the number of bacterial cells used was twice the highest dilution which gave clear haemagglutination  in a preliminary titration with erythrocytes. The mixtures were  incubated at room temperature for 30 min with shaking and then 100 ul of formalized red blood cells was added. The mixtures were incubated for 20 min and the titre determined.  (b)  Antisera against whole cells, the prepared haemagglutinin and the 43 kDa protein  were evaluated for their ability to inhibit haemagglutination. The antisera were serially diluted in two fold steps in P B S after they were standardized for protein (100 u.g/ml). The  concentration of purified HA or the number of bacterial cells used was twice the  highest dilution which gave clear haemagglutination in a preliminary titration with  38  erythrocytes.  The mixtures were gently shaken for 30 min after which 100 uJ of 2 %  erythrocytes was added to each well. After incubation for a further 20 min the plates were allowed to sit for 1h before the inhibitory titre of the antisera were recorded.  Enzymatic treatment Intact cells of B. gingivalis and the recombinant E. coli were resuspended ( A Q in  HEPES  (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic  and incubated with an equal volume of enzyme  6 0  acid) buffer ( pH  3.0) 7.2)  at 3 7 ° C for 60 min. The enzymes and  appropriate buffers were pronase, proteinase k, mixed glycosidases, papain, lysozyme, mutanolysin, chymotrypsin, phospholipase C ( H E P E S buffer, pH 7.2) trypsin, (20 mM phosphate buffer, pH 8.0)  and papain (0.05  M acetate buffer, pH 5.8). All enzymes  were used at a concentration of 1 mg per ml. After incubation, the reaction mixtures were  chilled  and the cells washed three times with ice-cold P B S (pH 7.2). The cells  were  resuspended in  haemagglutinating  PBS  to  their  original  volume  and  then  assayed  for  activity at 4 ° C . The purified haemagglutinin was treated in similar  fashion and its haemagglutinating activity was evaluated at 4 ° C without removal of the enzymes. Controls without bacterial cells or purified HA were included for each enzyme.  Sodium periodate treatment B. gingivalis cells and the purified HA were suspended in 0.12 M acetate buffer (pH 5.0) containing 0.05 M sodium periodate and incubated at 4^C for 2 h. Subsequently the cells were washed three times and suspended in P B S buffer to an A  6  6  0  n  m  of 3.0 and  assayed for haemagglutinating activity. The purified adhesin was also treated in similar manner and then assayed for activity.  Controls containing only sodium periodate were  incubated similarly and were assayed for haemagglutinating activity.  B-Mercaptoethanol Washed cells and purified adhesin were suspended in P B S (pH 7.2) containing  0.05 M  6-mercaptoethanol and incubated for 2 h at room temperature. The cells were washed  39  three times and resuspended in P B S buffer (pH 7.2) and assayed for HA activity. The purified haemagglutinin was also assayed for activity but without the removal of the reducing agent. Appropriate controls containing reducing agent but no bacterial cells or HA were included.  Heat cells or purified adhesin were suspended in P B S buffer (pH 7.2)  B. gingivalis  and  heated in a water bath at 7 0 ° C , 8 5 ° C and 1 0 0 ° C for 1, 5, 10 and 30 min after which they were assayed for haemagglutinating activity.  Electrophoretic techniques Sodium dodecyl sulphate polyacrymlamide gel electrophoresis was carried out with polyacrylamide concentrations of 12 % , 15 % or a gradient of 5 to 20 %  using the  buffer system of Laemmli (121). Non-denaturizing P A G E was carried out in the absence of S D S in the gel and buffer systems. For polypeptide analysis samples were solubilised in  0.125  M  Tris-HCl buffer  mercaptoethanol  and 0.01  containing  4  %  S D S , 20  % bromophenol blue (pH 6.8).  %  glycerol,  10%  8-  For lipopolysaccharides  (LPS), 20 % sucrose was added. The mixtures were boiled for 5 min unless stated otherwise.  Molecular weight standards were:  (97,400),  bovine  serum  albumin  myosin (200,000), phosphorylase B  (68,000),  chymotrypsinogen (25,700), 6-lactoglobulin  ovalbumin  (45,000),  (18,400) and cytochrome C  3-  (12,300).  Gels were stained for protein with Coomassie Brilliant blue or silver nitrate and  for  LPS by the method of Tsai and Frasch (236).  For  Western  blotting  nitrocellulose paper (pH 8.3).  (17),  material  was  in 25 mM Tris-HCI-192  transferred  electrophoretically  onto  mM glycine and 20 % methanol buffer  Transfer was carried out at 25 V for 18 h in a Bio-Rad Trans-Blot Cell after  which the voltage was increased to 60 V for 2 h. After blocking the unreactive sites with bovine serum albumin the transblotted material was reacted with specific antiserum and  40  visualized by the procedure described in the Bio-Rad Immuno-Blot (GAR-HRP) assay kit. Prestained markers  were used to calibrate molecular weights in Western blots.  Preparation of B. ainaivalis chromosomal DNA Chromosomal DNA from B. gingivalis was prepared by established methods. cells from 500  ml of a log phase culture B.  gingivalis  Briefly,  cells were pelleted  by  centrifugation and washed twice in T E S buffer (20 mM Tris-HCl, 5 mM E D T A , 100 mM NaCl, pH 7.0). Cells were resuspended in T E S buffer, S D S and RNase A were added to a final concentration of 1.6% and 50 ug/ml respectively and the mixture was incubated at 3 7 ° C until cell lysis was complete (30-60 min). Proteinase k was then added to a final concentration of 25 ug/ml and incubation was continued for 1h. The lysate was extracted twice with equal volumes of phenol and once with chloroform before the  DNA was  precipitated with 95 % ethanol. The DNA was further purified by cesium chloride density gradient centrifugation.  Plasmid DNA was isolated by an alkaline lysis procedure (143) and separated from RNA and chromosomal DNA by cesium chloride density gradient centrifugation in the presence of ethidium bromide. Cesium chloride and ethidium bromide were removed by dialysis and butanol  extraction  respectively. The  DNA was then  extracted  with phenol-  chloroform, ethanol precipitated and stored in T E buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) at 4 ° C .  Construction of genomic library Partial digestion  of B. gingivalis DNA for cloning was carried out as described by  Darzins et al (37) with some modifications. DNA  The optimal time for digestion of 50 ug of  with 5 units of Pst 1 restriction enzyme at 3 7 ° C was worked out by removing  aliquots from the reaction mixture at different times and analysing on a 0.7 % agarose gel.  Chromosomal DNA was partially digested with Pst 1 enzyme and separated  41  electrophoretically on a 0.7 % agarose gel. Fragments of 3-9 kb were electroeluted and ligated to alkaline phosphatase treated pUC 18 vector which had been linearised with Pst 1 enzyme. T4  DNA ligase (.1u.g), A T P and dithiothreitol  concentration of 0.1 and 1mM respectively.  were added to a final  Ligation was carried out at 1 2 ° C in a final  reaction volume of 20 u.l. The ligated DNA was used to transform  E. coli J M 83 or E.  coli J M 101 which had been made competent by calcium chloride treatment (143). After incubation at 3 7 ° C for 1 h in 1 ml of LB broth, the transformants were plated on LB  Agar containing  ampicillin  at  100  p.g per  ml, X-gal  (5-bromo-4  chloro-3-  indolyl-B-D-galactopyranoside)( 50 u.l of a 2 % solution) and IPTG (isopropyl-B-Dthiogalactopyranoside) ( 20 u.1 of a 2 % solution). White colonies were screened for haemagglutinating activity as follows: Colonies were individually picked and macerated in 100 U.I  of P B S . 100 u.l of erythrocytes were added to each well, the plates were  shaken for 20 min at room temperature and allowed to sit for 1 h before being scored for•>••haemagglutination.  Colony immunoblot assay White  colonies carrying recombinant  plasmids were  replica plated  on  LB agar  supplemented with ampicillin and X-gal and incubated at 3 7 ° C overnight. The cells were lysed by exposure to chloroform vapour for 15 min. Nitrocellulose filters  (BA85;  Schleicher &Schuell, Inc., Keene, N.H.) were blotted onto the agar plates for 1 h and air dried after which the filters were placed in a blocking albumin in P B S . Blots were then reacted with gingivalis. The antisera  solution of 3 % bovine serum  antibody raised to whole cells of B.  had been extensively adsorbed with E. coli  83 (pUC 18).  Following removal of the first antibody the blots were treated as described in the BioRad  Immuno-Blot (GAR-HRP) assay kit as described previously.  Restriction analysis of recombinant plasmids Plasmids were isolated from recombinant clones by the alkaline lysis method as described by Maniatis et al (143). Restriction endonuclease digestions using single and  42  double digestions were performed under the conditions described by the manufacturer (Bethesda Research Laboratories, Gaithersburg, MD.). The DNA fragments were resolved by electrophoresis in a 1.0 % agarose gel using 50 mM Tris-HCl, 50 mM borate, 0.002 M Na  2  E D T A buffer containing 0.5 ug P©  r  m  ' ° f ethidium bromide. The size of DNA  fragments was estimated by comparing the distance of migration to a logarithmic plot of the migration of standard restricted lambda DNA run on the same gel.  Southern blot analysis The recombinant plasmid was digested to completion with Pst 1 enzyme and the fragments  separated in a 0.7 % agarose gel. The insert DNA was electroeluted (Bio-Rad  Electroeluter)  and then  nick translated  using biotinylated  A T P nucleotide,  DNA  Polymerase 1 and DNA 1/DNase 1 as described by the manufacturer (Nick Translation System, Bethesda Research Laboratories, Gaithersburg, MD.). B. gingivalis,  S.  sanguis  and the recombinant plasmid DNA were digested with Pst 1 enzyme and the DNA fragments were separated by electophoresis on 1 % agarose gel. The transferred to a nitrocellulose membrane by Southern transfer (226) baked at 8 0 ° C for 2 h.  DNA was  and the filter was  After prehybridization for 4 h at 4 2 ° C in 50 % formamide and  5x Denhart's solution (1 % Ficoll, I % Polyvinylpyrrolidone, 1 % BSA) in the presence of 250 ug/ml denatured sperm DNA, the bound DNA was hybridized to the labelled probe at 4 2 ° C in 45 % formamide for 18 h. temperature  The membrane was washed twice at room  in 2 % S S C buffer (3 M sodium acetate, 0.3 M sodium citrate, pH 7.0)  containing 0.1 % S D S for 3 min each followed by washing twice in 0.16 % SSC-0.1 % S D S at 5 0 ° C for 15 min each. The membrane was incubated for 1h at 6 5 ° C in 3 % BSA in 0.1 M Tris-HCl , 0.15 M NaCl (pH 7.5) and then reacted with streptavidin-alkaline phosphatase (SAAP) conjugate for 20 min.  After washing in 0.1 M Tris-HCl buffer (pH  7.5) for 30 min the filter was incubated in nitroblue tetrazolium (NBT) and 5-bromo4-chloro-3-indolylphosphate (BCIP).  The incubation was carried out in the dark until  reactive bands appeared (10-40 min). for 5 min in 20 mM Tris-0.5 mM N a  2  To terminate the reaction, the filter was washed  EDTA pH 7.5.  43  Immunological procedures  (a) Serology B.  gingivalis  33277 and recombinant E. coli  cells in the late exponential phase of  growth were pelleted, washed twice in P B S buffer (pH 7.4) and resuspended in the same buffer to a concentration of 1.0 x 1 0  9  cells/ml. The cell suspensions were mixed with  an equal volume of Freunds complete adjuvant and injected intramuscularly into New Zealand white rabbits. Seven days later the rabbit was injected with antigen suspended in Freunds incomplete adjuvant. This was followed by three weekly injections of cells in P B S , serum was collected 5 days after the last injection. Antisera against the purified adhesin was prepared in similar fashion using 50 ug of protein per injection.  To obtain  antisera to the 43 kDa and 46 kDa proteins, rabbits were immunized with purified protein  eluted from S D S - P A G E . Gels were stained with Coomassie Brilliant blue and the  appropriate band was excised from the gel. The gel strips were destained in 25 % isopropanol-10 % acetic acid and the protein was eluted using the Bio-Rad electroeluter apparatus. The immunization protocol  was similar to that described above.  IgG  was  obtained by passage of the antisera over a column of Protein-A Sepharose C L 4B (Sigma St. Louis Mo.) followed by elution with 0.5 M glycine-HCI ( pH 2.3), All antisera were stored in small aliquots at  -20°C.  (b) Immunogold labelling of whole cells B. gingivalis give an A g 6  cells were washed twice in P B S and resuspended in the same buffer to 0  of 1.0.  200 u.l of the cell suspension was incubated with an equal volume  of the appropriate IgG for 1.5 h at room temperature. Unbound IgG was removed by washing twice in P B S , the cells were then resuspended in 200 uJ of P B S and 15 u,l of gold beads (5 nm) conjugated to goat anti-rabbit IgG (EM G A R G5; Janssen Life Sciences Products, Beerse Belgium) were added and incubated at 4 ° G overnight. Unbound secondary antiserum was removed by washing twice in P B S . Cells were resuspended in 100 u.l of P B S and 25 u.l was placed on a copper grid. After drying, the cells were negatively  stained with either 5 %  uranyl acetate  44  or 1 % phosphotungsic acid.  Observations were made with a Philips E M 300 transmission electron microscope.  For immunofluoresence, 200 u.1 of cells resuspended to an AggQ of 1.0  were incubated  with the appropriate antisera for 1 h at room temperature. The cells were washed free of  unbound primary antiserum and  incubated with FITC conjugated goat anti-rabbit IgG  for 1 h. Unbound antibody was removed by washing in P B S and the cells were visualized by epifluoresence microscopy.  (c) Thin sections Thin section immunoelectron microscopy,was done as follows. B. gingivalis cells (AggQ 0  f 1.0) were resuspended in 2.5 % gluteraldehyde and incubated  for 1 h at 4 ° C . The  cells were then pelleted by centrifugation, washed twice in P B S , (each washing was for 5 min)  resuspended in P B S and left at 4 ° C overnight. Cells were fixed in 1 % O s 0  30 min at 4 ° C , washed twice for 5 min  4  for  in water and then dehydrated progressively for  5 min each in 30, 50, 70, 95 and 100 % ethanol. They were then embedded in eponadaldite resin for 72 h at 5 7 ° C before sectioning. Thin sections were picked up on copper grids. The grids were  immersed in 3 % hydrogen peroxide for 10 min and then gently  washed in filtered 20 mM Tris-HCl buffer (pH 7.2) for 10 min. Grids were  immersed  in 10 % B S A for 10 min, transferred to primary antisera for 2 h, washed twice in 20 mM Tris-HCl buffer and placed in goat anti-rabbit IgG conjugated to gold beads for 2 h. The grids were washed in Tris buffer followed by distilled water and then stained with 5 % uranyl acetate. Observations were made with a Philips E M 300 electron microscope.  Binding to epithelial cells Human epithelial cells were collected by scraping oral and mucosal surfaces with a wooden applicator stick. Loosely bound bacteria were removed by filtration on a 8.0 um pore size filter (Nucleopore Corporation, Plessanton, CA)  as described by Gibbons et al  (74). The cells were suspended in saline solution to a final concentration of 1.0 x 1 0  45  5  cells per ml as determined by direct microscopic count. 1 ml of bacterial cells (1.0 x 10  8  per ml ) and 1 ml of epithelial cell suspension were incubated at room temperature  for 1 h after which the unattached and loosely bound cells were removed by the membrane filtration system. The epithelial cells were examined under phase contrast microscope and the number of bacteria attached per cell was determined. At least 50 epithelial cells were examined for each reaction mixture.  Scanning electron microscopy After the incubation of bacteria with  epithelial cells, the unbound and loosely bound  bacteria were removed by washing in the filtration system as described above  and the  epithelial cells processed for scanning electron microscopy by the method of Cimasoni et al (23). Briefly, the epithelial  cells were fixed with 2.5 % glutaraldehyde in P B S for  30 min, post fixed with 1 % osmium tetroxide for 30 min and washed in distilled water for 5 min. The cells were then tanned in 2 % tannic acid for 20 min, washed in water for 5 min and fixed again in 1 % osmium tetroxide for 30 min. After rinsing in distilled water, the cells were dehydrated in an ethanol series (30, 50, 70 %) for 5 min at 4 ° C . Final dehydration in 95 and 100 % ethanol was carried out at room temperature. After dehydration, the samples were transferred  to Teflon membrane filters (0.45  um,  Millipore Corp. Bedford Mass.) and dried with a Ladd critical point dryer. They were then coated with palladium gold using a Hummer V1 sputter coater and examined in a model S-100 Cambridge scanning electron microscope at 25 KV.  Dissociation of bacterial HA Attempts were made to dissociate the purified haemagglutinin into its subunits by variety of treatments.  a  Lyophilised samples were treated with 8 M urea (pH 7.0), 6 M  guanidine hydrochloride (pH 7.0), 1 % S D S , 3 M LiCI, 3 M sodium thiocyanate (pH 7.0), 0.1 N HCI, 0.1 N NaOH and 1 % octyl phenoxy polyethoxyethanol (Triton X-100). Treatments were carried out at 37^C for 18 h. Reagents were removed by dialysis against water before analysis by S D S - P A G E .  46  Enzvmes and reagents All restriction enzymes, polynucleotide kinase, T  DNA ligase and lambda DNA were  4  from Bethesda Research Laboratories, (Gaithersburg, MD). Calf intestinal phosphatase was from Boehringer-Mannheim (Indianapolis,  IN.).  IPTG, X-gal, naphthol A S - M X  phosphate, salmon sperm DNA, DNase, RNase, lysozyme, lipase, mixed glucosidase, proteinase k, trypsin, chymotrypsin, pepsin and papain were from Sigma Chemical C o . , (St. Louis, Mo).  Isolation of the bacterial aggregating factor B. gingivalis cells were pelleted by centrifugation and washed twice in The  P B S (pH 7.3).  cells were resuspended in a tenth of the original culture volume in 0.05  phosphate buffer (pH 7.4)  and agitated at room temperature  M  for 15 min, using a  magnetic stirrer. The cells were removed by centrifugation and ammonium sulphate was added to the supernatant  to give a final concentration of 40% (w/v). The mixture was  incubated at 4 ^ C overnight with stirring. centrifugation  The precipitated proteins were removed by  and resuspended in 20 mM Tris-HCl buffer (pH 8.0). The suspension  was dialysed extensively against 10 mM Tris-HCl pH 8.0 to remove the salt. Any material  which precipitated  during dialysis was removed by centrifugation. The  supernatant was used as the starting material for the purification of the bacterial binding adhesin.  The  coaggregating partner S. mitis was pelleted by centrifugation, washed twice in 50  mM H E P E S  buffer (pH 7.2) containing 10 mM C a C I - The cells were resuspended in the 2  same buffer to an AggQ of 5.0. the  40 %  5 ml of S. mitis suspension was incubated with 3 ml of  ammonium sulphate precipitate. The mixture  was  incubated at  room  temperature with shaking for 20 min. The aggregates were pelleted by centrifugation at 2000 x g for 5 min. The pellet was saved and 5 ml of S. mitis suspension was added to the supernatant which was shaken at room temperature for a further 20 min. Again the aggregates were pelleted by centrifugation and more S. mitis added to the supernatant.  47  This process was repeated until no more aggregates were formed.  The aggregates were pooled and washed once in H E P E S buffer. The adsorbed material was then eluted by incubating the aggregates in 10 ml of 0.15 M NaCI-0.1 M glycine pH 2.3 with shaking.  After 10 min, the pH was bought to neutrality by the addition of 0.15  NaCI-0.1 M N a H P 0 2  4  (pH 7.5).  The S. mitis was removed by centrifugation and the  supernatant was dialysed extensively against water  and  lyophilized. This material was  resuspended in 3 ml of H E P E S buffer and its bacterial aggregating activity was quantitated.  Bacterial aggregation assay Bacterial aggregation was measured as described by McBride et al (152).  Reaction  mixtures contained 100 ul of S. mitis resuspended in 50 mM H E P E S buffer (pH 7.2) (Ag  6 0  of 3.0 ) and 100 of of aggregating substrate. B. gingivalis used as aggregating  substrate was standardized to A ggQ of 2.0.  Reaction mixtures were shaken for 30 min  at room temperature and observed periodically. To establish an aggregation titre, B. gingivalis or any aggregating substrate was serially diluted and the last tube containing detectable aggregation was taken as the end point of the reaction. Since aggregates formed with the cell free substrate were smaller than those formed with whole B. gingivalis cells, evaluation of the degree of aggregation was made with a Leitz steromicroscope at 20 X magnification.  Bacterial aggregation inhibition A number of compounds including carbohydrates, amino acids and other substances were tested for their ability to inhibit the coaggregation reaction of  B. gingivalis with S.  mitis. Briefly, the putative inhibitor was serially diluted and 50 u.l of B. gingivalis or the visible  solubilised adhesin with a bacterial aggregating aggregation was added to each tube.  48  titre 2 times that required for  After shaking for 30 min at room  temperature, 50 ul of S. mitis was added and the mixture was shaken for a further 30 min before the results were determined.  A  similar assay was used to test the inhibitory activity of anti-adhesin serum.  Modification of cell surfaces (a)  Enzymes  B. gingivalis and S. mitis cell suspensions (A  6  6  0  of 3.0) were prepared in H E P E S  buffer at pH 7.2 for pronase and subtilisin treatment; phosphate buffer pH 8.0 for trypsin treatment; and 0.05M acetate buffer at pH 5.8 mixtures were  for pepsin treatment. The  incubated with the enzymes at 37^C for varying times. Cells were  washed 3 times with H E P E S buffer and then checked for their ability to coaggregate. Controls without enzymes were treated in similar fashion. The purified  bacterial  aggregating adhesin was also incubated with the different enzymes after which  BA  activity was evaluated at 4 ° C without the removal of the enzymes.  (b)  B-mercaptoethanol  Washed cells were suspended in H E P E S  buffer  pH 7.0  containing 0.05M  8-  mercaptoethanol and incubated for 2 h at room temperature. The cells were washed 3 times and resuspended in H E P E S buffer and their BA activity was measured. Control without reducing agent were included.  (c) Sodium periodate treatment Cells suspension of B. gingivalis  was treated for 60 min with 10 mg/ml sodium  periodate in 0.25 M potassium phosphate-buffer (pH 7.4) cells were washed in H E P E S buffer prior to assaying.  49  at room temperature.  The  (d)  Heat treatment:  Cell suspensions of B. gingivalis and S. mitis were placed in water baths at 50, 70, 85 and 1 0 0 ° C for 5, 10 or 30 min then evaluated for coaggregation .  Elisa-assav for bacterial coaggregation inhibition Briefly, S. mitis was resuspended to an A  6  6  0  n  m  of 1.0 and 0.1 ml was added to wells  of a microtitre plate (Dynatech) and incubated for 24 h at 4 ° C . The plate was washed three times with phosphate buffer saline containing 0.05% tween (PBS/T) (pH 7.5). 0.1 ml of 3 % B S A in P B S was added to each well and incubated for 1h at room temperature with shaking. The wells were washed 3 x in P B S / T and 100 u.l of a suspension of B. gingivalis prepared as follows: ( an equal volume of B. gingivalis cell suspension with 4 times the number of cells required for aggregation was added to serially diluted  anti-46 kDa serum followed  by incubation for 30 min at room  temperature) was added to each well and incubated for 30 min with shaking. The unbound bacteria  were removed by washing the wells  with PBS/T.  0.1 ml of anti-S. gingivalis  or anti-46 kDa was added to the wells and incubated with shaking for 1h. The wells were rinsed, washed  three times  with P B S / T  prior to adding  0.1 ml of a 1:1000 dilution of  goat anti-rabbit IgG alkaline phosphatase conjugate. Following incubation for 1h at room temperature.the wells were rinsed  3  times with P B S / T and then 0.1 ml of substrate  was added to the wells and the plate incubated at 3 7 ° C .  Adsorbance was measured at  405 nm on an Elisa plate reader. The percent inhibition was determined by relating the absorption readings to those obtained for non-immune serum.  Vesicles Preparation of vesicles Fifteen litres of a three-day old culture of B. gingivalis were concentrated to 250 ml by passage through an ultrafiltration  system (Millipore  with a membrane molecular weight cut off of 100,000.  Corporation, Bedford, MA.) Cells were removed by two  centrifugations at 10,000 x g for 30 min. The supernatant containing the vesicles was  50  dialysed overnight against 10  litres of 50 mM Tris-HCl , 1 mM dithiothreitol,  (pH  9.5). The vesicles were collected by centrifugation at 90,000 x g for 2 h and then lyophilized.  An outer membrane fraction from B. gingivalis was obtained by the method  of Boyd and McBride (11).  Adherence assay The assay was based on the method of Clark et al ( 26) with some modifications. 40 mg of washed and dried spheroidal hydroxyapatite (HA) beads (BDH, Poole, England) were added  to glass scintillation vials together  with 0.5 ml of pre-immune rabbit serum and  then shaken for 2 h at room temperature and left at 4 ° C overnight.  The excess serum  was aspirated and the beads were washed twice with distilled water (10 ml) and a third time with phosphate buffer (1mM K H P O , 5 0 mM NaCl, 1 mM C a C I , 0.01 mM MgCI 2  pH 6.5).  Two-hundred u.g  4  2  (dry weight) of vesicles from B.  2  33277 were  gingivalis  added in a volume of 200 ul of phosphate buffer, and the vials were shaken at 4 ° C for 1 h.  The unattached vesicles were removed by aspiration, the beads washed twice in  phosphate buffer and resuspended in 0.5 ml of the same buffer.  Radiolabeled bacteria (0.5  ml)  (specific activity 3.6  x 10  5  dpm/10  8  added and the mixture was incubated at 4 ° C for 1 h with constant agitation.  cells) were Unattached  bacteria were removed with three washes of phosphate buffer. On the third wash, the beads were transferred to plastic scintillation vials, excess buffer was aspirated and the beads were dried at 6 0 ° C for 1 h. Water (100  u,l)  a  n  d  tissue solubilizer (0.5  ml)  (Amersham Corp. Arlington Heights, IL.) were added and the vials were left at 6 0 ° C overnight. Scintillation fluid (10  ml) was added to each vial and the  radioactivity  monitored in a Tracor Analytic Scintillation counter to determine the number of attached bacteria.  All assays were performed in triplicate. For saliva-coated HA assays, 0.5 ml  of clarified, heat treated saliva replaced  the serum.  When indicated, the inhibitors  were pre-incubated for 1 h at 4 ° C with the serum-coated HA beads before the addition of  51  the bacteria.  Spectrophotometric assay In order to screen several species of oral streptococci for their ability to bind to vesicle-adsorbed SeHA beads, a modification of the adherence was used. The bacteria to be tested were resuspended in H E P E S buffer to an absorbance of 0.6 at 580 nm. The cells were added (1 ml) to SeHA beads (100 mg) which had been pre-incubated with vesicles (500 u,g) or buffer in a volume of 500 u,l. The mixture was shaken for  1 h at 4 ^ C .  After allowing the beads to settle, (5 min) the supernatant was removed and the absorbance measured at 580 nm.  Lipopolvsaccharide Purification of L P S Lipopolysaccharide from the nine different strains and species of BPB was prepared by the method of Darveau and Hancock (35) with minor variations. Briefly, cells from litres 7.2.  2  of culture were pelleted and washed twice in phosphate buffered saline (PBS) pH Ten grams of cells, wet weight, were resuspended in 15 ml of 10 mM Tris-HCl  buffer (pH 8.0) containing 2 mM M g C I , 100 u\g of pancreatic DNase (DN 100 Sigma 2  Chemical Co.) per ml and 25 ug of pancreatic RNase (R-4821 Sigma Chemical C o . St. Louis Mo.) per ml.  The cells were lysed by passage through a French pressure cell  (Amicon Corp) at 1500  lb per i n c h  2  three times and then sonicated (Sonifer  350  Branson Sonic Power C O . ) for 1 min at 70 % output to ensure complete breakage of cells. RNase and DNase were added to a final concentration of 100 and 200 ug/ml respectively and the suspension was incubated at 3 7 ° C for 2 h. After incubation, 5 ml of 0.5 M tetrasodium EDTA, 2.5 ml of 20 % S D S and 2.5 ml of 10 mM Tris-HCl (pH 8.0) were added, and the sample vortexed to ensure solubilization of the components. Pronase (Sigma Chemical Co.) was added to give a final concentration of 200 ug/ml and the sample was incubated at 3 7 ° C overnight. Two volumes of 0.375 M M g C I  52  2  in 95 %  ethanol were added and the mixture cooled to 0 C to precipitate  the L P S . After  U  centrifugation the pellet was resuspended in 25 ml of 2 % S D S , 0.1 M N a E D T A 4  in 10  mM Tris-HCl (pH 8.0) and briefly sonicated. After heating at 8 5 ° C for 10 min the pH was raised to 9.5 and pronase added (25 ug/ml) and the sample incubated overnight at 37°C.  The LPS was precipitated with two volumes of 0.375 M M g C I  2  in ethanol and  more ethanol was added to a final concentration of 80 % . The suspension was centrifuged, the pellet resuspended in water and dialysed extensively against water and lyophilized.  Characterization of the LPS Chemical analysis Protein  was determined by the method of Bradford et al (12)  using bovine serum  albumin as a standard. Total carbohydrates were measured by the phenol-sulphuric acid method (43) with glucose as the standard. Hexoses were quantified by the anthrone reaction (199)  using glucose as the standard. Hexosamines were determined by the  method of Elson and Morgan (5). The method of Dische and Schettles (108) was used to determine methylpentoses with rhamnose as the standard. Heptose and K D O were quantitated by the colorimetric methods of Wright and Rebers (257) and Karkhanis et al (112)  respectively.  Gas-liquid-chromatographic analysis of neutral and amino sugars The methods of Niedermeir (171) and Henry et al (92) were used to convert the sugars of the LPS to  alditol acetates.  Briefly, 10 mg of LPS was hydrolysed with 4 N HCI at  100^C for 1, 2 and 4 h in a sealed glass tube under nitrogen. The lipid A portion was removed by centrifugation standards.  and inositol and mannosamine were added as  internal  1.5 ml of distilled water was added to the vial and the mixture was mixed  thoroughly. The solution was transferred to a 50 ml beaker and the pH adjusted to between 6.8 and 7.2 with Dowex 1  HCO3"  resin. The resin was removed by filtration  through a Buchner funnel and the reaction mixture was lyophilized. The dried sample was then dissolved in 0.5  ml water and cooled to 0 ° C . 1 ml of 0.25  53  M sodium  borohydride was added and the reduction was allowed to proceed overnight at 4 ° C . One drop of 6 N HCI was added to decompose the sodium borohydride. After the solution was lyophilized, approximately 1 ml of methanol was added to the dry residue in order to convert the boric acid to the volatile trimethyl borate.  The methanol was removed under  reduced pressure at room temperature. The methanol treatment was repeated 6 times for each sample. residue .  0.2 ml of a 1:1 solution of pyridine and acetic anhydride was added to the  Acetylation was allowed to proceed for 30 min in a 1 0 0 ° C water bath. The  dried alditol acetates were dissolved in 0.1 ml of chloroform ( G . L C . grade) and analysed on a G P 3% SP-2340 on 100/200 Supelcoport (Supelco Chromatography Supplies) glass column ( 2000 x 1 mm) in a Perkin-Elmer Sigma 3B Dual FID Chromatograph. Reference alditol acetate derivatives of pure sugars were prepared in similar fashion.  Resolution of the alditol acetate derivatives was achieved by use of  the following  programme:  injector/detector  1 8 0 ° C , final  temperature  2 4 0 ° C , ramp rate 2 ° C / m i n , helium flow rate 30 ml/min.  temperature  2 5 0 ° C , initial temperature  Fatty acids analysis The method of Flesher et al (60) was used to analyse fatty acids.  LPS (3 mg) was  hydrolysed in 2 ml of 4 N NaOH for 5h at 100^C in glass vials sealed under nitrogen gas. After adding the internal standard (methyl-tetra decanoic acid) the hydrolysate was neutralized with 4 N HCI and extracted three times with 2 ml of chloroform. The organic phase was dissolved in 1.5 ml of 0.5 N NaOH in 95% methanol and incubated at room temperature for 30 min with shaking in a teflon capped test tube.  Boron trichloride  (Sigma Chemical C o . , St. Louis, MO.) was added and the mixture was heated to 5 0 ° C for 30 min, cooled and mixed with 1 ml water. The solution was shaken with 1 ml of hexane for 1 min and the hexane layer was removed and concentrated.  5 ul was injected into a  Perkin-Elmer Sigma 3B Dual FID Chromatograph with a 10% diethyeneglycol succinate column on Chromosorb W 80/100 mesh (Supelco, Belleforte, Pa.).  54  The carrier gas was  helium with a flow rate of 20 ml/min. The temperature of the injector was 2 0 0 C , the U  column temperature 1 8 0 ° C and the flame ionization temperature was 2 5 0 ° C . Individual fatty acids standards were treated in similar fashion and analysed under the same conditions. The NHI fatty acid methyl ether mixture (Supelco) was used as a standard.  55  Results  Isolation of the haemagglutinin Haemagglutinin  was removed from B. gingivalis  stirrer at room temperature.  by mixing the cells with a magnetic  Following removal of the cells, the HA  was recovered by  precipitation with ammonium sulphate . This material was applied to a DEAE-Sepharose column and eluted with a gradient of 0-0.3 M NaCl. protein and haemagglutinating  activity. S D S - P A G E  Figure 1 shows a single peak of analysis of the fractions with  haemagglutinating activity revealed that fractions eluted before 0.15 M NaCl ( fractions 28-31) had several protein bands (Fig. 2 lanes B and C) whereas fractions eluted between 0.18-0.2 M NaCl exhibited a single protein staining band  (Fig. 2 lanes D and  E ) at 43 kDa.  Those fractions containing a single protein band and strong haemagglutinating  activity  were pooled, dialysed against water and lyophilized. This material was chromatographed on a Sephacryl S-1000 column ( 80 x 1.5 cm). seen in Figure 3. The first peak which haemagglutinating activity,  Two protein peaks were obtained as  eluted in the void volume contained strong  the second peak  contained significant quantities of protein  but little haemagglutinating activity. All fractions with haemagglutinating activity were analysed by 12 % S D S - P A G E .  During the process of staining it was possible to identify  two closely spaced bands which tended to merge into a single band if staining was allowed to continue for too long. To try and separate these bands, the fractions were analysed on either a 15 % S D S - P A G E with electrophoresis emerged from the gel, or on a  continued for 4 h after the dye front  5 to 20 % gradient gel. Both methods resolved the two  proteins with the gradient gel giving the best results. On staining, two distinct bands one at 43 kDa and the other at 41 kDa were seen.  Fractions 20-25 contained mostly the 43  kDa band and good haemagglutinating activity, fractions 25-35 had a mixture of the 43  56  Figure 1. Elution of haemagglutinating activity (  •  ) and protein ( •  ) from the  40 % ammonium sulphate precipitate using a DEAE-Sepharose column. The column (16x1.5 cm) was eluted with a linear gradient of 0.0-0.3 M NaCl in 20 mM TrisHCl (pH 8.0). Fractions were collected and assayed for haemagglutinating activity after dialysis against water.  Fraction #  57  Figure 2. S D S - P A G E of fractions eluted from a DEAE-Sepharose column. Lanes: A, 40 % ammonium sulphate precipitate; B and C , fractions 29 and 31 eluted before 0.15  M NaCl; D,E and F, fractions 32, 33 and 35 eluted between  0.18  and  0.2 M NaCl. 10 ug of protein was loaded in lanes A and B, 8 u.g in lane C and 5 ug in lanes D,E and F.  58  Figure 3. Chromatography of HA on Sephacryl S-1000. Pooled and concentrated HA containing fractions from DEAE-Sepharose chromatography were applied to a Sephacryl S-1000 column (80 x1.5 cm) and eluted with 20 mM Tris-HCl (pH 8.0) containing 50 mM NaCl. Each fraction (3 ml) was assayed for haemagglutinating activity ( •  ) and absorbance at 280 nm ( 0 ) .  0  10  20  30  Fraction #  59  40  50  60  and 41 kDa bands and little haemagglutinating activity (Fig. 4). Fractions 40-43 had little haemagglutinating activity and small quantity of the 43 kDa protein. These results show that there is a correlation between the presence of the 43 kDa protein and haemagglutinating activity.  Additional evidence that the 43 kDa protein was the HA was obtained by adsorbing fractions from peaks 1 and 2 with erythrocytes. In all cases, haemagglutinating activity and most of the 43 kDa protein were removed by the red blood cells, the 41 kDa protein was not adsorbed.  It was difficult to elute  the adsorbed protein from the erythrocytes (0.15 M glycine-  0.15 M NaCl, pH 2.3) but the small percentage of the material that haemagglutinating  eluted displayed  activity and . There was a question as to whether the adsorbed HA  was inactivated during  elution  or simply did not elute. The latter seems to be  more  likely as it was shown that antisera to the 43 kDa protein reacted positively with the surface of red blood cells that had been subjected to the elution procedure following a  adsorption of the haemagglutinating activity.  Characterization of the haemannlutinin S D S - P A G E (12 %) of the purified adhesin of B. gingivalis  showed a single band at  kDa when the sample was boiled for 5 min in 4 % S D S - 2 %  43  3-mercaptoethanol. In the  absence of boiling no material entered the running gel but a band stained at the junction of the stacking and running gel. Heat treatment of the sample in the presence of S D S at 3 7 ° C , 7 0 ° C and 8 0 ° C for 30 min did not disassociate the molecule into a form which could migrate into the gel.  60  Figure 4. SDS-PAGE gradient gel analysis of fractions from Sephacryl S-1000 column. Lanes: A, molecular weight standards; B, fraction 24 showing the pure 43 kDa band; C, fraction 28 showing 43 and 41 kDa bands; D, fraction 45 showing the 41 kDa protein band. All fractions were boiled for 5 min in 4 % SDS. 5 u.g of protein was loaded in lanes B and C and 2 ng in lane D.  61  Dissociation of the haemagglutinin The inability of the HA to migrate into the gel without boiling in S D S suggested that it existed in a multimeric form of high molecular weight. The fact that the HA eluted in the void volume of the Sephacryl  column provided additional evidence for this. As heating  destroyed all haemagglutinating activity, other dissociating procedures were attempted in order to obtain non-denatured subunits and to gain some understanding of the bonds that held the subunits together. Urea (8M), guanidine hydrochloride (6M), (3M),  lithium chloride (1M),  deoxycholate  (0.5  %),  Triton X-100  (1  thiocyanate  %)  and  HCI  (0.1 N) and various combinations of these compounds were found to be ineffective in dissociating the HA. Reducing compounds such as 8-mercaptoethanol also had no effect on dissociating the HA.  Electrophoresis of an unboiled sample of the HA on a 7 % polyacrylamide gel under nondenaturing condition resulted in a smear running the length of the gel (data not shown). This suggests a mixture of varying size and may explain the presence of the minor quantities of the 43 kDa protein in peak 2 from the Sephacryl S-1000 column. When the  non-denaturing  gel was  electrophoresed after boiling in single 43 kDa  cut  into  1 cm strips and the  protein  eluted  and  S D S , the protein from each segment showed only a  band (results not shown).  Because of the confusion surrounding the structural properties of B. gingivalis HA, it was decided to look for fimbriae or other structural elements in the purified 43 kDa fraction. The purified HA was negatively stained with 5 % uranyl acetate and analysed by transmission electron microscopy. No fimbriae or any other polymerized structure was seen. Chemical composition of the HA Purified preparations of the HA were found to contain approximately 90 % protein and 10 % carbohydrate based on a dry weight basis. Staining boiled and unboiled samples for LPS proved to be negative.  62  Heat treatment Heating of the purified HA at 1 0 0 ° C for 1 min destroyed all haemagglutinating activity. The same result was obtained with whole cells of B.  gingivalis.  Inhibition of haemagglutination A number of putative erythrocyte  receptor analogues were  agglutination  exception of  by B.  gingivalis  tested for their  ability to  cells and by the purified HA.  inhibit  With the  fetuin at 100 ug per ml none of the compounds had any effect on  haemagglutinating activity (Table 1).  Sodium periodate, E D T A and C a C I  2  had no effect on haemagglutinating activity but the  reducing compound 6-mercaptoethanol was found to double the activity (Table Similar results were obtained for both whole cells and the purified HA of B.  2).  gingivalis.  This is not thought to be significant since it was within the realm of experimental error. Antisera raised against whole cells of B. gingivalis,  the purified haemagglutinin  the 43 kDa subunit were tested for their ability to inhibit haemagglutination of gingivalis.  serially diluted in P B S prior to adding the bacteria. Following incubation for  erythrocytes were added. As seen in Table 3, anti-43 antiserum had an  inhibitory titre of 128, the same as that for B. antisera.  B.  In the assay, the IgG fractions of these antisera were resuspended to 100 ug  per ml and 30 min,  and  Preimmune serum had a slight  gingivalis  and for the purified HA  effect with an inhibitory titre of 4. Similar  results were seen by Slots et al (214) with non-immune serum.  Immunoaold labelling The location and distribution of the HA on B. gingivalis  was determined  with antisera to  the purified 43 kDa as a probe. After incubation of the whole cells with the primary  63  Table 1.  Effect of a number of  putative inhibitors on haemagglutinating  activity.  effect on haemagglutinatin  activity  Substance  D-mannose  0  D-glucose  0  D-galactose  0  D-ribose  0  D-fucose  0  D-fructose  0  Lactose  0  Rhamnose  0  D-glucosamine  0  D-galactosamine  0  N-Acetylglucosamine  0  N-Acetylgalactosamine  0  L-lysine  0  L-Arginine  0  L- Leucine  0  Fetuin  -  a  (100u.g/ml)  Effect on activity designated as follows: enhancement (+),  no effect  inhibition  3  (-),  (0).  ^All the test substances were at an initial concentration of 100mM unless otherwise indicated.  64  Table 2.  Effect of various surface modifying substances on the haemagglutinating  activity of B. gingivalis.  effect on haemagglutinating activity  Substance  EDTA CaCI  0  (1 mM) 2  0  (10 mM)  0  Periodic acid (0.01 M) B-Mercaptoethanol (.2  a  %)  +  c  Effect on activity designated as follows: enhancement (+),  no effect  3  inhibition  (-),  (0).  '-'All the test substances were at an initial concentration of 100mM unless otherwise indicated. i n c r e a s e d HA activity two fold.  65  Table 3.  Effect of immune s e r a on haemagglutinating activity of B. gingivalis. 3  IgG  Anti-S. gingivalis Anti-43  kDa  Anti-HA Preimmune serum  a  Inhibitory  titre  1 28 128 128 4  l g G fractions were prepared by passage of the serum over a Protein A Sepharose  column. The IgG'S were resuspended to a final concentration of 100 u\g per ml for use in the inhibition assay. ^minimum antibody titre required to inhibit haemagglutination.  66  antiserum, the samples were reacted with goat-anti-rabbit IgG conjugated to gold beads. As seen in Figure 5 there was an even distribution of the adhesin over the cell surface as indicated by the gold bead labelling pattern. The labelling does not seem to be indicative of any structural entity such as fimbriae or pili. Nonimmune serum did not react  with  the surface of the B. gingivalis cells as no gold beads were seen when these preparations were viewed under the electron microscope.  Thin sections of B. gingivalis cells were prepared and reacted in similar manner  to  whole cells with primary and secondary antisera before being examined under the transmission electron microscope. As seen in Figure 6, most of the labelling is concentrated in the periplasm and on the outer membrane, while the cytoplasm is basically free of any labelling.  It is of interest to note that there appears to be clumping  of gold beads on the outside of the cells. These clumps may represent HA  which was  displaced during preparation of the specimen. A control using pre-immune serum as the primary antiserum was carried out and the result is seen in Figure 7; no labelling is visible. Enzvmatic treatment The effect of a variety of enzymes on the haemagglutinating activity of B. gingivalis was evaluated. B. gingivalis cells and the purified adhesin were incubated with the enzymes for 1 h after which samples were removed from the reaction mixtures and assayed for haemagglutinating activity. The result is summarized in Table 4. Pronase and proteinase K were the most effective in destroying the haemagglutinating activity of the whole cells and purified HA. The haemagglutinating activity titre dropped from 512 to 0 for the purified HA and from 256 to 32 for whole cells, a decrease of over 80 %. Of the other proteolytic  enzymes, trypsin  and  subtilisin were the  haemagglutinating activity reducing it by  most  active  in destroying  75 % for both whole cells and purified HA.  Papain, pepsin and chymotrypsin had no effect on the haemagglutinating activity of B.gingivalis cells but reduced the activity of the purified HA by 50 % .  67  Figure 5. Cells of B. gingivalis reacted with anti-43kd serum and then incubated with gold beads conjugated to goat anti-rabbit IgG. Cells are negatively stained with uranyl acetate. Bar 0.5 am.  Figure 6. Immunogold bead labelling of thin sections of B. gingivalis.  The sectioned  cells were reacted with anti-43 kDa serum and then incubated with gold beads conjugated to goat anti-rabbit  IgG. Thin sections were negatively  stained with  phosphotungstic acid. Arrow indicate clumps of gold beads on the outer membrane. Bar, 0.5 u.m.  69  Figure 7. Immunogold bead labelling of thin sections of B. gingivalis.  Sections were  reacted with preimmune serum and then incubated with gold beads conjugated to goat anti-rabbit IgG. Thin sections were negatively stained with phosphotungstic acid. Bar,  0.5 um.  70  Table 4. Effect of enzyme treatment on the and  haemagglutinating activity of intact cells  the purified haemagglutinin of B. gingivalis.  Haemagglutinating Titre Treatment 3  Intact cells  Purified  Haemagglutinin  Positive control  256  512  Pronase  32  0  Proteinase K  32  0  Papain  256  256  Pepsin  256  256  Trypsin  64  1 28  Chymotrypsin  256  256  Subtilisin  64  256  Neuraminidase  256  512  Lysozyme  1 28  256  Mixed glycosidases  256  512  Phospholipase C  64  1 28  Mutanolysin  256  512  Enzyme concentration was 1 mg per ml.  71  With  the  exception  haemagglutinating  of  phospholipase C  titre from 256  and  to 64 and 256  lysozyme to 128  which  reduced  the  for both cells and HA  respectively, none of the other enzymes tested had any inhibitory effect on the haemagglutinating activity on B. gingivalis. On S D S - P A G E , preparations of these latter two enzymes displayed several protein bands indicating that they were contaminated.  In addition to assaying for haemagglutinating digestions were  mixed with S D S - 6 M E ,  activity, samples from the  protein  boiled for 5 min and electrophoresed on 12 %  S D S - P A G E . The 43 kDa band was still present in  samples digested with pronase and  proteinase K. This was surprising in light of the fact that the haemagglutinating activity was destroyed in these latter two preparations. The meaning of this observation  is not  clear but it is possible that: (1) the proteolytic enzymes released a few amino or carboxy terminal amino acids but not enough for a difference in molecular weight to manifest itself; (2) the  enzymes prevented haemagglutinating activity by occupying the  binding site of the adhesin and not by hydrolysing the molecule; (3) the HA  is a minor  contaminant which coincidentally migrates with a similar sized non-haemagglutinin protein.  The  first  possibility was investigated by subjecting treated and untreated  isoelectric focusing.  samples to  In all cases the samples behaved in an identical manner, ie, they  had the same pi of 6.4.  The other possibility that the HA is a minor protein at 43 kDa is  doubtful for two reasons; first as explained previously in the absence of boiling no bands were seen on S D S - P A G E , and secondly in the adsorption experiments using red blood cells, all of the 43 kDa band was adsorbed as seen by S D S - P A G E .  To preclude the  possibility that the enzyme simply blocked the active site of the HA the  following set of experiments were carried out: (1)  the HA  was incubated with varying  amounts of proteinase K for 60 min after which the reaction mixtures were assayed for haemagglutinating activity, (2) the HA was incubated with 100 u.g of proteinase K and at  72  different time intervals samples were removed and assayed for haemagglutinationg activity.  As can be seen in Table 5, after 60 min, 10 ng of proteinase K reduced the HA  activity from a titre of 128 to 32 and 50 u.g totally destroyed all activity.  Table 6 ,  shows the time course of proteinase K inactivation of haemagglutination. There was no effect for 10 min, and then a gradual decline, 50 % of activity was lost at 20 min 88 % at 30 min and complete loss by 60 min. When these reactions were carried out with heat inactivated proteinase K or at 4^C there was no loss of haemagglutinating  activity.  The possibility that the enzyme caused steric hinderance and thereby inactivate the  HA  was not ruled out by these experiments. To address this problem protease inhibitors were used to inactivate proteinase K and the enzyme was then checked to see its effect on haemagglutinating activity. In preliminary experiments it was shown that hydrolysis of azocasein by 100 u.g of proteinase K was completely inhibited by 20 jxg of P M S F . The effect of P M S F treated proteinase K is shown in Table 7.  In the absence of inhibitor, all  HA activity was destroyed, but as the quantity of P M S F increased, so did the HA activity. P M S F by itself had no effect on HA activity. This indicates that proteinase K  had to be  active to destroy haemagglutinating activity.  Cloning of the haemagglutinin The surprising results observed when the HA was digested with proteolytic enzymes and analysed on S D S - P A G E introduced some confusion as to the exact identity of the HA and as such to elucidate this problem it was decided to clone the gene(s) encoding the HA from B. gingivalis into E. coli.  73  Table 5. Effect of increasing amounts of proteinase K on haemagglutinating activity of purified B. gingivalis haemagglutinin  ug proteinase K added  0  1 28  5  128  10  32  20  8  40  2  50  0  1 00  0  200  0  100  3  HA titre  3  (boiled)  1 28  Incubated for 60 min.  74  Table 6. Time course of HA inactivation by proteinase K.  Time of incubation (min)  HA titre.  0  1 28  5  1 28  10  1 28  20  64  30  16  45  2  60  0  1 20  0  3  3  Incubated with 100 ug of enzyme  75  Table 7. Effect of P M S F  a  on proteinase K digestion of the purified B.  haemagglutinin.  ug of PMSF added  HA titre  0  0  0  5  0  10  4  15  32  20  128  3 0  128  50  128  P M S F control  128  0  Boiled proteinase K + P M S F  a  D  c  128  Phenylmethyl sulfonylfluoride. Amount of P M S F added to 100 ug of proteinase k. No enzyme added, 20 ug of PMSF.  76  gingivalis  Genomic library Using Pst 1 enzyme to restrict B. gingivalis chromosomal DNA and the Pst 1 site of the vector p U C 18, a genomic library was constructed in E. coli JM 83. 5,500 transformants were  A total of  tested for the expression of antigens by the filter binding  enzyme immunoassay and for haemagglutinating activity by a biological assay.  Three  hundred and thirty seven clones were positive by the immuno assay ( Fig. 8), and two clones (1-3 and 1-49)  tested strongly positive for haemagglutination  in the biological  assay while a third clone gave weak activity ( Fig. 9).  Recombinant plasmids The size of the DNA inserts was determined by digesting the recombinant plasmids with Pst 1 enzyme followed by  electrophoresis on 1 % agarose gel .  both recombinant clones had  Figure 10 shows that  3.2 kb DNA inserts. Clone 1-49 was chosen for further  characterization.  Restriction analysis The 3.2 kb insert DNA was treated with a variety of restriction enzymes in single and double digestions. The physical map of restriction sites is shown in Figure 11. As can be seen Bam H1, EcoR 1, Pst 1 and several other common restriction enzymes did not restrict the insert DNA.  Southern blot analysis Southern blot analysis was performed to confirm that the DNA insert was derived from the B. gingivalis DNA. As can be seen in Figure 12, the 3.2 kb probe generated by Pst 1 digestion of the recombinant plasmid hybridized to identical size fragments in both B. gingivalis and the recombinant plasmid. not hybridize with the probe (Figure 12, lane 3).  77  DNA from  The control DNA from S. sanguis did  Figure 8. Colony immunoblot. White colonies, obtained by transforming E. coli JM 83 with B. gingivalis  DNA, were replica plated and lysed in situ.  Lysed colonies were blotted onto nitrocellulose paper and reacted with antiB. gingivalis  ATCC 33277 serum. A, B. gingivalis  (pUC18); C, E. coli  1-49;  78  cells; B, E. coli JM 83  Figure 9. Screening of recombinant clones obtained by transforming E. coli JM 83 with B. gingivalis  DNA for haemagglutinating activity. Recombinant colonies were  macerated in 0.1  ml of PBS and mixed with an equal volume of 2 % v/v erythrocytes.  The arrows indicate the positive clones.  i  A  A  3 1 4  2 A  5  5  y  8  9 M 0  11  A  B  ©©©©©0©0©©0@ ©©©©©©000® i>O©©©O0OO0O ©©©©©©0@@ ©©©©©©@©o©@@  79  Figure 10. Restriction endonuclease analysis of the haemagglutinin clones. 0.7 % agarose gel electrophoresis. Lanes :  recombinant  A, molecular weight markers,  Hind 111 digest of lambda DNA; B, pUC 18 digested with Pst 1,  C , E. coli  1-49  DNA; E, E. coli 1-49 digested with Pst 1, the arrow indicates the insert of 3.2 kb; F, E. coli 1-3 digested with  Pst 1.  80  Figure 11. Linear restriction map of the the cloned insert from recombinant  E. coli  1-49. Abbreviations: P, Pst 1; K, Kpn 1; S, Sma 1; B, Bgl 1; X, Xho 1; A, Sph 1; H, Hinc 11; C , Cla 1.  81  Expression of cloned antigen The polypeptide encoded by the insert DNA in recombinant 1 -49 was identified by S D S P A G E and Western immunoblotting. On 5 to 20 % S D S - P A G E a faint band at 43 kDa can be seen sometimes in the recombinant lysate which is not present in cell lysate of control  E . coli J M 83  (pUC  18).  Polyclonal antisera was  raised to  1-49  and  exhaustively adsorbed with control E. coli J M 83 (pUC 18). The adsorbed antisera reacted with a 43 kDa band  from the cell lysate of 1-49 (Fig. 13) but not with any  similar band in  E. coli JM 83 (pUC 18)  lysate. There are several bands that reacted  with the serum  although the serum was adsorbed extensively with E. coli J M 83 (pUC  18). These bands presumably represent E. coli antigens which are not expressed on the cell surface and would not bind antibody when whole cells were used to adsorb the antisera. There appears to be a band at about 18 kDa that is present in the recombinant lysate but not in the control lysate, however, in the original blot this was not evident. To determine where the majority of the 43 kDa protein was localized, outer membrane, periplasmic and cytoplasmic preparations of  1-49 were probed  with antisera to the  recombinant clone on Western blot. Most of the 43 kDa protein was found on the outer membrane and a small amount in the periplasm.  Antisera to  1-49 was  used  to  probe  B. gingivalis  cell lysate and the purified  haemagglutinin in a Western blot. As can be seen in Figure 14, this antisera only reacted with a  band at 43 kDa in both preparations. This demonstrates that immunologically  similar proteins are found in  1-49 and in B. gingivalis.  Prevalence of the 43 kDa protein in Bacteroides Several  species and strains of Bacteroides  were  checked by Western blot for the  presence of an immunologically similar 43 kDa protein using anti 1-49 antiserum and antiserum to the purified haemagglutinin.  Both antisera reacted with a 43 kDa  all strains of B. gingivalis but not to any band in the other species of Bacteroides (Fig.  14).  82  band in tested  Figure 12. Southern blot hybridization with the 3.2 kb Pst 1 restriction fragment of E. coli 1-49.  Panel A , DNA fragments separated by agarose gel electrophoresis.  Panel B, Hybridization with  the 3.2 kb biotinylated probe. Lanes: 1, molecular  weight markers, Hind 111 digest of lambda DNA; 2, Pst 1 digested B. DNA;  gingivalis  3, Pst 1 digested S. sanguis DNA; 4, Pst 1 digested E. coli 1-49 DNA.  B  83  Figure 13. Western blot of anti-1-49 antiserum against cell lysate of E. coli 1-49 using the GAR-HRP labelling system. Lanes: A, molecular weight markers (kDa); B, and  D, cell lysate of E. coli 1-49 10 ug and 20 ug of protein respectively; C, and E,  cell lysate of E. coli JM 83 (pUC18) 10 and 20 ug of protein respectively. The arrow indicates the 43 kDa band.  A B C 200  D  E  Figure 14. Western blot of BPB cell lysates and the the purified HA using anti-1-49 serum as the probe and using the G A R - H R P system for detection. Lanes:  A, cell  lysate of B. gingivalis 33277; B, cell lysate of B. intermedius; C, cell lysate of B. asaccharolyticus;  D, purified haemagglutinin of B. gingivalis 33277; E , F , G and H,  cell lysates of B. gingivalis W 12, W 50, W 83, and 2D respectively.  ABCD  E F G H  97.4  4 6  25.7  18.4  85  Immunogold electronmicroscopv To localize the cloned antigen and to provide additional evidence for its surface location in E. coli, antisera to B. gingivalis,  the haemagglutinin and to 1-49 were examined for  their ability to bind to the cell surface in immunofluoresence and immunogold labelling assays. Cells were incubated with one of the antisera, washed and then incubated with either gold beads conjugated to goat anti-rabbit IgG or FITC labelled goat anti-rabbit IgG. E. coli 1-49  reacted positively with all of the  antisera in immunofluorescence  labelling. There was uniform labelling of the cell surface, although the intensity of the labelling was low (results not shown). Immunogold labelling of the cell surface of 1-49 with anti-S. gingivalis serum is seen in  Figure 15. The recombinant protein appeared  to be distributed in a relatively even manner over the cell surface. The concentration of the  protein  appeared  low,  this  is  consistent  with  the  result  obtained  by  immunofluoresence, S D S - P A G E and Western blot analysis.  B. gingivalis was reacted with serum to 1-49 followed by gold beads conjugated to goat anti-rabbit IgG.  As can be seen in Figure 16, the gold labelling appeared to be uniform  over the cell surface. Another point of interest is the total absence of labelling of any structural material such as pili . and  Anti-S. sanguis serum did  not label B.  gingivalis  E. coli 1-49 cells. None of the antisera reacted with E. coli JM 83 (pUC 18)  control cells.  Similarly antisera to the 43 kDa subunit of the haemagglutinin reacted with surface antigen on E. coli 1-49 as seen in Figure 17. Again the labelling is sparse but uniform over the entire surface of the cell.  Inhibition by antisera Antisera to the purified HA, anti-43, anti-1-49 and anti-S. gingivalis were tested for their  inhibitory effect on the haemagglutinating activity of  result is shown in Table 8.  recombinant 1-49.  Anti-S. gingivalis anti 43 kDa and anti-HA  86  serum all  The  Figure 15. Immuno gold bead labelling of E. coli 1-49. Cells were reacted with anti-S. gingivalis serum followed by reaction with gold beads conjugated to goat anti-rabbit IgG and then negatively stained with uranyl acetate.  87  Bar, 0.5 u.m.  Figure 16. Immuno gold bead labelling of B. gingivalis. Cells were reacted with anti-E. coli 1-49 serum then incubated with gold beads conjugated to goat antirabbit IgG and negatively stained with 5 % uranyl acetate.  88  Bar, 0.5 am.  had inhibitory titres of 256. Anti-1-49 serum had an inhibitory titre of 64 whereas pre-immune serum had an inhibitory titre of 4.  Binding to buccal epithelial cells It is well established that B. gingivalis binds to epithelial cells. Thus it was of interest to know whether the HA is an epithelial cell binding adhesin. This question was answered by mixing bacteria with freshly prepared human epithelial cells. Microscopic analysis revealed that washed epithelial cells had an indigenous population of approximately 5 bacteria per epithelial cell (Table 9 ). E. coli JM 83 (pUC 18) did not possess adhesins and therefore did not bind  to the epithelial cells whereas E. coli 1 -49 which carried  the B. gingivalis HA bound in large numbers to the epithelial cells. Binding was strong as bacterial cells were not removed by vigorous washing. It was noted that there appeared to be two populations of epithelial cells, one which possessed receptors available to the adhesin and bound 1-49 and another population which either lacked the receptor or in which the receptor was masked.  The reaction of E. coli 1-49 with buccal epithelial cells was examined by scanning electron microscopy. As seen in Figure 18, E. coli 1-49 binds in large numbers to the cells whereas control E. coli JM 83 (pUC 18) did not bind at all. In another control it can be seen that B. gingivalis bound to the epithelial cells.  Epithelial cells with attached E. coli 1-49 and B. gingivalis cells  were sectioned and  viewed under transmission microscope. As seen in Figure 19 panel A , B. gingivalis cells bound to several villi-like structures on the epithelial cell surface. In panel B,  E. coli  1-49 bound to a number of points on the epithelial cell with plicate-like structures. Examination of the preparations under the electron microscope revealed the presence of two distinct populations of epithelial  cells; one supporting binding with the plicate  89  Figure 17. Immuno gold bead labelling of  E. coli 1-49. The recombinant E. coli  1-49 was reacted with anti-43 kDa serum and then incubated with gold beads conjugated to goat anti-rabbit IgG.  The cells were negatively stained with uranyl  acetate. Bar, 0.5 urn.  90  Table 8. Effect of immune s e r a  Antisera  on haemagglutinating activity of E. coli 1-49  3  Inhibitory  Anti-S. gingivalis  256  Anti-43  kDa  256  Anti-E.  CO//-1-49  64  Anti-HA  256  Preimmune serum  4  3  titre  IgG fractions were prepared by passage of the serum over a Protein A Sepharose  column. The IgG-s  were resuspended to a final concentration of 100 u.g per ml for  use in the inhibition assay. D  minimun antibody titre required to inhibit haemagglutination  91  Table 9. Adherence to buccal epithelial cells.  Organism added  Avo. no. of bacteria/epithelial cell -. 3  None  5 ±2  fl. gingivalis 33277  137±3 7  E. coli  b  1-49  70±2 6  E. coli J M 83 (pUC 18)  3  D  5±2  number of epithelial cells counted was 50. represents the number of bacteria bound to buccal epithelial cells after washing  and prior to addition of the test bacteria.  92  Figure  18. Scanning electron  micrograph of bacteria  bound to human buccal  epithelial cells. Bacteria and epithelial cells were incubated, washed and then coated with palladium gold. Panels : A, B. gingivalis JM 83 (pUC 18). Bar, 0.5 urn.  93  33277;  B, E. coli 1 -49;  C , E.  coli  like structures and those not supporting binding with smooth surfaces. The surface of the epithelial cell under went a morphological change when bacteria attached.  Characterization of the recombinant-epithelial cell adherence reaction The  nature of the the reaction of E. coli with buccal epithelial cells was investigated in a  series of experiments described in Table 10.  In all cases the recombinant was compared  to B. gingivalis.  of either  Proteinase K treatment  B. gingivalis  or E. coli 1 -49  destroyed their ability to attach to epithelial cells, similar treatment of the epithelial cells did not affect the number of bacteria that bound. This would indicate that a protein on the bacterial surface is responsible for the attachment.  Heating the bacteria and/or  epithelial  protein-protein  cells destroyed  all  binding  indicating  a  lectin  like  interaction. Several investigators have shown that arginine and lysine were capable of inhibiting the binding of B. gingivalis to erythrocytes. These two compounds were assayed for their ability to  inhibit the binding of B. gingivalis and E. coli 1-49 to epithelial cells. The  result is seen in Table 10. Neither of them proved to be effective in inhibiting the attachment of bacteria to epithelial cell.  Inhibition bv antisera Anti-fi. gingivalis, anti-43 kd, and anti-1-49 IgG were assayed inhibit the binding of B. gingivalis and E. coli 1-49  for their ability to  to buccal epithelial cells. The  result is shown in Table 11. The attachment of both bacteria was reduced by more than 70 % when incubated with 50 ug of each of the immunoglobulin. Pre-immune IgG had no effect on on the binding reaction.  Bacterial coaaareaatina adhesin A number of investigators have shown that B. gingivalis will bind to other species of bacteria. With the exception of Boyd and McBride (11) very little has been done to  94  Figure 19. Electron micrograph of bacteria attached to human buccal epithelial cells. The cells were embedded in epon-adaldite, sectioned and then stained with uranyl acetate. Panels: A, B. gingivalis;  B, E. coli 1-49. Bar, 0.5 um.  95  Table 10. Effect of protease, heat, and putative inhibitors on the attachment  to oral  epithelial cells.  Organism/treatment  Avg. no of bacteria bound per epithelial cell.  B. gingivalis (none)  137  E. coli 1 -49  70  (none)  E. coli JM 83 (pUC18) (none)  5  B. gingivalis (proteinase K 10 ug)  14  E. coli 1-49 (proteinase K 10 ug)  5  B. gingivalis (70°'C/10  16  E. coli  min)  1-49 ( 7 0 ° C /10 min)  5  B. gingivalis (lysine 100 mM) E. coli 1-49  117  (lysine 100 mM)  57  B. gingivalis (arginine 100 mM) E. coli  1-49  120  (arginine 100 mM)  56  96  Table 11.  Effect of immune l g G on binding to epithelial cells. a  Bacteria  Antisera  B. gingivalis  none  Avg. no. of bacteria/epithelial cell.  138  Anti-S. gingivalis  3 0  Anti-43 kDa  37  A n t i - E . coli 1-49  45  Preimmune  E. coli  a  1-49  124  none  68  Anti-S. gingivalis  18  Anti-43 kDa  20  A n t i - E . coli 1-49  21  Preimmune  64  l g G at 50 u.g per ml  97  identify the adhesin responsible. This study was coaggregating  adhesin  from  e.  gingivalis.  an attempt to isolate the bacterial  Prior to the isolation of the adhesin,  coaggregating tests were carried out with a number of oral bacteria. S. mitis was found to be the best coaggregating partner for B. gingivalis A T C C 33277.  Purification Chromatography The precipitate obtained after mixing B. gingivalis cells with a magnetic stirrer and precipitating the proteins from starting  material  the supernatant by ammonium sulphate  was used as the  for the purification of the bacterial coaggregating adhesin. This  material was fractionated on Sephadex G-100, G-200 and on Sephacryl S-400 columns in the presence and absence of sodium deoxycholate. None of the fractionation procedures resulted in the purification of the adhesin. In the absence of sodium deoxycholate in the eluting buffer the  coaggregating activity eluted in the void volume. On S D S - P A G E each  fraction displayed the same protein profile  as the starting material. In the presence of  sodium deoxycholate the material eluted in several peaks with bacterial aggregating activity distributed throughout the peaks. Bacterial coaggregating activity could not be recovered following chromatography on DEAE-Sepharose.  Adsorption assay The inability to effect a purification by standard chromatographic procedures led to the decision to try selective adsorption to S. mitis. Coaggregates form rapidly when S . mitis is mixed with B. gingivalis and for this reason it was thought that it would have good potential as a substrate for binding the solubilized adhesin. The dialyzed and concentrated 40 % ammonium sulphate the mixture  agitated for 10 min.  precipitate was incubated with S. mitis, and  Aggregates formed by interaction of the adhesin with  S. mitis were removed by centrifugation and more S. mitis cells were added to the supernatant. This process was repeated until no more aggregates formed. The  98  protein profiles of the adsorbed supematants are shown in Figure 20. As can be seen in lane A , the starting material contained several protein bands. After the first adsorption with S. mitis there was  a marked decrease in two bands, one at approximately 46 kDa  and the other at 53 kDA. After subsequent adsorptions, there was a further reduction in the concentration of these bands and after the fourth adsorption the 46 kDa band disappeared altogether. Other protein bands including the  band at 43 kDa show no  reduction .  The  aggregated  S. mitis cells were washed in H E P E S buffer and the  coaggregating adhesin was eluted. NaCl, 1 % S D S and  bacterial  Several different eluehts were tested including 1 M  0.15 M glycine-HCI-0.15 M NaCl pH 2.5. Incubation in the acidic  glycine buffer for 10 min gave the best results. Following neutralization, the eluate was dialysed and lyophilized. It was found to have had strong bacterial aggregating activity.  Only about 5 % of the bacterial aggregating activity was recovered in the glycine eluate. Attempts were made to remove more of the adhesin but it was found that  although this  was possible by prolonged exposure to 0.1 M glycine-HCI-0.15 M NaCl (pH 2.3), the activity of the eluted material was markedly reduced probably due to exposure to  the  low pH for protracted periods.  It seemed likely that the majority of the adhesin remained bound to the S. mitis. This possibility was investigated by immunofluoresence analysis. S. mitis cells recovered following glycine elution were incubated with antibody raised against B. gingivalis or the 46 kDa protein and then with FITC labelled goat anti-rabbit  IgG. The cells  fluorescesed strongly with either antibody indicating that significant quantities of the bacterial aggregating adhesin were still attached to the cells.  The eluate was analysed by S D S - P A G E and Western blot. On S D S - P A G E only two  99  Figure 20. Adsorption of the BA by S. mitis.  S D S - P A G E analysis of supernatant  recovered after removal of aggregates formed by incubating material containing solubilized the first  BA  with S. mitis.,  Lanes: A , starting material;  B, supernatant after  adsorption; C , supernatant after the second adsorption; D, supernatant  after the third adsorption; E, supernatant after the fourth  adsorption. 10 u.g of  protein was loaded in each lane except lane A which was loaded with 30 ug of protein.  A  B  100  C  D  E  bands were present, one at 43 kDa and another at 46 kDa. Similar results were seen when samples were immunoblotted against anti-B. gingivalis serum  (Figure 21). In  the absence of boiling no silver staining material entered the gel. The inability of the BA to migrate into the gel without boiling in S D S suggested that it existed in a multimeric form. As heating destroyed all bacterial aggregating  activity, other  solubilization  procedures were tried in order to obtain nondenatured subunits. Urea (8 M), guanidineHCI (6 M), LiCI (3 M), S C N " (3 M) and combinations of these compounds did not dissociate the BA molecule.  Characterization of the solubilized BA To try and gain an insight into the nature of the adhesin-receptor reaction a number of potential receptor analogues were tested to see if they were capable of blocking the reaction of the solubilized BA with S. mitis. As can be seen in Table 12 none of the amino acids tested at a  concentration of 100mM were effective in inhibiting the BA  activity of B. gingivalis. Of the sugars tested, only galactose and galactose containing compounds inhibited bacterial aggregation,  but the high  concentrations required ( 500  mM) suggests that they were acting in a non-specific manner.  Several surface modifying substances were tested for their effect on the aggregating  activity (Table  mercaptoethanol and  13).  EDTA,  CaCI  2  and  the  reducing  bacterial  agents  8-  dithiothreitol had no effect on the bacterial aggregating activity  whereas periodic acid decreased activity by 50 % . This is in direct contrast to the HA whose activity was enhanced by B-mercaptoethanol and unaffected by periodic acid.  Heating of B. gingivalis cells or the BA destroyed all bacterial aggregating activity.  101  at 8 5 ° C for 10 min or at 1 0 0 ° C for 1 min  Figure 21. Western-blot analysis of the supernatant remaining after adsorption of the BA with S. mitis. Samples were electrophoresed on 12 % S D S - P A G E . was  probed  with  anti-S.  gingivalis  serum.  Lanes:  A,  starting  The blot  material;  B,  supernatant after the second adsorption; C , supernatant after the fourth adsorption; D,  material  eluted from the aggregates with 0.15 M glycine-0.15 M NaCl, pH 2.3.  A B C D 9 7-4  •  6 8  •  4 5  •  2 5-7  •  1 8-4  •  102  Table 12. Effect of various compounds on aggregation.  Compound"  D- mannose  Effect on bacterial aggregating  0 _c  D-galactose D-ribose  0  D-fucose  0  D-fructose  0  Lactose  0  Rhamnose  0 _c  D-galactosamine D-glucosamine  0  N-acetylglucosamine  0  N-acetylgalactosamine  _c  L-Lysine  0  L-Arginine  0  L-leucine  0  Effect on activity designated as follows: enhancement  inhibition b  (-),  no effect  (+),  (0).  AII compounds were tested at a concentration of 100 mM unless indicated  otherwise. c  3  0  0  D-glucose  a  activity  Concentration: 500mM, inhibition 50 % .  103  Table  13. Effect of various substances on the bacterial aggregating activity of B.  gingivalis.  Substances  Effect on bacterial aggregating activity  EDTA (1 mM)  0  CaCI  (10 mM)  0  Periodic acid (0.01 M)  -  B-mercaptoethanol (.2 %)  0  Dithiothreitol  0  a  2  Effect on activity desugnated as follows: enhancement (+),  inhibition (-), D  50 mM  b  no effect  (0).  periodic acid inhibited the bacterial aggregating activity by 50 % .  104  3  Enzvmatic digestion of the adhesin The effect of  proteolytic enzymes on the  whole cells and the  bacterial aggregating activity of B. gingivalis  purified BA was evaluated. The enzymes were incubated with the  bacterial cells or adhesin at 3 7 ° C for 1 h after which samples were assayed for bacterial aggregating activity. For B. gingivalis, the enzymes were removed by washing the cells three times with H E P E S buffer. The enzymes were not removed from the reaction mixtures containing the purified adhesin. In these cases bacterial aggregating activity was measured at activity.  4 ° C . As can be seen in Table 14  Pronase and proteinase K  all of the proteases tested reduced  eliminated all the bacterial aggregating activity of  the purified adhesin. Of the other proteolytic enzymes trypsin was the most active, reducing the  bacterial aggregating titre by over 80 % for both the whole cells and the  purified adhesin. Neither lysozyme, mixed glycosidases nor phospholipase C had any effect on the activity. Treatment of S. mitis with the same enzymes had no effect on the bacterial aggregating activity.  S D S - P A G E analysis of the proteinase K and pronase digestion mixtures is seen in Figure 22. The 46 kDa band was hydrolysed completely leaving the 43 kDa band. This hydrolysis was accompanied by a loss in bacterial aggregating activity, indicating that the 46 kDa protein is associated with the bacterial binding activity of B. gingivalis.  Inhibition of bacterial aggregating activity bv antisera To provide more evidence that the 46 kDa protein is the BA, specific antiserum raised against the 46 kDa protein which had been eluted from an S D S - P A G E was assayed for its ability to block bacterial aggregation. The antisera was serially diluted and preincubated with B. gingivalis cells prior to  the addition of S. mitis. As can be seen in Table 15,  the inhibitory titre of the anti-46 kDa serum  and anti-6. gingivalis serum  was 256.  In comparison, pre-immune serum and anti-43 kDa serum had no inhibitory effect on the bacterial aggregating activity.  105  Table 14. Effect of enzymatic treatment on the bacterial aggregating activity of B. gingivalis and the purified BA .  Bacterial Aggregating Titre. Enzyme.  3  Intact cells.  Purified adhesin.  None  256  256  Pronase  16  0  Proteinase k  8  0  Papain  64  32  Pepsin  1 28  64  Trypsin  32  32  Chymotrypsin  1 28  1 28  Lysozyme  256  256  Mixed glycosidases  256  256  Phospholipase C  256  256  E n z y m e s concentration was  100 ug per ml.  3  106  Figure 22. SDS-PAGE analysis of the enzymatic digestion of the bacterial aggregating adhesin from B. gingivalis 33277. 10 ug of enzyme was used in each of the digestion mixture. Lanes: A, pronase digestion; B, proteinase K digestion; C, trypsin digestion; D, mixed glycosidase digestion; E, phospholipase C digestion; F, lysozyme digestion; G, pronase control; H, proteinase k control; I, trypsin control; J, phospholipase C control; L, undigested control of bacterial aggregating adhesin. The arrow indicates the 46 kDa protein.  107  Table 15. Effect of immune serum on bacterial aggregating activity.  Treatment  Inhibitory  Anti- B. gingivalis  Titre.  128  Anti- 43 kDa  2  Anti- 46 kDa  25 6  Preimmune serum  0  a  l g G fractions contained 100 ug per ml of protein.  108  An Elisa type assay was developed to provide inhibitory effect  of the antisera.  a more quantitative estimate of  the  Both antisera were resuspended to 100 ug per ml of  IgG and serially diluted in H E P E S buffer. B. gingivalis  was added and the mixtures were  incubated for 1 h at room temperature with shaking. 100 u,l of the  mixture was added to  wells of a microtitre plate coated with S. mitis cells and incubated for 1 h for binding to take place. The unbound cells were removed by washing and primary antiserum (anti46 kDa) was added. After incubation, unbound antisera was washed away and goat antirabbit  IgG conjugated to alkaline  preincubation of the B. gingivalis to the immobilized  phosphatase was added. As seen in Table  16  with anti-46 kDa serum eliminated its ability to bind  S. mitis. Preimmune serum had no effect.  Immunogold labelling To localize the BA and determine if it was associated with  any particular structure on  the cell surface, immunogold labelling was carried out on whole cells and thin sections of B. gingivalis.  Figure 23 shows whole cells of B. gingivalis  reacted with anti-46 kDa  primary sera followed by gold beads conjugated to goat anti-rabbit IgG.  In this electron  microphotograph it can be seen that gold beads are distributed evenly over the cell surface.  Numerous fields were examined but there was no evidence for the labelling of  fimbriae  or pili.  When thin section of B. gingivalis  were reacted with antisera to the 46 kDa protein  (Fig. 24) most of the gold beads were located on the outer membrane or the periplasmic space. Clumps of gold beads can be seen in areas around the thin sections suggesting that the adhesin may have been dislocated during cell processing.  109  110  Table 16. Effect of anti-46 kDa antiserum on the binding of B. gingivalis to immobilized S. mitis.  — 405 nm Dilution of antisera  Antisera  3  Anti-46 kDa  Preimmune  2  0.08  1.0  4  0.20  0.95  8  0.35  0.90  16  0.66  0.85  32  0.80  0.90  64  0.83  0.90  1 28  0.96  0.95  256  1.0  1.0  512  1.0  1.0  3  Initial concentration of antisera was 100 ug protein per ml.  110  Figure 23. Immuno gold bead labelling of B. gingivalis. Cells were reacted with anti- 46 kDa serum and then incubated with gold beads conjugated to goat anti-rabbit IgG. Cells were  negatively stained with uranyl acetate. Bar, 0.5 urn.  111  Figure 24. Immuno gold bead labelling of thin sections of B. gingivalis. The sections were reacted with anti-46 kd serum, incubated with goat anti-rabbit IgG conjugated to gold beads and then negatively stained with 5 % uranyl acetate. The arrow indicate the accumulation of the gold beads in the outermembrane. Bar, 0.5  112  urn.  Vesicles  The possibility that vesicles released by B. gingivalis retained the adhesive properties of the whole cell was investigated. It was decided to assess the role of vesicles in mediating attachment  of S. sanguis to model tooth surfaces. The model chosen was  hydroxyapatite (HA) to which had been adsorbed a pellicle derived from serum (SeHA). Serum was used to coat HA because it is  similar in  composition to  crevicular fluid  which bathes surfaces in the periodontal pocket. S. sanguis was chosen because it is an early colonizer and is found in significant numbers in subgingival plaque  Isolation of vesicles Vesicles of B. gingivalis 33277 were isolated from 3 day old culture supernatants by ultrafiltration followed by ultracentrifugation.  The yield was found to be 25 mg (dry  weight) of vesicles per litre of culture. Electron microscopic examination vesicular preparation revealed that it was 25) and free of fimbriae.  of the  homogeneous in regard to morphology (Fig.  The average size of the vesicles was approximately 25 nm.  The protein and LPS profiles of the  outer membrane and vesicles of B. gingivalis are  shown in Figure 26. They are similar supporting the contention that the vesicles are derived from the outer membrane.  Vesicles also have haemagglutinating and bacterial  aggregating activity.  Effect of vesicles on attachment of S. sanguis 12 to SeHA Figure 27 panel A shows the attachment of vesicles to vesicles to S e H A (SeHA-V) on the binding of  SeHA . The result of adding  S. sanguis is shown in Table 17.  In the  8 presence of vesicles the number of S. sanguis attached was 4.8 x 10  cells.  absence of vesicles the number attached decreased at least 10 fold to 4.6 x 1 0 SHA  beads had 8.0 x 1 0  7  In the 7  cells.  bacteria attached. When vesicles were included this number  113  Figure  25.  Transmission electron  micrograph  of  a  vesicle  preparation  of  B. gingivalis prepared by ammonium sulphate precipitation of 5 day old culture supernatant.  Bar 50 nm.  1 i  fifes  1 if  114  . '  •-  v  -A-  Figure 26. B. gingivalis stained  for  protein  SDS-PAGE profile of vesicles and outer membrane.. Gel 1, with  silver  nitrate.  Gel  2,  stained  with  silver  for  lipopolysaccharide. Lane A, vesicles; Lane B, outer membranes. 10 and 25 ug of material was loaded in lanes A and B respectively.  A B  A  B  was 2.8 x 1 0 ° cells, an increase of 3.5 times. The  electron micrographs seen in Figure  27 illustrates the differences described in Table 17.  In the absence of vesicles very few  bacteria attached to SeHA (Fig. 27 B). However, in the presence of vesicles the number of S. sanguis increased significantly (Fig. 27 C).  Kinetics of the bindino reaction A typical binding isotherm for the adherence of S. sanguis to S e H A vesicles is seen in Figure 28.  with and without  The number of S. sanguis which adsorbed to SeHA-V  beads increased as the number of bacteria added increased. Saturation of the beads did not occur until the number of S. sanguis added exceeded 2.5 x 1 0  8  cells. In the absence  of vesicles a similar pattern of binding was seen but the total number of S. sanguis bound was 10 fold less.  When the data for the adherence of S. sanguis to SeHA with and without vesicles were analysed by the Langmuir equation, the curve indicated independent and identical binding sites (Figure 29). Reaction parameters calculated from the Langmuir isotherm (Table 18 ) show that the number of binding sites for the vesicle treated SeHA was about four times higher than that of untreated SeHA. The dissociation constant for vesicle treated SeHA was more than twice that of untreated beads indicating stronger attachment of S . sanguis to the vesicle covered SeHA.  Analysis of the adherence data in the form of a Scatchard plot (Figure 30 ) revealed a curve with a negative slope indicating that the sites of interaction were homogenous and that there was no positive cooperativity between the sites.  The effect of increasing the quantity of vesicles on the attachment of S. sanguis to the SeHA was evaluated and the result is shown in Figure 31. As can be seen the number of bacteria bound increased as the quantity of vesicles added increased. The curve began to plateau after 100 u\g of vesicles was added and the reaction was  116  Table 17. Influence of vesicles on the binding of S. sanguis to SeHA and SHA  Treatment  # of bacteria attached  SeHA  4.6 x 10  7  SeHA-V  4.8 x 10  8  SHA  8.0 x 10  7  SHA-V  2.8 x 10  8  117  Figure 27.  Scanning electron micrograph of S e H A incubated with (A) B. gingivalis  vesicles, (B) S. sanguis, and (C) B. gingivalis vesicles followed by S. sanguis. Hydroxyapatite beads were incubated with serum followed by vesicles and bacteria then coated with palladium gold.Bar, 1 um.  Figure 28. Adsorption isotherm of S. sanguis  bound to SeHA with and without  vesicles. Hydroxyapatite beads were incubated with serum followed by vesicles and then bacteria. B, bound cells ( B  ) SeHA with vesicles; (  ( x 10 ) ; U, unbound cells (x 10 7  •  ) SeHA without vesicles.  30 -\  119  8  ).  Figure 29. Langmuir adsorption isotherm for the adherence of S. sanguis to SeHA, and S e H A pre-incubated with B. gingivalis vesicles. Hydroxyapatite  beads were  incubated with serum followed by vesicles and bacteria. B, bound cells x 10 ; U, 7  unbound cells x 10 . 8  ( •  ) SeHA with vesicles; ( •  o  H  0  >  ) SeHA without vesicles.  1  •  10  1  20 U  120  •  1  30  '  1  40  Table 18. Langmuir isotherm parameters for the adsorption of S. sanguis to SeHA and SeHA-V.  N  K  SeHA (without vesicles)  8.4 x 1 0  7  SeHA (with vesicles)  3.5 x 1 0  8  r  1.2 x 10  2.8 x 10"  0  9  9  2  0.98  N, Number of binding sites; K, dissociation constant; r, correlation coefficient.  121  Figure 30.  Scatchard adsorption isotherm for the adherence of S. sanguis to SeHA  pre-incubated with B. gingivalis vesicles. Hydroxyapatite  beads were  with serum followed by vesicles and bacteria. B, bound cells x 10 cells  x 10  8  7  ,  U, unbound  .  0.20-1  0.15-  0.10-  0.05-  o.oo-I 0.0  • '  i 1.0  1 2.0  i B  122  i  incubated  1— 3.0  Figure 31. Adhesion of S. sanguis to of B. gingivalis  SeHA  pre-incubated with  vesicles. Hydroxyapatite beads were incubated  by vesicles and radiolabeled bacteria.  30  -i  CO  o  mg of u e s i c l e s a d d e d .  123  increasing  quantities  with serum followed  Table 19. The effect of heat and putative inhibitors on the attachment of S. sanguis to SeHA-V beads.  Treatment  or inhibitor  # of cells attached  (x10 ) 7  none (without vesicles)  4.1  none (with vesicles)  25.7  heated bacteria ( 7 0 ° C / 1 0 min)  16.3  heated vesicles ( 7 0 ° C / 1 0 min)  4.1  sialic acid (50  lactose (100  arginine  (100  u,g/ml)  21.5  mM)  15.8  mM)  13.3  124  eventually  saturated after the addition of 400 u.g of vesicles to the serum coated  hydroxyapatite beads.  Experiments were carried out to find the optimum time of incubation for adsorption of vesicles to S e H A and the optimum time for attachment of S. sanguis to SeHA-V. Previous to this all incubations were carried out for 2 h. The optimum time for adsorption of vesicles  to S e H A and for the attachment of S. sanguis to S e H A - V was found to be 1 h.  3 7 ° C was found to be the optimum temperature for binding.  Pretreatment of the vesicles at 7 0 ° C for 10 min completely eliminated their ability to promote the attachment of S. sanguis to S e H A (Table 19).  The heat treatment did not  affect the ability of vesicles to adsorb to S e H A as evaluated by scanning electron microscopy.  Heating  S. sanguis at 7 0 ° C for 10 min reduced its ability to bind to  vesicle treated S e H A by 35 %.  Several substances were tested for their ability to inhibit the adherence of S. sanguis to SeHA-V.  Of these, arginine and lysine at 100 mM concentration reduced the binding by  48 and 38 % respectively (Table 19).  Sialic acid reduced binding by 16 % .  Spectrophotometric adherence assay A number of species and strains of oral streptococci were assayed to measure the effect of vesicles on their binding to SeHA. As can be seen in Table 20, the attachment of two of the three strains of S. salivarius were markedly enhanced by the presence of vesicles. Of the of S. mutans tested only M2B binding was enhanced. In the case of S. sanguis, only strain 12 showed enhanced binding.  This concludes the study on the adherence reactions of B. gingivalis. The HA has been isolated and cloned, the bacterial aggregating adhesin has been identified and purified and the role of vesicles in mediating attachment of oral streptococci to SeHA has been studied.  125  Table 20. Spectrophotometric assay of the adherence of oral streptococci to vesicles immobilized on SeHA.  Number of bacteria attached (x  10 ) 8  Organism without vesicles  S. sanguis 12  11.0  14.0  NY101  9.83  10.7  E  15.5  16.7  J11  8.73  9.4  15.5  17.8  K3  12.0  16.7  HB  10.5  14.4  19645  11.8  12.4  B13  9.4  9.4  M2B  7.5  11.4  S. salivarius  S. mutans  with vesicles  K8  Lipopolysaccharides  One of the original plan of this study was to determine whether LPS of B. gingivalis was directly involved in haemagglutination and bacterial aggregation. For this reason LPS were isolated and preliminary chemical characterization was completed. In terms of their biological properties no evidence could be found for a role in adherence.  Analysis of L P S from Bacteroides LPS from the oral BPB was prepared by the method of Darveau and Hancock (35). This material was found to constitute approximately 4 % of the bacterial dry weight. Spectra analysis of the L P S The  (250 u.g/m!) indicated that they were free of nucleic acid.  size range of L P S from B P B was investigated by sodium-dodecyl sulphate  polyacrylamide gradient gel electrophoresis (5 to 20 %). all of the LPS preparations displayed a prominent  As can be seen in Figure 32,  silver staining band which ran  close  to the dye front. This represent the lipid A portion plus the core region of the L P S . All of the B. gingivalis strains (lanes 1-5) display the typical ladder like  profile of smooth  type L P S . The molecular weight range between core material and the 200,000 molecular weight marker with a concentration of L P S material between the 18-25 kDa and 60-70 kDa range. Strain 2D appears to have more bands in the high molecular weight area than strains W 12 and W 50, this result was found to be the same for all LPS preparations of these two organisms.  The two strains of B. intermedius  ( lanes 6,7)  have very similar profiles on S D S -  P A G E , but they have fewer number of high molecular weight bands when compared to the LPS of the B. gingivalis  strains. B.  asaccharolyticus  (lane 8) has L P S of three  molecular weight ranges, between 10 and 12 kDa, 20 and 25 kDa and 60 and 70 kDa.  127  Figure 32. S D S - P A G E analysis of L P S from black pigmented Bacteroides. Lanes 1 through 5, B. gingivalis W 12, W 50, W 83, 2D and 33277 respectively; Lanes 6 and 7, B. intermedius BMA and 2332H respectively;  Lane 8, B. asaccharolyticus;  Lane 9, B. denticola; Lane 10, B. levii; Lane 11, B. melaninogenicus;  Lane 12,  E. coli.  12 11 10 9  8  7 6 5 4 3 2 1  200^  4 3*-  -I  18-4  12-3-  128  B. denticola has material.  a large amount of core and very little of the higher molecular weight  The same result was found for the  L P S of B. levii. This would indicate  that  the ratio of carbohydrates to lipid is low in the L P S of these two species when compared to the other BPB .  Chemical analysis of LPS The L P S were characterized  with regard to their carbohydrate and lipid composition. All  analysis were done in duplicate and on two different samples. The quantity of protein present was less than 0.2 % of the total dry weight.  Table 21 gives the results obtained when the nine LPS-s were assayed for carbohydrates and lipids. Total carbohydrates was measured by the phenol sulphuric method (43). total percentage of carbohydrates for the four strains of B. varying between 60 % for W 83 other BPB averaged from  to 67 % for W 12.  The  gingivalis  were  The  similar  total carbohydrates of the  35 % for B. denticola to a high of 55 % for B.  intermedius  BMA.  The methylpentoses B. intermedius  varied between a high of 25 % for B. denticola to a low of 4 % for  2332H.  The lipid portion of B. gingivalis displayed more variability when compared to the total carbohydrates with a high of 30 % for W 50 and a low of 19 % for 2D. All other BPB had higher percentage of lipid A with a high of 45 % for B.  denticola  to a low of 27 % for  t  B. intermedius BMA.  No 2-keto-3-deoxyoctonate (KDO) or heptose were found by chemical analysis in any of the B P B lipopolysaccharides analysed, whereas L P S from E. coli contained these two sugars.  129  130  Table 21. Chemical composition ( in percentages ) of LPS from oral B P B .  Organism  Composition (%) Pro . 3  B. gingivalis W 12  (CH 0)n  b  2  Me. Pent . 0  FA . d  0.1  67  13  21  W50  0.05  63  11  30  W 83  0.1  60  13  22  2D  0.1  60  17  19  B. asaccharolyticus  0.1  48  9  B. intermedius BMA  0.1  55  0.1  48  4  33  B. denticola  0.05  35  25  45  B. levii  0.1  40  13  40  2332H  3  protein,  D  total carbohydrates, ,  c  methylpentoses,  130  8  fatty acid.  30 27  Gas liquid chromatography of sugars from LPS of Bacteroides The L P S ' S were hydrolysed in 4 N HCI under nitrogen in glass vials, derivatised to their alditol acetates and analysed by gas liquid chromatography. 22.  The result is shown in Table  The sugars present in the core and O-antigenic side chains of LPS's were rhamnose,  mannose, galactose, glucose, galactosamine and glucosamine. B. denticola and  B. levii  were the exceptions in that they were devoid of mannose. In the L P S of B. gingivalis an unknown peak was found with retention time similar to that of the internal standard myoinositol,  but its identification was not known for certain  so it was classified as an  unknown.  Fatty acid composition The fatty acids from the L P S of 9 Bacteroides were hydrolysed and methylated before being analysed by gas liquid chromatography. As can be seen in Table 23, the major fatty acids  were  myristic ( C  1 4 : 0  )  palmitic ( C  1 6 : 0  ),  heptadeconoic ( C  1 7  IS0  )  and  stearic ( C - ^ . Q ) . For the B. gingivalis strain there was an unknown with retention time close to that of myristic acid which had a retention time of about 10 min.  Only B. gingivalis showed the presence of pentadeconic and heptadeconic acids although they were present in amounts less than 5 % of the total fatty acid content.There was considerable variation within the species in the quantity of any particular fatty acid. Myristic acid was present at a concentration of 32 % for B. levii and 25 % for B. intermedius  BMA, all of the other B P B had concentrations between  Palmitic acid was present in the highest concentration gingivalis  11 and 20 % .  in all of the B P B tested. B.  W 50 had the highest concentration at 40 % followed by B. gingivalis W 12  at 35 % and B. levii at 30 % . Stearic acid was present in B. gingivalis concentration of 43 % folloiwed  W 83 at a  by B. denticola at 40 % .  B. intermedius showed the presence of pentadeconoic acid which was not present in any other species.  131  Table 22. Carbohydrate compositiorTof lipopolysaccharides from B P B .  Bacteria  Rha  B.gingivalis W 12  6.5  W50  Man  h NH glu  h NH gal  Unl  Gal°  Glu  10.3  12  29  +  +  25  13.7  2.1  8.5  41  +  +  27  W83  4.5  4  28.5  12  +  +  30  W2d  5  4.4  13.3  60  +  +  13  B. asaccharolyticus  4  9  18  +  -  B. intermedius 2332  2  3  4.3  86  +  +  -  7.5  10.8  21  54  +  +  -  B. denticola  6  -  18.4  75  +  -  -  B. levii  23  3  55  +  BMA  a  0  32  d  2  +  e  2  As a percentage of the total carbohydrate present. a  rhamnose,  D  m a n n o s e , galactose, glucose, c  d  e  galactosamine, *-glucoasmine,  9 unidentified sugar with identical retention time. +, present. -, not present. h, glucosamine and galactosamine were not quantitated.  132  f  _  Table 23.  Fatty acid composition of LPS from BPB.  Bacteria  % of total fatty acid C 14  B. gingivalis  B.  a  C15:1  C16  D  c  C17:1 C l 7 d  e  C18  f  C18:1 U1 a  h  112'  W 12  1 3  5  35  3  7  1 7  -  1 7  -  W50  11  2  40  2  4  21  -  11  -  W83  15  7  1 2  2  6  43  -  1 0  -  2D  1 4  3  1 9  4  11  30  -  1 3  -  1 7  -  27  -  -  23  -  7  20  BMA 2 5  -  1 7  -  -  20  1 3  -  1 7  2332H 1 9  -  1 8  -  15  22  -  -  20  -  -  40  -  -  -  1 7  11  _  9  asaccharolyticus  B. intermedius  B.  denticola  20  -  27  B.  levii  32  _  30  -, not present. 3  myristic acid,  e  heptadecanoic acid, * stearic acid,  1  D  pentadecanoic acid, a  c  palmititic acid, oleic acid,  unidentified fatty acid.  133  n  d  methyl heptadecanoic acid,  unidentified fatty acid,  Western electroblottina analysis LPS  separated  by S D S - P A G E  and transferred  nitrocellulose paper by the Western blot  from the  electrophoretogram  technique (17) was reacted with polyclonal  antisera to whole cells of different strains and species of Bacteroides. gingivalis  strains reacted only with B. gingivalis  obtained on S D S - P A G E (Fig. 33). with  reacted  with LPS from  L P S from other strains.  134  Antisera to B.  L P S despite the similar profiles  when compared to other species such as B.  Antisera to 33277  to  asaccharolyticus  all B. gingivalis strains but not  Figure 33. Reaction of LPS from BPB with antisera to B. gingivalis through 4, B.  gingivalis  asaccharolyticus; Lane 8, B. denticola;  33277.  Lanes 1  W 50, W 83, 2D and 33277 respectively; Lane 5,  Lanes 6 and 7, B.  intermedius  BMA and 2332H respectively;  Lane 9, B. levii; Lane 10, E. coli.  135  B.  Discussion  Haemaqalutination Haemagglutination is a distinctive characteristic of B. gingivalis which  differentiates it  from other asaccharolytic B P B species (175). It is generally accepted that bacteria that haemagglutinate possess specific cell surface adhesins and that the presence of these adhesins can be an essential virulence determinant of  many infectious diseases. Since  the pathogenicity associated with B. gingivalis stems from adhesive interactions with eukaryotic and  procaryotic cells (214) the adhesins of this organism are of particular  interest.  Recently, there have been three reports on the purification of the B. gingivalis H A (100,163,176), but analysis of the purified material  by S D S - P A G E  revealed that  several protein bands were present and it was not possible to link a specific band to the HA.  In the study reported here B. gingivalis HA was purified and shown to be a protein  with an apparent molecular weight of 43 kDa.  The size of the native HA was found to be in excess of 1 0  6  daltons as was indicated by its  elution in the void volume of a Sephacryl S-1000 column and by its inability to enter a 5 % polyacrylamide gel if it had not been boiled in S D S . Molecules of this size are indicative of structures such as pili or fimbriae suggested that HA was associated with  and indeed previous reports had  pili (175). However, no evidence was found to  corroborate this, on the contrary evidence obtained from this study suggests that the HA is not on a pili. For example, transmission electron microscopic examination of the purified HA and cells of B. gingivalis 33277 did not revealed any fimbriae. Immunogold labelling of  B. gingivalis with either anti-43 kDa  or anti-HA sera did not show the  presence of these structures. Fimbriae were not detected in the purified preparations of the HA.  136  Attempts were made to obtain active subunits of the HA by methods which usually dissociate fimbriae of gram negative organisms. Fimbrial proteins from E. coli are insoluble in their native form but may be disaggregated by guanidine hydrochloride and HCI or by treatment with detergents and other chaotropic agents (16). However, none of the treatments tried had any effect on dissociating the B. gingivalis HA complex with the exception of boiling in S D S . Prolong exposure to this detergent at 37 ° C had no effect in dissociating the HA. This indicates that the bonds holding the subunits of the HA together are very strong and in that sense are similar to adhesins of A. salivarius  (242).  viscosus  and S .  The HA has all of the properties of a porin protein in terms of its  molecular weight and abundance on the cell surface, however there is not enough evidence to conclude that it is a porin. Pronase and proteinase K destroyed the haemagglutinating activity of the purified HA, yet on S D S - P A G E there was no perceivable change in the migration or quantity of the 43 kDa protein. In E. coli a similar phenomenon was seen for a non-fimbrial haemagglutinin with a molecular weight in excess of 1 0  6  daltons. Protease treatment destroyed the  haemagglutinating activity of the molecule but the by  S D S - P A G E (75).  molecule was not hydrolysed as seen  An explanation for this reaction could be (1) the'enzymes only  removed a few amino acids from either the amino or carboxy end of the protein and this is not enough for a difference in molecular weight to manifest itself on S D S - P A G E , (2) there was a minor protein at 43 kDa which was masked by the 43 kDa protein, (3) the proteolytic enzymes undergo self degradation and the fragments acted as inhibitors by occupying the active site of the adhesin, or (4) the enzymes do not hydrolyse the HA but in some manner block the active site. The removed  possibility that a few amino acids were  by the enzymes was not thought to be the answer since both digested and  undigested HA had the same pi although it must be noted that if the amino acids removed by the enzymes were not charged then there would be no change in the pi. The chance that a minor protein of 43 kDa is present is remote since the red blood cell were shown to adsorb all of the 43 kDa protein. The third and fourth possibilities  seems unlikely as  proteases preincubated before being added to the HA showed no inhibitory activity.  137  The strongest evidence implicating the 43 kDa protein as the HA was provided by cloning the HA gene into E. coli. Two clones with haemagglutinating activity were identified and one, 1-49 was characterized. This recombinant produced a B. gingivalis protein with a molecular weight of 43 kDa as seen by S D S - P A G E and Western blot analysis. The 43 kDa protein was immunologically similar to the 43 kDa subunit of the HA of B. gingivalis as shown by western blot. Immunogold and immunofluoresence staining with anti-43 kDa or anti-1-49 sera showed that this molecule was present on B. gingivalis and the recombinant  1-49. Anti 1-49 serum inhibited the haemagglutinating  activity of B.  gingivalis and likewise anti-43 kDa serum inhibited the haemagglutinating activity of 1-49. The two proteins, the 43 kDa from B. gingivalis 1-49  and the 43 kDa protein from  behaved similarly when exposed to protease. They both lost their ability to  agglutinate erythrocytes but their mobility in S D S - P A G E was not affected. Both were sensitive to heating.  The surface location of the HA in E. coli 1-49 suggest that B. gingivalis  surface  antigens can be processed as well as expressed in E. coli. The insert DNA probably codes for a leader sequence which was removed after which the protein was transported to the surface of the outer membrane of the recombinant.  B. gingivalis colonizes the sulcus a region which is continuously flushed by the serumlike crevicular fluid. Previous studies by Slots and Gibbons (214) had shown that serum was inhibitory to haemagglutinating activity of B. gingivalis and therefore cast doubt upon the significance of HA reaction in mediating binding in vivo. IgG prepared from nonimmune serum when compared to IgG prepared from serum to the HA, the 43 kDa, E. coli 1-49 and to B. gingivalis displayed only limited inhibitory activity and confirms the observation of Boyd and Mc Bride that serum has only a small effect on binding  (11).  138  The haemagglutinating activity of a number of  bacteria is inhibited by sugars such as  mannose, lactose and galactose (47,118 ) however, in this study none of the putative receptor analogues tested gingivalis.  were found to be inhibitory to the activity of the HA of B.  Arginine and lysine were reported to be receptor analogues for the  erythrocytes (100,176) but  the high concentration required suggests the reaction is  non specific.  The immunogold staining pattern seen when B. gingivalis was reacted with either anti43 kDa or anti-1-49 sera suggested that the HA was evenly distributed over the surface of  the cell. Thin section analysis of the cell revealed that the majority of the gold beads  were localized on the outer membrane and in the periplasm. This is consistent with the work of others who also reported that the adhesin was distributed in an even manner over the cell  surface (163,176).  Immunogold bead labelling with anti-43 kDa serum showed that the protein was present on the surface of all strains of B. gingivalis but not on other B P B . This is consistent with the inability of these cells to haemagglutinate and with the lack of immunological cross reactivity  between  species of B P B . There was variation  in the intensity of  fluoresence  suggesting that the different strains of B. gingivalis  levels of the  43,000 Mr protein  express  different  or that there is a difference in the accessibility of the  protein to the antibody.  In the oral cavity, the ability of B. gingivalis important in its colonization of the sulcus.  to attach to epithelial cells may be  B. gingivalis has been shown to be capable of  attaching to epithelial cells giving rise to the question of whether the HA and epithelial cell binding adhesin are the same. The results obtained from this study show that the HA and the epithelial cell binding factor conclusion  are:  haemagglutinating  (1)  anti-43  are one and the same. Evidence that supports this  kDa,  anti-HA  and  anti-1-49  and binding to epithelial cells; (2)  139  IgG  inhibited  heat destroyed both  both activity  simultaneously recombinant  (3)  protease  E. coli 1-49  destroyed  both  activity  simultaneously  (4)  the  gained both haemagglutinating and epithelial cell binding  capability. The binding of E. coli 1-49 to buccal epithelial cells was examined by phase contrast, scanning electron and transmission electron microscopy. There appeared to be two population of cells, one which supported binding and the other  (10 %) which did not .  Similar results were found by Gibbons et al (69) when they looked at the binding of oral bacteria to epithelial cells. They found that fibronectin was a potent  inhibitor of  attachment. Mild trypsin treatment of the epithelial cells enhanced binding by removing and unmasking the bacterial receptors.  Transmission electron microscopy of thin section of bacteria attached to epithelial cells showed that bacteria attached to plicate like extensions on the epithelial cell from their surface.  Epithelial cells which did not support binding did not have these projections,  they had smooth surfaces. Similar results have been reported by Williams et al studying the attachment of  (252)  enteropathogenic E. coli attached to gut epithelial cells. The  explanation for this phenomenon is not known but it appears as if on recognition of the adhesin by the receptor, the cytoskeleton of the epithelial cells are transformed to produce these extensions.  Bacterial agglutinin In the oral cavity  bacteria accumulate by binding to host tissue or to other bacteria  (214). This latter is extremely accumulation of bacteria  important as most of the interaction which lead to  involve bacteria-bacteria  interaction.  B. gingivalis is an  organism which colonize in areas where microflora has been established, thus it can be imagined that the ability to bind to other plaque organism is important in the ecology of the organism.  In the studies reported here it was shown that bacterial aggregating  activity can be attributed to a surface protein (M 46 kDa).  The purified protein is able  to induce aggregation of several species of oral bacteria.  Purification of this adhesin  r  140  proved to be relatively difficult since none of the conventional biochemical procedures tested were effective. However, specific adsorption of BA with S. mitis yielded a pure and active fraction. recovered from  There was a problem in that only 5 % of the material  S. mitis.  Immunofluoresence  of S. mitis  could be  with anti-46 kDa serum  revealed that much of the BA remained associated with the cells even after extreme attempts to remove it. Whether a covalent bond is formed between the adhesin and the receptor is not known but it is conceivable that a peptide bond can be formed between an NH  2  group on the adhesin and a - C O O H group on the receptor such a system was recently  described for the binding of C. albicans  to epithelial cells.  The molecular weight of the adhesin molecule on S D S - P A G E was approximately 46 kDa and it always copurified with a protein of approximately 43 kDa.  Since the  material  with BA activity had HA activity, this suggests that both haemagglutinating and bacterial aggregating adhesins are part of a complex. An explanation for this association could be that the bacteria placed more than one adhesin in a complex with the majority of proteins in the complex responsible for haemagglutination, since this protein is in much greater abundance than the 46 kDa protein.  Immunogold electron microscopy with anti-46 kDa serum showed that the bacterial coaggregating adhesin was evenly distributed located on the surface of the cell and did not appear to be associated with fimbria.When B. gingivalis with anti-46 antiserum, most of the  was sectioned and labelled  labelling was concentrated in the periplasm and  outer membrane. There were no fimbriae  present in purified preparations of the  bacterial coaggregating adhesin as seen by electron microscopy.  Anti-46 antiserum inhibited BA activity of B. gingivalis anti-S. gingivalis  to the same extent as that of  antiserum, whereas preimmune serum had no inhibitory  effect.  Anti-43 antiserum had no effect on BA activity, thus proving that BA and HA activities are found on two immunologically distinct molecules. On Western blot analysis there was  141  no cross-reactivity between the 46 kDa BA and antiserum to E. coli 1-49. Several reports have identified bacteria-bacteria adhesin-receptor reactions which are inhibited by sugars. Kolenbrander et al (118 ) has identified a number of coaggregation reaction of oral bacteria which can be inhibited by galactose and lactose. In this study several sugars were tested as putative inhibitors for their ability to inhibit  bacterial  aggregation between B. gingivalis and S. mitis. None of the sugars tested proved to be inhibitory with the exception of galactose and galactose containing sugars, but the high concentration needed indicate a non-specific effect.  Many gram negative bacteria coaggregate with gram positive bacteria. This includes the BPB  B. loeschii which causes the aggregation of S. sanguis and A. israelii (245).  Using anti-46 kDa serum several BPB were tested for the presence of  immunologically  similar molecules on their cell surface. All strains of B. gingivalis tested expressed an immunologically similar 46 kDa protein as determined by Western blot analysis.  None  of the other B P B tested reacted with the antiserum indicating the uniqueness of the B. gingivalis coaggregation reaction.  The purified bacterial aggregating adhesin caused the aggregation of several species of bacteria tested including S. sanguis and S. salivarius. This would indicate that B. gingivalis has only a single adhesin  for coaggregation of a number of organisms rather  than an adhesin for each coaggregating partner. This is not the case with some organisms which possess a number of adhesins, each capable of reacting with a different bacteria (245).  Vesicles  Bacterial binding to organic matrices adsorbed to hydroxyapatite is a complex process. Most studies have involved looking at saliva-coated hydroxyapatite but more recently the  142  role of serum and other organisms has been evaluated (131,214). In trying to assess the role of a receptor source such as saliva or serum it is important to note that other constituents  may  play  a  role,  eg  dextran,  glucosyltransferase  (242).  These  supplementary factors will increase the diversity of organisms able to bind by increasing the range of potential receptors. Presumably they will also eliminate some potential binding sites by occupying receptors.  One such modifier of the binding activity of saliva or serum derived pellicle could be the vesicles released by the Gram negative plaque microflora. These structures resemble the outer membrane in terms of their chemistry and it is expected that they would possess those binding properties associated with the whole cell outer membrane. These vesicles would represent a concentrated array of immobilized putative receptors if they were bound to the pellicle surface. The B. gingivalis vesicles reported here were found to bind avidly to serum- or saliva-coated HA. The bound vesicles then served as a source of receptors for other oral organisms. The vesicles were tightly bound to the insoluble substratum as they resisted removal by washing even when they were acting as a bridge between HA and the streptococci. The experiments reported here show that vesicles can act as a supplementary form of receptor with the potential to mediate the attachment of a heterogeneous group of organisms to plaque and would therefore contribute to the microbial complexity of plaque. The vesicle mediated binding reaction would not take place at all subgingival sites but rather would only occur at specific sites and at those times when an appropriate flora was present. At those times the vesicles could have a significant effect on colonization.  The electrophoretic analysis of the vesicles supports the contention that vesicles are similar to outer membranes. Williams and Holt (251)  showed that vesicle protein  profiles were somewhat more complex than outer membranes but contained the same major proteins. It is possible that the minor bands seen in vesicles are  partial  degradation products which arise from the proteolytic activity which is known to be  143  present in vesicles (80,81). The increase in binding of S. sanguis, S. mutans and S. salivarius to vesicle treated S e H A correlated with the ability of these cells to coaggregate with B. gingivalis whole cells. Those strains which did not coaggregate were not affected by the presence of vesicles adsorbed to SeHA whereas those that coaggregate very strongly e.g. S . salivarius,  showed a marked increase in binding.  While the assay demonstrates the  potential of the vesicles to mediate binding there are obviously other factors that will determine whether an organism can utilize this binding mechanism in vivo. S. sanguis can be isolated from the sulcus in significant numbers and therefore would be in a position to avail itself of this receptor. On the other hand S. salivarius is not commonly found in the sulcular flora and presumably would not bind via this mechanism. Vesicles also induce aggregation of S. sanguis and thus it can be envisaged that accumulation of S. sanguis within the plaque matrix would be mediated by vesicles derived from B. gingivalis. Adsorption of vesicle by organisms such as S. sanguis would have the added beneficial effect of removing a potentially  harmful factor. The vesicles would be  prevented from coming into contact with the gingival tissues where they could exert this pathogenic effect.  Langmuir and Scatchard analysis of S. sanguis binding to the SeHA immobilized vesicles shows a single type of interaction. This suggests that the binding as measured in these assays is to the vesicle and not to serum or saliva derived receptors implying that the vesicles block the pellicle receptors. The electron micrographs show that vesicles cover a significant portion of the SeHA surface and that binding of S. sanguis to the vesicles would exclude a high percentage of potential pellicle binding sites.  The coating of HA beads with serum was done to more closely mimic the subgingival hard tissue surfaces which are bathed in crevicular fluid.  Bacterial binding to serum or  crevicular fluid coated beads differs from binding to S H A . Cimasoni et al. (23)  and  Liljemark et al. (127,128) found that in contrast to saliva, human crevicular fluid and  144  serum inhibited binding of S .  sanguis  mechanism for S. sanguis to adhere. interfered  to HA. The vesicles would thus provide a Slots and Gibbons (214)  with the binding of B. gingivalis  reported that serum  to S H A , by extrapolation  expected that the vesicles would be affected in a similar way. this study that vesicles readily attach to SeHA.  it would be  However it was shown in  This confirms earlier work by Cimasoni  et al (23). which showed by scanning electron microscopy that B. gingivalis cells bound to SeHA.  The enhanced binding to immobilized vesicles demonstrates the potential importance of bacterial vesicles as mediators of adherence to hard tissues and indicates that the pellicle can be an extremely complex structure containing a diverse array of host and bacterially derived receptors. This type of interaction could also be important in promoting the succession of microorganisms within the sulcus. Accumulation of B. gingivalis within the sulcus would be accompanied by the release of large numbers of vesicles which would have the opportunity to attach to the serum coated enamel or cementum surfaces. This in turn would create improved opportunities for colonization by other organisms. In the right circumstances, organisms such as S. sanguis or Actinomycoces  viscosus  (R.G.  Ellen and D.A. Grove, J . Dent. Res. 67:268) would colonize and might eventually displace B. gingivalis thus contributing to the cyclical nature of periodontal infections.  Lipipolvsaccharides  Lipopolysaccharides  are  known  to have  a broad spectrum  of biochemical  and  immunological activities. There are conflicting reports about the LPS of the BPB-s with regard to their chemical constituents and biological activities.lt has been reported that the composition of LPS from BPB are unique; they lack K D O , heptose and B-hydroxy myristic  acid  enterobacterial  and  their  LPS  biological potency  is considerably  (102,103,113,117,145,167).  145  In  this  lower  study,  than  the  that of  LPS  from  several strains of BPB were analysed physically, chemically and immunologically. Chemical analysis of LPS of the B P B revealed that they have much in common but dissimilarities are present as well. Similar to previous reports (102,117,34,95) the majority of the sugars present were hexoses, followed by hexosamine and pentoses. No KDO  nor heptoses were found by chemical analysis which is in agreement with most of  the earlier reports in this area. However, in a recent publication (103)  it was reported  that K D O was found in minute quantities in some strains of Bacteroides.  One way of  explaining this discrepancy is that the KDO may be masked by phosphate groups and not liberated by acid hydrolysis, thus  it is not recognized by the thiobarbituric acid assay of  Karkhani et al (112).  The study described here underscores the complexity and specificity of the reaction involved in the attachment of B. gingivalis to host tissue and to other bacteria. The epithelial  cell binding adhesin have been  purified and cloned and the  bacterial  coaggregating adhesin purified and characterized. The role of vesicles in the adherence of B.  gingivalis  in the sulcus was elucidated and the L P S from several B P B were  characterized. 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