Plaque-inhibiting protein from bacteroides loeschei and methods for using the same

A purified, characterized surface protein from Bacteroides loeschei is an adhesin which is useful in inhibiting plaque formation. The adhesin is also useful in a diagnostic assay for gingivitis, a diagnostic indicator for changes in the surface components of certain human tissues, and as a binding agent to purify polysaccharides.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a surface protein from Bacteroides 
loeschei which is an adhesin useful in preventing plaque formation in the 
oral cavity. More specifically, the present invention relates to a 
purified, characterized adhesin isolated from B. loeschei and its use in a 
method for preventing or retarding the formation of plaque in the oral 
cavity as well as other uses based on the binding capabilities of the 
adhesin. 
2. Description of Related Art 
The present technologies used to clear microorganisms from the oral cavity 
rely on non-specific agents to disrupt prior colonization and plaque 
formation. These agents also pertubate the normal flora which actually 
protect the hard surfaces in the oral cavity. Such agents include 
toothpastes, mouthwashes, chewing gum, etc., which all work in a 
non-specific manner, primarily by means of detergents and abrasives. Thus, 
it is desirable to obtain a better means for clearing specific unwanted 
microorganisms from the oral cavity so as to avoid pertubating the normal 
flora. 
Most bacteria isolated from the human oral cavity possess the ability to 
participate in intergeneric coaggregation, i.e. bacteria from different 
genera bind to each other primarily via a protein on one attaching to a 
saccharide component on the other. Coaggregation is characterized by a 
highly specific binding between stable surface components found on two 
different bacterial types. Intergeneric coaggregation is thought to play 
an important role in the formation of dental plaque deposits. 
Streptococcus sanguis is one of the earliest colonizers of the clean tooth 
surface and is found in significant numbers in dental plaque. Other 
primary colonizers of oral hard tissue and the soft tissues of the host 
(i.e. neuraminidase treated erythrocytes and epithelial cells) include S. 
oralis and Gamella morbillorum. 
Bacteroides loeschei PK1295, a human oral isolate, synthesizes an adhesin 
which mediates its coaggregation with S. sanguis 34 (Weiss et al, 
"Fimbria-associated proteins of Bacteroides loeschei PK1295 mediate 
intergeneric coaggregations," J. Bacteriol., 169, pp. 4215-4222 (1987)) 
and hemagglutination of a variety of neuraminidase-treated mammalian 
erythrocytes (Weiss et al, "Fimbria-associated adhesin of Bacteroides 
loeschei that recognizes receptors on procaryotic and eucaryotic cells," 
Infect. Immun. 57, pp. 2912-2913 (1989)). Adhesin-specific monoclonal 
antibodies (MAb) prepared by immunizing mice with adhesin-bearing fimbriae 
were screened for inhibition of coaggregation (Weiss et al, 
"Characterization of monoclonal antibodies to fimbria-associated adhesins 
of Bacteroides loeschei PK1295," Infect. Immun., 56, pp. 219-224 (1988)). 
Subsequently, the MAbs were used to estimate the number of adhesin 
molecules per cell and immunoelectron microscopy revealed that the 
adhesins were associated with the distal portion of the microorganisms 
fimbriae (Weiss et al, "Localization and enumeration of fimbria-associated 
adhesins of Bacteroides loeschei," J. Bacteriol., 170, pp. 1123-1128 
(1988)). Although some of the coaggregation properties of adhesins from B. 
loeschei are known, a substantially purified and characterized adhesin has 
heretofore not been prepared. 
SUMMARY OF THE INVENTION 
Therefore, it is an object of the present invention to provide a purified, 
characterized adhesin isolated from Bacteroides loeschei which exhibits 
plaque-inhibiting properties in a specific manner without disturbing the 
normal flora in the oral cavity. 
It is a further object of the present invention to provide a method for 
inhibiting plaque formation in the oral cavity by applying a composition 
containing the adhesin to plaque-prone areas of the teeth or gingivae in 
the oral cavity. 
Another object of the present invention is to provide an oral composition, 
such as a dentifris, mouthwash or toothpaste, for applying the 
plaque-inhibiting adhesin to the oral cavity. 
Still a further object of the present invention is to provide a diagnostic 
assay for gingivitis wherein antibodies prepared against the adhesin are 
used as a diagnostic aid to detect the presence of B. loeschei in plaque 
or saliva. 
It is still a further object of the present invention to employ the adhesin 
isolated from B. loeschei as a binding agent for use a diagnostic 
indicator for changes in the surface components of certain human tissues. 
It is yet a further object of the present invention to provide a method of 
using the adhesin isolated from B. loeschei as a binding agent to purify 
polysaccharides. 
The foregoing objects and others are accomplished in accordance with the 
present invention, generally speaking, by providing a purified, 
characterized surface protein isolated from Bacteroides loeschei which 
exhibits plaque-inhibiting properties. The present invention further 
encompasses a method for inhibiting plaque formation by the application of 
the adhesin in an appropriate composition to the oral cavity, as well as 
the oral composition which includes the adhesin. The present invention 
also encompasses providing a diagnostic assay for gingivitis using 
antibodies prepared against the adhesin; employing the adhesin as a 
diagnostic indicator for changes in the surface components of certain 
human tissues; and providing a method for using the adhesin as a binding 
agent to purify polysaccharides. 
Further scope of the applicability of the present invention will become 
apparent from the detailed description and drawings provided below. 
However, it should be understood that the detailed description and 
specific examples, while indicating preferred embodiments of the 
invention, are given by way of illustration only, since various changes 
and modifications within the spirit and scope of the invention will become 
apparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION 
The purified, characterized surface protein from Bacteroides loeschei in 
accordance with the present invention is an adhesin which mediates the 
organism's attachment to bacteria which are primary colonizers of oral 
hard and soft tissues. B. loeschei is a gram negative bacterium found in 
the oral cavity. Primary colonizers of oral hard tissue and soft tissues 
of the host (i.e. neuraminidase treated erythrocytes and epithelial cells) 
include Streptococcus sanguis, S. oralis and Gamella morbillorum. The B. 
loeschei streptococcal-specific adhesin of the present invention is a 
basic protein with an isoelectric point (pI) of 7.4-8.4 composed of six 75 
kD subunits so as to have a native molecular mass of 450 kD. The 
N-terminal amino acid sequence is as follows: 
ala-tyr-ser-his-val-lys-asn-ala-thr-gly-glu-asp-ileu-glu-arg 
ileu-lys-glu-gly-asp-val-asp-asp-asp-ileu-glu-val-asn 
An amino acid analysis of the adhesin of the present invention provided the 
results summarized in Table 1 below. 
TABLE 1 
______________________________________ 
Amino Acid Analysis of the Surface Protein 
from Bacteroides loeschei 
Amino Acids Residues per 100 Residues 
______________________________________ 
Asparatate.sup.1 
10.8 
Glutamate.sup.2 
10.9 
Serine 6.8 
Glycine 8.0 
Histidine 1.2 
Arginine 3.9 
Threonine 6.0 
Alanine 9.0 
Proline 4.7 
Tyrosine 10.0 
Valine 6.8 
Methionine 2.4 
Isoleucine 3.0 
Leucine 7.2 
Phenylalanine 
3.6 
Lysine 8.0 
Cystine 0.4 
______________________________________ 
.sup.1 Residue may be either Asp or Asn. 
.sup.2 Residue may be either Glu or Gln. 
The protein of the present invention was purified by a one step procedure 
which employed an anti-adhesin gG Sephraose 4B affinity column wherein 
both monoclonal and polyclonal antibody prepared against the adhesin 
worked satisfactorily. A more detailed description of this procedure is 
provided below in the Examples. 
The purified, characterized protein in accordance with the present 
invention may be used in a method for inhibiting plaque formation in the 
oral cavity. When added to suspensions of streptococci and gamella 
possessing the appropriate carbohydrate receptor, the adhesin causes them 
to agglutinate thereby providing a mechanism for clearance from the oral 
cavity. In addition, the adhesin prevents other oral bacteria, which might 
ordinarily colonize the oral cavity by attaching to the streptococcal 
primary colonizers, from becoming established in the mouth. This group of 
bacteria includes strains of B. loeschei, Veillonella atypica, Actinomyces 
viscosus, A. naesludii, and other strains of S. sanguis and S. mitis. 
Removal of these secondary colonizing bacteria advantageously prevents the 
late colonizers from accreting to plaque deposits. The polyvalent effects 
of the protein are due to the fact that the various adhesins on the 
heterologous microbes recognize the same carbohydrate receptor on the 
primary colonizer. Thus, the protein, or a specific peptide (the binding 
site) derived from it, can prevent attachment and colonization of bacteria 
to hard and soft oral tissues by competing for common receptors. The 
mechanism of the adhesin is advantageous since it inhibits plaque 
formation in a very specific manner without disturbing the normal flora 
within the oral cavity. 
In accordance with the method of the present invention, the adhesin, or an 
active peptide derived from it, may be used as a disinfectant, applied via 
an oral composition, such as a dentifris, mouthwash, toothpaste, etc. 
Alternatively, the adhesin or active peptide may be applied topically to 
plaque-prone areas of the teeth or gingivae. In this capacity, the adhesin 
functions in two distinct ways. First, the adhesin has the ability to 
agglutinate three different species of streptococci that are primary 
colonizers in the oral cavity allowing salivary flow to clear the 
complexes. Second, the adhesion can attach to primary colonizers already 
attached to teeth thereby preventing attachment of other gram positive and 
gram negative bacteria, including species of Streptococcus, Actinomyces, 
Bacteroides and Veillonella. In vitro testing showed that between 10 and 
20 .mu.g of adhesin was sufficient to block 10.sup.8 streptococcal cells 
completely in a volume of 200 ml and prevented them from interacting with 
the above mentioned heterologous strains of oral bacteria. In formulating 
oral compositions incorporating the adhesin in accordance with the present 
invention, the compositions may include conventional excipients or 
carriers in addition to the adhesin, such as excipients found in 
conventional dentrifices, mouthwashes, and toothpastes. Conventional 
ingredients include dilute alcohols, abrasives, glycerin, sodium benzoate, 
polysorbate, sodium borate and flavorings. It must be noted that an 
anionic detergent cannot be included in the oral composition since this 
ingredient may denature the protein or peptide. The concentration of the 
adhesin in the oral composition is generally in the range of 80 to 100 
.mu.g per ml volume of the composition (for all affected species of 
bacteria) and in the range of 50 to 80 ug per ml. In formulating the oral 
composition of the present invention, the pH may be acidic since the 
protein is stable to acid. For example, the pH may be in the range of 
about 3 to 7, more preferably in the range of about 3 to 5.5. The oral 
composition may be in the form of a dentifris, mouthwash, toothpaste, etc. 
so as to provide a means for delivering the adhesin to plaque-prone areas 
in the oral cavity. 
In addition to the above oral composition and method of use employing the 
adhesin, antibodies prepared against the adhesin may be used in a 
diagnostic assay to detect the presence of species of B. loeschei in 
plaque or saliva as an indicator of gingivitis. Such an assay may be based 
generally on enzyme-linked immunosorbent assays (ELISAs). For example, a 
sample of plaque or scraping from the gingivae is placed into microtiter 
plate wells. The sample is first treated with purified monoclonal 
antibody, then treated with antimouse IgG conjugated to alkaline 
phosphatase and then developed with a phosphatase substrate. The adhesin 
may be bound to conventional markers, such as radiolabels. The protocol is 
based on the procedures outlined in Weiss et al, "Characterization of 
monoclonal antibodies . . . ," Infect. Immun., January, 1988, pp. 219-224. 
For a sample of about 2 to 4 mg, the monoclonal antibody is used in the 
range of I to 10 .mu.g. This diagnostic assay may also be in the form of a 
kit for detecting bacterial growth on teeth which includes the adhesin and 
a non-human erythrocyte. 
In addition to the above-noted uses, the adhesin may also be employed as a 
diagnostic indicator for changes in the surface components of certain 
human tissues based on the adhesin's interactive properties with certain 
saccharide groups, such as GalNAc. This diagnostic procedure is 
essentially the same as the assay for gingivitis as described above. A 
tissue sample is immobilized on a slide or microtiter plate, and treated 
with the adhesin. The alkaline phosphate can be conjugated to the adhesin 
for a direct assay, or the "sandwich" technique may be employed by 
applying MAb after the adhesin and anti-mouse IgG (conjugated to alk. 
phosphatase) as described above with regard to the assay for gingivitis. 
The adhesin from B. loeschei may also be used as a binding agent in a 
method for purifying GalNAc or lactose-containing polysaccharides of 
biological interest. In this procedure for purifying a desired 
polysaccharide, the adhesin can be conjugated to a carrier, such as 
CNBr-Sepharose 4B beads in the presence of GalNAc to protect the active 
site. The bead conjugate is placed into a small column, such as an 
affinity column, and the polysaccharide solution poured over it. The 
polysaccharide will bind to the column matrix and the contaminating 
material is washed out. The material bound to the column is released by 
washing the column with GalNAc or lactose and the sugars can be dialyzed 
away leaving only the purified polysaccharide. 
EXAMPLES 
Purification and Characterization of B. loeschei Adhesin 
Bacterial strains and culture conditions. Bacteroides loeschei PK1295, 
Streptococcus sanguis 34, Capnocytophaga ochracea 25 and Streptococcus 
sanguis H1 were grown in screw cap tubes containing Schaedler broth (BBL, 
Cockeysville, Md.) at 37.degree. C. under anaerobic conditions. Large 
scale cultures of B. loeschei were grown in 1-L bottles; 20 L of medium 
yielded roughly 40 g (wet weight) of cells. Cell pastes were stored at 
-20.degree. C. until used. 
Purification of the Streptococcal-Specific Adhesin. Affinity gels were 
prepared by activating 1 g of CNBr-Sepharose 4B (Pharmacia LKB, Sweden) 
and reacting it with 15 to 20 mg of MAb 5BB1-1 or 3AD6 (24) according to 
the manufacturer's instructions. The washed Sepharose beads bearing the 
coupled antibody were stored in 200 mM sodium borate buffer, pH 8.0, 
containing 0.8 percent NaCl (BBS) at 4.degree. C. until used. 
The adhesin was released from B. loeschei PK1295 by suspending 8 to 10 g of 
cells (wet weight) in 20 ml of BBS containing the protease inhibitors 
phenylmethyl sulfonyl fluoride (PMSF (5 mM)) and ethylenediamine 
tetracetic acid (EDTA (10 mM)) and subjecting the continuously cooled cell 
suspension to ultrasonic disruption with a Branson model 350 sonifer (Heat 
Systems, Plainsview, N.Y.) operating at 70 percent of maximum power output 
for 4 minutes. Unbroken cells and membrane fragments were removed by 
centrifugation at 240,000.times.g for 60 minutes in a Sorval model ADT75B 
ultracentrifuge (Dupont, Newton, Conn.). The supernatant was concentrated 
to a volume of 4 to 5 ml in an Amicon filtration unit (Amicon, Danvers, 
Mass.) equipped with a YM-10 low protein binding membrane and the 
concentrate was added to a vial containing 300 mg of the MAb conjugated 
beads. The suspension containing beads and sonic concentrate was mixed by 
end-over-end rotation for 14 hours at 4.degree. C. After mixing, the beads 
were separated from the concentrate by low speed centrifugation, 
4000.times.g for 3 minutes and the concentrate was saved for further 
analysis to determine whether all of the adhesin had been removed. The 
Sepharose beads carrying the immune complex, MAb and adhesin, were rinsed 
once with 3 ml of BBS containing protease inhibitors, three times with 3 
ml of BBS containing protease inhibitors plus 2 mM 
(3-(3-cholamidopropyl)-dimethylammonio)-1-propane sulfonate (CHAPS), once 
with 3 ml of BBS containing 2 mM CHAPS plus 1M NaCl, twice with 3 ml of 
BBS containing 2 mM CHAPS and six times with 3 ml of 20 mM (Tris 
(hydroxymethyl) aminomethane) buffer (Tris-HCl), pH 8.0 to remove 
contaminating adherent material. Adhesin was eluted by treating the beads 
three times with 3 ml of 5 percent acetic acid. Acetic acid washes were 
concentrated to a volume of 0.5 to 1 ml by centricon filtration (Amicon, 
Danvers, Mass.). The pH of the solution was then increased to 4.6 by 
several passages of the adhesin solution through the concentrating filter 
replacing the lost volume with 50 mM sodium acetate buffer, pH 4.6 
containing 0.02 percent azide. This procedure yielded between 200 to 300 
ug adhesin protein. In some instances, the pH of the adhesin solution was 
raised to 6.8 by the addition of NaOH and 0.1M Tris-HCl buffer, pH 6.8. 
The adhesin was stored at -20.degree. C. 
The efficiency of the recovery was estimated by resolving samples of the 
purified adhesin, supernatant fluid following adsorption to the affinity 
beads and the pellet of the centrifuged ultrasonic sample on SDS gels, 
immunobloting the separated polypeptides and developing the nitrocellulose 
filters with the appropriate antibody-conjugate system (see below). 
Scanning the intensity of the stains on dried gels with an Ultroscan XL 
laser densitometer (LKB, Uppsala, Sweden) and calculating the area under 
the peaks indicated that between 85 and 90% of the adhesin had been 
recovered by the MAb affinity matrix. Assuming that B. loeschei is similar 
to other gram negative bacteria, water comprises 70% of the cells' dry 
weight and 55% of the dry weight is protein. Thus, of a 10 g (wet weight) 
cell pellet, 1.65 g is protein. With a yield of 300 .mu.g of adhesin 
protein, the adhesin represents roughly 0.02% (300 ug/1.65.times.106 
.mu.g.times.100) of the cell's total protein. Protein concentration was 
determined by the commercially available BioRad protein assay kit (BioRad 
Labs, Richmond, Calif.). 
Preparation of Rabbit Polyclonal Anti-Adhesin Antibody. After withdrawing a 
30 ml sample of blood, a four month old white, female New Zealand rabbit 
was given three intradermal injections consisting of a total of 150 .mu.g 
of the streptococcal-specific adhesin emulsified in ml Ribi adjuvant (Ribi 
Immunological Research, Hamilton, Mont.) over a period of 6 weeks. Blood 
was subsequently withdrawn from the central ear artery of the immunized 
rabbit, allowed to clot overnight and the serum was stored at -20.degree. 
C. until used. Rabbit IgG was purified by a two step procedure employing 
ammonium sulfate precipitation and DEAE ion exchange chromatography. The 
IgG was ultimately made up in 0.02M phosphate buffer, pH 7.2 containing 
0.78 percent NaCl (PBS) and stored at -20.degree. C. until needed. 
Coaggregation Inhibition and Hemagglutination Tests. Quantitative 
coaggregation experiments were performed using the spectrophotometric 
procedure of McIntire et al ("Mechanism of coaggregation between 
Actinomyces viscosus T14V and Streptococcus sanguis 34," Infect. Immun., 
21, pp. 978-988 (1978)). Inhibition of coaggregation studies with 
anti-streptococcal specific adhesin serum were performed as follows. A 
series of test tubes containing a suspension of 10.sup.9 B. loeschei cells 
in coaggregation buffer (CAB (0.001M Tris-HCl, pH 7.4; 0.15M NaCl; 0.0001M 
CaCl,; 0.0001M MgCl.sub.2 and 0.002% NaN.sub.3)) received anti-adhesin IgG 
(in PBS) in the range of 98 to 200 .mu.g of protein to a volume of 400 
.mu.l. The mixture was incubated at room temperature with constant shaking 
for 30 minutes. After the incubation period, 200 .mu.l of a suspension of 
S. sanguis 34 (10.sup.9 cells) was added to each tube to a final volume of 
600 ul, the tubes were mixed vigorously for 5 minutes and the tubes were 
centrifuged for 1 minute at 500.times.g. The supernatants were diluted 1:2 
with CAB and optical density at 600 nm was measured with a Gilford model 
2400 spectrophotometer (Gilford, Oberlin, Ohio). Controls consisted of 
mixture of both suspensions containing PBS instead of IgG or 200 .mu.l 
suspensions of each organism brought to a volume of 600 .mu.l with CAB. 
The percent inhibition of coaggregation was determined using the procedure 
of McIntire et al. 
Qualitative studies using the purified adhesin as the inhibitor were 
performed as follows. A suspension of 2.5.times.10.sup.8 S. sanguis cells 
(50 .mu.l) in CAB and adjusted to a pH of 5.0 with acetate buffer was 
incubated with 2.75 to 22 .mu.g of adhesin protein at room temperature. 
After gentle agitation for 30 minutes, 5.times.10.sup.8 B. loeschei cells 
were added to the mixture and shaken for several minutes until the control 
assay without adhesin added showed strong coaggregation. If the pH of the 
adhesin solution and the streptococcal cell suspension had been adjusted 
to 6.8 or greater, the cells quickly aggregated after mixing. The assays 
were scored visually assigning values between 0 to +4 or the results were 
recorded photographically. To establish the specificity of the purified 
adhesin, controls were performed using the coaggregation pair S. sanguis 
H1 and C. ochracea 25. These assays were performed as described above. 
Hemagglutination studies were carried out with sheep erythrocytes (RBCs) 
stored in Alsevers solution. RBCs were washed three times in Hank's 
buffered saline solution and packed cells were diluted 1:5 in phosphate 
buffered saline (PBS). One ml of RBCs was treated with 10 .mu.l of 
clostridial type X neuraminidase (Sigma Chemical Co., St. Louis, Mo.) 
containing 10 units per ml PBS for 1 hour at 37.degree. C. RBCs were 
washed twice in PBS containing 0.1% bovine serum albumin and 0.02% sodium 
azide and adjusted to a density of 5.times.10.sup.9 cells per ml in the 
same buffer. Hemagglutination assays were performed in microtiter plates; 
each well contained 80 .mu.l of neuraminidase treated or untreated RBCs 
and between 2.5 to 16 .mu.g purified adhesin (pH 6.8) in a final volume of 
140 .mu.l. Controls consisted of untreated cells plus adhesin or treated 
cells containing buffer only. Where required, the galactosides, 
N-acetyl-D-galactosamine, D-glucosamine, D-galactose and lactose were 
added to a final concentration of between 6 and 25 mM. The microtiter 
plates were incubated at room temperature with vigorous mixing for 30 
minutes. Plates were then centrifuged and the size and nature of the 
resultant pellets were scored visually and photographed. The same protocol 
was used to test for agglutination of suspensions of S. sanguis 34 or S. 
sanguis H1. Suspensions containing 5.times.10.sup.8 cells (in 25 mM sodium 
acetate, pH 5.6) were incubated with between 5 and 10 .mu.g of adhesin 
protein with mixing at ambient temperature. When required, the 
galactosides described above were added to the agglutination assay at 
levels of between 6 and 25 mM. 
Polyacrylamide Gel Electrophoresis and Isoelectric Focusing. Native anionic 
PAGE was carried out on 14.times.13 cm, 7 percent polyacrylamide gel slabs 
using the Tris-HCl glycine buffering system of Davis ("Disc 
electrophoresis. II. Method and application to human serum," Ann. N.Y. 
Acad., 12, pp. 404-427 (1964)); between 0.5 and 8 .mu.g of protein were 
added to the sample wells. Denaturing gel electrophoresis was carried out 
according to the method of King and Laemmli ("Polypeptides of tail fibers 
of bacteriophage T4", J. Mol. Biol., 62, pp. 465-477 (1971)); similar 
protein loads were added to the sample wells. Bands on the gels were 
visualized with Coomassie blue R250 stain in accordance with the King and 
Laemmli method. 
Isoelectric focusing was performed on commercially available pH 3 to 10 
ampholine-containing polyacrylamide slabs according to the manufacturers 
instructions (Novex Corp., Encinitas, Calif.). Samples containing between 
2 and 10 .mu.g of adhesin and a solution containing proteins with known 
isoelectric points (cytochrome C (pI=9.6), chymotrypsin (pI=7.0), whale 
myoglobin (pI=8.05), equine myoglobin (pI=7.0), human carbonic anhydrase 
(pI=6.5), bovine carbonic anhydrase (pI=6.0), lactoglobulin B (pI=5.1) and 
phycocyanin (pI=4.65)) were run concomitantly on the gels. The solution of 
standard proteins was used to determine the linearity of the pH gradient 
in the gel and, following staining, the position of the adhesin on the gel 
was used to estimate its pI. 
The native molecular weight of the adhesin was estimated by anionic 
gradient (3 to 20 percent) gel electrophoresis according to the method of 
Lambin and Fine ("Molecular weight estimations of proteins by 
electrophoresis in linear polyacrylamide gradient gels in the absence of 
denaturing agents," Anal. Biochem., 98, pp. 160-168 (1979)). Oligomeric 
and monomeric forms of apoferritin (M.sub.r =886,000 and 443,000), urease 
(M.sub.r =545,000 and 272,000) and bovine serum albumin (M.sub.r =132,000 
and 66,000) served as molecular weight standards. 
Immunoblot analyses were performed by separating adhesin polypeptide(s) SDS 
gel, transferring the protein components to nitrocellulose filters, 
treating the filters with 1:1000 dilutions of MAb 3AD6 IgG (3 mg/ml) or 
1:2000 dilutions of polyclonal IgG (5 mg/ml) and visualizing the immune 
complex with a commercially available alkaline phosphatase goat anti-mouse 
IgG conjugated secondary antibody and dye indicator system (Promega, 
Madison, Wis.). 
N-terminal Amino Acid Sequencing of the Adhesin. Lyophilized samples 
containing between 0.5 and 1 nmoles of adhesin were dissolved in 1 percent 
trifluoroacetic acid and processed by automated Edman degradations using a 
Model 470A gas phase sequencer (Applied Biosystems, Foster City, Calif.) 
in conjunction with the standard "NoVac" program supplied by the 
manufacturer. Phenylthiohydantoin derivatives were identified by HPLC on 
an IBM cyano column. The system used with this column consisted of a 
Perkin Elmer Series 4 liquid chromatograph, a LC-85B spectrophotometric 
detector and a LCI 100 computing integrator. These procedures were 
performed under contract with the University of California, San Diego. 
Amino Acid Analysis of the Adhesin. Approximately 2 nmole of adhesin was 
hydrolyzed with constant boiling 6N HCL at 110.degree. C. for 16 or 24 
hours. The dried residue was dissolved in Beckman sample buffer diluent 
and analyzed with a Beckman Gold System HPLC amino acid analyzer using 
ninhydrin postcolumn derivatization as the detection system. Amino acid 
standards were obtained from Beckman. 
RESULTS 
Purification of the B. loeschei Streptococcal-Specific Adhesin. 
FIG. 1 is a demonstration of the purity of the streptococcal-specific 
adhesin by immunoblot analysis, denaturing and native anionic gel 
electrophoresis. Lanes A-C are denaturing SDS gel. Lane A, 2 .mu.g 
purified adhesin. Lane B contains the following molecular weight standards 
(top to bottom); phosphorylase A, (M.sub.r =97,400); bovine serum albumin, 
(M.sub.r =66,200); ovalbumin (M.sub.r =42,700), carbonic anhydrase 
(M.sub.r =31,000), trypsin inhibitor (M.sub.r =21,400), lysozyme (M.sub.r 
=14,400). Lane C contains the following molecular weight standards (top to 
bottom); myosin, (M.sub.r =200,000), .beta.-galactosidase, (M.sub.r 
=116,500), phosphorylase A, bovine serum albumin and ovalbumin. Lane D, 
immunoblot developed with MAb 3AD6, 0.5 .mu.g adhesin run in gel. Lane E, 
immunoblot developed with rabbit polyclonal anti-adhesin, 0.5 .mu.g 
adhesin run in gel. Lane F, 6 .mu.g adhesin resolved in a native anionic 
gel (positive electrode at bottom). The batchwise purification of the 
adhesin with the MAb 5BB1-2 or 3AD6 conjugated to a Sepharose 4B matrix 
yielded preparations that were greater than 95% pure as determined by 
scanning laser densitometry of native (FIG. 1, lane F) and denaturing 
(FIG. 1, lane A) gel electrophoretograms. The minor contaminants which 
migrated more rapidly than the adhesin monomer, M.sub.r =75,000, (FIG. 1, 
lane D and E) reacted with the MAb or polyclonal antiserum specific for 
the adhesin (FIG. 1, lane C) suggesting that some small fraction of the 
native protein was slightly degraded during the purification procedure. 
These large fragments of the molecule must have retained the epitope(s) 
recognized by MAb 5BB1-2 and 3AD6 since they remained associated with the 
affinity matrix during the purification process and reacted with other 
MAbs (i.e. 5DB5 and 3BC5) capable of visualizing the adhesin on 
immunoblots. On native anionic polyacrylamide gels (FIG. 1, lane F), the 
adhesin migrated as a single band remaining near the top of the gel. The 
relatively slow migration rate in the electrical field was the first 
suggestion that the adhesin was a basic protein possessing a net positive 
charge. The protein exhibited the property of aggregating at a pH above 
neutrality. If the pH of the protein solution was increased to between 7.5 
and 8.0, the protein precipitated out of solution and could not be 
solubilized by making the suspension acid (pH 3) or by treatment with 
either 6M urea or 4% SDS. 
Properties of the Adhesin. The native molecular weight of the adhesin was 
estimated by comparing the migration rate of the protein on 3 to 20 
percent gradient anionic polyacrylamide gels with monomeric and polymeric 
forms of reference proteins. FIG. 2 is an estimation of native molecular 
weight of the streptococcal-specific adhesin by anionic gradient gel 
electrophoresis. The open circle denotes the relative position of the 
adhesin in the gel. FIG. 2 shows that the adhesin migrates like a protein 
with a M.sub.r =450,000. Thus, in its native form, the adhesin appears to 
exist as a hexamer. The pI of the adhesin was determined by isoelectric 
focusing. FIG. 3 shows a distribution of the streptococcal-specific 
adhesin in a pH gradient. Standards (closed circles) are (1) cytochrome C, 
(2) a-chymotrypsin, (3) whale myoglobin, (4) equine myoglobin, (5) human 
carbonic anhydrase, (6) bovine carbonic anhydrase, (7) b-lactoglobin B and 
(8) phycocyanin. The open circles indicate the positions of the adhesin 
bands (6 .mu.g added, bands of near-equal intensity). FIG. 3 shows that 
the affinity purified adhesin preparations migrated in an apparent 
polydispersed fashion over a pH range of 7.4 to 8.4. The pI of the adhesin 
in crude preparations was determined by transferring the proteins to 
nitrocellulose filters; the adhesin was visualized by immunoblot staining. 
The latter preparations appeared to be less polydispersed and exhibited a 
pI of between 8.2 and 8.7. Thus, the autoagglutination observed as the pH 
of the adhesin solution rises above neutrality probably reflects the 
protein-protein interactions that occur among the adhesin molecules as 
they approach their pI. 
N-terminal amino acid sequencing identified the initial 28 amino acids of 
the mature protein; this portion of the adhesin was shown to consist of 
the following amino acid residues: 
ala-tyr-ser-his-val-lys-asn-ala-thr-gly-glu-asp-ileu-glu-arg-ileu-lys-glu-g 
lu-asp-val-asp-asp-asp-ileu-glu-val-asn 
The sequenced segment of the adhesin contains a relatively large number of 
charged amino acids including 6 aspartate residues. In contrast to the 
proteins overall basic character, 10 of the N-terminal charged amino acids 
are acidic in nature. Of the 28 amino acids, only one (tyr) is strongly 
hydrophobic. The results of an amino acid analysis of the protein adhesin 
are summarized in Table 1 above. Glx and Asx account for 22 percent of the 
protein's amino acid residues while basic amino acids (arg, his and lys) 
constitute 13 percent of the total residues. A significant number of the 
potentially acidic amino acid residues, i.e. asx and glx, probably exist 
as asn or gln since the pI of the adhesin ranges between 7.4 and 8.4. 
Coaggregation Inhibition Studies. To establish that the purified protein 
was indeed the streptococcal specific adhesin, an anti-adhesin polyclonal 
antiserum was produced in rabbits and its ability to inhibit coaggregation 
between B. loeschei and S. sanguis was determined. Since the MAbs 
originally prepared against the adhesin were potent inhibitors of 
coaggregation and erythrocyte agglutination by B. loeschei, it was 
reasoned that the polyclonal antiserum would duplicate, at least in part, 
the action of the MAbs. FIG. 4 is a graph showing the inhibition of 
coaggregation by preincubation of B. loeschei cells with polyclonal 
anti-adhesin prior to adding S. sanguis cells. A preimmune IgG preparation 
was not inhibitory up to a concentration of 1 mg ml.sup.-1. FIG. 4 shows 
that the purified IgG fraction of the antiserum inhibited the 
interactions, however, it was not as effective as the MAbs which inhibited 
coaggregation in the range of 0.5 to 5 ug antibody protein. IgG prepared 
from preimmune serum had no effect on the coaggregation reaction at levels 
of 1 mg protein ml.sup.-1. At similar concentrations (between 98 and 200 
.mu.g IgG protein), the polyclonal antiserum also inhibited 
hemagglutination of sheep RBCs by cell suspensions of B. loeschei. In 
addition, the purified polyclonal IgG reacted with purified preparations 
of adhesin in a fashion identical to the MAbs on immunoblots (FIG. 1, lane 
E). The IgG of the polyclonal antiserum functionally resembled the MAbs in 
that it did not agglutinate B. loeschei cells nor did it produce 
precipitin reactions with crude or purified adhesin preparation in 
immunodiffusion plates. 
The purified adhesin itself was capable of inhibiting coaggregation at an 
acid pH (4.6) when preincubated with S. sanguis cells prior to the 
addition of B. loeschei cells. FIG. 5 shows the inhibition of 
coaggregation between B. loeschei and S. sanguis by preincubating S. 
sanguis cells with streptococcal-specific adhesin. All reaction mixtures 
contain 5.times.10.sup.8 S. sanguis cells. Panel A, control; panel B, 5.5 
.mu.g adhesin added; panel C, 11 .mu.g adhesin added; panel D, 22 .mu.g 
adhesin added. Concentrations between 11 and 22 ug (equivalent to 0.2-0.4 
.mu.M native protein) of adhesin protein completely inhibited 
coaggregation between these two microorganisms (FIG. 5, panels C and D). 
However, constant mixing of the cell suspension containing 11 ug of 
adhesin protein (FIG. 5, panel C) eventually allowed some aggregation to 
occur after 30 minutes (approximately 25 percent of the control (A) as 
estimated visually); at 22 .mu.g, no coaggregation was observed after an 
overnight incubation. Since the concentration of cells used in the assay 
was known and since blocking occurred in levels between 11 and 22 .mu.g of 
adhesin, a rough approximation of the number of adhesin molecules required 
to block each streptococcal cell was determined. An estimated 5.6 to 
11.times.10.sup.5 molecules per cell resulted in complete abolition of 
coaggregation. To establish that the effect of the adhesin preparation on 
coaggregation was specific and not result of electrostatic interactions 
between the basic protein and streptococcal cells, a suspension of S. 
sanguis H1 was incubated with 22 .mu.g of adhesin prior to addition of its 
partner cell, C. ochracea strain 25 (26). The adhesin had no effect on 
the coaggregation reaction. 
FIG. 6 indicates hemagglutination of neuraminidase-treated sheep 
erythrocytes by the purified adhesin. Each well contained 5.times.10.sup.7 
cells. Column A, line 1 (control), untreated sheep RBCs plus 11 .mu.g 
adhesin; column A, line 2 (control) treated sheep RBCs plus PBS buffer; 
column A, line 3; treated RBCs plus 2.75 .mu.g adhesin; column A, line 4, 
treated RBCs plus 5.5 .mu.g adhesin; column B, line 2, treated RBCs plus 
11 .mu.g adhesin; column B, line 3, treated RBCs plus 16.5 .mu.g adhesin; 
column B, line 4, treated RBCs preincubated with 6 mM 
N-acetyl-D-galactosamine plus 11 .mu.g adhesin; column C, line 2, 
(control) treated RBCs plus 40 .mu.l acetate buffer. In contrast to the 
experiment described above, addition of similar levels of purified adhesin 
preparations (5-10 ug (equivalent to a concentration of 0.1-0.2 .mu.M of 
native adhesin)) to neuraminidase-treated RBCs resulted in their rapid 
agglutination (FIG. 6, column B, lines 2 and 3). However, it is important 
to note here that these experiments were carried out at a pH of 6.8 with 
the adhesin being diluted in PBS prior to mixing with the RBCs. No 
immediate hemagglutination was observed with untreated erythrocytes, but 
with prolonged incubation (30 to 60 minutes at RT), a slight formation of 
particulate material was noted in the reaction mixture (FIG. 6, column A, 
line 1). Adding N-acetyl-D-galactosamine at a final concentration of 6 mM 
to the erythrocytes prior to introducing adhesin prevented 
hemagglutination (FIG. 6, column B, line 4). At concentrations of 10 to 25 
mM, galactosamine, lactose and galactose also inhibited agglutination. 
FIG. 7 shows photomicrographs of adhesin mediated hemagglutination of sheep 
RBCs and aggregation of the S. sanguis 34 cells. A. Sheep RBCs plus 20 
.mu.g adhesin protein, B. RBC control (untreated RBCs plus 20 .mu.g 
adhesin), C. streptococcal cells plus 10 .mu.g adhesin protein and D. 
streptococci control (cells in buffer only). Magnification is 480.times.. 
These photomicrographs of reaction mixtures containing adhesin demonstrate 
conclusively that the adhesin mediated extensive agglutination of the 
neuraminidase-treated RBCs (FIG. 7, panel A), the adhesin had no effect on 
untreated RBCs (FIG. 7, panel B). If an aliquot of the neutral adhesin 
solution (5 .mu.g protein) was added to S. sanguis 34 cells suspended in 
PBS, the streptococci agglutinated immediately upon mixing (FIG. 7C). The 
appearance of the suspension was identical to that seen in conventional 
coaggregation experiments (see FIG. 5, panel A); photomicrography revealed 
that, like the RBCs, the streptococcal cells had undergone extensive 
aggregation (FIG. 7, panel C). In the controls, adjusted to a Ph of 6.8, 
no aggregation occurred in the absence of adhesin (FIG. 7, panels B and 
D). Like the RBCs, addition of the galactosides in the same concentration 
range inhibited or reversed the adhesin mediated aggregation. FIG. 8 shows 
the specificity of the adhesin-mediate aggregation of streptococcal cells. 
A. S. sanguis 34 (5.times.10.sup.8 cells ml.sup.-1) plus 10 ug adhesin 
protein. B. S. sanguis H1 (5.times.10.sup.8 cells per ml ) plus 10 .mu.g 
adhesin protein. Incubated for 5 minutes at ambient temperature with 
mixing. FIG. 8 shows that ten micrograms of adhesin failed to aggregate a 
cell suspension of S. sanguis H1, a strain closely related to S. sanguis 
34, indicating that the interaction was specific for a receptor on the 
latter. 
The lectin-like protein on the surface of B. loeschei that mediates 
coaggregation with S. sanguis and hemagglutination with a wide variety of 
erythrocytes is one of the few bacterial adhesins to be purified in any 
significant quantity and partially characterized with regard to both 
structure and function. The adhesin was related from cells by ultrasonic 
disruption and purified to electrophoretic homogeneity by affinity 
chromatography. From the adhesin's amino acid composition and pI, it 
appears to be a hydrophilic protein with a comparatively strong positive 
charge. In its native state, the adhesin exist as a relatively large 
hexameric molecule with an estimated M.sub.r of 450,000. The unequivocal 
identification of the adhesin's 28 amino terminus residues suggests that 
this region of the six monomers comprising the native protein is 
homologous. However, the amino acid sequence of the remainder of the 
monomer may not be so highly conserved since isoelectric focusing data 
indicated that the protein migrates as 4 distinct bands. The apparent 
polydispersed nature of the purified preparation may reflect 
postranslational modifications of the adhesin monomers or it may be a 
result of acid hydrolysis of the amide groups in glutamine and asparagine 
during purification. A comparison of N-terminal amino acid sequences of 
the E. coli type 1, P and bacteroides adhesin showed that each of the E. 
coli proteins matched with the B. loeschei protein at two or four 
non-consecutive positions (FIG. 9), respectively; the two enteric adhesins 
only showed six non-consecutive cross-matches. Thus, there appears to be 
little or no homology in this portion of the three proteins. 
It was essential to establish that the protein of M.sub.r =450,000 
associated with the B. loeschei fimbriae was the adhesin because the 
distinction between the adhesin and fimbrillin or pilin subunits in other 
systems had not been clearly delineated in many earlier studies. The 
following evidence supported the conclusion that the purified protein was 
the adhesin; (a) the polyclonal antiserum prepared against the adhesin 
inhibited both coaggregation and hemagglutination, (b) the purified 
adhesin itself inhibited coaggregation with streptococcal cells at an acid 
pH or agglutinated neuraminidase-treated erythrocytes and streptococci at 
a neutral pH, and (c) electron micrographs of purified adhesin 
preparations shows no fimbrial structures. The coaggregation inhibition 
studies established that the adhesin readily binds to partner cells in 
relatively large numbers. Rough estimates indicated that each cell may 
bind as many as 4.times.10.sup.5 adhesin molecules. This number is 
approximately three orders of magnitude greater than the value obtained 
for the number of adhesin molecules found on individual B. loeschei. It is 
clear that the adhesin recognizes the same sugars on the procaryote and 
eucaryote receptors since the same set of galactosides inhibit blocking of 
coaggregation and agglutination of streptococcal cells and RBCs. However, 
the nature of those receptors is most probably very different. The 
bacterial receptor may be similar to the S. sanguis 34 
N-acetyl-D-galactosamine and galactose-containing cell wall polysaccharide 
that an adhesin Actinomyces viscosus T14V recognizes, while the 
erythrocyte receptor is apt to be a glycoprotein or glycolipid. S. sanguis 
H1, which failed to interact with the adhesin and, thus, served as an 
aggregation control, possesses a polysaccharide receptor that is 
structurally distinct from that described in strain 34. 
The invention being thus described, it will be obvious that the same may be 
varied in many ways. Such variations are not to be regarded as a departure 
from the spirit and scope of the invention, and all such modifications as 
would be obvious to one skilled in the art are intended to be included 
within the scope of the following claims.