Adhesion of staphylococcus aureus to fibrinogen, collagen and lectin in relation to cell surface structure

Zbl. Bakt. Hyg. A 268, 325-340 (1988)

Adhesion of Staphylococcus aureus to Fibrinogen, Collagen and Lectin in Relation to Cell Surface Structure TOSHICHIKA OHTOMO and KOSAKU YOSHIDA Department of Microbiology, St. Marianna University School of Medicine, 2-16-1, Sugao, Miyamaye-ku, Kawasaki 213, Japan

With 2 Figures' Received July 15, 1987 . Accepted September 28, 1987

Summary The adherence of an encapsulated strain of Staphylococcus aureus, S-P, and its variants to fibrinogen-, collagen-, and lectin-coated hydroxyapatite were compared. The parent strain, S-P, possesses a large capsule while its variants S-A and S-B possess a small capsule and microcapsule, respectively. The third variant, S-C, has no capsule. Adherence to proteinaceous substances varied according to the strains. While all four strains showed a similar degree of adhesion to collagen, the adhesion of strains S-A, S-B and S-C to fibrinogen and lectin differed from those of strain S-P. The effect of physical and enzymatic pretreatment of the strains on adhesion characteristics was measured. Generally, these results suggest that both carbohydrate and protein moieties on cell surface may be involved in adherence. In addition, the inhibition of adhesion by cell-surface polymers and monosaccharides was measured. The inhibition of adhesion of large capsulated (s-P) and unencapsulated (S-C) strains by proteinaceous substances differed. The large capsulated strain (S-P) of S. aureus had different adherence capacities in early-, mid-, or late log phases of growth, whereas the adherence capacities of the unencapsulated strain S-C remained nearly constant.

Zusammenfassung Es wurde die Haftfahigkeit eines verkapselten Starnmes von Staphylococcus aureus S-P und seiner Varianten an mit Fibrinogen, Kollagen und Lectin beschichtetem Hydroxyapatit verglichen. Der Ausgangsstamm S-P weist eine grofe Kapsel auf, wahrend seine Varianten S-A und S-B eine kleine Kapsel bzw. eine Mikrokapsel aufweisen. Die dritte Variante S-C hat keine Kapsel. Die Haftfahigkeit an proteinhaltigen Substanzen war von Stamm zu Stamm unterschiedlich. Wahrend aile vier Stamme einen ahnlichen Grad der Haftung an Kollagen zeigten, war die Adhasion der Stamrne S-A, S-B und S-C an Fibrinogen und Lectin anders als beim Stamm S-P. Die Wirkung einer physikalischen und enzymatischen Vorbehandlung der Stamme auf die Adhasion wurde gemessen. Allgemein weisen diese Ergebnisse darauf hin, daf sowohl Kohlenhydrat- als auch Proteinanteile der Zelloberflache an der Haftung beteiligt sein konnen, Daruber hinaus wurde die Hemmung der Adhasion durch Polymere und Monosaccharide der Zelloberflache gemessen. Die Hemmung der Haftung an Stammen mit grofser Kapsel (S-P) und ohne Kapsel (S-C) durch Proteinsubstanzen war unterschiedliche. Die S. aureus-Stamme mit grofer Kapsel (S-P) zeigten eine unter-

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schiedliche Hafrungsfahigkeit in der friihen, mittleren oder spiiten logarithm ischen Phase des Wachstums, wogegen die Haftungsfahigkeit des nicht verkapselten Stammes S-C nahezu gleich blieb.

Introduction Microbial adhesion to host materials must be mediated by macromolecules associated with the outermost layer of bacteria (9,2 1, 45). However, differences in cell surfa ce structure, media and extracellular substances influence adhesion capacity, including the number of binding sites (1, 38). While quantitative studies concerning the adhesion of staphylococcal strains to various types of cells have been performed (4,1 1, 14, 17, 27), problems still remain concerning the detailed mechanisms involved in the adhesion process. Fibrinogen, collagen and soy bean lectin were studied as these are high molecular weight, multifunctional and adhesive glycoproteins with a distribution in eukaryotic cells. In an earlier report (48) we described the relationship between four types of S. aureus that exhibit different colonial morphology and cell surface structure. In the present study, the adhesion to hydroxy apatite and to fibrinogene, collagen-, and lectin-coated hydroxyapatite of an S. aureus strain and its variants possessing different cell surface stru ctures was investigated.

Materials and Methods

Strain Strain S-P of S. aureus (Smith diffuse) and its variants, S-A, S-B, and S- C were used. These strains were reformed from the original strain, S-7, S-1 8, and S-12 and S-C due to the result of seru m-soft agar (SSA) application as noted described previously (48) and as below. The parent strain (S-P, large encapsulated strain ) and the variant S-A strain (small encapsulated strain ) show streaming and thin-type colonial morphol ogies in SSA medium and they exhibit the highest and second highest virulence in mice, respectively (48). Ultrathin sections of the parent strain stained by uranyl acetate showed that the orgaisms were surro unded by a zone of oval to round polymorphous vesicular structures in the outermost layer, the structures being covered by a zone of high electron density (Fig. I-a). The variant ' S-A lacked the oval to round polymorphous vesicular structures as well as the high electron density zone (Fig. I-b). The variant S-B strain, which was designated as microcapsular, in accordance with the description by Caputy and Costerton (12), possessed an extramicrocapsule, large round colonial morph ology in SSA and exhibited low virulence in mice. A faint electron dense area extended aro und the outermost layer of the cell wall (Fig. I-C). The variant, S-C unencapsulated, showed small compact type growth in SSA, was avirulent in mice and had no external layer (Fig. I-d) . The biological and serological activities of these strains were determined by the methods described by Yoshida and Takahashi (48).

Growth conditions

3rc

The organisms were cultured in brain heart infusion (BHI; Difco) broth at for 14 h with constant shaking. S. aureus cells were labeled with tritium by culture for 4-24 h in BHI broth containing 24 f.l Ci [methyl-l ', 2-3H] thymidine (Radiochemical, Center) per ml. The cells were washed with pho sphate buffered saline, (pH 7.2), and resuspended in 3 ml of.the same buffer to give appro ximately 107 cells per ml, The final concentration of the thymidine-labelled cells was determined by counting the number of viable bacteria. A suspension cont aining 107 3H-thymidine-Iabelled bacteria rnl" had a radioactivity of 10 to 15 f.l Ci ml"! as counted in a Beckman liquid scintillation system (LS-230).

S. aureus Adhesion and Cell Structure

327

Preparation of cellular substances Extraction and purification of the cell wall (CW) and cell wall teichoic acid (CWTA) from strain S-C (29), and capsule (CF) and capsule teichoic acid (CTA) fractions from the parent S-P (31) were performed as previously described. Lipoteichoic acid (LTA) was extracted and purified from the parent strain S-P according to the methods of Ofek et al. (28) and Alkan and Beachey (3). Slime materials from strain S-P were prepared as described elsewhere (35). Surface polysaccharide antigen (SPA) extracted with 5% trichloroacetic acid (32,36) was purified by a modification of the methods of Smith (41) and Liau et al. (26). Protein A (PA) was isolated from variant S-C by the method of Jensen (23). Highly purified PA was obtained by column chromatography using DEAE-cellulose (Sigma) and Sephadex G-200 (Pharmacia) as described by Forsgen and S;oquist (20). Compact colony forming active substance (CCFAS) was isolated from variant S-C and was prepared according to the methods described previously (37, 50).

Enzymatic treatment of large capsulated strain (S-P) and the unencapsulated variant (S-C) of S. aureus The effects of physical or enzymatic treatment on the adherence of the strains to lectin, collagen and fibrinogen was studied. Samples of the bacteria (2 ml; 5 X 10 8 cell ml') were treated with the following enzymes: 200 ug trypsin (type 11 twice crystalized; Sigma) in phosphate buffered saline (PBS), pH8.2; 100 ug pronase (Type V1, Sigma) in PBS, pH 7.8; and 200 ug papain (Type 11, Sigma) in PBS,pH 6.0, containing 30 mMCaCI 2• The mixtures were incubated for 1 h at 37 °C. The cell surface was also treated with glucose oxidase, galactose oxidas~ (Sigma) and mixed ?lycosidase (Seikagaku, To~yo) . Sample of S-P and S-C cell suspensions (2 ml; 4-6 x 10 cells. ml') were treated WIth 100 to 200 ~g of the above enzymes in 0.15 M phosphate buffer, pH 7.6, for 30 min at 37 °C. Bacteria were then collected by centrifugation at 3000 g for 15 min and washed twice with 0.15 M NaC! . They were resuspended in 0.15 M sodium phosphate buffer, pH 703 , and the adhesion assay was performed. As a control, bacteria were treated with buffer solutions lacking the enzymes.

Physical and periodate treatments of large capsulated strain (S- P) and unencapsulated variant strain (S- C) of S. aureus Physical treatments consisted of incubating bacteria in OJ M tris-buffer at pH 8.5 and pH 6.0 for 30 min and heating cells at 80 °C and 100 °C in 0.03 M tris-buffer, pH 7.0, for 15 min. Bacteria were suspended in 10% sodium periodate in PBS at pH 6.5 and incubated for 10 h at 4 °C. Treated organisms were collected by centrifugation (3000 g for 15 min) and washed twice with 0.05 M KCI in PBS before the adhesion assay. Control cells were incubated in PBS lacking the periodate.

Preparation of [ibrinogen-, collagen- and lectin-coated hydroxyapatite beads Hydroxyapatite beads (HA-b) with a diameter of 50-100 urn and an approximate surface area of 21 cm 2 g-l were coated with fibrinogen, collagen and lectin by the method of Appelbaum et al. (6). HA-b were washed twice with 15 volumes of PBS in Hopkins tubes and beads of uniform size were obtained by decantation. The beads were then allowed to equilibrate in 0.01 M PBS buffer at 30 °C for 12 h. Proteins were absorbed onto HA-b (lg) by mixing the HA-b with 0.3 % (w/v) sterile solutions of the fibrinogen (human, KABI, Sweden ), collagen (Type III, acid soluble, from calf skin, Sigma) and Soy-bean lectin (Type VI, from Glycine max, affinity for N-acetyl-D-galactosamine, Sigma) in O.OlM Phosphate buffer (pH 7.4) containing 0.01 M MgCI2> 0.05M CaCl 2 and 0.05 M KCl. The suspensions were incubated for 10 h at 20 °C and then freeze-dried to the protein-coated HA-b which were then stored at 4 °C. The protein content of the coated beads wa s determined by the Lowry method to be 100-200 mg per g of beads. The various coated hydroxyapatite materials were designated FHA, CHA and LHA, respectively. 20

Zbl. Bakt. Hyg. A 268/3

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T. Ohtomo and K. Yoshida

Adhesion assay The assay was a modification of the procedures described by Clark et al. (15) and Staat et al. (42). Protein coated HA-b (15 mg) were mixed with 4.2 x 105 3-H-labelled bacteria in PBScontaining 0.1 M MgS0 4 and CaCl z• After equilibration by shaking for 2 h at 37°C the beads were allowed to settle. The supernatant fluid containing unabsorbed organisms was then removed and the radioactivity contained in 1 ml samples was counted in a Beckman liquid scintillation system (LS-230). A known number of 3H-labelled bacteria were counted by the same method so that the counts per minute related to the number of bacteria present. Unabsorbed and poorly absorbed bacteria were removed from the protein-coated HA-b by two rapid washings with PBS buffer. After the final washing, the beads were dried at 50°C overnight and then bound radioactivitiy was determined. Bacterial adherence to the plastic vials in the absence of protein-coated HA-b or in the presence of uncoated HA-b was determined for all experiments. The data were corrected to determine only binding to protein-coated HA-b. Binding to the vials was usually less than 10 % of the amounts bound to the protein-coated HA-b.

Inhibition experiments Inhibition of adherence was measured after pretreatment of the protein-coated HA-b with cell wall (CW), capsular fraction (CF), cell wall teichoic acid (CWTA), capsular teichoic acid (CTA), compact colony forming active substance (CCFAS), surface polysaccharide (SP), lipoteichoic acid (LTA), and protein A (PA). Protein-coated HA-b (lg) were washed with PBS, PH 7.6, and then incubated for 1 to 2h at 37 °C with 50 to 100 mg of CW, CF, CWTA, CCFAS, LTA and PA. The HA-b were centrifuged at 1000 g for 30 min, washed with PBS buffer to remove unattached cellular products and then used in adherence assays. As controls, protein-coated HA-b were treated with PBS buffer alone. Similarly, inhibition of adherence by D-glucose, D-galactose, L-fucose, Lrhamnose, N-acetylgalactosamine, glucosamine, a-methylgalactoside, and B-methylgalactoside were determined after preincubation of protein-coated HA-b with solutions of each sugar in 0.01 M trishydrochloride buffer, pH 7.6 was added to 1 mg protein-coated HA-B. The mixtures were incubated for 1 h at 37 °C and then centrifuged at 100 g for 3 min. The inhibition percent were compared to control values obtained with no substances added .

Electron microscopy study Cells of S. aureus grown in BHI agar medium at 37 °C for 18h were harvested by centrifugation at 8,000 g at 4 °C for 10 min. They were fixed initially with 5% (w/v) glutaraldehyde in 0.1 M phosphate buffer, pH 7.0 at 4 °C for 18h, and then fixed with 0.1 % osmic acid at room temperature for 5 h according to Ohtomo et al (33). The fixed material was first dehydrated with ethanol and acetone, and then embedded with Epon 812 by the standard procedure. The sections were prepared with an LKB microtome, mounted on carbon coated grids, and stained with aqueous uranyl acetate. These sections were observed by a JSM 100B transmision electron microscope (Japan Electron Optics Lab., Tokyo, Japan) operating at 80 KV.

Results

Adhesion of S. aureusand cellsurface variants to [ibrinogen-, collagen- and lectincoated HA-b The comparative adherence of strain SoP, and its variants, S-A, SoB and SoC, to FHA, CHA and LHA or HA-b were investigated in vitro. Different degrees of adhesion to HA-b coated with various proteins (Table 1) were observed among the different strains

S. aureus Adhesion and Cell Structure

329

Table 1. Relative adhesion capacity of 3H thymidine-labelled S. aureus cellsfrom strain S-P and its variants, S-A, S-B and S-C, to fibrinogen-, collagen- and lectin-coatedhydroxyapatite beads Strain

HA-beads only

S-P S-A S-B S-C

43.3 38.4 53.9 74.9

a

b C

d

e

± 4.8e ± 5.3 ± 9.9 ± 8.2

Adherence capacity (binding cpm FHA" CHAb 36.8 78.9 254.5 398.7

± 3.9*** ± 8.9** ± 29.3*** ± 32.9*

197 219 252 230

± 14.9ns ± 16.8ns ± 21.5* ± 19.2*

X

10-2 ) ± SEMd LHAc 352.9 192.3 110.9 78.9

± ± ± ±

32.6* * 17.9** 6.8* 6.7**

Fibrinogen-coated hydroxyapatite beads. Collagen-coated hydroxyapatite beads. Lectin-coated hydroxyapatite beads. The results are the mean ± the standard error of the mean of three experiments. The significance (p value) of differences between control and experimental group was evaluated according to student's t test. ns, no significant difference, ", p < 0.05, **, p < 0.01, ***, p < 0.001. Data obtained by non-coated proteins HA-b only were used as control.

(Fig. 1). A striking difference in the adhesion of the large capsulated strain S-P (Fig. i-a) and the non-capsulated variant (S-C) (Fig. I-d) to FHA and LHA was noted. The greatest adhesion to FHA was observed with the non-capsulated variant (S-C) rather than with the S-P strain. The reverse was true when adhesion to LHA was examined. In this case the greatest adhesion was shown by strain (S-P) whereas the small-capsulated cells (Fig. l-b) adhered less well. However, the adhesion of non-capsulated (S-C) cells to LHA was about 5 times (p < 0.05) less than that of the large capsulated strain S-P with a polysaccharide rich outermost layer. The adhesion of S-C cells to FHA was about 9-10 times (P < 0.05) greater than that of S-P cells. On the other hand, strain SP and variants, S-A, S-B (Fig. i-c) and S-C, possessed similar adhesion capacities to CHA. Binding of all strains to uncoated HA-b was low (Table 1).

Adhesion as function of the concentration of S. aureus cells and incubation temperature

In comparing different adhesion conditions in this system, we examined the effects of cell concentration and incubation temperature. Increasing the cell number of variants S-C and parent S-P cells in the PBS buffer (up to a cell concentration of approximately 3.4 X 10 9. mI-1 in PBS buffer containing 0.05 M KCI) resulted in a linear increase in adhesion to the protein-coated HA-b. When variants S-C and parent S-P cells were gently incubated at 37°C with each proteincoated HA-b, adhesion of the cells to FHA, CHA and LHA reached maximal levels within the first 30 min incubation period. Further incubation for 1 h or more showed no increase in adherence. Also, the adhesion of all strains was identical following incubation at temperatures of 10°, 20°, 30°, and 37°C (Results not shown).

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T.Ohtomo and K.Yoshida

a

b

S. aureus Adhesion and Cell Structure

331

c

d

Fig. 1. Ultra-thin sections of (a) large encapsulated (S-P), (b) small encapsulated (S-A), (c) microcapsulated (S-B) and (d) unencapsulated strains of S. aureus stained with uranyl acetate; scale-bar indicated 0.2 11m.

332

T. Ohtomo and K.Yoshida

Effect of physical and enzymatic treatment of S-P and S-C strains of S. aureus on adhesion to fibrinogen-, collagen-, and lectin-coated HA-b These experiments were designed to compare the effects of physical or enzymatic pretreatment of strains S-P (large capsulated) or S-C (non-capsulated) on their adhesion capacity to FHA, CHA and LHA. Pretreatment of S-C cells with proteolytic enzymes such as trypsin and papain significantly decreased their capacity to adhere to FHA, much smaller effects were shown on the adhesion of S-Pcells (Table 2). Pretreatment of strain S-P with galactose oxidase and glycosidase for 30 min markedly increased its capacity to adhere to FHA but decreased adherence to LHA. Adhesion to CHA was not obviously affected. The effects these enzymes on adhesion of strain S-C to any of the protein-coated beads was not marked. When S-P cells were treated with 10% sodium periodate, their adherence capacities to LHA were markedly affected whereas adherence to CHA and FHA was not or no affected. However, the effect was observed after treatment of S-C strain with periodate to FHA and CHA but LHA had a little effect. These suggest that both carbohydrate and protein moieties on cell surface may be involved in adherence. Heating S-P and S-C cells at 80° or 100°C for 15 min, did not change their adhesion to FHA whereas changes were shown in the adhesion of both strains to CHA and LHA. After 30 min incubation at pH 8.5 and 6.0, the adhesion capacity of both S-P and S-C cells to CHA was unaffected, and LHA had little affected. However, the adherence capacity of S-C cells to FHA was lost after incubation at pH 6.0 (results not shown).

Effect of pretreatment of FHA, CHA and LHA with various cellular materials on bacterial adhesion The adhesion of S-P and S-C strains to protein-coated HA-b which had been pretreated with cellular materials was investigated (Table 3). The inhibition of adhesion of S-P cells to CW- and CF-treated FHA differed from that of S-C cells. The most potent inhibition of adhesion was the decrease in strain S-C binding to FHA after pretreatment with CCFAS. Pretreatment of FHA and LHA with CW resulted respectively in about 62 and 42% (p < 0.05) reductions in adhesion of S-C cells whereas the adherence of S-P cells to CW-treated FHA and CHA decreased only 16 and 27% (p < 0.05), respectively. The greatest inhibition of adhesion to LHA occurred when the coated HA-b were pretreated with CTA in the case of S-P cells and with CWTA in the case of S-C cells. CP and PA were less effective than the cellular substances in inhibiting adherence of S-C and S-P cells to FHA, CHA and LHA. Pretreatment of CHA with the cellular substances only inhibited the adhesion of both S-P and S-C cells, and the level of inhibition by each substance was similar in both strains.

Effect of sugar on adhesion The adhesion of S-P and S-C cells to FHA, CHA and LHA treated with a range of sugars and aminosugars was investigated. Treatments with glucose, L-rhamnose, Nacetylglucosamine and glucosamine caused no significant inhibition of adhesion of S-P or S-C cells to FHA, CHA and LHA. However, galactose, ~-methylgalactoside and Nacetyl-galactosamine inhibited the adhesion of both strains to FHA and LHA, whereas less inhibition was observed with u-methylgalactoside and galactosamine as shown in Table 4. Adhesion to CHA did not change significantly after treatment with these sugars. Pretreatment of FHA with ~-methylgalactoside and N-acethylgalactosamine resulted in a 49-70% inhibition of adherence of both S-P and S-C strains (Table 4).

d

C

b

a

37.9 40. 9 47.9 29.5 68.9 234 .6 289.5 28.9

FHA"

± 1.9 ± 6.9 ± 8.7 ± 6.3 ± 4.8 ± 37 .6 ± 42 .9 ± 3.3 205 .3 178.4 193 .8 163 .8 134 .9 176 .9 186.4 162 .9

± ± ± ± ± ± ± ± 7.9 34.8 33.4 27.8 25.2 22 .9 24.6 12.3

314.6 247.8 23 8.4 216.4 184.3 67 .8 45.6 33.3

± 13.8 ± 29.9 ± 38.5 ± 33 .3 ± 27.8 ± 4.6 ± 4.1 ± 4.1 369.5 38.9 87.4 93.9 169 .9 288.4 247.9 62.3

± 16.9 ± 4.8 ± 3.5 ± 4.4 ± 17.9 ± 27.9 ± 29 .9 ± 5 .3

210 .2 183 .5 87.4 198 .3 177.5 109.2 142 .3 93.2

± ± ± ± ± ± ± ±

10.3 17.6 19.9 13.4 16.6 19.5 13.9 8.3

Adherence capacity (binding cpm x 10- 2 ) ± SEM d S-C (unencapsulated) S-p (large capsulated) FHA CHA CHAb LHA'

63.4 20.5 39.8 47.8 5 6.9 41.5 38.4 33 .4

LHA

± 2.6 ± 3.9 ± 7.4 ± 4.9 ± 3.9 ± 5.8 ± 3.6 ± 4.2

Fibrinogen-coated hydroxyapatite beads. Collagen-coated hydroxyapatite beads. Lectin-coated hydroxyapatite beads. The results are the mean ± the standard error of the mean of thr ee experiments. Th e significance (p valu e) of differenc es betw een control and all experimental group was evaluated according to student , s t test, p < 0.05 .

Control 200 ug trypsin 100 ug pronase 200 ~g papain 100 ug glucose ox idase 100 ~g galactose ox ida se 200 ug glycosidase Sodium periodate

Pretreatment

Table 2. Effects of physical and enzymatic treatment of 3H thym idine-labelled S-P and S-C strains of S. au reus on adhe sion to fibrinogen-, coJlagen- and lectin -coated hydroxyapatite beads

w

w w

@

a

(")

2

Vl

n 2::

'"

=' =' 0-

'" o'

n>

'" >§:

n> C

..,'"c

!J"

2.4 9.6 0.4 13.6 ± 9.6 ± 4.2

± ± ± ±

7.9 ± 1.1 11.3 ± 3.2 1.2 ± 0.3

16.7 73.8 6.2 87.9 93.5 38.9

FHAa

± ± ± ± ± ± 1.9 1.8 0.7 1.6 3.1 2.6

8.3 ± 0.6 9.3 ± 0.8 0.7 ± 0.07

14.5 16.4 9.7 14.9 24.7 29.8

± ± ± ± ± ± 5.2 6.5 0.6 14.4 9.9 2.9

39.5 ± 5.3 5.5 ± 0.6 1.2 ± 0.4

27.5 62 .6 6.7 94.7 88.6 19.9

± 14.9

± 1.7 ± 11.3

± 9.3 ± 6.4 ± 9.3

.1 ± 0.7 6.7 ± 0.7 0.9 ± 0.1

62.8 23 .5 82 .8 13.3 73.5 96.8

± 2.9

± 0.7 ± 2.4

± 2.1 ± 1.5 ± 0.9

7.6 ± 0.5 11.3 ± 0.6 0.7 ± 0.1

17.3 12.3 11.3 9.6 28.5 34 .5

Percent inhibition of adherence ± SEM Unencapsulated (S-C) Large capsulated (S-P) FH A CH A CHAb LHN

± 3.1

± 4.4 ± 9.6

± 6.2 ± 1.5 ± 14.2

4.5 ± 0.6 4.8 ± 0.3 1.8 ± 0.4

42.5 16.8 91.9 17.3 84.3 25.4

LHA

b

a Fibrinogen-coated hydroxyapatite . Collagen-coated hydroxyapatite. C Lectin-coated hydroxyapat ite. d The results are the mean ± the standard error of the mean of three experiments. The significa nce (p value) of differences between contro l and all experimental group was evaluated according to student, s t test, p < 0.05.

Cell wall (CW) Capsular fraction (CF) Cell wall teichoic acid (CWTA) Capsular teichoic acid (CTA) Lipoteichoic (LTA) Compact-colony forming active sub stance (CCFAS) Capsular po lyaccharide (CP) Protein A (PA) Control

Pretreatment

d

Table 3. Inh ibition of adhesion of 3H thymidine-labelled S-C and S-P strains of S. aureus to fibrinogen-, collagen- and lectin-coated by various cellular substances

w

'"

::r 0.:

?" ~ en

0-

~

g

8

o ::r

:-I

~

w

d

C

b

a

66.5 20.1 70.9 52.9 29.4 2.4

± ± ± ± ± ±

FHA"

7.3d 2.1 6.8 7.4 4.2 0.4 14.3 18.9 21.3 13.4 10.1 1.9

± ± ± ± ± ± 1.3 0.2

1.5

1.1 3.3 1.9 78.9 37.5 73.9 79.8 36.7 2.0

± ± ± ± ± 4.3 8.3 11.4 4.1 0.5

± 7.8 76.9 20.9 68.6 49.4 31.2 0.9

± ± ± ± ± ± 8.1 4.1 6.8 3.7 3.6 0.1

17.3 16.3 13.1 6.9 13.4 0.7

± ± ± ± ± ±

1.4 2.3 1.2 0.5 1.1 0.09

Percent inhibition of adherence ± SEM d Large capsulated (5-P ) Unencapsulated (5-C) CHA b LHN FHA CHA

49.9 19.3 76.8 53.9 28.3 1.8

LHA

± ± ± ± ± ±

3.9 3.3 6.2 5.5 2.5 0.3

Fibrinogen-coated hydroxyapatite beads. Collagen-coated hydroxyapatite beads. Lectin-coated hydroxyapatite beads. The results are the mean ± standard error of the mean of three experiments. The significance (P value) of difference s between control and all experimental groups was evaluated according to student, s t test, < 0.01.

D-galactose a -methylgalactoside j3-methylgalactoside N-acetylgalactosamine Galactosamine Control

Pretreatment

Table 4. Inhibition by sugar of adhesion of 3H thymidine-labelled S-C and 5-P strains of S. aureus to fibrinogen-, collagen-and lectin

Y"

v,

w w

~

2

8 n

Vl

~

ll>

=' =' 0n

o'

~

e-

'" > 0-

g

C

ll>

336

T. Ohtomo and K.Yoshida

Effect of various phases of growth on adhesion to FHA, CHA and LHA Strains S-P and S-C were grown at 37°C (culture conditions were as described above) and harvested at 4 h intervals up to 24 h. The adhesion capacity of S-C cells at various phases of growth to FHA, CHA and LHA were nearly constant. However, the growth phases of the S-P cells (large capsulated) showed a marked influence on their capacity to adhere to each type of protein-coated HA-b (Fig. 2).

Discussion Potential interaction between bacterial surface polymers and the surface of the substrate must be investigated in order to clarify the mechanism of bacterial adhesion. One important interaction in adhesion involves the polysaccharide of the so-called "glycocalyx" moiety of the bacterial surface (16, 21). Such molecules will contribute to the physical properties of the bacterial surface and by their effects on charge density, hydrophobicity and polymer interactions, must influence the cohesive properties of any specific mediators of microbial adhesion (2,45). However, the function of this surface polymer in S. aureus is known to be biochemically complicated (30, 34, 46, 47). The results presented in this paper concerning the in vitro adhesion of staphylococci to protein substances such as fibrinogen, collagen, and soy bean lectin show that cell surface structure variants possess different capacities to adhere to proteins, suggesting specific adhesion between the bacterial surface and macromolecules.

Fig. 2. Effect of growth phase on adhesion of 3H thymidine labeled S-C and S-P strains of S. aureus to fibrinogen-, collagen- and lectin-coatedhydroxyapatite. (-.6.-) fibrinogen-coated hydroxyapatite, (-0-) collagen-coated hydroxyapatite, (-e-) lectin-coatedhydroxyapatite. Each point represents the mean ± standard deviation of four determinations.

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Lectins are proteins or glycoproteins of non-immune origin which bind with specific sugars (18, 45). In the present experiments, the adhesion to soy bean-lectin of the S-P strain, which is rich in polysaccharides in its outermost layer (32), differed from that of strains S-A (small capsule with abundant cell surface protein), S-B (microcapsule) and S-C (unencapsulated). In addition the capacity to adhere to various proteins after physical and enzymatic treatments of the bacteria or pretreatment with cell surface substances derived from S. aureus suggest a possible direct interaction between cell surface components and protein substances. This is shown by the effects of galactose, ~-methylgalactoside and N-acetylgalactosamine which inhibited adherence of the encapsulated S-P strain of S. aureus to LHA. Inhibition of this type of adhesion was also shown by treatment of the bacteria with galactose oxidase and glycosidase. Thus adhesion of strain S-P to LHA may be due to the occurrence of multiple galNAc units in components of staphylococcal capsule. It is known that the unencapsulated strain SC dose not contain galNAc in the cell surface fraction (32). Recently, fibrinogen or fibronectin was shown to bind to S. aureus (25, 38, 44,50). Our experiments suggest that this binding occurs to a lesser degree with the encapsulated S-P strain. The adhesion of the unencapsulated (S-C) strain to fibrinogen was much greater than observed with the encapsulated S-P strain. Furthermore, trypsin, pronase and galactose oxidase treatments which remove proteinaceous components and polysaccharides including a clumping factor (22) or CCFAS (37), markedly reduced the adherence of S-C or S-B strains of S. aureus to fibrinogen. On the other hand, the adhesion of the S-P strain (large capsule) to FHA was not affected by treatment with the above enzymes. Clumping factor is known to be trypsin sensitive (22) and CCFAS is known to be stable to alkali and is associated with polysaccharides containing galactose (37,50). Fibrinogen, collagen and lectin were chosen in order to obtain a wide range of isoelectric points, in the belief that isoelectric points might affect adhesion (1, 19,40,43) suggested that bacterium-collagen fiber interaction is mediated by a physicochemical interaction with either the collagen or the mucopolysaccharide cementing matrix between individual collagen or fiber. Our studies show that the S. aureus cell surface variants have similar adhesion capacities of collagen-coated HA-b, specifically to type IV (acidic soluble calf skin collagen). Early studies suggested that LTA or TA from Gram-positive bacteria were the principal components involved in the adhesion process (4, 5, 8, 28). Our study presents evidence that cell surface LTA and CTA bind to FHA, CHA and LHA and inhibit the adhesion of both encapsulated (S-P) and non-capsulated (S-C) S. aureus to these proteins. Similar reductions in adhesion of both encapsulated and non-capsulated strains were observed after LTA-treatment of the protein-coated HA-b. In contrast, the capacity of non-capsulated S-C cells was reduced 60-93% when the protein substances were treated with CWTA whereas CWTA pretreatment had little effect on the adhesion of the encapsulated S-P strain. These results suggest that cell surface LTA is a component of adhesion sites common to both encapsulated and non-capsulated strains of S. aureus while CWTA is a component of receptor sites in the non-capsulated strain but not in the encapsulated strain. At present, the mechanism of specific interaction between CWTA, CTA or LTA from S. aureus and various protein substances is not known. However, several papers (3, 9, 10,28) have discussed similarities of agglutination activity of TA from bacterial strains and that of various lectins (18). An experimental model for adhesion of bacteria to HA-b or protein coated HA-b was used in studies of dental plaque (15). This system has several limitations which

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must be understood to avoid misinterpretation of the data (8). In our binding experiments, results in different experiments varied by approximately 10%, although the coefficient of variation for the parameters was much higher in experiments involving enzymatic pretreatments of the Sop strain. On the other hand, an encapsulated stra in of S. aureus (S-P) was shown to possess different adhesion capacities in early, mid-, or late log phases. There was no change of adhesion capacity of non-capsulated S-C cells in any stage of growth. These results suggest that cell surface structures are involved in the adhe sion capacity of bacteria to host cell or materials (27 , 38, 39 , 45). However, further investigations are required concerning adhesion in vivo and these investigations are currently in progress in our laboratory.

References

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Toshichika Ohtomo, Ph. D., M.D., Dept. of Microbiology, St. Marianna University, School of Medicine, 2-16-1, Sugao, Miyamaye-ku, Kawasaki 213, Japan