Cell Surface Antigen of Encapsulated Staphylococcus epidermidis SE-360 Protects Mice from Homologous Infection

Zbl. Bakt. Hyg. A 270, 219-227 (1988)

Cell Surface Antigen of Encapsulated Staphylococcus epidermidis SE-360 Protects Mice from Homologous Infection* YUKIO OHSHIMA Department of Microbiology, St. Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-Ku, Kawasaki 213, Japan

With 2 Figures

Summary Cell surface antigen was mechanically extracted from encapsulated strain SE-360 of

Staphylococcus epidermidis and purified by DEAE-SephadexA 25 (CI- form) ion exchange chromatography. This antigen manifested type-specific activity and major sugar constituents were galactose, glucose and N-acetyl-giucosamine at the molar ratio 1.00: 9.05: 1.65. a-D-giucosyl- and N-acetyl-giucosaminyl-residueswere closely correlated to the antigenic determinant. In mice, protection against homologous microorganisms could be achieved by active immunization with thus purified antigen. Type-specific opsonin in rabbit anti-SE-360 serum could also be absorbed.

Zusammenfassung Das Zelloberflachenantigen des bekapselten Staphylococcus epidermidis-Stammes SE360 wurde mechanisch extrahiert und mittels DEAE-Sephadex A 25 (Cl-Porml-Ionenaustauschchromatographie gereinigt. Dieses Antigen vermittelte eine typspezifische Aktivitat. Als hauptsachliche Zuckerbestandteile konnten Galaktose, Glukose und N-Acetyl-Glukosamin in einem molaren Verhiiltnis von 1.00: 9.05: 1.65 respektive nachgewiesen werden. aD-Glukosyl- und N-Acetyl-Glukosaminyl-Bestandteile waren mit der antigenen Determinante eng verbunden. In Mausen konnte mit diesem Zelloberfliichenantigen eine aktive Immunisierung gegen den homologen Staphylokokkenstamm durchgfiihrt werden, auch konnten damit typspezifische Opsonine im Kaninchen-Anti-SE-360-Serum absorbiert werden.

Introduction Coagulase-negative staphylococci (CNS) have been recognized as human pathogens being able to cause septicemia and urinary tract infection (3, 4, 6, 15, 20, 27, 30). Although more than 20 species of CNS have been identified, S. epidermidis was most

* Dedicated to Professor Dr. G. Henneberg on occasion of his 80th birthday.

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frequently isolated from clinical specimens (3,4,20,27). Some of them could be shown to be encapsulated (1, 10, 12, 13,33) and proved to be more virulent than unencapsulated bacteria as well as encapsulated Staphylococcus aureus (7, 12, 16, 17,21,32). Slime or capsule components of those organisms may be considered to be the main virulence factors of S. epidermidis (12, 14, 32). These surface substances are able to mask opsonin-binding receptors on cell walls of bacteria (11, 29). Opsonin mediated bacterial adhesion to phagocytic cells may be considered an important step in phagocytosis. Bacterial endocytosis eventually may occur more rapid after this procedure. Especially, the third component (C3 b, C3 bi and C3 d molecules) of the complement cascade plays an important role in promoting phagocytosis and killing of bacteria (11, 26,28). To achieve in vivo clearance of encapsulated bacteria, specific opsonins (i. e. antiserum against cell surface substances) are required for the promotion of bacterial killing. Similarly, antigen inducing production of specific opsonin against encapsulated bacteria (i. e. as a vaccine) might also be useful for the in vivo clearance of certain bacteria (7, 12, 16, 23). Here, the isolation and biochemical characterization of an antigen of the encapsulated strain SE-360 of S. epidermidis protecting mice from homologous infection is discussed.

Materials and Methods

Bacterial strain. Encapsulated strain SE-360 of Staphylococcus epidermidis isolated from clinical specimen, as described by lchiman and Yoshida (12), was used in this study. To obtain cell surface antigen, S. epidermidis SE-360 was grown in dialyzate of brain heart infusion (BHI, Difco Laboratories, Detroit, Mich.) supplemented with 10% (w/v) glucose at 37°C for 20 h. Cells were harvested by centrifugation (2800 g for 30 min) and suspended in phosphate buffered saline (PBS; 0.1 M, pH 7.0). Preparation ofthe cellsurface antigen. Bacterialsuspensionswere sonicated at 10 KC for 5 min in an ice bath and centrifuged at 2800 g for 30 min. The supernatant was filtered (Milliporefilter, 0.45 J.UIl) and treated with ribonuclease (Sigma) and pronase (Sigma), respectively. After enzyme digestion, chloroform was added and the mixture was kept overnight at 4°C under agitation to remove free fatty acids and protein. The water-phase containing the cell surface antigen was obtained by centrifugation, dialyzed against dist. water, lyophilized and designated as crude cell surface antigen (CSA). Crude CSA (100 mg) was dissolvedin 3 mIlO mM Tris-HCI buffer pH 7.0, and applied to DEAE-Sephadex A 25 column (Cl- form; 1.0 by 30.0 em, Pharmacia) equilibrated with same buffered solution. After elution with 10 mM Tris-HCI buffer, binding substances were eluted by linear gradient elution with sodium chloride (0 to 1.0 M). Each fraction was collected, dialyzed against dist, water, lyophilized and used in subsequent experiments. Chemical analysis. Total carbohydrate (5), hexosamine (8), protein (19) and phosphorus (2) contents were determined as previously described. Each sample was hydrolyzed with 2 N HCI at 100°C for 3 h. To assess sugar and aminosugar composition of hydrolysate, these sugars were analyzed by gas-liquid chromatography after preparing alditol acetate derivatives of the hydrolysate as previously described (22). Preparation of rabbit antiserum and determination of its activity. Preparation of rabbit antiserum and determination of its activity (12) as well as techniques to measure immunochemical properties (23, 25) were recently described.

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Quantitative precipitin reaction of cell surface antigen against rabbit antiserum and concanavalin-A. Quantitative precipitin reaction of CSA against rabbit antiserum was performed as follows: 50 III of antiserum was mixed with 450 III PBS containing varying amounts of the antigens. After incubation at 37 °C for 1 h, the mixture was kept overnight at 4°C. Precipitates were collected by centrifugation at 1500 g for 15 min. To measure the precipitin reaction of CSA against concanavalin-A (Con-A; Sigma), 100 I-l1 of Con-A (5 mg/ml) was mixed with 400 III of PBS containing varying amounts of antigen. After incubation as described, the resulting precipitates were collected by centrifugation at 1500 g for 15 min. Both precipitates (against antiserum and Con-A) were washed twice with PBS, and dissolved in 1% (w/v) Na2CO~. Protein contents of the precipitates were measured by the method of Lowry et aI. (19). Precipitin inhibition with monosaccharides. Inhibition of the quantitative precipitin reaction of CSA against rabbit antiserum with monosaccharides was carried out as follows; 100 ul PBS containing 10 mM monosaccharides (fucose, rhamnose, glucose, a-methyl-Dglucoside, ~-methyl-D-glucoside, galactose, mannose, Nsaceryl-glucosamine, N-acetylgalactosamine, glucuronic acid and galacturonic acid) were added to 50 111 rabb it antiserum and incubated at for 1 h. After preincubation, 150 I1g of CSA (optimum concentration to 50 III antiserum) was added and the final volume was made up to 500 III per tube with PBS. These mixtures were incubated at 37°C for 1 h, and stored overnight at 4°C. The protein contents of precipitates were determined as described above. Absorption ofpassive protective activity in rabbit antiserum with cell surface antigen. To rabbit antiserum (1 ml) containing two units of protective activity, 100,300 and 1000 I1g of the antigen were added. The mixtures were maintained at 37°C for 2 h, and centrifuged at 6000 g for 15 min at 4 °C. The supernatant (0.5 ml absorbed serum) was intraperitoneally injected into mice (n = 5). Thirty minutes later, the mice were challenged with 0.5 ml of 8% (w/v) mucin containing 1 X 108 colony forming units (CFU) of strain SE-360. The number of dead animals was recorded during a period of 10 days. Active immunization with cell surface antigen. 3, 10 and 30 ug of CSA were intraperitoneally injected into mice (n = 5). Ten days after active immunization, the micewere challenged intraperitoneally with 0.5 ml of strain SE-360 as described above and the number of dead mice was recorded over a period of 10 days.

3rc

Results Biochemical properties of cell surfa ce antigen s After applying cell surface antigenic substance to DEAE-Sephadex A 25 (Cl- form) column, six fractions could be obtained as shown in Fig. 1. Three out of six (subfractions 4 to 6) reacted with antiserum as well as with Con-A (Table 1). Serologically active fractions did not show cross reacti vity to other CSAs isolated from encapsulated S. epidermidis and S. aureus in immunodiffusion tests. Quantitative analysis of the serologically active fractions (subfractions 5 and 6) manifested high amounts of total sugar whereas high amounts of hexosamine could be detected in subfraction 5 (Table 1). Concerning carbohydrate components of serologically active fractions (subfractions 4 to 6), galactose and glucose were detected as common monosaccharides at molar ratios (galactose to glucose) of 3.94, 9.05 and 1.31, respectively . However, glucosamine was detected in subfractions 4 and 5, whereas sub fraction 6 contained negligible amounts only (Table 2). Glycerol could be detected in sub fractions Sand 6, exclusively.

Y.Ohshima

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1.4

2

8

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to

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cQ8

j

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.....

:E

--

,/

...

1.0

si

~Q6

JJ c(

Q4

I I

I

Q2

40 eo 80 Fraction number

20

100

Fig. 1. Elution pattern of crude cell surface antigenic substance from encapsulated Staphylococcus epidermidis SE-360 on a DEAE-Sephadex A25 (Cl- form) ion exchange chromatography. - 0 -, -

e-- and ----x- --- indicate protein, carbohydrate and phosphorus, respectively.

Table 1. Quantitative analysis of subfractions from DEAE-Sephadex A 25 ion exchange chromatography Fraction Number

Tot al Sugar

Hexosamine content

Protein content

Phosphate content

Reactivity to Con-A

Fr-l Fr-2 Fr-3 Fr-4 Fr-5 Fr-6

18.5· 31.8 23.7 34.0 76.1 54.7

15.27 18.71 20.61 19.09 22.91 11.83

466.5 318.8 304.7 302.4 234.4 128.9

0.72 1.44 15.26 24.05 75.38 75.56

+•• + + +++ +++ +

• Micrograms per one mg of sample Precipitin reaction

••

Absorption of passive protective activity with cell surface antigen To determine the absorbing activity of CSA to rabbit antiserum, 0.5 ml of the antiserum was used per mouse which could be shown to protect mice (100% ) after str ain SE-360 challenge. After absorbing the antiserum with CSA (subfractions 4 to 6), the protective activity against challenge with strain SE-360 was extremely decreased. As shown in Table 3, the protective activity of the antiserum was completely absorbed with 1.0 and 0.1 mg of the ant igen (subfractions 4 and 5); more than 80 % of the mice died after experimental infection.

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Table 2. Sugar components sugar of subfractions from DEAE-Sephadex A25 ion exchange chromatography Fraction number

Mannose

Molar Ratio" Galactose Glucose

Glucosamine

Fr-1 Fr-2 Fr-3 Fr-4 Fr-5 Fr-6

1.65 1.18 0.54 trace ND** ND

1.00 1.00 1.00 1.00 1.00 1.00

2,53 2.77 4.11 1.55 1.65 trace

1.59 0.73 1.54 3.94 9.05 1.31

* Molar ratio was determined by using gas-liquid chromatography ** ND: Not detected

Table 3. Absorption of passive protective activity in rabbit antiserum with the subfractions obtained from DEAE-Sephadex A25 ion exchange chromatography Absorbing dose (ug)

Fr-1

Fr-2

1000 100 Unabsorbed (as control)

0/5*

2/5

Subfractions Fr-3 Fr-4 5/5 3/5

4/5 5/5

Fr-5

Fr-6

5/5 5/5

5/5 2/5

9/10

* Number of dead/Number of challenged

Active immunization of mice with cell surface antigen 10 days after active immunization with CSA (subfractions 4 to 6), mice were challenged with strain SE-360. Immunization of mice with 3, 10 and 30 ug of antigen induced protection against the challenge. Especially, subfraction 5 could be shown to protect more potent than sub fractions 4 and 6 (Table 4).

Table 4. Protective effect of active immunization in mice with the subfractions obtained from DEAE-Sephadex A25 ion exchange chromatography Immunizing dose (ug)

Fr-4

3 10 30 Unimmunized as control

5/5* 3/5 1/5 9/10

* Number of deadlNumber of challenged

Subfractions Fr-5 Fr-6 1/5 0/5 0/5

5/5 5/5 3/5

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Quantitative precipitin reaction of the antigen against rabbit antiserum and its inhibition with monosaccharides Quantitative precipitin reaction of the antigen (subfraction 5) against rabbit antiserum was carried out to determine the optimal ratio. Addition of 50 \11 antiserum to 150 \1g of antigen was found to produce optimal antigen-antibody precipitates {Fig.2). Concerning the antigenic part (of the subfraction 5), quantitative precipitin inhibition was performed with monosaccharides. Thus, a-methyl-D-glucoside and Neacerylglucosamine most effectively inhibited (80% and 52% inhibition, respectively) the precipitin reaction (Table 5).

100..---------,

o

100

200 300

Antigen (~g)

Fig. 2. Quantitative precipitin curve of cell surface antigen (subfraction 5) against anti-SE-360 serum.

Table 5. Quantitative precipitin inhibition of monosaccharides with protection inducing antigen and anti-SE-360 serum Monosugars

Percent inhibition

Fucose Rhamnose Glucose a-Methyl-D-Glucoside ~-Methyl-D-Glucoside Galactose Mannose N-Acetyl-Glucosamine N-Acetyl-Galactosamine Glucuronic acid Galacturonic acid

35.98 2.74 43.84 79.45 28.77 31.51 5.48 52.05 32.88 0.00 5.48

Discussion The bacterial capsule is considered the most important and ubiquitious antiphagocytic substance (Ll). Most extracellular bacteria causing infection are encapsulated and their virulence apparently is closely associated with encapsulation (Ll). Accordingly,

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encapsulated staphylococcal strains have been found to be more resistant to phagocytosis than unencapsulated (1, 7, 12, 21, 32). It might be considered that the antiphagocytic capacity of encapsulated staphylococci is due to a lack of complement fixation on the surface of these organisms. Thus, complement is not available at the surface of these bacteria to interact with phagocyte complement receptors mediating phagocytosis. In experimental infection of mice with encapsulated staphylococci, complement and! or specific antibodies are obligate for attachment to and ingestion by phagocytic cells (26, 28, 29, 34). The attachment of certain particles (i. e. bacteria) to the surface of phagocytes apparently triggers the organization and coordination of contractile elements at the region of attachment. Whereas unencapsulated staphylococci might be ingested in the absence of specific opsonin molecules, encapsulated strains require specific opsonins on the bacterial surface. Generally, CSA such as capsule induce specific antibody production after active immunization in mice (7, 12, 16,23,32). In this study, the isolation and biochemical characterization of an antigen from encapsulated S. epidermidis SE-360 is discussed. This antigen showed protecting activity as a vaccine as well as absorbing activity for specific opsonins against strain SE-360 in rabbit antiserum. Furthermore, type-specific antigenicity could be shown since no cross-reactivity to other CSAs from encapsulated staphylococci was observed. These data suggest that the SE-360 CSA contain high amounts of carbohydrates. The major carbohydrate constituents were galactose, glucose, and N-acetylglucosamine, and the strong reactivity with Con-A suggested that the configuration of glucose is a-linkage. In addition, precipitin inhibition with monosaccharides suggested that a -D-glucosyl- and N-acetyl-glucosaminyl-residues might be the antigenic determinants. Recently, it could be shown that IgM production in mice and human granulocyte function were enhanced after treatment with CSA from encapsulated S. epidermidis ATCC-31432 containing galactose, glucose, N-acetyl-glucosamine and two so far unidentified carbohydrates (23, 24). Since antigen from SE-360 did not show serological cross-reactivity to the antigen of strain ATCC-31432, it might also be considered as immunomodifier activating phagocytic cells similar to the antigen of strain ATCC31432 (24). Interestingly, unique aminosugars such as 2-acetamino-2-deoxyglucuronic acid, 2-acetamino-2-deoxy-galacturonic acid, 2-acetamino-2-deoxy-mannuronic acid and N-acetyl-fucosamine which are common constituents of S. aureus capsule (9, 18,31), could not be detected in CSA of strain SE-360. In conclusion, our experimental data suggest that the antigen isolated from encapsulated S. epidermidis SE-360 mainly consists of carbohydrates; a-D-glucosyl- and Naceryl-glucosaminyl-residues apparently are related to antigenicity whereas protein in the molecule is involved in the production of specific opsonins to promote bacterial killing in mice. Furthermore, it might be supposed that phagocytic cells can be directly activated by this antigen as could be shown for the antigen from S. epidermidis ATCC31432.

Acknowledgements. I am deeply indebted to Professor Dr. Kosaku Yoshida, Chairman, Department of Microbiology, St. Marianna University School of Medicine, for advice and encouragementduring the course of this work; and to ProfessorDr. Gerhard Puluerer, Dr. Josef Beuth and Dr. Hong Lioe Ko, Institute of Hygiene, University of Cologne, for their useful discussion and critical reading of the manuscript.

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