Nutritionally Variant Streptococcus Serotype II - Infection and Immunity

INFECTION AND IMMUNITY, Feb. 1991, p. 592-599 0019-9567/91/020592-08$02.00/0 Copyright © 1991, American Society for Microbiology

Vol. 59, No. 2

Purification and Characterization of Streptococcus adjacens (Nutritionally Variant Streptococcus Serotype II) Group Antigen P. A. SIELING AND I. VAN DE RIJN* Wake Forest University Medical Center, 300 S. Hawthorne Road, Winston-Salem, North Carolina 27103 Received 26 September 1990/Accepted 19 November 1990

Nutritionally variant streptococci (NVS) possess amphiphiles which are serologically distinct from lipoteichoic acid and which serve as group-specific antigens for NVS. The objective of this study was to purify and characterize the NVS serotype II (Streptococcus adjacens) amphiphile. Amphiphile was isolated from stationary-phase culture supernatants of NVS strain 81 (NVS serotype II). Phenol-water extracts of culture supernatants were subjected to hydrophobic interaction chromatography and gel filtration chromatography. A homogeneous preparation of amphiphile (22 mg; 8.5 x 106 hemagglutination units) was recovered, and its approximate molecular size (23,000 to 24,000 Da) and chemical composition were determined. Purified S. adjacens amphiphile contained phosphorus, ribitol, galactose, galactosamine, alanine, and fatty acids in molar ratios of 1.00:0.88:1.39:1.10:0.08:0.24. Since ribitol, galactose, and galactosamine were the primary carbohydrate components, the amphiphile may exist as a polyribitol phosphate with galactose and galactosamine substituents. Preliminary structural analysis demonstrated the presence of phosphodiester bonds within the amphiphile structure. Finally, the amphiphile serves as the S. adjacens group antigen.

identity. These immunological, biochemical, and genetic differences indicate that NVS types I and II are distinct organisms. On the basis of these differences and their distinction from other streptococci, Bouvet et al. (2) have designated serotype I NVS as S. defectivus and serotype II as S. adjacens. Therefore, the serotype antigens now serve as group antigens for the two streptococcal species. NVS are important human pathogens, as evidenced by their occurrence in 5% of streptococcal endocarditis infections (27) and their presence in numerous other bacterial infections (12). Identification of NVS from endocarditis patients can be hindered because they may exist in so-called culture-negative isolates (27). Appropriate antimicrobial therapy may be less effective in the event of delayed diagnosis of NVS infection. Precise identification of NVS would be enhanced through the purification and characterization of group-specific antigens. This report describes the purification and characterization of the S. adjacens amphiphile from NVS. S. adjacens strains were compared for production of both intracellular and extracellular amphiphiles. Maximal amphiphile production during the growth phase was also examined. The S. adjacens amphiphile was purified by hydrophobic interaction chromatography (HIC) and gel filtration chromatography. The purified amphiphile was demonstrated to contain phosphorus, ribitol, galactose, galactosamine, alanine, and fatty acids in a 1.00:0.88:1.39:1.10:0.08:0.24 molar ratio.

Amphiphiles of gram-positive bacteria have various chemical compositions, though each maintains a common structural pattern of hydrophilic and hydrophobic domains (43). Lipoteichoic acids (LTAs) of many streptococci are composed of a 1-3-linked poly(glycerol phosphate) backbone substituted periodically at the C-2 position of the glycerol molecule with glucose, galactose, or D-alanine in ester linkage. Covalently attached to a terminal glycerol residue in phosphodiester linkage is either a glycolipid, phosphatidylglycolipid, or a fatty acid (8, 42). Streptococcus mitis is one example of a number of gram-positive bacteria lacking the poly(glycerol phosphate) of LTA (29). Nutritionally variant streptococci (NVS) possess amphiphiles with serological properties distinct from those of LTA (3). The serotype I amphiphile from NVS was previously purified and characterized as a lipid-substituted poly(ribitol phosphate) (10). Presumably, the amphiphile's hydrophobic portion anchors in the cytoplasmic membrane while the hydrophilic region extends through the cell wall and beyond, giving amphiphiles a cell surface location (38). Amphiphiles have also been found in the culture supernatants of gram-positive bacteria (17, 25). Their heterogeneous chemical nature and surface location make amphiphiles useful serological markers. Indeed, the group antigens of Streptococcus groups D and N and Lactobacillus group F are LTAs (6, 20, 41). NVS have been classified serologically by van de Rijn and George, with serotypes I and II comprising 97% of isolates (37). These serological differences corresponded to immunological differences when examined in an experimental animal model of bacterial endocarditis (35). Also, serotypes I and II were distinguishable by the ability of serotype I to produce ,-galactosidase and serotype II to produce 0-glucuronidase (4). Further evidence for differentiation of the two NVS types was recently published by Bouvet et al. (2). In their investigation, serotypes I and II were compared by chromosomal DNA hybridization and found to share less than 10%

MATERIALS AND METHODS Bacterial strains. Stock cultures of NVS strains were kept frozen at -70°C in a 50% (vol/vol) solution of chemically defined medium supplemented with Todd-Hewitt dialysate (CDMT) (3) and glycerol. The identity of NVS isolates was determined by the ability of the strains to form satellite colonies around colonies of Staphylococcus epidermidis and by the presence of a pH-dependent chromophore (36). Bacterial maintenance. NVS strains were grown in CDMT at 37°C without shaking. The growth of streptococcal cul-

* Corresponding author.

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tures was measured in 18-mm tubes by using a Spectronic 20 spectrophotometer at a wavelength of 650 nm. Purification of amphiphile. Amphiphile purification was based on the method used to purify the NVS serotype I antigen (now S. defectivus group antigen) (11). Bacteria (360 liters) were grown to stationary phase (30 h) at 37°C and were sedimented in a refrigerated high-speed centrifuge (Sharples-Stokes Div., Pennwalt Corp., Warminster, Pa.). The culture supernatant was then concentrated (with a DC101 concentrator and a HlOP30-20 hollow fiber cartridge; Amicon Corp., Lexington, Mass.) and dialyzed against deionized water. The concentrated supernatant was centrifuged (7,000 x g, 20 min), filtered through a 0.45-,um-poresize filter, resedimented to remove any remaining bacteria, and then lyophilized. Lyophilized culture supernatant was rehydrated in 800 ml of sodium phosphate buffer, pH 7.0. DNase and RNase (40 mg each; Sigma Chemical Co., St. Louis, Mo.) were added, and the solution was incubated for 2 h at 37°C with stirring, followed by an identical 2-h treatment with 40 mg of trypsin (Sigma). Enzyme-treated culture supernatant was then extracted with an equal volume of 90% phenol at 68°C by the method of Kessler and Shockman (18). Lyophilized phenol-water extracts of amphiphile were rehydrated in 0.1 M sodium acetate-15% 1-propanol, pH 6.0, at a concentration of 10 mg/ml and loaded onto a column (2.5 by 34 cm) of octyl-Sepharose (CL-4B; Sigma) for HIC. After loading the column with extract (10 ml/h), unbound material was removed with two column volumes of the column buffer and eluted at a flow rate of 20 ml/h with two column volumes of 0.1 M sodium acetate-50% 1-propanol, pH 6.0. Fractions (5 ml) were collected throughout the chromatographic process and analyzed for sheep erythrocyte (SRBC) sensitization activity, phosphorus, and hexose. Column fractions containing SRBC sensitization activity were pooled and diluted with 0.1 M sodium acetate buffer (pH 6.0) to restore the propanol concentration to 15%. HIC was repeated on the pooled fractions under the conditions described above until phosphorus and hexose were no longer detected in fall-through fractions (three times total). Fractions with SRBC sensitization activity were pooled, dialyzed against deionized water (48 h at 4°C with six changes), and lyophilized. Amphiphile preparation isolated via HIC was rehydrated in 0.1 M sodium acetate-0.5% Chapso, pH 6.5, (4 mg/ml) and loaded onto a Sephadex G-75 (Sigma) gel filtration column (2.5 by 39 cm). Amphiphile was eluted with the same buffer (10 mlIh), and fractions (5 ml) were collected. SRBC sensitizing fractions were pooled, dialyzed against water, and lyophilized. Amphiphile was rechromatographed on octylSepharose as described above to remove detergent from the preparation. Determination of amphiphile homogeneity. (i) RIE and CIE. Rocket immunoelectrophoresis (RIE) and crossed immunoelectrophoresis (CIE) of S. adjacens amphiphile were performed with sodium barbital buffer (ionic strength, 0.02; pH 8.8) in the presence or absence of 1% (vol/vol) Triton X-100 (19). The immunoprecipitates were visualized by staining with 0.5% (wt/vol) Coomassie brilliant blue in 45% ethanol10% acetic acid (vol/vol). (ii) SDS-PAGE. S. adjacens amphiphile was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by the system of Laemmli (21). Resolving gels (12.5% acrylamide) were prepared in Laemmli buffers containing 0.002 M EDTA. Standards (lipopolysaccharide [LPS] and LTA) and samples mixed with sample buffer (0.15 M

S. ADJACENS GROUP ANTIGEN

593

Tris [pH 6.8], 5% SDS, 25% glycerol, bromphenol blue) were boiled for 3 min before loading onto the gel. Samples were electrophoresed at 40 mA and silver stained (26). Amphiphile molecular size determination. Purified NVS-81 amphiphile (53 ,ug) was dissolved in 0.1 M sodium acetate0.5% Chapso, pH 6.5, and loaded onto a column of Sephacryl S-200 (0.7 by 28.5 cm) equilibrated in the same buffer. Amphiphile was eluted from the column with equilibration buffer at a flow rate of 6 ml/h. Fractions (0.5 ml) were analyzed for antigenic activity by RIE and SRBC sensitization activity. Molecular size determination was performed by chromatographing dextrans (average molecular weights, 66,300, 40,200, and 10,000 Da) on the S-200 column under the same conditions as the amphiphile. Peak elution volumes were converted to Kav, and a standard curve was generated from these values. Amphiphile chemical composition. (i) Hexose. Hexose was determined by the phenol-sulfuric acid method of Dubois et al. (5) by using a glucose standard. The standards and samples were measured in triplicate and read against a buffer blank. (ii) Phosphorus. The acid-soluble and inorganic-plus-labile phosphorus content of the samples was determined by the method of Lowry et al. (23) with a KH2PO4 standard. The standards and samples were measured in triplicate. (iii) Protein. Protein quantity was determined by the Lowry protein assay (24) with bovine serum albumin as a standard. (iv) Nucleic acid. Nucleic acid content was measured by scanning the A850 to A200 of amphiphile preparations. Streptococcal DNA was used as a standard for quantitation purposes. (v) Glycosides and glycitols. Neutral sugars were identified and quantitated as their alditol acetates (13) after hydrolysis of the amphiphile in 2 N trifluoroacetic acid for 90 min at 120°C. Fatty acids were extracted with hexane before derivatization of the sample. To ensure complete quantitation of polyalcohols and sugars, the hydrolyzed samples were treated with phosphomonoesterase (see below). Ribitol was the only new peak detected after this treatment. Monosaccharides released by trifluoroacetic acid hydrolysis were reduced with 0.6 N NaBH4 in 1 N NH40H (60 min, room temperature). Excess borohydride was removed with glacial acetic acid, and the resulting borate was removed by dissolving in 1 ml of methanol followed by evaporation (repeated five times). Acetylation was performed by incubating samples in the presence of acetic anhydride (250 ,lI) for 30 min at 120°C. Derivatized sugars were then chromatographed on a 15-m fused silica capillary column (SP2330; Supelco, Inc., Bellefonte, Pa.) in a Carlo Erba Strumentazione gas chromatograph with helium as the carrier gas (2.7 ml/min). The chromatograph was equipped with a flame ionization detector set at 295°C and a manual injection port set at 245°C. Oven temperatures were programmed as gradients from 100 to 245°C (30°C/min) with a 1-min initial hold and an 18-min final hold. The identification of sugars was based on comparison of retention times of known standards. Amphiphile components were quantitated with the Maxima 820 software system (Waters Associates, Inc., Milford, Mass.) with xylose as the internal standard. (vi) Fatty acids. Fatty acids were detected and quantitated as their methyl esters by a modification of the method of

Rogozinski (28). Samples

were

chromatographed on a 30-m

fused silica column coated with DB-225 (J and W Scientific, Inc., Folsom, Calif.) in a Hewlett-Packard 5890A gas chromatograph with helium as the carrier gas (1 ml/min). The

594

SIELING AND VAN DE RIJN

chromatograph was equipped with a flame ionization detector at 280°C and a manual injection port at 230°C. Oven temperatures were programmed as gradients from 165 to 226°C (3°C/min) with an initial hold of 2.0 min. Unknowns were identified by comparisons with reference standards. Heptadecanoic acid, added as an internal standard, allowed for quantitation of identified fatty acids. (vii) Amino acids and amino sugars. Amino acids and amino sugars were identified as their phenylisothiocarbamyl (PITC) derivatives and quantitated by high-performance liquid chromatography (HPLC) by using the PicoTag method (1). Amphiphile (100 pug) was hydrolyzed in 6 N HCI (vapor phase) for 18 to 24 h at 110°C. Hydrolysates were labeled with PITC and chromatographed on an RCM column (8 by 10) (Waters) with a Nova-Pak C18 packing at 42°C according to the procedure of Bidlingmeyer et al. (1). PITC derivatives were detected at 254 nm. All samples were assayed in triplicate and identified and quantitated on the basis of retention times and peak areas of reference standards. The limit of quantitation for amino acids was 25 pmol. A standard curve for quantitation of galactosamine was derived by treating known amounts of galactosamine under the same conditions as amphiphile. Phosphomonoesterase treatment of S. adjacens amphiphile. S. adjacens amphiphile (100 nmol of phosphorus) was dissolved in a buffer of 0.1 M glycine, 1 mM MgCl2, and 1 mM ZnCl2 (pH 10.4). Phosphomonoesterase (0.5 U) (bovine alkaline phosphatase; Sigma) was added, and the mixture (500 ,ul) was incubated at 37°C for 60 min. Phosphatasetreated amphiphile was examined for the release of inorganic phosphate, as described above, compared with the amount of inorganic phosphate present in untreated amphiphile (incubated with buffer alone). Preparation of antisera. (i) Streptococcal whole cell sera. Bacterial strains to be used as inocula for immunization were grown to stationary phase and killed by exposure to a UV light source for 30 min. Rabbits were immunized by the protocol described by Rotta et al. (30). (ii) NVS culture supernatant antisera. Animals were vaccinated with 1.0 ml (administered in four aliquots at four different sites) of an emulsion of the culture supernatant (5 mg/ml in water) and Freund complete (first vaccination) or incomplete (subsequent vaccinations) adjuvant. Immunization was performed weekly for 3 weeks. At week 3, the rabbits were bled and immunized for 6 to 9 additional weeks. Culture supernatant antisera were also produced by the double adjuvant method described below. (iii) Antisera against partially purified amphiphile. Amphiphile (1 mg/ml) isolated by HIC was mixed with methylated bovine serum albumin (1 mglml). The conjugate was then mixed with Freund incomplete adjuvant (1:1, vol/vol), followed by the addition of 2% Tween 80 in saline (1:1 ratio, vol/vol) (7). The double adjuvant mixture (1 ml) was administered to rabbits intravenously once a week for the first 3 weeks. Rabbits were bled at week 3 and bled and immunized for 6 to 9 additional weeks. HA assays. Hemagglutination (HA) titers of antiamphiphile antisera were determined in duplicate by previously published methods (16). Crude amphiphile preparations (10 ,ug/ml, final concentration) were added to a 2% solution of SRBCs. Sensitization of the SRBC solution was performed at 37°C for 60 min, followed by 4°C for 16 h. Sensitized SRBCs were then washed with saline to remove unbound amphiphile and were resuspended at a 1% concentration. Anti-S. adjacens antiserum titers were determined by serial dilution of antisera in microtiter plates followed by addition

INFECT. IMMUN.

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80

120

160

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Fraction No. FIG. 1. HIC of NVS-81 amphiphile. Octyl-Sepharose columns (2.5 by 34 cm) were prepared as described in Materials and Methods. Phenol-water extracts (1.3 g per column) of NVS-81 were dissolved in 0.1 M sodium acetate-15% 1-propanol, pH 6.0, and loaded onto the first octyl-Sepharose column. After loading, the column was washed with equilibration buffer followed by elution buffer (0.1 M sodium acetate, 50% 1-propanol, pH 6.0), as indicated by the arrow. Fractions were analyzed for total phosphorus (A), hexose (0), and SRBC sensitization activity (0) (A). Peak SRBC sensitizing fractions were pooled, diluted to 15% 1-propanol with 0.1 M sodium acetate, and loaded onto the next column. Removal of unbound material and elution were repeated as described above. HIC was performed three times until a single peak of phosphorus, hexose, and SRBC sensitization activity Was eluted (B).

of the sensitized SRBCs. The suspension was mixed and then incubated for 60 min at 37°C. Titers were quantitated as the reciprocal of the highest dilution producing complete HA. Amphiphile concentration was determined semiquantitatively by using a microtiter plate method (31). Serially diluted amphiphile preparations were incubated with a 1% suspension of SRBCs for 20 min at 37°C with agitation. Microtiter pla-tes were then transferred to 4°C for 10 min and subsequently washed with saline. Anti-S. adjacens antiserum equivalent to 4 HA units was added to the sensitized SRBCs and incubated for 60 min at 37°C. Amphiphile was quantitated as the reciprocal of the highest dilution causing complete HA.

S. ADJACENS GROUP ANTIGEN

VOL. 59, 1991

RESULTS

30

30

595

Purification of S. adjacens amphiphile. Preliminary experiments were designed to ascertain the S. adjacens strain 0 which produced the most amphiphile, the growth phase for 20 optimal amphiphile production, and the quantity of cellassociated versus extracellular amphiphile. On the basis of results of these preliminary experiments, S. adjacens amwas purified from 360 liters of strain NVS-81 phiphile .0 10 stationary-phase culture supernatant. Enzyme-treated phenol-water extracts were subjected to HIC on octyl-Sepharose. Figure 1A (first HIC column) illustrates that fall0~ through fractions contained contaminating hexose and a* phosphorus. The SRBC-sensitizing fractions were pooled X _0X'_* and rerun on two successive HIC columns. By the third HIC 40 24 32 0 8 16 column (Fig. 1B), amphiphile migrated as a single peak Fraction No. composed of SRBC sensitization activity, hexose, and phosphorus. FIG. 2. Gel filtration chromatography (Sephadex G-75) of NVS-81 amphiphile. HIC-isolated NVS-81 amphiphile (4 mg) was Though HIC yielded a single peak, gel filtration chromarehydrated in 0.1 M sodium acetate-0.5% Chapso (pH 6.5) and tography on Sephadex G-75 revealed two distinct peaks (Fig. loaded onto a column of Sephadex G-75 (2.5 by 39 cm) equilibrated 2) when examined by the SRBC sensitization assay and CIE. in the same buffer. Amphiphile was eluted from the column, and A possessed SRBC sensitization activity, indicating the Peak fractions (5 ml) were collected and analyzed for antigenic activity by of amphiphile, whereas peak B had less than 1% of presence CIE (O and A), expressed as peak height in millimeters, and SRBC the SRBC sensitization activity of peak A. Amphiphile sensitization activity (0). Fractions containing both SRBC sensitizfractions (14 to 17, peak A) were pooled and examined ing activity and antigenic activity (peak A, fractions 14 to 17) were further to determine the homogeneity of the purified ampooled as amphiphile, whereas fractions 18 to 20 were pooled and phiphile. Fractions containing both amphiphile and contamrechromatographed to recover additional amphiphile. Peak B contained less than 1% of the SRBC sensitization activity of peak A and inating antigen (fractions 18 to 20) were pooled and rechrowas not analyzed further. VO, Voided volume; VT, total volume. matographed to isolate additional amphiphile. 4

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FIG. 3. CIE analysis of NVS-81 amphiphile preparations. Samples of NVS-81 culture supernatants (1.5 ,ug of amphiphile in 5 ,ul) were added to a slide containing 1% agarose prepared in sodium barbital buffer with 1% Triton X-100. Electrophoresis in the second dimension took place in antiserum-containing agarose (anti-NVS-81 culture supernatant antiserum [panels A and C to El and anti-NVS-81 phenol-water extract [panel B]). (A) NVS-81 culture supernatant; (B) phenol-water extract; (C) HIC preparation; (D) Sephadex G-75-purified amphiphile; (E) Sephadex G-75-purified amphiphile in the absence of Triton X-100.

SIELING AND

596

FIG. 4.

SDS-PAGE

amphiphile (1.5 amide

p.g;

VAN DE

of NVS-81

right lane)

gel. Electrophoresis

was

INFECT. IMMUN.

RIJN

was

Purified

amphiphile. loaded onto

performed

a

NVS-81

12.5% polyacryl-

at 40 mA, after which the

by the method of Morrissey (26). LPS (1.5 p.g; left lane) and LTA (0.25 jig; center lane) were electrophoresed adjacent to the amphiphile for comparison.

gel

was

silver stained

Demonstration of a homogeneous preparation of S. adjacens amphiphile. CIE was performed to detect antigenic species of the amphiphile preparation throughout the purification process. Amphiphile preparations were electrophoresed in agarose containing anti-S. adjacens antiserum. The CIE slides (Fig. 3) demonstrate that more than 15 antigens were precipitated in crude culture supernatant (Fig. 3A) and at least 3 were precipitated in the phenol-water extract (Fig. 3B). CIE of HIC-isolated amphiphile in the presence of Triton X-100 (Fig. 3C) revealed two antigens in the amphiphile preparation. The use of Triton X-100 in CIE of HIC-purified amphiphile was necessary, since in the absence of detergent the contaminating antigen was undetectable. A similar phenomenon was observed in the purification of the S. defectivus amphiphile (11). Analysis of G-75-purified amphiphile by GTE revealed a single antigen in the presence or absence (Fig. 3D and E, respectively) of Triton X-100. CIE analysis with four additional anti-NVS-81 antisera also

precipitated a single antigen from the G-75-purified material, confirming the antigenically homogeneous nature of the amphiphile (data not shown). RIE analysis also detected a single antigen when purified amphiphile was titrated to a quantity at which 1% protein contamination would be detectable. To further analyze the homogeneity of the purified amphiphile, SDS-PAGE was performed (Fig. 4). Amphiphile (1.5 ,ug; Fig. 4, right lane) was run on a 12.5% polyacrylamide gel, and the gel was silver stained by the method of Morrissey (26). SDS-PAGE of LPS (left lane) produced a characteristic stepladder-like staining pattern, whereas LTA (center lane) produced only a single, diffuse band. S. adjacens amphiphile produced a staining pattern which resembled that of LPS. Purification summary. S. adjacens amphiphile was quantitated at each purification step for its HA activity (SRBC sensitization) and RIE peak height. Additionally, total phosphorus and hexose were examined at each purification step. The results from these analyses from one of two independent preparations are presented in Table 1. From 360 liters of NVS-81 stationary-phase culture supernatant, 1.8 x 108 HA units of the S. adjacens amphiphile was present in crude form. Nuclease and protease treatments of the culture supernatant, followed by a phenol-water extraction, reduced the protein level to less than 0.2% of the amphiphile. The amount of amphiphile recovered, 138 mg, represented 23% of the amphiphile present in the starting material. HIC and gel filtration chromatography were used to remove phosphorus- and hexose-containing contaminants. These chromatographic methods also reduced nucleic acid below quantifiable levels (